PhD Chang Xian Zhang
email: chang.zhang@lyon.unicancer.fr

Muriel Le Romancer
email: muriel.leromancer-cherifi@lyon.unicancer.fr

MS Ya-Kun Luo
email: yakun.luo@lyon.unicancer.fr

Virginie Vlaeminck-Guillem
email: virginie.vlaeminck-guillem@inserm.fr

Silvère Baron

Sarah Dallel

Romain Teinturier

Myriam Decaussin-Petrucci

Francis Vacherot

Virginie Firlej

Rémy Bonnavion

Razan Abou
email: razan.abouziki@lyon.unicancer.fr

Samuele Gherardi

Isabelle Goddard

Nicolas Gadot

Philippe Bertolino

MS Razan A Ziki

JunD and β-catenin 116

Keywords: Prostate cancer, the MEN1 gene, AR regulation, MYC, EZH2, PCAT1 breast cancer, prostate cancer, estrogen receptor alpha, androgen receptor, the MEN1 gene Mouse models, The MEN1 gene, Tumorigenesis

MEN1 mutations predispose patients to multiple endocrine neoplasia type 1 (MEN1), a genetic syndrome associated with the predominant co-occurrence of endocrine tumors. Accumulating evidence suggests that menin, the MEN1 gene product, could also be involved in the development of prostate cancer, a major hormone-related cancer. Intriguingly, our previous study found that menin plays dual role in prostate cancer (PCa) cells.

The androgen receptor (AR) acts as a vital transcription factor in development and progression of PCa. Our previous results unveiled that menin is involved in the transcriptional regulation of AR in AR-dependent PCa cells. Nevertheless, the role and mechanism of menin in prostate cancer still remain poorly understood.

The aim of my thesis project was to dissect the molecular mechanisms underlying the biological role of menin respectively in AR-dependent and AR-independent prostate cancer cells. In the first part of my works, our data demonstrated that menin exerts oncogenic effects by modulating AR transcription specifically in AR-dependent PCa cells. Furthermore, different analyses revealed that menin exerted a critical role on the MYC binding site on the AR promoter and was essential for the regulation of MYC-mediated AR transcription. Interestingly, my work also showed that menin was involved in the regulation of MYC-related LncRNA PCAT1 in AR-dependent PCa cells, through both its positive regulation of EZH2 transcription and its physical interaction with EZH2, as well as their co-binding on the regulatory sequence of PCAT1 in AR-dependent PCa cells.

In the second part, my work was to determine its role in AR-independent PCa cells. Our results indicated that MEN1 silencing in AR-independent cells resulted in an increase in cell anchorage independence and cell migration, accompanied by sustained MYC expression and the nuclear translocation of both JunD and β-catenin.

Moreover, the expression of several molecular markers of EMT and stemness was altered after MEN1 silencing in AR-independent cells. In addition, the analyses using cultured cell and PC3-GFP xenografts in the mouse demonstrated that both JunD and β-catenin are necessary for the altered tumorigenic potential triggered by MEN1 inactivation in AR-independent PCa cells. Thus, firstly, my thesis works uncovered an essential role of menin in regulating MYC transcription, MYC-mediated AR transcription and the MYC pathway, involving PCAT1 regulation via EZH2, specifically in AR-dependent PCa cells.

Furthermore, the work highlighted an unrecognized oncosuppressive role of menin specifically in AR-independent PCa cells, via regulating the JunD and β-catenin pathways.

Résumé

Les mutations du gène MEN1 prédisposent les patients à la néoplasie endocrinienne multiple de type 1 (MEN1), un syndrome génétique qui peut entrainer la survenue de tumeurs endocrines. De plus en plus de preuves ont suggéré que la ménine, la protéine codée par le gène MEN1, pourrait également être impliquée dans le développement de cancers de la prostate, un cancer hormono-dépendant majeur. De manière intéressante, une étude que nous avons précédemment menée a révélé que la ménine joue un double rôle dans les cellules du cancer de la prostate (PCa).

Le récepteur aux androgènes (AR) agit comme un facteur de transcription essentiel dans le développement et la progression du PCa. L'étude précédemment que nous avons menée a démontré que la ménine est impliquée dans la régulation transcriptionnelle d'AR dans les cellules PCa AR-dépendantes. Néanmoins, le rôle et le mécanisme de la ménine dans le PCa restent encore mal compris. L'objectif de mon projet de thèse était de disséquer les mécanismes moléculaires sous-jacents du rôle biologique de la ménine dans les cellules cancéreuses de la prostate AR-dépendantes et AR-indépendantes. La première partie mes travaux a démontré que la ménine exerce des effets oncogènes en modulant la transcription d'AR, spécifiquement dans les cellules PCa AR-dépendantes. De plus, différentes analyses ont révélé que la ménine a un rôle critique sur le site de liaison de MYC sur le promoteur d'AR, et qu'elle est essentielle pour la régulation de la transcription de AR médiée par MYC. De manière intéressante, mes travaux ont également démontré que la ménine est impliquée dans la régulation du ARNlnc PCAT1 lié à MYC dans les cellules PCa AR-dépendantes, à la fois par sa régulation positive de la transcription d'EZH2 et par son interaction physique avec EZH2, ainsi que par leur co-liaison sur la séquence régulatrice de PCAT1 dans les cellules PCa AR-dépendantes.

La deuxième partie de mes travaux vise à déterminer le rôle de la ménin dans les cellules PCa AR-indépendantes. Mes résultats ont indiqué que l'inactivation de MEN1 dans les cellules AR-indépendantes entraîne une augmentation de la croissance cellulaire sans adhésion et de la migration cellulaire, accompagnée d'une surexpression de MYC, et de la translocation nucléaire à la fois de JunD et de la β-caténine. De plus, l'expression de plusieurs marqueurs moléculaires de la TEM et des cellules souches a été modifiée dans les cellules AR-indépendantes suite à l'inhibition de l'expression de MEN1. En outre, les analyses dans des lignées cellulaires en culture et dans un modèle murin xénogreffé PC3-GFP ont démontré que JunD et la β-caténine sont nécessaires à l'altération du potentiel tumorigène induite par l'inactivation de MEN1 dans les cellules PCa AR-indépendantes.

Ainsi, mes travaux de thèse ont permis de mettre en évidence un rôle essentiel de la ménine dans la régulation de la transcription de MYC, de la transcription d'AR médiée par MYC, et de la voie MYC, impliquant la régulation PCAT1 via EZH2, spécifiquement dans les cellules PCa AR-dépendantes. De plus, mes travaux ont révélé un rôle oncosuppresseur jusqu'ici inconnu de la ménine spécifiquement dans les cellules PCa AR-indépendantes, via la régulation des voies JunD et la β-caténine.

ChapterⅠ Introduction

The prostate 1.1 Anatomy

The prostate is the male sexual accessory gland. It is located on the floor of the pelvis and surrounds the neck of the bladder and urethra (Fig. 1.1). In men, the urethra serves two purposes; urination and ejaculation. It runs from the bladder through the prostate and to the tip of the penis. The section of the urethra running through the prostate is known as the prostatic urethra. After being produced in the testicles, sperm moves into a coiled mass, known as the epididymis for maturation. It then goes into two muscular tubes known as the vas deferens, which coil around the bladder and seminal vesicles. The seminal vesicle can house the sperm for several days until ejaculation. During ejaculation, the prostate muscles contract and the sperm into the prostatic urethra towards the tip of the penis [START_REF] Blandy | The Prostate[END_REF][START_REF] Humera | Determinants of prostate cancer: the Birmingham Prostatic Neoplasms Association study[END_REF].

The average weight of a healthy prostate is approximately 11 grams, ranging between 7 and 16 grams [START_REF] Leissner | The weight of the human prostate[END_REF]. It is encapsulated by a fibroelastic tissue layer, leading to septa extending inwards and dividing the prostate into different lobes. The lobes accommodate nearly 50 irregularly branched saccular glands, https://www.cancer.gov/types/prostate/understanding-prostate-changes excretory ducts, stroma (connective tissue cells), blood vessels and nerves. This transitional epithelium or urothelium has the ability to contract and expand according to the volume of fluid within [START_REF] Cohen | Central zone carcinoma of the prostate gland: a distinct tumor type with poor prognostic features[END_REF].

It has been described four histologically distinct zones within the prostate (Fig. 1.2). The peripheral zone is found postero-laterally and forms 70% of the prostate.

Approximately 70-80% of prostate cancers arise in the peripheral zone. The central zone forms 25% of the prostate and is positioned anterior to the peripheral zone.

Fewer than 5% of prostate cancers arise in the central zone.

The transitional zone surrounds the prostatic urethra and is the exclusive site of origin of BPH. It forms only 5% of the prostate but approximately 10% of prostate cancers arise here. Finally, the anterior fibro-muscular zone is devoid of glandular components, and forms the anterior surface of the prostate [START_REF] Cohen | Central zone carcinoma of the prostate gland: a distinct tumor type with poor prognostic features[END_REF].

The main male hormone is testosterone and is produced in the testicles. The prostate is regulated by dihydrotestosterone, which is synthesized from testosterone in the peripheral tissue [START_REF] Blandy | The Prostate[END_REF].

Prostate Histology

Within the prostatic epithelium, there are at least three distinct cell types that can be distinguished by their morphological characteristics, functional significance and relevance for carcinogenesis (Fig. 1.3). The glandular lumen is lined by secretory luminal epithelial cells which express the androgen receptor (AR). These tall, columnar epithelial cells secrete prostatic acid phosphatase (PAP), PSA and human kallikrein-2 into the lumen of the gland to form seminal fluid. Basal epithelial cells separate luminal epithelial cells from the basement membrane and are thought to secrete components of the basement membrane. These low, cuboidal epithelial cells have low expression of AR. Amongst the basal cell population, it is proposed that progenitor stem cells produce prostatic epithelial cells via intermediate cell stages [START_REF] Van Leenders | Demonstration of intermediate cells during human prostate epithelial differentiation in situ and in vitro using triple-staining confocal scanning microscopy[END_REF]. Neuroendocrine cells, the third epithelial cell type found within the prostate, are irregularly distributed throughout the glands. They do not express AR and their role is not fully understood.

Prostate function

The prostate gland is located in front of the rectum, just below the urinary bladder, and surrounds the urethra. The main function of the prostate is to synthesize and secrete proteins and fluids that, together with contributions from the seminal vesicles, form most of the ejaculate. Although the prostate is involved in fertility, it is not required for reproduction. The major protein produced by the prostate is a protease, prostate specific antigen (PSA) that helps to liquefy the semen so that the sperms way to the egg is facilitated (Balk et al., 2003). Normally PSA is secreted into the prostate Note that the rare neuroendocrine cells are morphologically indistinguishable from basal cells. Obtained from: [START_REF] Abate-Shen | Molecular genetics of prostate cancer[END_REF] lumen, transported to the urethra and removed during ejaculation. During conditions such as prostate cancer, benign prostate hyperplasia (BPH) and inflammation, the basal epithelial layer and basal membrane are disrupted and PSA leaks into the surrounding stroma and vasculature. Thereby PSA can be elevated in the blood and used as a diagnostic marker for prostate diseases [START_REF] Brawer | Prostate-specific antigen in management of prostatic carcinoma[END_REF].

Prostate cancer 2.1 General background

Prostate cancer, with its near 1,414,259 new cases and more than 375, 304 deaths reported in 2020 in the world (Sung et al., 2021), represents continuously a major cause of cancer-related mortality and morbidity in men worldwide. Prostate cancer is mainly a disease of the elderly and most of the men are diagnosed with prostate cancer between ages of 70-74 years, while the majority of prostate cancer deaths occur in men over 79 years. The single most significant risk factor for developing prostate cancer is advanced age. The incidence and mortality for prostate cancer varies in different regions around the world and the environment and diet/lifestyle could be important factors that may explain those differences. In addition, the genetic background may also affect the disease risk [START_REF] Lichtenstein | Environmental and heritable factors in the causation of cancer--analyses of cohorts of twins from Sweden, Denmark, and Finland[END_REF][START_REF] Sun | Genetic variability in inflammation pathways and prostate cancer risk[END_REF][START_REF] Rebbeck | Prostate cancer genetics: variation by race, ethnicity, and geography[END_REF][START_REF] Pernar | The epidemiology of prostate cancer[END_REF].

Clinical and histopathological aspects of prostate hyperplasia, neoplasia and cancer

Benign Prostatic Hyperplasia (BHP)

The prostate is the only internal organ in men that continues to grow throughout adulthood. In BPH, there is benign proliferation of both stromal and epithelial components of the prostate, occurring exclusively in the transitional zone (Figure 1.4).

As the transitional zone enlarges, there is compression of the peripheral zone.

Androgens are known to play a role in BPH; castrated pre-pubescent males do not develop BPH in later life. BPH is so common in men, that it is viewed by many as a normal part of the ageing process. It is estimated that 20% of men in their 40s will have BPH, rising to 80-90% of men in their 70s and 80s [START_REF] Roehrborn | Benign prostatic hyperplasia: an overview[END_REF]. The clinical manifestation of BPH is with bothersome lower urinary tract symptoms (LUTS) comprised of urinary frequency, urinary urgency, hesitancy and incomplete bladder emptying. Complications of BPH include recurrent urinary tract infection (UTI), bladder calculi and acute urinary retention. There is no association between BPH and the later development of prostate cancer [START_REF] Schenk | Association of symptomatic benign prostatic hyperplasia and prostate cancer: results from the prostate cancer prevention trial[END_REF]. Treatment for BPH includes conservative management with lifestyle advice initially where appropriate, before progressing to medical treatment and/or surgery if required.

Medical management is with 5-alpha reductase inhibitors, which inhibits the conversion of testosterone to the more potent dihydrotestosterone (DHT), and alpha-blockers, which cause prostatic smooth muscle relaxation. The mainstay of surgical management in BPH is transurethral resection of the prostate (TURP), however this is becoming less common with the success of medical management.

High-grade Prostatic Intraepithelial Neoplasia (HGPIN)

Known as prostatic intraepithelial neoplasia (PIN) [START_REF] Mcneal | Intraductal dysplasia: a premalignant lesion of the prostate[END_REF], this lesion can be classified into four common architectural types: tufting, micropapillary, http://www.auanet.org/education/auauniversity/education-products-and-resources/pathologyfor-urologi cribiform, and flat [START_REF] Bostwick | Prostatic intra-epithelial neoplasia and early invasion in prostate cancer[END_REF][START_REF] Nagle | Phenotypic relationships of prostatic intraepithelial neoplasia to invasive prostatic carcinoma[END_REF]Bostwick, 1999).

PIN is recognized as a continuum between low-grade and high-grade forms, with high-grade PIN thought to represent the immediate precursor of early invasive carcinoma.

High-grade prostatic intraepithelial neoplasia (HGPIN) is an asymptomatic, pathological entity that is a premalignant lesion of prostate adenocarcinoma. There is increased proliferation of luminal epithelial cells, with complete or partial preservation of the basal cell layer but no invasion into the surrounding stroma. In addition, it is characterised by the presence of atypical epithelial cells with prominent nucleoli, increased nuclear-to-cytoplasmic ratio and increased nuclear size. (Figure 1.5). As with prostate cancer, HGPIN is most commonly identified in the peripheral zone and can be multifocal. HGPIN is more common with advancing age [START_REF] Bostwick | High-grade prostatic intraepithelial neoplasia[END_REF]. There is no evidence that HGPIN causes elevation of serum PSA.

HGPIN has been found to be independently associated with increased risk of developing prostate cancer, particularly if multifocal [START_REF] Merrimen | Multifocal high grade prostatic intraepithelial neoplasia is a significant risk factor for prostatic adenocarcinoma[END_REF]. As such, patients with HGPIN in ≥3 biopsy sites on an otherwise negative prostate biopsy are recommended to have repeat biopsy [START_REF] Mottet | EAU-ESTROSIOG Guidelines on Prostate Cancer. Part 1: Screening, Diagnosis, and Local Treatment with Curative Intent[END_REF]. [START_REF] Bostwick | High-grade prostatic intraepithelial neoplasia[END_REF] The majority of primary PCa, approaching to 99%, is prostatic adenocarcinoma. Its histological diagnosis is based on the assessment for the three major criteria: glandular architecture, loss of basal cells and nuclear features of the glandular lining cells (Table 1.1) [START_REF] Mostofi | A pathologist's view of prostatic carcinoma[END_REF][START_REF] Humphrey | Histological variants of prostatic carcinoma and their significance[END_REF].

Primary PCa

For primary PCa, histologically, basal cells are lost and epithelial cells become more proliferative and less differentiated. The emerging atypical cells develop aberrant nuclei and nucleoli, and are less able to form glandular structures, instead clumping into sheets of malignant cells. There is also a change in protein and receptor expression, leading to altered communication with surrounding cells. Indeed, stromal paracrine signals can promote malignant proliferation and control anti-survival signals in cancer cells [START_REF] Picard | Fibroblast-dependent tumorigenicity of cells in nude mice: implication for implantation of metastases[END_REF]Miller et al., 1989).

PCa invasion and metastasis

Prostate cancer invasion is the spread of the cancer cells out of the prostate glands into nearby tissues, and to further distant sites such as lymph nodes, lung and bone.

The process begins with biological changes within the cancer cells, which enhance the capacity of, or excite the potential for, cancer cell movement and lead to Adapt from: Humphrey, 2017 local invasion. This is followed by intravasation into blood and lymphatic vessels, transit through the invaded circulatory system, and extravasation from the vessel into the parenchyma of distinct tissue. In the secondary site, small nodules of cancer cells form and grow from micro-metastases into large macro-metastatic lesions (Hanahan and Weinberg, 2011;[START_REF] Leach | Stromal androgen receptor regulates the composition of the microenvironment to influence prostate cancer outcome[END_REF][START_REF] Leach | Stromal Androgen Receptor in Prostate Cancer Development and Progression[END_REF].

Within the environs of the prostate, invasion and metastasis is limited by cancer cell movement, which can be achieved by collective-cell or single-cell mechanisms.

Collective cell movement involves a multitude of cells moving as large coordinated mass, or as small cohorts of cells [START_REF] Friedl | Tumour-cell invasion and migration: diversity and escape mechanisms[END_REF]. Singular cell movement occurs without stress fibre formation or protease activity, squeezing through extracellular matrix (ECM) components [START_REF] Sabeh | Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited[END_REF]. Singular cell movement is unequivocally the most noted form of movement in prostate cancer, which may be facilitated by epithelial mesenchymal transition (EMT) of cancer cells. As a result of EMT, cancer epithelial cells exhibit changes in cadherin expression (E-/N-cadherin), acquire mesenchymal properties and detach from surrounding cells and the ECM.

Cells that have undergone EMT secrete proteases that degrade the ECM, facilitating their movement out of the prostate [START_REF] Nauseef | Epithelial-to-mesenchymal transition in prostate cancer: paradigm or puzzle?[END_REF][START_REF] Leach | Stromal androgen receptor regulates the composition of the microenvironment to influence prostate cancer outcome[END_REF].

CRPC and metastasis CRPC

CRPC

In general, PCa is strongly driven by androgen receptor (AR) regulated transcription [START_REF] Sehgal | Tissue-specific quantification and localization of androgen and estrogen receptors in prostate cancer[END_REF]. However, PCa can become "castration resistant" (CRPC) after escaping conventional androgen deprivation treatment (ADT, with luteinizing hormone releasing hormone (LHRH) agonists and/or the AR antagonist enzalutamide (ENZ)). CRPC status is defined as the presence of castrate serum testosterone levels (<50 ng/dL or 1.7 nmol/L) plus either biochemical progression (three consecutive rises in prostate-specific antigen (PSA) 1 week apart, resulting in two 50% increases over the nadir, and PSA >2 ng/mL) or radiologic progression [START_REF] Cornford | EAU-ESTRO-SIOG Guidelines on Prostate Cancer. Part II: Treatment of Relapsing, Metastatic, and Castration-Resistant Prostate Cancer[END_REF].

As prolonged and intense targeting AR in CRPC patients results in a survival benefit, "castration resistance" often remains AR driven [START_REF] Luo | Role of Androgen Receptor Variants in Prostate Cancer: Report from the 2017 Mission Androgen Receptor Variants Meeting[END_REF]. However, histological and clinical resistance cancer types can emerge after AR-inhibition, in which the tumors become androgen independent prostate cancer (AIPC), neuroendocrine (NEPC) and other rare histological variants [START_REF] Graham | Targeting persistent androgen receptor signaling in castration-resistant prostate cancer[END_REF]) (Table 1.2).

mCRPC

In mCRPC patients, prolonged AR pathway inhibition can alter the typical course of the disease, manifest by histological differentiation to a small cell/high-grade neuroendocrine phenotype in 11-17% of cases [START_REF] Aggarwal | Clinical and genomic characterization of treatment-emergent small-cell neuroendocrine prostate cancer: a multi-institutional prospective study[END_REF]Abida et al., 2019).

Despite recent advances in the treatment of patients progressing with mCRPC, average survival remains approximately three years [START_REF] Roviello | Role of the novel generation of androgen receptor pathway targeted agents in the management of castration-resistant prostate cancer: A literature based meta-analysis of randomized trials[END_REF]. The heterogeneous appearance of these changes underlies the multifocal nature of PCa. It is thought that AR insensitive clones will survive the therapeutic selection pressure by AR targeting treatments. Clinical evaluation for adverse prognostic indicators in mCRPC involves either histopathologic/immunohistochemical assessment of biopsy specimens or molecular analysis of biopsy material or circulating tumor cells. Some adverse prognostic indicators include small cell/high-grade neuroendocrine transformation on histopathologic/IHC evaluation, alterations such as AR splice Table 1.2 Clinical, histological and molecular characteristics of most common CRPC variants. Adapt from: [START_REF] Vlachostergios | Emerging Variants of Castration-Resistant Prostate Cancer[END_REF] variant 7, those involving TP53, RB1, DNA damage response genes, and AR/PI3K pathways on molecular profiling [START_REF] Antonarakis | AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer[END_REF][START_REF] Conteduca | Androgen receptor gene status in plasma DNA associates with worse outcome on enzalutamide or abiraterone for castration-resistant prostate cancer: a multi-institution correlative biomarker study[END_REF][START_REF] Annala | Circulating tumor DNA genomics correlate with resistance to abiraterone and enzalutamide in prostate cancer[END_REF].

NEPC

The diagnosis of neuroendocrine prostate cancer (NEPC) is histologically defined, encompassing morphologies that overlap with neuroendocrine tumors arising from other primary sites (eg., lung, bladder), and may occur with mixed or overlapping features with prostate adenocarcinoma (Epstein et a;[START_REF] Shin | A RUNX2-Mediated Epigenetic Regulation of the Survival of p53 Defective Cancer Cells[END_REF][START_REF] Vlachostergios | Emerging Variants of Castration-Resistant Prostate Cancer[END_REF]. Immunohistochemical detection of neuroendocrine markers such as synaptophysin, chromogranin, and CD56 may help support the diagnosis of NEPC but are not required in the presence of morphologic characteristics (Watson et al., 2015).

Pure small-cell neuroendocrine prostate carcinoma is a subtype of NEPC that has the most aggressive biologic behavior and poor outcomes. The clinical significance of other NEPC subtypes, especially mixed or hybrid phenotypes, is not well defined (Watson et al., 2015;Epstein et a;[START_REF] Shin | A RUNX2-Mediated Epigenetic Regulation of the Survival of p53 Defective Cancer Cells[END_REF][START_REF] Vlachostergios | Emerging Variants of Castration-Resistant Prostate Cancer[END_REF].

Although NEPC rarely arises de novo and accounts for less than 2% of patients at the time of diagnosis of prostate cancer [START_REF] Parimi | Neuroendocrine differentiation of prostate cancer: a review[END_REF], NEPC is enriched in the advanced disease setting and reported as high as 10-20% in CRPC [START_REF] Nadal | Small cell carcinoma of the prostate[END_REF].

Clinically, NEPC is often suspected in patients that progress in the setting of low or moderately rising PSA levels (suggesting less AR driven disease) with a predominance of visceral and/or lytic bone metastases. Morphologically, NEPC shares features with other high grade neuroendocrine cancers, including presence of small cells with 'salt and pepper' chromatin, high mitotic count and nuclear molding (Wang et al., 2008). In the majority of cases, at least one neuroendocrine immunohistochemical (IHC) marker, such as neuron-specific enolase, synaptophysin, chromogranin, or CD56 stains positive. NEPC is often negative for classical luminal markers of prostatic glandular differentiation (eg., prostate-specific antigen (PSA) and prostatic acid phosphatase). Uncommonly, polypeptide hormones may be present (including ACTH, antidiuretic hormone (ADH), and corticotropin-releasing factor) (Wang et al., 2008;[START_REF] Vlachostergios | Emerging Variants of Castration-Resistant Prostate Cancer[END_REF].

Prostate cancer staging and grading

Staging: TNM classification system

Once a patient has been diagnosed with a prostate tumor, the latter must be staged to determine if it has spread beyond the prostate. Staging also provides a better insight into the risk of the disease spreading further, so the correct treatment option is selected. The TNM stage was developed by the American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC) (Wallace et al., 1075). It is used to evaluate the extent of the primary tumor (T), the affected regional lymph nodes (N) and if it has spread or metastasized (M). There are four stages: in stage I only a small part of the prostate is cancerous, most of the cells are normal and the gland feels normal. In stage II, a lump can be felt in the prostate to the examining finger and a larger part of the prostate is affected. In stage III, the tumor has spread beyond the prostate but very locally, and in stage IV, it has spread to lymph nodes or nearby organs. A more detailed view can be found in Table 1.3.

Grading: Gleason Grading System

Ultrasound guided needle biopsies are taken from the prostate in patients with elevated PSA levels, and if a biopsy contains cancer, it should be examined by a pathologist using microscopic analysis and scored according to the Gleason system, which is the strongest prognostic tool available today for prostate cancer (Gleason and Mellinger, 1974). The differentiation pattern of the tumor is scored on a scale ranging from 1 to 5, where 5 represents the less differentiated and most aggressive tumor pattern. The most common areas of differentiation are summarized into the Gleason score (GS) [START_REF] Coffey | Control of prostate growth[END_REF][START_REF] Westin | Castration therapy rapidly induces apoptosis in a minority and decreases cell proliferation in a majority of human prostatic tumors[END_REF]. GS is a good predictor of outcome in patients with low GS < 6 or high GS 8-10, but the majority of patients have where the outcome is very variable and today largely unpredictable [START_REF] Andren | How well does the Gleason score predict prostate cancer death? A 20-year followup of a population based cohort in Sweden[END_REF]. The Gleason patterns are detailed in Fig. 1.6.

Diagnosis and prognosis of prostate cancer

DRE

Digital rectal examination (DRE) is an important part of the assessment of a patient suspected of having prostate cancer. Since most prostate cancers arise in the peripheral zone [START_REF] Mcneal | Zonal distribution of prostatic adenocarcinoma. Correlation with histologic pattern and direction of spread[END_REF] (i.e. the posterior part of the prostate), larger or advanced lesions can be palpable on DRE. An abnormal DRE may be defined by asymmetry, a palpable nodule or fixed craggy mass. An abnormal DRE initial assessment is highly predictive for high grade prostate cancer (odds ratio 6:1) and estimating prostate size also improves diagnostic accuracy when combined with PSA [START_REF] Roobol | Prediction of prostate cancer risk: the role of prostate volume and digital rectal examination in the ERSPC risk calculators[END_REF].

Previous work by Epstein et al [START_REF] Epstein | Pathologic and clinical findings to predict tumor extent of nonpalpable (stage T1c) prostate cancer[END_REF] had already highlighted the importance of assessing the estimated prostate volume in conjunction with the patient's PSA reading, finding that a PSA density of less than 0.1 ng/ml per gram was predictive of no adverse pathological findings on needle biopsy and a PSA density of 0.1-0.15 ng/ml per gram indicative of low or intermediate grade cancer smaller than 3 mm in one needle core biopsy specimen. Thus, DRE still plays an important role in the diagnostic evaluation of men suspected of having prostate cancer, although the positive predictive value of DRE in primary care remains extremely variable [START_REF] Hoogendam | The diagnostic value of digital rectal examination in primary care screening for prostate cancer: a meta-analysis[END_REF].

PSA

The prostate-specific antigen (PSA) is a glycoprotein enzyme responsible for liquefaction of semen thus enabling fertilisation. It is produced by columnar acinar and ductal prostatic epithelial cells, being present in both benign and malignant cells.

PSA is mainly secreted into the semen with small quantities found in the urine and blood. Normally there are significant tissue barriers (basal cell layer, basement membrane, stromal layer and the capillary wall itself), between prostatic cells and capillaries. In prostate cancer these barriers are compromised and thus PSA leaks into circulating capillaries and hence serum PSA values rise.

Stamey et al first published results of 699 patients who underwent both PSA testing and prostatic acid phosphatase (PAP) testing (this was the only serum marker for prostate cancer prior to PSA), concluding that PSA was more sensitive than PAP in detecting prostate cancer [START_REF] Stamey | Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate[END_REF]. The original work on PSA thresholds conducted by Catalona et al in the early 1990s [START_REF] Catalona | Comparison of digital rectal examination and serum prostate specific antigen in the early detection of prostate cancer: results of a multicenter clinical trial of 6,630 men[END_REF] suggested a PSA cut-off value of 4ng/ml when guiding whether to perform prostate biopsy in absence of any positive examination findings. They found that prostate cancer was detected in 26% of men with a PSA in the range 4-10 ng/ml and in 53% of men with PSA > 10 ng/ml. Oesterling et al [START_REF] Oesterling | Serum prostate-specific antigen in a community-based population of healthy men. Establishment of age-specific reference ranges[END_REF] conducted work which concluded that age-specific PSA thresholds were more useful. They suggested the following cut-off points to determine a raised PSA based on age (Table 1.4):

Furthermore, PSA has been shown to predict pathological stage in prostate cancer, even allowing for the increased diagnosis of low-risk, localised prostate cancers [START_REF] Freedland | PSA in the new millennium: a powerful predictor of prostate cancer prognosis and radical prostatectomy outcomes--results from the SEARCH database[END_REF]. PSA is currently used in the NICE guidelines for risk stratification for men with localised prostate cancer [START_REF] Chun | Prostate-specific antigen improves the ability of clinical stage and biopsy Gleason sum to predict the pathologic stage at radical prostatectomy in the new millennium[END_REF] (Table 1.5).

PSA has also been used in assessing response to treatment and disease progression.

Androgen receptor (AR)

The androgen receptor (AR) is a transcriptional factor, belonging to the steroid receptor superfamily and activated by testosterone and dihydrotestosterone, and required for prostate development and normal prostate function (Cunha et al., 2004). AR and the modulators of AR activity remain important in prostate cancer, even in castration-resistant prostate cancer (CRPC) [START_REF] Knudsen | Outsmarting androgen receptor: creative approaches for targeting aberrant androgen signaling in advanced prostate cancer[END_REF].

Approximately 80-90% of prostate cancers are dependent on androgen at initial diagnosis, and endocrine therapy of prostate cancer is directed toward the reduction of serum androgens and inhibition of AR [START_REF] Denis | Endocrine treatment in prostate cancer[END_REF]. Studies of AR in CRPC revealed that AR was still active in CRPC, and it remains as a potential target to treat CRPC.

Structure of the AR gene

The AR gene is located on chromosome X (Xq11-12) and consists of 8 exons coding The NTD has glutamine repeats (CAG repeats) varying in size (most men have 19-25 repeats), approximately 919 amino acids, which results in the variation of all amino acids in AR [START_REF] Gottlieb | The androgen receptor gene mutations database: 2012 update[END_REF]. Shorter glutamine repeats are associated with high transcriptional activity of AR [START_REF] Beilin | Effect of the androgen receptor CAG repeat polymorphism on transcriptional activity: specificity in prostate and non-prostate cell lines[END_REF]. It has been reported that men with shorter glutamine repeats have a higher risk of prostate cancer [START_REF] Giovannucci | The CAG repeat within the androgen receptor gene and its relationship to prostate cancer[END_REF]. In contrast, patients with Kennedy disease have long CAG repeats (>40), which results in low AR-transcriptional activity with gynecomastia, erectile dysfunction, testicular atrophy, and muscular atrophy [START_REF] Finsterer | Bulbar and spinal muscular atrophy (Kennedy's disease): a review[END_REF]. The NTD includes the transcriptional regulatory region, activation function-1 (AF-1), and the LBD includes activation function-2 (AF-2) [START_REF] Jenster | Domains of the Human Androgen Receptor Involved in Steroid Binding, Transcriptional Activation, and Subcellular Localization[END_REF]. Upon DNA binding, the AR dimer forms a complex with coactivator and coregulatory proteins at the AF-1 and AF-2 regions [START_REF] Jenster | Domains of the Human Androgen Receptor Involved in Steroid Binding, Transcriptional Activation, and Subcellular Localization[END_REF][START_REF] Antonarakis | Androgen receptor variant-driven prostate cancer: clinical implications and therapeutic targeting[END_REF][START_REF] Fujita | Role of Androgen Receptor in Prostate Cancer: A Review[END_REF], including SRC1, SRC2, SRC3, p300/CBP, and AEA54, among many others.

The hinge region between the DBD and LBD is involved in nuclear localization and degradation. The N-terminus has a unique LxxLL-like motif, which binds to a hydrophobic cleft of the C-terminus generated by ligand binding to the receptor. The initial N-C interaction occurs in the cytoplasm, which stabilizes the ligand binding caused by physical interaction between the N-terminal and C-terminal of the receptor a b [START_REF] Bai | A positive role of c-Myc in regulating androgen receptor and its splice variants in prostate cancer[END_REF]. In the absence of androgens, heat shock protein (HSP) binds to AR, therefore, AR remains inactive in the cytoplasm. Binding of androgens with AR induces a conformational change, resulting in the dissociation of HSP from AR. Then, the AR dimer translocates into the nucleus where it binds to androgen responsive elements of genomic DNA and regulate its target genes involved in growth and proliferation [START_REF] Masiello | Recruitment of beta-catenin by wild-type or mutant androgen receptors correlates with ligand-stimulated growth of prostate cancer cells[END_REF]Mills, 2014) (Fig. 1.8).

AR regulates the gene expressions with diverse functions located downstream of the androgen-response element, including secreted proteins (KLK3, KLK2), fusion genes (TMPRSS2-ERG), growth stimulators (IGF1R, APP), PI3K modulation (FKBP5), transcription factors (NKX3.1, FOXP1), metabolic enzyme (CAMKK2), cell cycle regulators (UBE2C, TACC2), and glucuronidation (UGT1A1) [START_REF] Takayama | Transcriptional network of androgen receptor in prostate cancer progression[END_REF]. DHT binds to the ligand binding pocket of AR and promotes its dissociation from the heat shock protein (HSP). Free AR then translocates into the nucleus and binds to androgen receptor element (ARE) present in the promoter region of AR responsive genes. At the promoter, AR recruits components of basal transcriptional machinery such as TATA binding protein (TBP), transcription factor IIF (TFIIF), and cAMP responsive element binding protein (CRBP) which ensure the transcription of AR responsive genes. Adapt from: Heinlein and Chang, 2014.

AR variants

It has been known for more than two decades that AR has splicing variants (Bryce et al., 2016) (Table 1 .6). More than 20 AR variants (AR-Vs) have been reported, and most are missing some C-terminal domain including LBDs, thus functionally active without androgens (Hu et al., 2009;van der Steen et al., 2013) (Fig. 1.9).

Most AR-Vs contain an intact DNA-binding domain (DBD), whereas AR-V3

(aka AR6) lacks the second zinc finger of the DBD. Nevertheless, it contains the first zinc finger of DBD, can constitutively activate AR-responsive promoters in prostate cancer cells [START_REF] Dehm | Splicing of a novel androgen receptor exon generates a constitutively active androgen receptor that mediates prostate cancer therapy resistance[END_REF]. Some of these variants are constitutively active, such as AR-V7 and ARv567es

(aka AR-V12), whereas some others are conditionally active, depending on the cellular context, such as AR-V1 [START_REF] Hu | A snapshot of the expression signature of androgen receptor splicing variants and their distinctive transcriptional activities[END_REF]). The latter is truncated at the end of exon 3 and contains 19 amino acids from cryptic exon 1. AR-V1 and AR-V7 were the most abundant variants, with 20-fold higher expression in CRPC compared with hormone-naïve prostate cancer (Hu et al., 2009).

AR-V567es has exons 5-7 spliced out and only contains a small portion of the LBD (Shafi et al., 2013). ARv567es was found to be enriched in prostate epithelium of patients with prostate cancer and in CRPC bone metastasis where they were associated with increased nuclear AR, aberrant cell cycle regulation, and reduced overall survival [START_REF] Sun | Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant[END_REF][START_REF] Hornberg | Expression of androgen receptor splice variants in prostate cancer bone metastases is associated with castration-resistance and short survival[END_REF]. Expression of AR-V567es

and/or AR-V7 was associated with poor survival [START_REF] Sharp | Androgen receptor splice variant-7 expression emerges with castration resistance in prostate cancer[END_REF].

AR-V7 is truncated at the end of exon 3 and contains 16 amino acids from cryptic exon 3. AR-V7 is located in the nucleus under androgen-depleted conditions and is constitutively active in androgen-responsive genes. More recently, ARv7 expression was detected in circulating tumor cells of patients treated with antiandrogen enzalutamide (12 of 31) or Cyp17A1 inhibitor abiraterone (6 of 31), and its expression was correlated with therapy-resistance [START_REF] Antonarakis | AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer[END_REF]. These studies suggested the potential of using AR-V7 as a predictive marker of response to enzalutamide and abiraterone.

AR45 is truncated in the N-terminal domain, while all the other variants contain an intact N-terminal domain but lack portions of the ligand-binding domain (LBD).

As the N-terminal domain harbors the two trans-activating regions (Tau1 and Tau5/AF5) [START_REF] Need | A novel androgen receptor amino terminal region reveals two classes of amino/carboxyl interaction-deficient variants with divergent capacity to activate responsive sites in chromatin[END_REF], AR45 loses its transactivating ability and acts as a dominant-negative variant to inhibit the function of the full-length AR (AR-FL) by forming a heterodimer with AR-FL. Moreover, overexpression of AR45 in LNCaP cells inhibited proliferation (Ahrens-Fath et al., 2005). Together, these data indicate that AR45 is a negative regulator of AR signaling.

AR mutations

Somatic AR mutations may occur selectively in response to androgen deprivation (Hoang et al., 2017). A review of 27 clinical studies found that AR mutations in androgen-dependent tumors ranged from 2-25%, while the incidence in CRPC tumors was slightly higher at 10-40% (Koochekpour, 2010;Hoang et al., 2017). Additional work has identified the AR LBD as a mutational hotspot, placing the incidence of AR LBD point mutations in CRPC at ~15-20% (Grasso et al., 2012;Robinson et al., 2015). Two well-known examples are AR-T877A and AR-W741C, originally described in LNCaP cells, which convert the antiandrogens flutamide and bicalutamide to partial agonists, respectively [START_REF] Otsuka | Androgen receptor W741C and T877A mutations in AIDL cells, an androgen-independent subline of prostate cancer LNCaP cells[END_REF]. Recently, emergence of AR-T878A and AR-L702H was observed in 13% of CRPC tumors progressing on abiraterone treatment, while total AR copy number remained unchanged pre-and post-treatment with abiraterone [START_REF] Jernberg | Clinical relevance of androgen receptor alterations in prostate cancer[END_REF].

Mechanistically, these AR LBD mutationss were demonstrated to sensitize AR to activation by progesterone (T878A) (Chen et al., 2015) and glucocorticoids (L702H) [START_REF] Attard | Clinical and biochemical consequences of CYP17A1 inhibition with abiraterone given with and without exogenous glucocorticoids in castrate men with advanced prostate cancer[END_REF], circumventing the effects of CYP17A1 inhibition on AR signaling.

AR dependent mechanisms driving prostate cancer progression

AR amplification, mutations and variants

AR amplification/overexpression is the most common genomic aberration in CRPC patients; up to 80% of these patients show AR overexpression [START_REF] Edwards | Androgen receptor gene amplification and protein expression in hormone refractory prostate cancer[END_REF].

This type of adaptation is more common in patients who progressed during or after the treatment with new generation hormonal therapy than in treatment-naïve patients, so it has been considered as a potential resistance mechanism [START_REF] Crona | Androgen receptor-dependent and -independent mechanisms involved in prostate cancer therapy resistance[END_REF].

AR mutations can be found in up to 30% of CRPC patients treated with ADT.

Their incidence may increase during treatment with abiraterone and enzalutamide because, when AR signalling is more effective suppressed, clonal selection of tumour cells can enhance AR somatic mutations and the consequent aberrant transcription.

Most AR point mutations are clustered in the LBD, altering the steroid-binding pocket and enabling its activation by alternative ligands including progesterone, hydrocortisone, oestradiol, and some AR antagonists [START_REF] Taplin | Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer[END_REF].

Constitutively active AR splice variants (AR-Vs), lacking the LBD, play a critical role in the development and progression of CRPC, even without androgen binding [START_REF] Dehm | Alternatively spliced androgen receptor variants[END_REF]. ARV expression is significantly increased during ADT and is related to PC progression. The most common AR splicing variant, AR-V7, is associated with resistance to both abiraterone and enzalutamide and with short survival [START_REF] Antonarakis | AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer[END_REF].

Treatment of castration-resistant prostate cancer

Given that AR signaling remains active in CRPC patients, it is recommended that ADT should be continued by adding an AR antagonist such as bicalutamide to the castration therapy (combined androgen blockade). The first-line treatment for patients who relapse after ADT are chemotherapy in form of docetaxel [START_REF] Tannock | Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer[END_REF][START_REF] Kellokumpu-Lehtinen | 2-Weekly versus 3-weekly docetaxel to treat castration-resistant advanced prostate cancer: a randomised, phase 3 trial[END_REF] (Parker et al., 2013) (Fig. 1.10).

AR gene regulation

Regulation of the AR gene by AR itself

Grad with colleagues found that the AR gene itself is regulated by AR in osteoblast-like cells. Mechanistically, a 350-bp fragment consisting of two androgen responsive elements (ARE) have been identified in exons 4 and 5 of the AR gene.

These elements selectively control the auto-activation of the AR promoter, being responsible for androgen-mediated upregulation of AR mRNA in U2OS cells (Grad et al., 2001). Furthermore, it was reported that the expression of the AR gene in PCa was directly suppressed by itself through recruitment of lysine-specific demethylase 1 to the enhancer in the second intron of AR and demethylation of H3K4me1 and H3K4me12 [START_REF] Cai | Androgen receptor gene expression in prostate cancer is directly suppressed by the androgen receptor through recruitment of lysine-specific demethylase 1[END_REF]. [START_REF] Dang | MYC on the path to cancer[END_REF]. All MYC proteins heterodimerize with the obligatory bHLHZip protein MAX, which enables the MYC: MAX heterodimer to bind so-called E-box DNA sequences (CACGTG and similar sequences) situated in regulatory regions of target genes [START_REF] Eilers | Myc's broad reach[END_REF][START_REF] Meyer | Reflecting on 25 years with MYC[END_REF][START_REF] Larsson | The Yin and Yang functions of the Myc oncoprotein in cancer development and as targets for therapy[END_REF][START_REF] Hydbring | MYC Modulation around the CDK2/p27/SKP2 Axis[END_REF].

The MYC gene is present on human chromosome 8q24 and encodes MYC protein which is a transcription factor that plays a key role in regulating a number of cellular processes including cell cycle progression, metabolism, ribosome biogenesis, protein synthesis, mitochondrial function, and stem cell self renewal [START_REF] Knoepfler | Why myc? An unexpected ingredient in the stem cell cocktail[END_REF][START_REF] Eilers | Myc's broad reach[END_REF][START_REF] Dang | MYC on the path to cancer[END_REF]. Mechanistically, MYC has the ability to regulate target genes by recruiting different cofactors participating in chromatin modification and remodeling and/or in the initiation and elongation of RNA Pol I, II, and III-mediated transcription. Recently, two components of the multisubunit COMPASS/mixed lineage leukemia (MLL) histone H3 methylase complexes, ASH2L [START_REF] Ullius | The interaction of MYC with the trithorax protein ASH2L promotes gene transcription by regulating H3K27 modification[END_REF] and WRD5 [START_REF] Thomas | Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC[END_REF], were found to interact with MYC. The WRD5 seems to stabilize MYC's interaction with chromatin [START_REF] Thomas | Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC[END_REF], while the interaction between the ASH2L and MYC was shown to promote histone H3 lysine 27 (H3K27) demethylation and subsequent H3K27 acetylation [START_REF] Ullius | The interaction of MYC with the trithorax protein ASH2L promotes gene transcription by regulating H3K27 modification[END_REF]. H3K27 acetylation is, like histone H3 lysine 4 (H3K4) methylation, a mark for actively transcribed genes (Shilatifard et al., 2012). However, accumulating results showed that MYC mediated also repression of transcription through interacting with MIZ1 [START_REF] Staller | Repression of p15INK4b expression by Myc through association with Miz-1[END_REF][START_REF] Seoane | TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b[END_REF], SP1 [START_REF] Gartel | Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3[END_REF][START_REF] Parisi | Identifying synergistic regulation involving c-Myc and sp1 in human tissues[END_REF] and SMADs [START_REF] Seoane | TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b[END_REF].

MYC protein is very short-lived, having a half-life of around 30 min. A number of E3 ubiquitin ligases have been implicated in ubiquitylation of MYC, including SKP2 [START_REF] Carrano | SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27[END_REF], FBW7 [START_REF] Popov | Ubiquitylation of the amino terminus of Myc by SCF(β-TrCP) antagonizes SCF(Fbw7)-mediated turnover[END_REF], HUWE1/HECTH9 (Zhao et al., 2008), FBX29 [START_REF] Koch | Large-scale identification of c-MYC-associated proteins using a combined TAP/MudPIT approach[END_REF], TRUSS [START_REF] Yada | Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7[END_REF]) and FBXL14 [START_REF] Thomas | Proteolytic control of the oncoprotein transcription factor Myc[END_REF][START_REF] Farrell | MYC degradation[END_REF]. In addition, MYC protein levels are regulated at the level of mRNA translation through the 5′ cap mRNA-binding eIF4F complex but also through internal ribosomal entry site elements that are bound by eIF4A-containing complexes, both of which are controlled by mammalian target of rapamycin (mTOR) signaling [START_REF] Wolfe | RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer[END_REF][START_REF] Bhat | Targeting the translation machinery in cancer[END_REF][START_REF] Castell | Targeting MYC Translation in Colorectal Cancer[END_REF][START_REF] Wiegering | Targeting Translation Initiation Bypasses Signaling Crosstalk Mechanisms That Maintain High MYC Levels in Colorectal Cancer[END_REF].

b. MYC in prostate cancer

The MYC oncogene is key player in cancer initiation and progression, being critical for maintaining the tumorigenic state in numerous cancer types, including prostate cancer. The overexpression of MYC in PCa has been a well-recognized phenomenon since 1986, when Fleming et al. showed a significantly higher level of its expression in adenocarcinoma of the prostate than in benign prostate hyperplasia by Northern blotting (Fleming et al., 1986). Furthermore, Sato et al reported that MYC amplification is strongly associated with higher histopathological grades and

Gleason scores, as well as with earlier disease progression and cancer-associated death (Sato et al., 2006). Gene amplification of MYC is a key event at the precancer stage (i.e. PIN) of the PCa development [START_REF] Ellwood-Yen | Myc-driven murine prostate cancer shares molecular features with human prostate tumors[END_REF][START_REF] Gurel | Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis[END_REF]Koh e al., 2010;).

Since MYC is so commonly overexpressed at the protein level in PCa, the current findings raise the possibility that MYC might be an excellent therapeutic target in this disease. New approaches to target MYC in MYC-related tumors are being developed [START_REF] Goga | Inhibition of CDK1 as a potential therapy for tumors over-expressing MYC[END_REF], and if these, or similar approaches, reach the clinic trial stage, it will be important to test their therapeutic efficacy in prostate cancer patients.

c. AR regulation by MYC

The MYC has a well-documented role in driving AR oncogenic functions in PCa cells [START_REF] Grad | Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA[END_REF], and was recently exposed as a key activator in the regulation of AR transcription [START_REF] Nadiminty | MicroRNA let-7c suppresses androgen receptor expression and activity via regulation of Myc expression in prostate cancer cells[END_REF]. They identified miR-let-7c as a key regulator of expression of AR in human prostate cancer cells by targeting its transcription via MYC [START_REF] Nadiminty | MicroRNA let-7c suppresses androgen receptor expression and activity via regulation of Myc expression in prostate cancer cells[END_REF].

More recently, Bai et al reported a role of MYC in accounting for coordinated AR-full length (AR-FL) and AR-vriants (AR-Vs) expression [START_REF] Bai | A positive role of c-Myc in regulating androgen receptor and its splice variants in prostate cancer[END_REF]. Using primary prostate tumors showed that the level of MYC is positively correlated with that of individual AR isoforms. More importantly, inhibiting MYC sensitizes enzalutamide-resistant cells to growth inhibition by enzalutamide [START_REF] Bai | A positive role of c-Myc in regulating androgen receptor and its splice variants in prostate cancer[END_REF].

Taken together, these results highlight a critical role of MYC in regulating the coordinated expression of AR-FL and AR-Vs that is commonly observed in CRPC, suggesting the utility of targeting MYC as an adjuvant to AR-directed therapy.

However, the mechanism of AR regulation by MYC is still not clear.

EZH2

a. The function of EZH2

Enhancer of Zeste Homolog 2 (EZH2), a key component of the Polycomp repressive complex 2 (PRC2) that helps to maintain cell identity during development [START_REF] Czermin | Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites[END_REF][START_REF] Müller | Histone methyltransferase activity of a Drosophila Polycomb group repressor complex[END_REF][START_REF] Simon | Mechanisms of polycomb gene silencing: knowns and unknowns[END_REF], has been found to contribute to gene repression via the histone H3 lysine 27 trimethylation (H3K27me3) [START_REF] Cao | The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3[END_REF] catalyzed by its SET domain. EZH2 regulates several cellular processes, including cell fate determination, cell cycle regulation, senescence, cell differentiation and carcinogenesis [START_REF] Sauvageau | Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer[END_REF]. Furthermore, EZH2 has been reported to be overexpressed and to function as an oncogene in various cancers by mediating the expression of target genes [START_REF] Sauvageau | Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer[END_REF][START_REF] Chase | Aberrations of EZH2 in cancer[END_REF], including prostate cancer [START_REF] Varambally | The polycomb group protein EZH2 is involved in progression of prostate cancer[END_REF], breast cancer [START_REF] Chang | EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-β-catenin signaling[END_REF][START_REF] Yoo | EZH2 methyltransferase and H3K27 methylation in breast cancer[END_REF], hepatocellular carcinoma [START_REF] Feng | A CCRK-EZH2 epigenetic circuitry drives hepatocarcinogenesis and associates with tumor recurrence and poor survival of patients[END_REF], colorectal cancer [START_REF] Fornaro | EZH2 polymorphism and benefit from bevacizumab in colorectal cancer: another piece to the puzzle[END_REF], gastric cancer [START_REF] Bai | Inhibition enhancer of zeste homologue 2 promotes senescence and apoptosis induced by doxorubicin in p53 mutant gastric cancer cells[END_REF], ovarian cancer (Wang et al., 2015), melanoma [START_REF] Tiffen | Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes[END_REF] and cervical cancer [START_REF] Ding | The polycomb group protein enhancer of zeste 2 is a novel therapeutic target for cervical cancer[END_REF]. Additionally, EZH2 has been found to act as an epigenetic modifier during the TGF-β-induced epithelial-mesenchymal transition (EMT) and to control the p38 mitogen-activated protein kinase (MAPK) signaling pathway during breast cancer cell migration, invasion and metastasis [START_REF] Moore | EZH2 inhibition decreases p38 signaling and suppresses breast cancer motility and metastasis[END_REF].

b. AR regulation by EZH2

EZH2 has recently been shown to coordinate with AR on transcriptional repression. Through ChIP-seq analysis of the key transcription factors and histone marks, they identified an integrative transcriptional network involving AR, EZH2, and ETS-related gene-1 (ERG1) in PCa cells (Yu et al., 2010). ERG belongs to the ETS family of transcription factors and is frequently fused with the 5' untranslated region of the TMPRSS2 gene in PCa [START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF]. They showed that ERG cooccupies with AR on many ARE-containing genomic loci to inhibit gene expression, which may be mediated, at least partially, by direct induction of EZH2 expression and recruitment to the target sites for epigenetic silencing. Further, integrating genome-wide AR localization data with androgen-induced gene expression data, they have identified a large number of genes whose expression is directly inhibited by AR but can be rescued through EZH2 inhibition (Zhao et al., 2012;Fong et al., 2017) Collectively, their data demonstrated that EZH2 facilitates AR-transcriptional repression through catalyzing H3K27me3.

More interestingly, it has been reported that EZH2 activates AR transcription through direct occupancy at its promoter [START_REF] Kim | Polycomb-and Methylation-Independent Roles of EZH2 as a Transcription Activator[END_REF]. Importantly, this activating role of EZH2 is independent of PRC2 and its methyltransferase activities.

The authors also demonstrated an enhanced efficacy of enzymatic EZH2 inhibitors Furthermore, they also demonstrated that EZH2 regulates AR protein expression and AR downstream targets, such as, PSA, TMPRSS2 and FKBP5, in AR positive PCa cell lines [START_REF] Liu | Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer[END_REF]. Taken together, the above data showed EZH2 as a transcriptional activator, a key target of which is AR, suggesting a drug-combinatory approach to treat advanced prostate cancer.

Pioneering factors: FOXA1, GATA2 and HOXB13

By analyzing enriched motifs around the histone modifications in AR binding sites (ARBSs), AR-associated transcription partners such as Forkhead box protein A1 (FOXA1) [START_REF] Gao | The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes[END_REF], GATA2 [START_REF] He | GATA2 facilitates steroid receptor coactivator recruitment to the androgen receptor complex[END_REF], HOXB13 [START_REF] Huang | Posterior Hox gene expression and differential androgen regulation in the developing and adult rat prostate lobes[END_REF] have been mapped to the prostate cancer genome and these studies suggested that these factors activate AR-driven transcriptional program. Among them, a chromatin-opening transcription factor, FOXA1, is able to directly bind to the chromatin to open up the local nucleosomal domain [START_REF] Cirillo | Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4[END_REF]. In prostate cells, FOXA1 protein has been shown physically interact with AR and plays critical roles in regulating the transcription of prostate genes such as PSA [START_REF] Gao | The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes[END_REF].

Besides FOXA1, several other transcription factors such as GATA2 and HOXB13

may have similar pioneering cofactor effects on AR-chromatin binding and transcriptional regulation. Similar as FOXA1, GATA family transcription factors have also been shown to open compact chromatin through their conserved zinc finger domains [START_REF] Boyes | Perturbation of nucleosome structure by the erythroid transcription factor GATA-1[END_REF]. In addition, GATA2 expression is also essential for AR-mediated transcription of prostate genes such as PSA and TMPRSS2 [START_REF] Perez-Stable | A role for GATA transcription factors in the androgen regulation of the prostate-specific antigen gene enhancer[END_REF][START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF]. Likewise, HOXB13 is a member of the homeodomain family of sequence-specific transcription factors. Mouse studies have shown that HOXB13 plays an essential role in prostate development [START_REF] Huang | Posterior Hox gene expression and differential androgen regulation in the developing and adult rat prostate lobes[END_REF]. Through analysis of several candidate genes, HOXB13 has been shown as a regulator of AR-chromatin interaction, similar as FOXA1 [START_REF] Norris | The homeodomain protein HOXB13 regulates the cellular response to androgens[END_REF].

Specifically, HOXB13 was found to inhibit transcription of genes regulated by AR binding to ARE, but enhance AR binding to cis-regulatory regions containing HOX elements juxtaposed to AREs, thereby inducing corresponding gene expression. How GATA2 and HOXB13 regulate genome-wide AR binding profile, however, are yet to be carefully examined.

PCAT1

Prostate Cancer Associated Transcript 1 (PCAT1) is a long non-coding RNA with a length of ~1900 nt, made of 2 exons. Exon 1 contains a retroviral long terminal repeat (LTR) sequence and exon 2 contains sequences from the HSMAR1 mariner family transposase bisected by an AluY repeat element [START_REF] Prensner | Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression[END_REF]. The PCAT1 gene locates at chromosome 8q24 and 725 kb upstream of the MYC gene. Chr8q24 locus is an area commonly studied in prostate cancer due to frequent amplification and the presence of prostate cancer susceptibility SNP loci [START_REF] Gudmundsson | Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24[END_REF][START_REF] Albadine | TMPRSS2-ERG gene fusion status in minute (minimal) prostatic adenocarcinoma[END_REF].

A recent study demonstrated that PCAT1 was regulated by an SNP located in its enhancer region (Guo et al., 2016). Specifically, the prostate cancer risk-associated SNP rs7463708 was associated with increased expression of PCAT1 through modulating ONECUT2 and AR binding at a distal enhancer. In addition, PCAT1

interacted with AR and lysine-specific demethylase 1 (LSD1) and was required for their recruitment to the enhancers of the glycine N-methyltransferase (GNMT) and 24-dehydrocholesterol reductase (DHCR24) genes (two androgen late-response genes implicated in prostate cancer development and progression) to activate their transcription (Guo et al., 2016) (Fig. 1.11).

PCAT1 promotes cell proliferation and is upregulated in major types of cancers [START_REF] Yan | Prognostic significance of long non-coding RNA PCAT-1 expression in human hepatocellular carcinoma[END_REF]Zhao et al., 2015;[START_REF] Qiao | Down regulation of the long non-coding RNA PCAT-1 induced growth arrest and apoptosis of colorectal cancer cells[END_REF][START_REF] Cui | Up-regulation of long non-coding RNA PCAT-1 correlates with tumor progression and poor prognosis in gastric cancer[END_REF], including prostate cancer [START_REF] Fagerberg | Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics[END_REF], particularly mCRPC [START_REF] Salinas | Prostate cancer in young men: an important clinical entity[END_REF]. In addition, PCAT1 overexpression was closely associated with clinicopathological characteristics, including tumor size, lymphatic metastasis, distant metastasis, tumor-node-metastasis stage and poor prognosis [START_REF] Salinas | Prostate cancer in young men: an important clinical entity[END_REF]. These results suggested that PCAT1 may function as a potential prognostic biomarker and therapeutic target in prostate cancer. According to recent reports by Rotinen et al and Guo et al, ONECUT2 (one cut homeobox 2) plays a critical role, as a master transcriptional regulator, in poorly differentiated neuroendocrine prostate tumors [START_REF] Rotinen | ONECUT2 is a targetable master regulator of lethal prostate cancer that suppresses the androgen axis[END_REF][START_REF] Guo | ONECUT2 is a driver of neuroendocrine prostate cancer[END_REF]. As a survival factor in mCRPC models, ONECUT2 depresses AR transcription-related program and activates NE differentiation genes, accelerating tumor progression to lethal disease [START_REF] Rotinen | ONECUT2 is a targetable master regulator of lethal prostate cancer that suppresses the androgen axis[END_REF]. Besides, overexpression of ONECUT2 in prostate adenocarcinoma under hypoxia condition is able to inhibit AR signaling and induce NE phenotype [START_REF] Guo | ONECUT2 is a driver of neuroendocrine prostate cancer[END_REF]. Given the crucial role of hypoxia in angiogenesis, the authors postulate that ONECUT2 may also contribute to the angiogenic phenotype of NEPC. Indeed, one study in ovarian cancer demonstrated that silencing ONECUT2 reduced VEGF expression and vascularization in xenografted tumors [START_REF] Lu | Blockade of ONECUT2 expression in ovarian cancer inhibited tumor cell proliferation, migration, invasion and angiogenesis[END_REF].

b. Sp1

Sp1, the first mammalian transcription factor identified and initially named according to the purification procedure adopted (Sephacryl and phosphocellulose columns), is now more commonly named Specificity protein 1 [START_REF] Kadonaga | Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain[END_REF][START_REF] Berg | Sp1 and the subfamily of zinc finger proteins with guanine-rich binding sites[END_REF]. It is a ubiquitously expressed transcription factor and belongs to a zinc finger family [START_REF] Lania | Transcriptional regulation by the Sp family proteins[END_REF][START_REF] Philipsen | A tale of three fingers: the family of mammalian Sp/XKLF transcription factors[END_REF][START_REF] O'connor | The Role of the Ubiquitously Expressed Transcription Factor Sp1 in Tissue-specific Transcriptional Regulation and in Disease[END_REF]. It is reported that Sp1 regulates gene transcription by binding to a GC rich element (consensus sequence: GGGGCGGGG), as well as GT/CACC (GGTGTGGGG) boxes in the promoter of target genes [START_REF] Hagen | Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes[END_REF][START_REF] Kingsley | Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression[END_REF][START_REF] Matsumoto | Cloning the cDNA for a new human zinc finger protein defines a group of closely related Krüppel-like transcription factors[END_REF]. Sp1 not only regulates the expression of AR but also acts as its coregulator. Yuan et al. showed that Sp1 can enhance transcriptional activity of the AR promoter and of androgen upregulated gene promoters (PSA and hK2) [START_REF] Yuan | Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells[END_REF]. Additionally, Hay et al. also demonstrated that Sp1 binds to GC-rich motifs of the AR promoter and activates AR transcription, whereas the associated antagonistic transcription factor pur-α can bind to the same region and inhibit AR transcription in PCa cells (Hay et al., 2015). WNT signaling is an evolutionary highly conserved signaling system throughout the eukaryotic kingdom [START_REF] Nelson | Convergence of Wnt, beta-catenin, and cadherin pathways[END_REF]. During embryonic and postnatal development, WNT signaling controls many cellular processes, including proliferation, survival and differentiation [START_REF] Grigoryan | Deciphering the function of canonical wnt signals in development and disease: Conditional loss-and gain-of-function mutations of beta-catenin in mice[END_REF][START_REF] Klaus | Wnt signalling and its impact on development and cancer[END_REF][START_REF] Lien | Wnt some lose some: Transcriptional governance of stem cells by WNT/β-catenin signaling[END_REF]. Deregulation in WNT signaling leads to an imbalance of such processes, often resulting in aberrant development or disease [START_REF] Clevers | Wnt/ β-catenin signaling in development and disease[END_REF][START_REF] Polakis | The many ways of WNT in cancer[END_REF]. In particular, deregulated WNT signaling is common in human cancers, including malignancies of the intestine [START_REF] Taketo | Shutting down wnt signal-activated cancer[END_REF][START_REF] White | Dysregulation of Wnt/β-catenin signaling in gastrointestinal cancers[END_REF], liver [START_REF] Satoh | AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1[END_REF], skin [START_REF] Chan | A common human skin tumour is caused by activating mutations in beta-catenin[END_REF], breast [START_REF] Shulewitz | Repressor roles for TCF-4 and SFRP1 in WNT signaling in breast cancer[END_REF] and prostate [START_REF] Hu | AXIN2 expression predicts prostate cancer recurrence and regulates invasion and tumor growth[END_REF].

The WNT signaling transduction can be mediated by three different, essentially separating but interacting pathways, the canonical Wnt/β-catenin, non-canonical planar cell polarity and the non-canonical Wnt/Calcium pathways (Fig. 1.12) [START_REF] Polakis | Wnt signaling in cancer[END_REF]. The best-studied Wnt signaling pathway is the canonical Wnt/β-catenin pathway in which Wnt ligands form a complex with Frizzled (FZD) receptor and coreceptors such as the low-density lipoprotein receptor-related protein 5 (LRP5) or LRP6. Upon receptor activation, the "destruction complex" which includes adenomatous polyposis coli (APC) protein and Axin is inhibited, thereby blocking the phosphorylation of β-catenin by both casein kinase Iα and glycogen synthase kinase-3β (GSK-3β), which is prerequisited for β-catenin degradation [START_REF] Gómez-Orte | Multiple functions of the noncanonical Wnt pathway[END_REF][START_REF] Sprowl | Past visits present: TCF/LEFs partner with ATFs for β-catenin-independent activity[END_REF]. [START_REF] Polakis | Wnt signaling in cancer[END_REF][START_REF] Gómez-Orte | Multiple functions of the noncanonical Wnt pathway[END_REF][START_REF] Sprowl | Past visits present: TCF/LEFs partner with ATFs for β-catenin-independent activity[END_REF].

A hallmark of canonical

Accumulating evidence supports that the WNT/β-catenin pathway plays an important role in CRPC, by interacting with AR signaling [START_REF] Chesire | Detection and analysis of beta-catenin mutations in prostate cancer[END_REF]Wang et al., 2008;Schweizer et al., 2008;[START_REF] Yokoyama | Wnt signaling in castration-resistant prostate cancer: implications for therapy[END_REF]. Findings of a Fig. 1.12 Overview of the Wnt signaling pathway. In the "Wnt-Off" state, Wnt/receptor interactions are interrupted by secreted Wnt antagonists, including sFRPs, DKKs and WIF1; and β-catenin is degraded by the formation of the "destruction complex" consisting of APC protein, Axin and others. In the "Wnt-On" state, Wnts are are lipid modified by the acyl transferase porcupine in the endoplasmic reticulum, and act in an autocrine and paracrine fashion. The Wnts form a complex with Frizzled receptor and coreceptors LRP. Upon receptor activation, the "destruction complex" which includes APC, Axin and others is inhibited thereby blocking β-catenin phosphorylation for degradation. This inhibition results in cytoplasmic β-catenin stabilization and accummulation which facilitates its translocation into the nucleus. Nuclear β-catenin acts as a transcriptional co-activator for LEF1/TCF, leading gene transcription of Wnt target genes, such as JUN, Cyclin-D1, and MMP7. In addition, Wnts bind to tyrosine-protein kinase transmembrane receptors ROR2 and RYK to activate other non-canonical planar cell polarity, the Wnt/JNK and the Wnt/Calcium pathways. Adapt from: [START_REF] Yokoyama | Wnt signaling in castration-resistant prostate cancer: implications for therapy[END_REF] protein-protein interaction between AR and β-catenin suggested the biological significance of β-catenin in PCa cells. [START_REF] Truica | Beta-catenin affects androgen receptor transcriptional activity and ligand specificity[END_REF] showed that β-catenin could directly bind to AR to enhance its transcriptional activity stimulated by androgen, androstenedione, or estradiol in LNCaP cells [START_REF] Truica | Beta-catenin affects androgen receptor transcriptional activity and ligand specificity[END_REF]. In 2002,

Yang et al demonstrated that β-catenin preferentially and directly bound to the LBD of AR in the presence of DHT over several other steroid hormone receptors [START_REF] Yang | Linking beta-catenin to androgen-signaling pathway[END_REF]. Further studies revealed that β-catenin bound to the AF-2 region of the AR LBD, and modulated the transcriptional effects of the AR NTD as well as the p160 coactivator transcriptional intermediary factor 2 (TIF2); importantly, a single AR lysine residue (K720) has been shown to be necessary for the AR/β-catenin and TIF2/β-catenin interactions [START_REF] Song | Beta-catenin binds to the activation function 2 region of the androgen receptor and modulates the effects of the N-terminal domain and TIF2 on ligand-dependent transcription[END_REF][START_REF] Masiello | Recruitment of beta-catenin by wild-type or mutant androgen receptors correlates with ligand-stimulated growth of prostate cancer cells[END_REF]. In β-catenin,

early mapping experiments suggested that the NH2 terminus and the first six armadillo repeats of β-catenin were involved in its interaction with AR. In particular, deletion of repeat 6 fully abolished the physical interaction between AR and β-catenin, suggesting a key role of this repeat in the interaction [START_REF] Yang | Linking beta-catenin to androgen-signaling pathway[END_REF].

Phenotypically, transient over-expression of β-catenin in AR-positive PCa cell lines CWR22-Rv1 and LAPC-4 enhanced AR-mediated transcription of its target genes, in an androgen-dependent manner (Chesire et al., 2004). Hence, β-catenin (wild-type or mutated) is considered as a ligand-dependent co-activator of the AR-driven transcription. Binding of β-catenin to ligand-engaged AR also facilitates the movement of β-catenin into the nucleus (Mulholland et al., 2005). Furthermore, it was shown that WNT/β-catenin signaling could increase AR transcription via the TCF/LEF-1 binding sites in the AR promoter [START_REF] Li | LEF1 in androgen-independent prostate cancer: regulation of androgen receptor expression, prostate cancer growth, and invasion[END_REF]. Thus, in hormone-naïve PCa, WNT/β-catenin signaling serves as a positive regulator of AR signaling in an androgen-dependent manner.

β-catenin is also one of the three AR coactivators (other two AR specific coactivators are ARA70 and ARA55) that can enhance AR transcriptional activity in LNCaP cells when treated with 17β-estradiol [START_REF] Yang | Linking beta-catenin to androgen-signaling pathway[END_REF][START_REF] Song | Beta-catenin binds to the activation function 2 region of the androgen receptor and modulates the effects of the N-terminal domain and TIF2 on ligand-dependent transcription[END_REF][START_REF] Masiello | Recruitment of beta-catenin by wild-type or mutant androgen receptors correlates with ligand-stimulated growth of prostate cancer cells[END_REF]. In addition, β-catenin can function as a coactivator with altered AR with mutations W741C and T877A in prostate cancer cell lines [START_REF] Song | Beta-catenin binds to the activation function 2 region of the androgen receptor and modulates the effects of the N-terminal domain and TIF2 on ligand-dependent transcription[END_REF]. These AR mutations were detected in CRPC patients that have been treated

with bicalutamide leading to the W741C mutation and also in CRPC patients with lymph node metastatic lesions containing the T877A mutation [START_REF] Song | Beta-catenin binds to the activation function 2 region of the androgen receptor and modulates the effects of the N-terminal domain and TIF2 on ligand-dependent transcription[END_REF]. In rodent studies, Chesire et al. reported that castrated mice receiving androgen treatment exhibited nuclear co-localization of AR and β-catenin in normal prostatic epithelium (Chesire and Isaacs, 2003). Nuclear β-catenin localization was found to occur concomitantly with androgen-induced regrowth of normal rat prostate from androgen deprivation induced regression. Furthermore, Wang et al observed increased expression and nuclear colocalization of AR and β-catenin as well as the interaction between endogenous AR and β-catenin in CRPC from castrated mice (Wang et al., 2008). However, they found no interaction or colocalization of AR and β-catenin in xenografts from noncastrated mice. Mutations of β-catenin are uncommon in prostate cancer (< 5%) (Voeller et al., 1998;[START_REF] Chesire | Detection and analysis of beta-catenin mutations in prostate cancer[END_REF].

PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR signaling pathway has a diverse array of functions, including the regulation of cellular survival, differentiation and stem cell-like properties, growth, proliferation, metabolism, migration, and angiogenesis (Guba et al., 2002;Dubrovska et al., 2009;[START_REF] Liu | MLL fusions: pathways to leukemia[END_REF]Courtney et al., 2010;Furic et al., 2010;Hsieh et al., 2012). There are three classes of PI3K that are differentiated by their structural characteristics and substrate specificities (Courtney et al., 2010).

Class I PI3Ks are activated by receptor tyrosine kinases (RTKs), G-protein-coupled receptors, and some oncogenes, such as rat sarcoma oncogene (RAS), and can be further subdivided into class IA and IB, of which class IA PI3Ks are most frequently implicated in cancer. Class IA PI3Ks consist of two subunits: a regulatory subunit, p85, and a catalytic subunit, p110. There are three isoforms of p85 (p85α, p85β, and p55γ) encoded by the genes PIK3R1, PIK3R2, and PIK3R3 respectively and three isoforms of class IA p110 (α, β, and δ) encoded by the genes PIK3CA, PIK3CB, and PIK3CD respectively (Courtney et al., 2010). Activation of PI3K leads to the phosphorylation of phosphatidylinositol 4,5-bisphosphate [PI (4,5)P2] to phosphatidylinositol 3,4,4,5)P3] and subsequent recruitment of AKT to the plasma membrane where Akt is activated [START_REF] Liu | MLL fusions: pathways to leukemia[END_REF], Courtney et al., 2010). AKT activation is mediated through phosphorylation at two residues: T308 by phosphoinositide-dependent kinase-1 and S473 by the mTOR complex 2 (mTORC2). Both phosphorylation events are required for full activation of AKT (Sarbassov et al., 2005). Upon activation, AKT phosphorylates a host of other proteins including glycogen synthase kinase 3 (GSK3), FOXO transcription factors, and tuberous sclerosis complex (TSC) and thereby regulates a range of cellular processes, including protein synthesis, cell survival, proliferation, and metabolism [START_REF] Liu | MLL fusions: pathways to leukemia[END_REF]Courtney et al., 2010). In prostate cancer, PI3K signaling has been shown to repress AR transcriptional activity, illustrating the lineage-specific complexity of this pathway [START_REF] Carver | Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer[END_REF].

mTOR is a serine threonine kinase and participates in the formation of the catalytic subunit of the functionally distinct mTORC1 and mTORC2. Depending on available nutrients and the cellular environment, mTORC1 controls the growth of the cell through phosphorylation of S6K and the eukaryotic initiation factor 4E-binding protein et al., 2006;Carracedo et al., 2008;Rodrik-Outmezguine et al., 2011).

The PI3K/AKT/mTOR and AR signaling pathways have recently been shown to regulate each other through complex reciprocal feedback mechanisms (Wen et al., 2000;Lin et al., 2001;[START_REF] Liu | A transcription-independent function of FOXO1 in inhibition of androgen-independent activation of the androgen receptor in prostate cancer cells[END_REF]Sarker et al., 2009;[START_REF] Carver | Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer[END_REF][START_REF] Mulholland | Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth[END_REF]. PI3K/AKT/mTOR signaling inhibits AR signaling via feedback inhibition of human epidermal growth factor 2/3 (HER2/3) kinases, which have been shown to promote AR stability and transcriptional activity [START_REF] Carver | Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer[END_REF]. In addition, PTEN increases the transcriptional activity of AR by negatively regulating the expression of the transcription factors Egr1 and c-JUN, which inhibit AR-targeted gene expression, such as FKBP5 [START_REF] Mulholland | Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth[END_REF]. Meanwhile, AR signaling downregulates PI3K/AKT/mTOR signaling through FK506-binding protein-5 (FKBP-5)-mediated stabilization of the Akt phosphatase PHLPP [START_REF] Carver | Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer[END_REF]. Activation of the PI3K/AKT/mTOR pathway as a result of treatments targeting AR signaling or due to PTEN loss may, therefore, enable prostate cancer cells to survive and proliferate in androgen-reduced conditions. Conversely, PTEN controls the transcription of NKX3.1, which negatively regulates the AR promoter and reduces AR pathway signaling. When PTEN is lost, this brake on AR signaling is also lost (Lei et al., 2006). Therefore, inhibition of the PI3K/AKT/mTOR pathway may lead to a compensatory increase in AR activity and may promote prostate cancer progression and development of resistance to single-agent PI3K pathway inhibitor therapy. This concept of reciprocal inhibition is further supported by the recent finding that use of antiandrogens for chemoprevention actually accelerates progression to invasive prostate cancer in a Pten-null mouse model (Jia et al., 2013).

More importantly, PI3K-AKT-mTOR pathway inhibition is associated with augmented AR signaling that can contribute to drug resistance and promote prostate cancer progression [START_REF] Carver | Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer[END_REF][START_REF] Thomas | Synergistic targeting of PI3K/AKT pathway and androgen receptor axis significantly delays castration-resistant prostate cancer progression in vivo[END_REF][START_REF] Qi | Reciprocal feedback inhibition of the androgen receptor and PI3K as a novel therapy for castrate-sensitive and -resistant prostate cancer[END_REF][START_REF] Audet-Walsh | Nuclear mTOR acts as a transcriptional integrator of the androgen signaling pathway in prostate cancer[END_REF]. Carver and colleagues showed that PI3K/mTOR inhibition activates AR signaling in human xenograft and transgenic mouse models of prostate cancer, and that co-treatment with the PI3K/mTOR inhibitor BEZ235 and the antiandrogen MDV3100 (enzalutamide) significantly reduced tumor burden relative to BEZ235 monotherapy [START_REF] Carver | Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer[END_REF][START_REF] Shorning | The PI3K-AKT-mTOR Pathway and Prostate Cancer: At the Crossroads of AR, MAPK, and WNT Signaling[END_REF]. In corroboration, resistance to the AKT inhibitor capivasertib (AZD5363) in LNCaP prostate cancer xenografts is also associated with elevated AR signaling, and combining AZD5363 treatment with the antiandrogen bicalutamide prolonged disease stabilization [START_REF] Thomas | Synergistic targeting of PI3K/AKT pathway and androgen receptor axis significantly delays castration-resistant prostate cancer progression in vivo[END_REF]. Furthermore, mTOR and EGFR co-inhibition with everolimus and gefitinib has shown limited sensitivity in patients owing to enhanced AR activity and PSA levels (Audet- [START_REF] Audet-Walsh | Nuclear mTOR acts as a transcriptional integrator of the androgen signaling pathway in prostate cancer[END_REF], providing further rationale for combining AR and PI3K-AKT-mTOR blockade to treat prostate cancer [START_REF] Shorning | The PI3K-AKT-mTOR Pathway and Prostate Cancer: At the Crossroads of AR, MAPK, and WNT Signaling[END_REF].

Several distinct molecular mechanisms have been identified that underpin AR reactivation upon AKT inhibition. Notably, AKT inhibition can prevent FOXOs from AKT-mediated nuclear exclusion, which can lead to augmented transcription of FOXO-target genes such as RTKs (e.g., ERBB2/3 encoding HER2/3) (Figure 3B) [START_REF] Yao | Suppression of HER2/HER3-mediated growth of breast cancer cells with combinations of GDC-0941 PI3K inhibitor, trastuzumab, and pertuzumab[END_REF][START_REF] Carver | Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer[END_REF][START_REF] Chandarlapaty | AKT inhibition relieves feedback suppression of receptor tyrosine kinase expression and activity[END_REF]. HER2/3 activity has been shown to promote AR signaling by protecting AR from ubiquitination and proteasomal degradation, and by enhancing AR binding to ARE target sequences and stimulating AR transcriptional activity [START_REF] Yeh | From HER2/Neu signal cascade to androgen receptor and its coactivators: a novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells[END_REF][START_REF] Mellinghoff | HER2/neu kinase-dependent modulation of androgen receptor function through effects on DNA binding and stability[END_REF].

Nonetheless, the role of FOXO-dependent signaling in PI3K/AKT/mTOR and AR pathway crosstalk is complex. Although FOXO transcription factors upregulate the expression of RTKs [START_REF] Chandarlapaty | AKT inhibition relieves feedback suppression of receptor tyrosine kinase expression and activity[END_REF] causing a subsequent increase in AR signaling [START_REF] Mellinghoff | HER2/neu kinase-dependent modulation of androgen receptor function through effects on DNA binding and stability[END_REF], the ectopic expression of FOXO1 conversely dampens AR activity [START_REF] Liu | A transcription-independent function of FOXO1 in inhibition of androgen-independent activation of the androgen receptor in prostate cancer cells[END_REF][START_REF] Shorning | The PI3K-AKT-mTOR Pathway and Prostate Cancer: At the Crossroads of AR, MAPK, and WNT Signaling[END_REF].

RAS/RAF/MEK pathway

The RAS/RAF/MEK pathway is also involved in extensive cross talk with the PI3K/AKT/mTOR pathway (Fig. 1.13), and activation of this pathway has been associated with decreased sensitivity to PI3K/AKT/mTOR pathway inhibitors (Ihle et al., 2009). Recently, a study of prostate cancer tissue microarrays found that the RAS/RAF/MEK pathway was significantly elevated in both primary and metastatic lesions (Mulholland et al., 2012). In murine models, PTEN deletion results in the development of prostate cancer (Wang et al., 2003); while the combination of PTEN deletion and RAS activation significantly accelerated prostate cancer progression caused by PTEN loss, and this was accompanied by EMT and macrometastasis (Mulholland et al., 2012). Furthermore, inhibition of the RAS/RAF/MEK pathway with a MEK inhibitor (PD325901) significantly reduced metastatic progression initiated by PTEN-deficient and KRAS-activated stem/progenitor cells (Mulholland et al., 2012). Thus, cross talk between the PI3K/AKT/mTOR pathway and the RAS/RAF/MEK pathway in prostate cancer is likely clinically important in promoting metastasis.

Receptor tyrosine kinases (RTKs), which activate both the PI3K/AKT/mTOR and the RAS/RAF/MEK pathways, are in turn negatively regulated by mTORC1 activity; inhibition of mTORC1 leads to a relief of an inhibitory signal from S6K to IRS-1, a receptor substrate for multiple cell surface receptors, such as insulin and IGF, HER2/3 and epidermal growth factor receptor (EGFR), and others (O'Reilly et al., 2006;[START_REF] Carver | Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer[END_REF], Rodrik-Outmezguine et al., 2011). Relief of this feedback inhibition with single-agent rapamycin analogs may lead to rapid compensatory PI3K

and Akt re-activation and limit the pharmacodynamics and clinical impact of these agents on tumor cell survival and invasion/metastasis (Rodrik-Outmezguine et al., 2011). Combined inhibition of cell surface receptors with mTORC1 inhibitors has demonstrated an ability to overcome this feedback loop and is an area of ongoing investigation. Whether combination therapy with PI3K, AR, cell surface receptor, and/or RAS/RAF/MEK pathway inhibition is needed for optimal therapy in preclinical or clinical prostate cancer is not known yet.

The function of AR

AR in normal tissues

In humans, AR is expressed in all organs with the exception of the spleen and bone marrow. It is essential for the development of male reproductive organs, puberty, male fertility and male sexual function. In the prostate, androgens are essential for normal development and function. AR is expressed in both epithelial cells and stromal cells. In epithelial cells, AR has a role in cellular differentiation, survival and the (Wen et al., 2000;Lin et al., 2001).

expression of secretory proteins (Cunha et al., 2004;[START_REF] Olsen | Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes[END_REF]. Stromal AR has a role in embryonic prostate development, epithelial differentiation and determining secretory protein expression [START_REF] Wen | Stromal androgen receptor roles in the development of normal prostate, benign prostate hyperplasia, and prostate cancer[END_REF].

Cell cycle regulator

Several papers have already shown that AR is a critical regulator of G1-S transition in AR-dependent cell cycle progression in prostate cancer cells. The p21 cip has been validated as a direct AR target, and the induction of p21 cip upon androgen stimulation may assist in assembling active CDK4/cyclin D1 complexes (Lu et al., 1999;[START_REF] Alt | p21(Cip1) Promotes cyclin D1 nuclear accumulation via direct inhibition of nuclear export[END_REF][START_REF] Sherr | Living with or without cyclins and cyclin-dependent kinases[END_REF]. This supposition is consistent with the findings revealing that p21 cip expression was enhanced in tumors, and correlated with a higher proliferative index and Gleason grade (Aaltomaa et al., 1999;Baretton et al., 1999). Furthermore, Knudsen et al. showed that androgen depletion induces p27 Kip1 , which likely contributes to the observed reductions in CDK2 activity (Knudsen et al., 1998). And, p27 Kip1 loss in the context of a PTEN mutation promotes a tumorigenic phenotype in the prostate [START_REF] Cristofano | Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse[END_REF][START_REF] Gao | A critical role for p27kip1 gene dosage in a mouse model of prostate carcinogenesis[END_REF].

Apoptosis regulator

Kerr et al first suggested the term "apoptosis" and confirmed it as a general mechanism of controlled cell deletion, which is complementary to mitosis in the regulation of animal cell populations [START_REF] Kerr | Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics[END_REF]. Rokhlin et al found androgen/AR signaling could directly regulate p53 to suppress apoptosis.

Mechanistically, androgen suppresses TNF-α/Fas-induced apoptosis through inhibition of p53 expression and caspase-2 activation in LNCaP and C4-2 cells (Rokhlin et al., 2005). Other studies also showed that androgen could block apoptosis induced by Fas activation and TNF-α [START_REF] Kimura | Androgen blocks apoptosis of hormone-dependent prostate cancer cells[END_REF]. Besides, p21 has been proven to be able to protect against p53-mediated apoptosis [START_REF] Gorospe | p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells[END_REF].

Asada found that overexpression of p21 may induce cell cycle arrest and led to an apoptosis-resistant phenotype [START_REF] Asada | Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation[END_REF]. Furthermore, AR stimulated endogenous p21 mRNA and protein levels and functioned as an apoptosis inhibitor to promote PCa LNCaP cell growth (Lu et al., 1999). Interestingly, Frezza et al found that co-treatment with Bortezomib and the AR antagonist Casodex caused significant decrease in AR expression and led to caspase-3 activity increase in LNCaP and PC3-AR cells, suggesting that AR might suppress caspase-3 expression (Frezza et al., 2011). Liao et al also showed that knockdown of AR via siRNA led to apoptotic death in PCa cells (Liao et al., 2005). androgen may be critical for prostate physiology and disease [START_REF] Wu | CCN3/NOV gene expression in human prostate cancer is directly suppressed by the androgen receptor[END_REF].

Histone modifiers: acetyltransferases and methyltransferases

Acetyltransferases

Some of these histone modifications are associated with transcriptional activation (e.g., acetylation), and others are indicative of active or repressed genes (e.g. methylation) [START_REF] Mellor | It takes a PHD to read the histone code[END_REF][START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF]. Dynamic changes in the modification state of covalent histones are related to androgen-stimulated transcription. These include modifications that activate histone 3 such as acetylation of lysine 9 and lysine 14, dimethylation of arginine 17, phosphorylation of serine 10, and dimethylation as well as trimethylation of lysine 4 [START_REF] Kang | Coregulator recruitment and histone modifications in transcriptional regulation by the androgen receptor[END_REF]. Fu et al. demonstrated a direct interaction between p300, CBP, P/CAF and AR. The potentiation of ligand-induced AR transactivation by these three coactivators relies on the presence of a functional histone acetylase domain. Furthermore, p300 and P/CAF acetylate AR at three lysine residues in its DBD-hinge region. They conclude that CBP and p300 function as a direct bridge between DNA-bound AR and the basal transcriptional machinery (Fu et al., 2000).

Methyltransferases

AR-dependent transcription relies on methyltransferase activities. Androgen stimulation leads to recruitment of CARM-1 (coactivator-associated arginine methyltransferase-1) to androgen-responsive enhancers [START_REF] Majumder | Involvement of arginine methyltransferase CARM1 in androgen receptor function and prostate cancer cell viability[END_REF].

Moreover, the presence of CARM-1 enhances AR transactivation by p160 family members. Loss of CARM-1 reduces transcription of androgen-responsive genes [START_REF] Chen | Regulation of transcription by a protein methyltransferase[END_REF][START_REF] Majumder | Involvement of arginine methyltransferase CARM1 in androgen receptor function and prostate cancer cell viability[END_REF]. CARM-1 also recruited to the AR transcriptional complex through p44, a component of the methylosome. p44, found on the promoters of AR target genes upon androgen stimulation, interacts directly with AR and CARM-1 and stimulates the transcription of some AR-target genes such as, NKX3.1. Lee et al. showed that G9a, a histone H3 Lys-9 methyltransferase localized in euchromatin, functions as a coactivator for AR, although weakly. G9a cooperates synergistically with CARM-1 and p300 in activating transcription by the AR. This synergy is strongly dependent on the activity of CARM-1 arginine-specific protein methyltransferase, which indicates a link between histone arginine and lysine methylation during AR-mediated transcription [START_REF] Lee | Histone H3 lysine 9 methyltransferase G9a is a transcriptional coactivator for nuclear receptors[END_REF]. 2008). Previous study showed that NKX3.1 expression is completely lost in advanced cancers [START_REF] Bowen | Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression[END_REF], more recent analyses using a highly sensitive antibody indicate that low levels of NKX3.1 expression can be demonstrated in nearly all prostate cancers and metastases examined [START_REF] Gurel | NKX3.1 as a marker of prostatic origin in metastatic tumors[END_REF] Interestingly, several studies reported NKX3.1 as a critical regulator of prostate epithelial differentiation and stem cell function in mouse models. During development, NKX3.1 is expressed in all epithelial cells of the nascent prostate buds from the urogenital sinus, and represents the earliest known marker for the prostate epithelium (Bhatia-Gaur et al. 1999). In the absence of NKX3.1, there is a significant decrease in prostatic ductal branching, as well as in production of secretory proteins (Bhatia-Gaur et al. 1999;Schneider et al. 2000). Notably, young adult Nkx3.1 heterozygous and homozygous mutants frequently display prostate epithelial hyperplasia and dysplasia, and often develop Prostatic Intraepithelial Neoplasia (PIN) by 1 year of age (Bhatia-Gaur et al. 1999;Schneider et al. 2000). In particular, Nkx3.1 inactivation in mice results in a defective response to oxidative damage, while its expression in human prostate cancer cell lines protects against DNA damage and is regulated by inflammation [START_REF] Ouyang | Loss-of-function of Nkx3.1 promotes increased oxidative damage in prostate carcinogenesis[END_REF][START_REF] Markowski | Inflammatory cytokines induce phosphorylation and ubiquitination of prostate suppressor protein NKX3.1[END_REF][START_REF] Bowen | NKX3.1 activates cellular response to DNA damage[END_REF]. These findings are consistent with the tumor suppressor activity of NKX3.1 observed in cell culture and xenograft assays [START_REF] Kim | Nkx3.1 mutant mice recapitulate early stages of prostate carcinogenesis[END_REF]Lei et al. 2006).

TMPRSS2-ERG translocations

One of the earliest genetic alterations in prostate cancer is overexpression of the ERG (ETS-related gene) oncogene, which occurs in over 50% of prostate cancers. In the majority of tumors, this is driven by fusion of the ERG gene with transmembrane protease, serine 2 (TMPRSS2), a prostate-specific and androgenregulated gene that maps very close to ERG on the same chromosome [START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF][START_REF] Perner | TMPRSS2:ERG fusion-associated deletions provide insight into the heterogeneity of prostate cancer[END_REF]Wang et al., 2006;[START_REF] Clark | Diversity of TMPRSS2-ERG fusion transcripts in the human prostate[END_REF]. This generates an androgen-responsive fusion oncoprotein, leading to overexpression of the respective ETS family member.

ERG is a member of the large family of ETS transcription factors [START_REF] Hollenhorst | Genomic and biochemical insights into the specificity of ETS transcription factors[END_REF]. This gene fusion has never been found in normal prostate but is present in tumor adjacent to PIN. The frequency of these TMPRSS2-ERG fusions is 15% in HGPIN lesions and 50% in localized prostate cancer [START_REF] Clark | Complex patterns of ETS gene alteration arise during cancer development in the human prostate[END_REF][START_REF] Mosquera | Characterization of TMPRSS2-ERG fusion high-grade prostatic intraepithelial neoplasia and potential clinical implications[END_REF][START_REF] Albadine | TMPRSS2-ERG gene fusion status in minute (minimal) prostatic adenocarcinoma[END_REF]Taylor et al. 2010) [START_REF] Brase | TMPRSS2-ERG -specific transcriptional modulation is associated with prostate cancer biomarkers and TGF-β signaling[END_REF]. Although the data reported in different studies are variable, a consistent association with ERG overexpression of more than ten genes, including CACNA1D, TDRD1, PLA2G7, and NCALD, has been found [START_REF] Iljin | TMPRSS2 fusions with oncogenic ETS factors in prostate cancer involve unbalanced genomic rearrangements and are associated with HDAC1 and epigenetic reprogramming[END_REF], Jhavar et al. 2008, Taylor et al. 2010[START_REF] Brase | TMPRSS2-ERG -specific transcriptional modulation is associated with prostate cancer biomarkers and TGF-β signaling[END_REF][START_REF] Boormans | Identification of TDRD1 as a direct target gene of ERG in primary prostate cancer[END_REF].

This does not mean that these genes are direct ERG target genes. They might be indirectly regulated by ERG or they might represent a common prostate cell type in which TMPRSS2-ERG fusion occurred. Interestingly, a recent study revealed that TMPRSS2-ERG was also involved in PCa bone metastasis via its regulation of the expression of osteoblastic markers [START_REF] Delliaux | TMPRSS2:ERG gene fusion expression regulates bone markers and enhances the osteoblastic phenotype of prostate cancer bone metastases[END_REF]. The mechanism of expression of other ERG-associated genes remains to be investigated.

EZH2

The EZH2 gene, situated on 7q35-36, encodes EZH2 protein, and EZH2 protein levels are significantly increased in prostate cancer compared with BPH tissue [START_REF] Varambally | The polycomb group protein EZH2 is involved in progression of prostate cancer[END_REF]. EZH2 increases histone deacetylation, which is a repressor of transcription in prostate cancer. Elevated levels of EZH2 mRNA and protein in clinically localised prostate cancer predicts poor prognosis. In addition, repression of EZH2 by small interfering RNA (RNAi) decreases the rate of cell proliferation in PCa cell lines [START_REF] Varambally | The polycomb group protein EZH2 is involved in progression of prostate cancer[END_REF]. This gene represents an interesting development both as a prognostic marker and also as a therapeutic target.

PTEN

Germline disruption of Pten in heterozygous mutants or conditional ablation in the prostate epithelium in the mouse results in PIN and/or adenocarcinoma development (Di Cristofano et al. 1998a;[START_REF] Podsypanina | Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems[END_REF][START_REF] Trotman | Pten dose dictates cancer progression in the prostate[END_REF]Wang et al. 2003). Fluorescence in situ hybridization (FISH) has been used to identify genomic PTEN loss, which is found in 9-23 % of HGPIN lesions [START_REF] Schmitz | Complete loss of PTEN expression as a possible early prognostic marker for prostate cancer metastasis[END_REF]Sircar et al., 2009) and 10-70 % of prostate cancers [START_REF] Suzuki | Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues[END_REF][START_REF] Yoshimoto | FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome[END_REF], and is correlated with an overall poor prognosis [START_REF] Yoshimoto | FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome[END_REF]Taylor et al., 2010).

Notably, PTEN reduction or loss in prostate cancer is correlated to the emergence of castration-resistant prostate cancer [START_REF] Mulholland | PTEN and GSK3beta: key regulators of progression to androgen-independent prostate cancer[END_REF]Shen and Abate-Shen 2007). In particular, perturbation of PTEN expression in human prostate cancer cell lines or targeted Pten disruption in the mouse is sufficient for the development of castration resistance (Lin et al. 2004;[START_REF] Bertram | Loss of PTEN is associated with progression to androgen independence[END_REF]Gao et al. 2006;Wu et al. 2006). PTEN loss is more common in hormone refractory and metastatic prostate cancer than in hormone-dependent primary tumors, with homozygous PTEN loss in 10 and 50 % of hormone-dependent and metastatic/hormone refractory cases, respectively [START_REF] Verhagen | The PTEN gene in locally progressive prostate cancer is preferentially inactivated by bi-allelic gene deletion[END_REF][START_REF] Yoshimoto | Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias[END_REF][START_REF] Mulholland | PTEN and GSK3beta: key regulators of progression to androgen-independent prostate cancer[END_REF][START_REF] Schmitz | Complete loss of PTEN expression as a possible early prognostic marker for prostate cancer metastasis[END_REF]Sircar et al., 2009;Han et al., 2009).

Moreover, loss of PTEN and AR expression has been correlated clinically with increased mortality in CRPC patients (Sircar et al., 2009). Microarray-based

Comparative Genomic Hybridization (aCGH) analysis on metastatic prostate cancer samples has also demonstrated frequent amplification of AR (73%), coinciding with aberrant deletion of PTEN (87%) [START_REF] Friedlander | Common structural and epigenetic changes in the genome of castration-resistant prostate cancer[END_REF]. Therefore, PTEN could serve as a prognostic marker for hormone refractory and metastatic disease.

The AKT/mTOR signaling pathways

As noted above 3.5.3.2, considerable evidence indicates that Pten loss of function results in up-regulation of the AKT/mTOR signaling pathway in prostate cancer, primarily through activation of AKT1 [START_REF] Thomas | Antibody-based profiling of the phosphoinositide 3-kinase pathway in clinical prostate cancer[END_REF][START_REF] Chen | The deficiency of Akt1 is sufficient to suppress tumor development in Pten+/-mice[END_REF][START_REF] Mulholland | PTEN and GSK3beta: key regulators of progression to androgen-independent prostate cancer[END_REF]Shen and Abate-Shen 2007). Upregulation of this pathway in prostate cancer can take place through activating mutations of AKT1 [START_REF] Boormans | An activating mutation in AKT1 in human prostate cancer[END_REF], or through activation of the p110β isoform of PI3K [START_REF] Hill | The role of PI3-kinase p110beta in AKT signally, cell survival, and proliferation in human prostate cancer cells[END_REF][START_REF] Lee | A constitutively activated form of the p110beta isoform of PI3-kinase induces prostatic intraepithelial neoplasia in mice[END_REF]. The consequences of AKT/mTOR pathway activation are particularly relevant for castration-resistant prostate cancer, as has been shown in genetically engineered mouse models, in gain-of-function studies with orthotopic grafting or tissue recombination models, as well as in human cell lines [START_REF] Majumder | Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: the MPAKT model[END_REF][START_REF] Uzgare | Enhanced redundancy in Akt and mitogen-activated protein kinase-induced survival of malignant versus normal prostate epithelial cells[END_REF]Gao et al. 2006a;[START_REF] Xin | Progression of prostate cancer by synergy of AKT with genotropic and nongenotropic actions of the androgen receptor[END_REF]). In addition, the consequences of Akt activation are mediated in part by activation of NF-κB signaling via stimulation of IKK [START_REF] Dan | Akt-dependent regulation of NF-kB is controlled by mTOR and Raptor in association with IKK[END_REF]. Conversely, functional studies in mouse models and correlative studies in human prostate cancer have implicated deregulated NF-κB signaling in mediating androgen responsivity, metastasis, and disease outcome [START_REF] Fradet | Nuclear factor-kappaB nuclear localization is predictive of biochemical recurrence in patients with positive margin prostate cancer[END_REF][START_REF] Ismail | Expression of NF-kappaB in prostate cancer lymph node metastases[END_REF][START_REF] Lessard | Nuclear localization of nuclear factor-kappaB p65 in primary prostate tumors is highly predictive of pelvic lymph node metastases[END_REF][START_REF] Luo | Nuclear cytokine-activated IKKalpha controls prostate cancer metastasis by repressing Maspin[END_REF]Zhang et al. 2009).

TGFβ

Several reports showed that AR crosstalks with transforming growth factor β (TGFβ) in prostate cancer (Culig et al., 2002;[START_REF] Yoon | Direct activation of TGF-beta1 transcription by androgen and androgen receptor complex in Huh7 human hepatoma cells and its tumor in nude mice[END_REF]Zhu and Kyprianou, 2008).

TGFβ cooperates with AR signaling to promote prostate cancer cell growth, in part through the regulation of the Runx2 transcription factor (van der [START_REF] Van Der Deen | The cancer-related Runx2 protein enhances cell growth and responses to androgen and TGFbeta in prostate cancer cells[END_REF].

Runx2 enhances PCa cell growth in response to both androgen and TGFβ signaling.

TGFβ typically is growth stimulatory in stromal cells, but inhibitory in epithelial cells in vitro. However, there are a number of mechanisms by which PCa cells overcome this response including loss of TGFβ receptor expression [START_REF] Guo | Down-regulation of protein and mRNA expression for transforming growth factor-beta (TGF-beta1) type I and type II receptors in human prostate cancer[END_REF]Shafi et al., 2013), DHT mediated repression of receptor or Smad3 expression [START_REF] Song | DHT selectively reverses Smad3-mediated/TGF-beta-induced responses through transcriptional down-regulation of Smad3 in prostate epithelial cells[END_REF], over-expression of COUP-TFII resulting in inhibition of TGFβ signaling through SMAD4 [START_REF] Qin | COUP-TFII inhibits TGF-β-induced growth barrier to promote prostate tumorigenesis[END_REF] or induction of cytokines such as IL-6 [START_REF] Park | Transforming growth factor-beta1 activates interleukin-6 expression in prostate cancer cells through the synergistic collaboration of the Smad2, p38-NF-kappaB, JNK, and Ras signaling pathways[END_REF].

TGF-β induces epithelial-mesenchymal transition (EMT) to facilitate tumor progression and metastasis [START_REF] Jones | Targeting TGF-beta in prostate cancer: therapeutic possibilities during tumor progression[END_REF]. In prostate cancer, TGF-β1 induces EMT in prostate tumor epithelial cells and in a mouse model of tumorigenesis with a targeted SMAD3 disruption, supporting a contributing role of TGF-β signaling in EMT and prostatic cancer metastasis [START_REF] Ao | Transforming growth factor-beta promotes invasion in tumorigenic but not in nontumorigenic human prostatic epithelial cells[END_REF][START_REF] Roberts | Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis[END_REF]. TGF-β induces EMT in prostate tumor through constitutively active Akt that inhibits translocation of SMAD3 and p21 to the nucleus [START_REF] Ao | Transforming growth factor-beta promotes invasion in tumorigenic but not in nontumorigenic human prostatic epithelial cells[END_REF]. In PC-3 prostate cancer cells, blockade of NF-κB signaling leads to decreased vimentin expression and inhibition of their invasive capability, indicating functional involvement of NF-κB in mediating TGF-β-induced EMT (Zhang et al., 2009).

AR independent mechanisms driving prostate cancer progression

Progression to CRPC results from escape of ADT, which targets the AR signaling axis and inhibits AR-driven proliferation and survival pathways. The exact mechanisms underlying the transition from androgen-dependent PC to CRPC remain incompletely understood. At the same time, the presence of AR-negative cell populations in CRPC has also been identified. While AR signaling has been proposed as the primary driver of CRPC, AR-independent signaling pathways may represent additional mechanisms underlying CRPC progression.

Alternative steroid receptors: GR and PR

Steroid receptors, including AR, progesterone receptor (PR) and glucocorticoid receptor (GR) are high homology, particularly in the DBD binding region [START_REF] Laudet | Evolution of the nuclear receptor gene superfamily[END_REF][START_REF] Karmakar | Interaction of glucocorticoid receptor (GR) with estrogen receptor (ER) α and activator protein 1 (AP1) in dexamethasone-mediated interference of ERα activity[END_REF]. Almost all PCa were discovered to express AR, whereas GR is only present in 30% of PCa cases; however, GR expression increases in patients following ADT [START_REF] Szmulewitz | Serum/glucocorticoid-regulated kinase 1 expression in primary human prostate cancers[END_REF]. Recent studies indicated that AR and GR possess the same chromatin binding sites and regulate the expression of AR-specific genes, such as FKBP5 [START_REF] Sahu | FoxA1 specifies unique androgen and glucocorticoid receptor binding events in prostate cancer cells[END_REF]. ADT can increase GR expression and the potential for GR signaling (Arora et al., 2013;[START_REF] Xia | A Novel Triazole Nucleoside Suppresses Prostate Cancer Cell Growth by Inhibiting Heat Shock Factor 1 and Androgen Receptor[END_REF].

GR can also act as a substitute for AR signaling. Montgomery et al reported a series of clinical trials to assess the contributions of glucocorticoids and GR in patients with CRPC [START_REF] Montgomery | Glucocorticoids and prostate cancer treatment: friend or foe?[END_REF][START_REF] Montgomery | Impact of baseline corticosteroids on survival and steroid androgens in metastatic castration-resistant prostate cancer: exploratory analysis from COU-AA-301[END_REF]. In these trials, analysis of patients in clinical cohorts who received glucocorticoids showed poor prognostic features, compared with patients who did not receive such treatment; therefore, stimulation of GR signaling may pose negative effects to patients who have received androgen-targeted therapies (Song et al., 2014;[START_REF] Montgomery | Impact of baseline corticosteroids on survival and steroid androgens in metastatic castration-resistant prostate cancer: exploratory analysis from COU-AA-301[END_REF].

Mechanistically, endogenous GR was strongly expressed in DU145, weakly in PC3

but not in LNCaP cells. AR was strongly expressed in LNCaP but not in DU145 cells.

DHT treatment enhanced STAT5 phosphorylation (pSTAT5), GR translocating into the nucleus and promoted proliferation of all CRPC cell lines, including LNCaP, DU145 and PC3 cells. Mifepristone treatment, that is a remarkably active antiprogesterone and antiglucocorticosteroid agent, abolished DHT-induced cell proliferation and STAT5 activation in both DU145 and PC3 cells but not in LNCaP cells. Similarly, GR-specific siRNA completely suppressed STAT5 activation. They also confirmed direct complex formation between the GR and pSTAT5 by immunoprecipitation (Song et al., 2014). In such conditions, the presence of increased glucocorticoids in serum and absence of androgen possibly enables the selection of 'promiscuous' AR variants with mutations in the LBD, allowing their activation by glucocorticoids [START_REF] Lorente | Tumour responses following a steroid switch from prednisone to dexamethasone in castration-resistant prostate cancer patients progressing on abiraterone[END_REF]; therefore, GR can bypass AR pathways and promote the development of CRPC.

On the other hand, PR is also a member of the steroid hormone nuclear receptor family, and it is structurally associated with AR [START_REF] Levin | Nuclear receptors outside the nucleus: extranuclear signalling by steroid receptors[END_REF]. As with GR, PR may have the ability to transcriptionally regulate a subset of AR target genes in PCa and thereby bypass AR [START_REF] Chen | Progesterone receptor in the prostate: A potential suppressor for benign prostatic hyperplasia and prostate cancer[END_REF]. A large retrospective analysis carried out recently demonstrated the association of high PR staining in primary PCa with its clinical recurrence [START_REF] Grindstad | High progesterone receptor expression in prostate cancer is associated with clinical failure[END_REF]. A research on a cohort of > 500 patients also revealed the association of high PR expression in cancer cells with reduced clinical failure-free survival [START_REF] Grindstad | High progesterone receptor expression in prostate cancer is associated with clinical failure[END_REF]. This evidence indicates that PR antagonists may possess therapeutic effects, suggesting that PR signaling can be an important carcinogenic target in the treatment of PCa. However, the mechanism still needs to be investigated.

Histologic transformation

Neuroendocrine transformation

Recent genomic profiling studies have shown a significant overexpression and gene amplification of Aurora Kinase A (AUR-KA) and N-MYC in 40% of NEPC and 5% of advanced PC, and a loss of AR target gene expression (Beltran et al., 2016).

AUR-KA is best known for its role in mitosis, but its interactions with the oncogene N-myc are not completely understood. In neuroblastoma, a positive feedback loop has been described in which AUR-KA induces and stabilizes N-MYC (Beltran et al., 2011). A phase II trial was recently conducted to evaluate if AUR-KA could be a new potential target in the treatment of NEPC. 60 patients were treated with alisertib, an oral AUR-KA inhibitor. Although the study did not meet its primary endpoint of 6-month radiographic progression free survival, a subset of patients with molecular features supporting AUR-KA and N-MYC activation achieved a significant clinical benefit. In particular, two patients achieved a remarkable response, with complete regression of liver lesions (Beltran et al.,219). Alisertib was also studied in combination with abiraterone in patients with mCRPC progressing during abiraterone treatment [START_REF] Lin | A phase I/II study of the investigational drug Alisertib in combination with Abiraterone and Prednisone for patients with metastatic castration-resistant prostate cancer progressing on Abiraterone[END_REF].

Epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) process is crucial for physiological embryonic development and differentiation of the urinary genital system, but it is also activated in pathological conditions, such as fibrosis or cancer progression [START_REF] Lamouille | Molecular mechanisms of epithelial-mesenchymal transition[END_REF]. While the epithelial phenotype is characterized by apical-basal cell polarity and strong intercellular adhesions, mesenchymal cells are totally different, being neither adherent nor polarized [START_REF] Larue | Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3' kinase/AKT pathways[END_REF]. EMT can be induced by ADT in metastatic PC and it confers invasive potential to tumour epithelial cells.

Snail and Twist are transcription factors able to reduce the expression of E-cadherin, a key event in EMT [START_REF] Montanari | Epithelial-mesenchymal transition in prostate cancer: an overview[END_REF]. The TGF-β superfamily members induce Snail transcriptional factor. Aberrant TGF-β pathway and the protein kinase C (PKC)/Twist1 signalling activation are putative mechanisms for the occurrence of enzalutamide resistance. There are currently no direct Twist inhibitors available, but Shiota et al investigated a PKC inhibitor (Ro31-8220) to switch back resistance in PC cell lines with promising results [START_REF] Shiota | Inhibition of protein kinase C/Twist1 signaling augments anticancer effects of androgen deprivation and enzalutamide in prostate cancer[END_REF]. Therapeutic targeting of EMT by a proteasome inhibitor suppressing Snail seems to be promising in mCRPC [START_REF] Baritaki | Inhibition of epithelial to mesenchymal transition in metastatic prostate cancer cells by the novel proteasome inhibitor, NPI-0052: pivotal roles of Snail repression and RKIP induction[END_REF].

AR-negative prostate cancer

Incidence of AR-negative prostate cancer

In a recent paper, Bluemn with colleagues showed that AR-directed therapies are resulting in the emergence of prostate metastases devoid of AR signaling and discover an effective pathway to target in these AR-null cancers (Bluemn et al., 2017). They characterized metastatic CRPCs from 84 patients for AR status and neuroendocrine status (NE). Neuroendocrine tumors express markers of neuroendocrine differentiation and are a subtype of prostate carcinoma separate from the more common epithelial type. The CRPCs collected from patients between 1997-2011 were 85% AR-positive, 10% NE-positive, and 5% negative for both. However, in samples collected between 2012-2016, when AR-targeting therapies became available, 69%

were AR-positive, 10% were NE-positive, and 21% were double-negative, demonstrating that double-negative metastases were four times as prevalent in recent cases (Bluemn et al., 2017).

AR-negative cell populations: stem-like cells

Small CSC populations have also been detected in PCa (van Leenders et al., 2001). Although the main hypothesis is that prostate cancer arises from terminally differentiated luminal cells, there is growing evidence that PCa arises from more undifferentiated cells with a basal phenotype [START_REF] Nagle | Cytokeratin characterization of human prostatic carcinoma and its derived cell lines[END_REF][START_REF] Marzo | Prostate stem cell compartments: expression of the cell cycle inhibitor p27Kip1 in normal, hyperplastic, and neoplastic cells[END_REF][START_REF] Stoyanova | Prostate cancer originating in basal cells progresses to adenocarcinoma propagated by luminal-like cells[END_REF]. Studies demonstrated that androgen-independent tumours arise from AR-negative cells at a frequency of 1:10 5 -10 6 AR-positive cells [START_REF] Craft | Evidence for clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors through a two-step process[END_REF]. Furthermore, several putative populations of prostate cancer stem-like cells (PCSCs) have been reported in the literature, most commonly identified through fluorescence-activated cell sorting (FACS) of bulk tumor cell lines, xenografted tumors or clinical samples for stem-like cell surface markers. Reported marker profiles include CD44+/α2β1+/CD133+ [START_REF] Collins | Prospective identification of tumorigenic prostate cancer stem cells[END_REF], ALDH [START_REF] Van Den Hoogen | High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer[END_REF], TRA-1-60/CD151/CD166 [START_REF] Rajasekhar | Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-kappaB signalling[END_REF], and PSA-/lo (Qin et al., 2012), among others.

A number of reports have suggested that PCSCs are AR-negative or express very low levels of AR, predicting lack of responsiveness to antiandrogens and other therapies targeting the AR signaling axis. Patrawala and colleagues demonstrated that CD44+ PCSCs were negative for AR expression. Moreover, putative CD44+/α2β1+/CD133+ PCSCs isolated from clinical PCs following prostatectomy were found to be AR-negative and exhibited a prostate basal cell phenotype [START_REF] Collins | Prospective identification of tumorigenic prostate cancer stem cells[END_REF]. More recent work supports enrichment of a basal cell gene expression profile in metastatic CRPC, establishing a link between expansion of a stem-like cell population and castrate resistance [START_REF] Zhang | Onco-miR-24 regulates cell growth and apoptosis by targeting BCL2L11 in gastric cancer[END_REF].

Importantly, at least one study demonstrated that tumours containing luminal, basal and neuroendocrine cells could be reconstituted in mice by using AR -and p63 - negative cells expressing the stem cell markers CD44, PROM1, nestin (NES) and KIT [START_REF] Gu | Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo[END_REF]. CRPCs. They suggested conducting a clinical trial of FGFR or MAPK antagonists in patients selected for AR activity status to conclusively determine if targeting these pathways will help patients with AR-negative prostate cancer (Bluemn et al., 2017).

Multiple endocrine neoplasia type 1 (MEN1): form gene to disease

MEN1 disease

Multiple endocrine neoplasia type 1 (MEN1) (MIM#131100) is a hereditary syndrome transmitted as an autosomal dominant trait. It is characterized by multiple occurrences in a same patient or in several members of the same family of tumors in endocrine organs. The major endocrine organs affected in MEN1 comprise the parathyroids, the endocrine pancreas (and the duodenum) and the anterior pituitary.

In MEN1 patients, hyperplasic and tumor lesions of the parathyroids affect virtually all patients (90%), pancreatico-duodenal endocrine tumors occur in 40% of patients, followed by anterior pituitary lesions (functional prolactinomas in 20% of patients, GH secreting, GH/PRL secreting, ACTH secreting, non-functional in 17% of patients) [START_REF] Falchetti | Multiple endocrine neoplasia type 1 (MEN1): not only inherited endocrine tumors[END_REF][START_REF] Thakker | Multiple endocrine neoplasia type 1 (MEN1)[END_REF]. Non endocrine cutaneous tumors are frequent in MEN1 patients with prevalence at 40 years old of age 85% for facial angiofibromas and 70% for collagenomas (Table 1.7).

The MEN1 gene

The MEN1 gene was mapped to chromosome 11q13 in 1988 [START_REF] Larsson | Multiple endocrine neoplasia type 1 gene maps to chromosome 11 and is lost in insulinoma[END_REF] and positionally cloned in 1997 (Chandrasekharappa et al., 1997;Lemmens et al., 1997). It is located at chromosome 11q13, spans 9.8 kb with 10 exons, the exon1 and distal part of exon10 are non-coding sequence. The human MEN1 coding sequence consists of 1830bp. Two MEN1 transcripts were detected by northern-blotting in human tissues, a main 2.8kb transcript expressed in most organs, and a 4.2 kb transcript expressed in thymus and pancreas (Lemmens et al., 1997). In the mouse, the Men1 gene is localized in the pericentromeric region of the chromosome 19, in a region sythenic to human 11q13 locus, with at least two reported transcripts, a 2.8kb and a 3.2 kb transcripts that result from differential alternative splicing of the noncoding exon1, containing a coding sequence of 1833bp, and are expressed in most tissues excepted skeletal muscle [START_REF] Stewart | Characterization of the mouse Men1 gene and its expression during development[END_REF][START_REF] Bassett | Studies of the murine homolog of the multiple endocrine neoplasia type 1 (MEN1) gene, men1[END_REF].

The MEN1 gene encodes a 610 amino acid protein referred to as menin, with no homology to any know protein [START_REF] Guru | the product of the MEN1 gene, is a nuclear protein[END_REF]. MEN1 mRNA is the expressed in the majority of the tissues and cell lines examined, although its expression levels are variable among different tissues. Menin orthologues have been identified in vertebrates and also invertebrates, including fruits fly and snail, but not in the yeast Saccharomyces crevisiae or nematode Caenorhabditis elegans. Menin is mainly located in nuclei. Half-life of protein menin is about 6-8 h in hEK293T cells (Yaguchi et al. 2004), and 10 h in COS cells [START_REF] Ikeo | Characterization of the MEN1 gene product, menin, by site-specific polyclonal antibodies[END_REF].

MEN1 mutations

An extensive landscape of MEN1 mutations has been described to date, with more than 1000 germinal mutations and more than 200 somatic mutations (occurring in sporadic tumors) having been reported. About 41% mutations are frameshift deletions or insertions, 23% are nonsense, 20% are missense, 9% affect splice-site and 6% are in-frame deletions or insertions. Interestingly, some mutations in MEN1 patients are not localized in the coding sequence, but rather in the promoter or other untranslated regions. Germline deletions of MEN1 exons have also been reported. Most mutations are inactivating mutations, consistent with the tumor suppressing functions of the MEN1 gene. No phenotype-genotype correlations have been reported so far, except the work reported by the French GTE showing that mutations affecting the interaction domain of the protein with JunD had a higher risk of death [START_REF] Thevenon | Higher risk of death among MEN1 patients with mutations in the JunD interacting domain: a Groupe d'etude des Tumeurs Endocrines (GTE) cohort study[END_REF].

In MEN1 endocrine tumors (90%), the loss of heterozygosity (LOH) of the MEN1 wild-type allele, leading to complete MEN1 inactivation, can be systematically detected. The LOH seems, therefore, to be an essential event to initiate tumorigenesis, even if a report suggests that heterozygous Men1-disruption in the mouse may lead to a happloinsufficiency [START_REF] Lejonklou | Accelerated proliferation and differential global gene expression in pancreatic islets of five-week-old heterozygous Men1 mice: Men1 is a haploinsufficient suppressor[END_REF].

Menin is a scaffold protein that controls gene expression and cell signaling

During the past two decade, it has also been reported that menin plays a role in suppressing hyperplasia or tumors in several other organs, such as the lung, prostate, and breast, and it exacerbates diabetes in mouse models. Menin also influences the function of other organs such as bone and live. Detailed mechanisms for how menin impacts these organs are less clear, but accumlating evidence showed that menin is a key scaffold protein that functionally crosstalks with various partners to regulate gene transcription and interplay with multiple signaling pathways (Table 1.8). Here we review the progress in understanding the molecular mechanisms by which menin functions.

Table 1.8 Summaries for different partners of menin.

Regulation of histone modifying enzymes by menin

Menin-interacting chromatin modifying proteins include histone methyltransferase MLL (H3K4me3), protein arginine methyltransferase 5 (PRMT5) (H4R3me2s), SUV39H1(H3K9me3), HDAC/SIRT and EZH2, and menin affects the binding of these histone modifiers to the promoter of its target genes and influences their function.

MLL and H3K4me3

Chromosomal rearrangements of the MLL (mixed lineage leukemia) gene located at chromosome band 11q23 are found in patients with de novo acute myeloid (AML)

and acute lymphoblastic (ALL) leukemias [START_REF] Pui | Outcome of treatment in childhood acute lymphoblastic leukaemia with rearrangements of the 11q23 chromosomal region[END_REF][START_REF] Liu | MLL fusions: pathways to leukemia[END_REF]Zeisig et al., 2014;Milne et al., 2015), and in therapy related leukemia or myelodysplastic syndrome (MDS) [START_REF] Rowley | International workshop on the relationship of prior therapy to balanced chromosome aberrations in therapy-related myelodysplastic syndromes and acute leukemia: overview report[END_REF]. The MLL proteins of the lysine methyltransferase (KMT) family, MLL1 (KMT2A) and MLL2 (KMT2B) are part of a protein complex that catalyzes a specific histone modification for gene activation, histone H3 lysine-4 trimethylation mark (H3K4me3) (Shilatifard 2012). MLL1

translocation with several different genes that encode transcription factors leads to the formation of MLL1-fusions that activate the expression of genes including the HOX genes [START_REF] Ayton | Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9[END_REF][START_REF] Yokoyama | Transcriptional activation by MLL fusion proteins in leukemogenesis[END_REF]). The role of HOXA genes in leukemic transformation has been observed both in vitro [START_REF] Argiropoulos | Hox genes in hematopoiesis and leukemogenesis[END_REF] and in vivo [START_REF] Rice | HOX deregulation in acute myeloid leukemia[END_REF]) models, demonstrating that MLL fusion protein mediated upregulation of HOXA9 and MEIS1 genes resulted in enhanced proliferation and blockage of hematopoietic differentiation, ultimately leading to acute leukemia [START_REF] Armstrong | MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia[END_REF][START_REF] Ayton | Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9[END_REF]. Menin was identified as an oncogenic co-factor of MLL fusion proteins in leukemia [START_REF] Yokoyama | The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis[END_REF]Chen et al., 2006). Loss of menin has been shown to coincide with the loss of H3K4me3 and the gain of H3K27me3, that is an epigenetic mark of gene repression, at specific genes [START_REF] Agarwal | Genome-Wide Characterization of Menin-Dependent H3K4me3 Reveals a Specific Role for Menin in the Regulation of Genes Implicated in MEN1-Like Tumors[END_REF][START_REF] Lin | Dynamic epigenetic regulation by menin during pancreatic islet tumor formation[END_REF][START_REF] Scacheri | Genome-wide analysis of menin binding provides insights into MEN1 tumorigenesis[END_REF]. The interaction of menin in the Trithorax-like MLL protein complex has been shown to be essential for the oncogenic activity of MLL1-fusion proteins to cause leukemia [START_REF] Hughes | Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus[END_REF][START_REF] Yokoyama | The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis[END_REF]. Evidence from studies conducted by using bone marrow of mouse models has confirmed this pro-oncogenic action of menin (Chen, et al. 2006).

Therefore, menin-MLL interaction inhibitors have been developed, facilitated by the deciphering the 3D structure of menin with the MLL1 menin-bindng motif (MLL1 MBM) (Thiel et al., 2012;Grembecka, et al. 2012). These inhibitors named MI and further improved versions of these inhibitors (MI-463, MI-503, MI-538 and compound 27) have been shown to block the proliferation of MLL1-fusion leukemia cells from patients in vitro and in mouse xenografts in vivo [START_REF] Borkin | Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein-Protein Interaction between Menin and Mixed Lineage Leukemia (MLL)[END_REF]. Such menin-MLL interaction inhibitors hold promise to conduct future clinical trials as a potential treatment for patients with MLL1-fusion leukemia. Menin-MLL interaction inhibitors have also been studied in mice to block the growth of specific types of prostate cancer, Ewing sarcoma, and childhood gliomas, respectively [START_REF] Funato | Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation[END_REF][START_REF] Malik | Targeting the MLL Complex in Castration-Resistant Prostate Cancer[END_REF][START_REF] Svoboda | Tumorigenicity of Ewing sarcoma is critically dependent on the trithorax proteins MLL1 and menin[END_REF].

On the other hand, in endocrine cells or mouse embryonic fibroblasts (MEFs), menin is required for MLL1 binding to the cyclin-dependent kinase (CDK) inhibitors, p27Kip1 and p18Ink4c loci to increase H3K4me3 and induce their expression (Milne et al. 2005). Moreover, menin forms a complex with MLL and several other cofactors, including WDR5 [START_REF] Song | WDR5 interacts with mixed lineage leukemia (MLL) protein via the histone H3-binding pocket[END_REF] and ASH2L [START_REF] Hughes | Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus[END_REF]), leading to H3K4me3 at the promoter of the target genes. Interestingly, menin is also required for recruiting MLL1 to the GATA3 locus to regulate GATA3 expression and Th2 cytokine production in T helper type 2 (Th2) cells (Onodera et al. 2010). These studies all strongly suggested that the target genes regulated by menin-MLL methyltransferase complex vary in different cells and tissues.

Besides directly binding to the N-terminus of MLL1 via the central pocket [START_REF] Borkin | Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein-Protein Interaction between Menin and Mixed Lineage Leukemia (MLL)[END_REF], menin also directly binds chromatin-associating protein LEDGF (lens epitheliumderived growth factor) (Yokoyama & Cleary 2008[START_REF] Van Nuland | Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes[END_REF]. LEDGF is crucial for co-localization of menin and wild-type MLL1 or MLL fusions to the loci of menin/MLL target genes such as HOX and CDKIs (Yokoyama & Cleary 2008). Importantly, crystallographic studies indicate that menin, the N-terminal part of MLL, and LEDGF form a ternary complex in which a helical structure from LEDGF sits on a 'V' shape structure formed from a MLL helix and menin. This structure clearly shows that menin acts as a scaffold to recruit both MLL and LEDGF (Huang et al. 2012). Moreover, although menin has no DNA-binding domains, it was reported that menin could associate with chromatin (Jin et al. 2003),

and directly bound to genomic DNA via the positively charged amino acid residues in the nuclear localization signals (NLSs) of menin [START_REF] Oh | Estrogen-Regulated Genes Predict Survival in Hormone Receptor-Positive Breast Cancers[END_REF]). These findings indicate that menin acts as a scaffold by recruiting the MLL complex to its target genes partly via its binding to genomic DNA.

PRMT5

Menin also regulates PRMT5 function (Gurung et al. 2013b), a member of PRMT family (Bedford & Clarke 2009;[START_REF] Stopa | The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond[END_REF]. PRMT5, a type II enzyme, is found in both the cytoplasm and the nucleus of cells [START_REF] Pasternack | Evolutionarily divergent type II protein arginine methyltransferase in Trypanosoma brucei[END_REF][START_REF] Banasavadi-Siddegowda | PRMT5-PTEN molecular pathway regulates senescence and self-renewal of primary glioblastoma neurosphere cells[END_REF], and involved in many physiological processes, including adipogenesis [START_REF] Leblanc | Protein arginine methyltransferase 5 (Prmt5) promotes gene expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and its target genes during adipogenesis[END_REF], hematopoiesis [START_REF] Liu | Arginine methylation of SREBP1a via PRMT5 promotes de novo lipogenesis and tumor growth[END_REF], and spermatogenesis (Wang et al., 2015;Wang et al., 2018). The overexpression of PRMT5 in various tumors and in leukemia has also been reported [START_REF] Yoshimatsu | Dysregulation of PRMT1 and PRMT6, type I arginine methyltransferases, is involved in various types of human cancers[END_REF][START_REF] Karkhanis | Versatility of PRMT5-induced methylation in growth control and development[END_REF]Wang et al., 2018). PRMT5 works together with its co-factor MEP50 to mediate the methylation of histones H2A and H4 at arginine 3 and histone H3 at arginine 8 [START_REF] Karkhanis | Versatility of PRMT5-induced methylation in growth control and development[END_REF]. In addition, PRMT5 can repress globin gene expression through recruitment of DNA methyltransferase 3A (DNMT3A) (Zhao et al., 2009), indicating a potential crosstalk between histone arginine methylation and DNA methylation [START_REF] Girardot | PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G+C-rich regions of the mouse genome[END_REF]. Posttranslational histone modifications catalyzed by PRMT5 significantly affect gene expression and regulate cell growth and proliferation [START_REF] Scoumanne | PRMT5 is required for cell-cycle progression and p53 tumor suppressor function[END_REF]. Menin regulates PRMT5's function by several modes. First, menin can suppress glucagon-like peptide-1 (GLP1)-induced and PKA-mediated phosphorylation of both FOXO1 and cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), likely through PRMT5 or PRMT5-like enzyme-mediated methylation of FOXO1 and CREB [START_REF] Muhammad | Menin and PRMT5 suppress GLP1 receptor transcript and PKA-mediated phosphorylation of FOXO1 and CREB[END_REF]. Second, menin recruits PRMT5 to the promoter of the Gas1 gene, increases repressive H4R3me2s and suppresses Gas1 expression.

Third, menin can also recruit PRMT5 to the promoter of Gli1 and subsequently repress Gli1 expression and thus Hedgehog signaling (Gurung et al. 2013a). However, further molecular details remain to be investigated.

H3K9me3 and Daxx

Several reports have revealed that menin silences the transcription of target genes by interacting with the suppressor of variegation 3-9 homologous protein 1 (SUV39H1), a histone 3 lysine 9 (H3K9) methyltransferase [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF], and increasing histone 3 lysine 9 trimethylation (H3K9me3) at the promoter of the target genes [START_REF] Yang | Menin mediates epigenetic regulation via histone H3 lysine 9 methylation[END_REF], Song et al. 2014[START_REF] Feng | Menin and Daxx Interact to Suppress Neuroendocrine Tumors through Epigenetic Control of the Membrane Metallo-Endopeptidase[END_REF]. Menin interacts with SUV39H1 through 360-445 amino acid region of menin and recruits SUV39H1 to the promoters of GBX2 [START_REF] Yang | Menin mediates epigenetic regulation via histone H3 lysine 9 methylation[END_REF]) and IL6 (Song et al. 2014) and represses their expression via enhancing H3K9me3.

Interestingly, previous works reported that menin directly binds to Daxx/ATRX complex and further recruits SUV39H1 to the promoter of membrane metallo-endopeptidase (MME, or CD10) and represses MME expression by enhancing H3K9me3 at the MME promoter [START_REF] Feng | Menin and Daxx Interact to Suppress Neuroendocrine Tumors through Epigenetic Control of the Membrane Metallo-Endopeptidase[END_REF]. Daxx is a H3.3-specific histone molecular chaperone. It deposits H3.3 in specific chromatin regions together with ATRX (Drane et al., 2010;[START_REF] Lewis | Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres[END_REF], and is often mutated in neuroendocrine tumors (Jiao et al., 2011). Menin and Daxx are required for each other to recruit SUV39H1 to the MME promoter. Daxx also directly binds to ATRX, which may also bind SUV39H1-binding domain [START_REF] Tang | A novel transcription regulatory complex containing death domain-associated protein and the ATR-X syndrome protein[END_REF][START_REF] Yang | Menin mediates epigenetic regulation via histone H3 lysine 9 methylation[END_REF][START_REF] Feng | Menin and Daxx Interact to Suppress Neuroendocrine Tumors through Epigenetic Control of the Membrane Metallo-Endopeptidase[END_REF]. These findings suggest that menin and Daxx/ATRX firstly form a complex and recruit SUV39H1 to the MME promoter, leading to the suppression of MME expression.

Histone deacetylases: HDAC/SIRT

The silencing by menin of JunD-mediated transcription has been attributed to repressive chromatin modifications. Gobl and coworkers showed that repression of JunD-dependent transcription could be reversed using the histone deacetylase (HDAC) inhibitor trichostatin A [START_REF] Gobl | Menin represses JunD-activated transcription by a histone deacetylase-dependent mechanism[END_REF]). Kim and coworkers found an association of menin with SIN3A-HDAC complexes [START_REF] Kim | a tumor suppressor, represses JunD-mediated transcriptional activity by association with an mSin3A-histone deacetylase complex[END_REF], which could not be confirmed by quantitative proteomic analysis of HeLa cells (van Nuland et al.

2013)

. In a more recent study, an interaction between menin and another histone deacetylase sirtuin 1 (SIRT1) was reported. Menin was shown to be required for the recruitment of SIRT to the CD36 gene promoter in mouse hepatocytes [START_REF] Cao | Hepatic menin recruits SIRT1 to control liver steatosis through histone deacetylation[END_REF]).

Menin regulates EZH2

Unlike the SET domain in MLL that methylates H3K4 (Yokoyama et al.,2004), the EZH2 SET domain specifically methylates H3K27, and the methylated H3K27 can be recognized by other specific binding proteins to compress chromatin structure, leading to repression of gene transcription [START_REF] Cao | Role of histone H3 lysine 27 methylation in Polycomb-group silencing[END_REF][START_REF] Sparmann | Polycomb silencers control cell fate, development and cancer[END_REF]. Gao et al. reported that menin bound to the promoter of Pleiotrophin (PTN) and recruited the Polycomb group (PcG) complex to the locus, resulting in H3K27 trimethylation, PTN suppression, and inhibition of proliferation of lung cancer cells (Gao et al., 2009). Moreover, they also reported that menin and EZH2 co-occupy a large number of promoters from ChIP-on-ChIP analysis in HCC specimens [START_REF] Gao | The functional and mechanistic relatedness of EZH2 and menin in hepatocellular carcinoma[END_REF]. On the other hand, in endocrine tissues, the important genetic interaction between MEN1, EZH2 and Inhbb (activinB) in β-cell proliferation has been revealed by Gherardi et al (Gherardi et al 2017). They showed that activin B expression is mediated through a direct modulation of H3K27me3 marks on the Inhbb The hallmark of the AP-1 protein complex c-Fos/c-Jun is its role in regulation of proliferation responses [START_REF] Jochum | AP-1 in mouse development and tumorigenesis[END_REF]. Additional study has shown that JunD is involved in negative control of cell proliferation in human intestinal epithelial cells or rat tumor cell lines [START_REF] Xiao | Induced JunD in intestinal epithelial cells represses CDK4 transcription through its proximal promoter region following polyamine depletion[END_REF][START_REF] Fries | CEBPbeta, JunD and c-Jun contribute to the transcriptional activation of the metastasis-associated C4.4A gene[END_REF];

Hernandez et al. 2008).

The transcription factor JunD was the first to be identified as a direct-interacting partner of menin by using yeast two-hybrid screening (Agarwal et al., 1999;[START_REF] Gobl | Menin represses JunD-activated transcription by a histone deacetylase-dependent mechanism[END_REF]. Menin interacts with JunD and represses the JunD activity (Agarwal et al. 1999, Huang et al. 2012). The crystal structural study showed that menin binds JunD via its central pocket, which can also bind MLL1 (Huang et al. 2012). In other words, menin uses the same pocket to bind either MLL1 or JunD, but having much more affinity with the former. A conserved sequence (FPXXP) is found in the menin-binding domain (MBD) of both JunD and MLL (Huang et al. 2012). The region of JunD that binds menin spans 27-47 amino acid residues (Agarwal et al. 1999, Huang et al. 2012). Earlier biochemical studies have suggested that menin binds to JunD to repress JunD-induced transcription through interaction with co-repressors mSin3A and HDAC 1 or 2 in cultured cells (Agarwal et al. 1999[START_REF] Gobl | Menin represses JunD-activated transcription by a histone deacetylase-dependent mechanism[END_REF][START_REF] Kim | a tumor suppressor, represses JunD-mediated transcriptional activity by association with an mSin3A-histone deacetylase complex[END_REF]. Furthermore, a new mechanism was reported whereby menin binds JunD and thus block c-Jun N-terminal kinase (JNK)-mediated phosphorylation of JunD at residues S90, S100 and T117, an event crucial for activating JunD function in transcriptional regulation (Huang et al. 2012).

Coincidently, both the basic residues and the leucine residues in JunD are bound by JNK to phosphorylate JunD, and these residues are also bound by menin. As such, menin binding to JunD effectively blocks JNK's interaction with JunD and thus inhibits JNK-mediated phosphorylation and activation of JunD. This finding well explains why JunD activates proliferation of MEFs in the absence of menin, but suppresses proliferation in the presence of menin [START_REF] Agarwal | Transcription factor JunD, deprived of menin, switches from growth suppressor to growth promoter[END_REF]. In addition, JunD has two isoforms: full-length and truncated isoforms, and menin only can bind to full-length isoform of JunD [START_REF] Yazgan | Differential binding of the Menin tumor suppressor protein to JunD isoforms[END_REF].

MYC

MYC (c-Myc) is a transcription factor that binds to E-boxes in the DNA to activate transcription. Increased MYC expression and activity is correlated with cell proliferation rates, and this is observed in many cancer types. Both activating and repressive effects of menin on MYC action have been reported. Menin has been suggested to interact with MYC and the SKI-interacting protein (SKIP) coactivator at the HIV-1 promoter (Bres et al. 2009). In HEK293 embryonic kidney cells, menin and the mixed-lineage leukemia (MLL) complex were found to interact with the transcription factor FUSE-binding protein 1 (FBP1/FUSBP1) to stimulate MYC expression (Zaman et al. 2014). More in line with its tumor suppressive function, loss of menin was shown to result in the upregulation of the MYC mRNA in mouse-derived pancreatic cells, possibly via increased Hedgehog signaling pathway activity (Gurung et al. 2013).

A recent report shows that menin can directly interact with the transactivation domain (TAD) of MYC and then bind to E-boxes to enhance transcription of Myc target genes. This enhanced transcription of MYC target genes depends on P-TEFb, a key factor to facilitate transcription regulation by MYC (Wu et al. 2017). The above findings suggest that, by transcriptionally promoting the expression of MYC target genes, menin can stimulate cell proliferation, cellular metabolism and cancer progression in certain types of cancers cells.

Nuclear receptors: ERα and AR

Several works showed that menin is a generic co-regulator for nuclear receptor-mediated transcription, as it interacts with many other NR family members including the vitamin D receptor (VDR) (Dreijerink et al., 2009a), the retinoid X receptor (RXR) [START_REF] Cheng | Menin prevents liver steatosis through co-activation of peroxisome proliferator-activated receptor alpha[END_REF], the peroxisome proliferator-activated receptors (PPAR) alpha (Dreijerink et al., 2009b), the liver X receptor alpha (LXR ) [START_REF] Cheng | Tumor suppressor Menin acts as a corepressor of LXRα to inhibit hepatic lipogenesis[END_REF] and the androgen receptor (AR) (Malik et al. 2015;Teinturier et al., 2021).

ERα

Menin is able to interact with the estrogen receptor alpha (ERα) ligand-binding domain in an estradiol (E2)-dependent fashion. Menin interacts with hormone activated estrogen receptor alpha (ERα) and links ERα to MLL1 and MLL2 methyltransferase complexes. This interaction is important for transcription of ERα target genes through recruitment of H3K4me3 methyltransferase activity, such as the estrogen regulated TFF1 gene (Dreijerink et al. 2006). Menin also regulates transcription of the ESR1 gene via H3K4me3 histone marker in MCF-7 cells (Dreijerink et al. 2017). Interestingly, they revealed that menin-dependent ESR1 gene promoters display looping to distal enhancers that are bound by menin, FOXA1 and GATA3 in MCF-7 cells.

More importantly, studies hypothesized that the expression of menin in breast cancer cells might be associated with resistance to antiestrogen therapy. The analysis of ER positive breast cancer samples revealed that patients with menin-positive tumors had a worse outcome than those with menin-negative tumors (Imachi et al., 2010;Imachi et al., 2011). Therefore, inhibition of the menin interaction with MLL methyltransferases may impair co-activation of the estrogen receptor and may offer a therapeutic benefit for patients with breast cancers dependent on ER signaling.

AR

Almost a decade after discovering the interaction between menin and ERα, menin was identified as an important co-factor for AR signaling, by its physical interaction with AR-NTD and the recruitment of the MLL histone methyltransferase complex to AR target genes. Inhibition of menin-MLL interaction with a small-molecule inhibitor (MI) impaired AR signaling and inhibited the growth of castration-resistant tumors in xenograft experiments in mice (Malik et al., 2015). Hence, these results suggest that menin can facilitate tumor growth through AR signaling in prostate cancer.

More recently, our work suggested that menin could play a tumor suppressor role during the tumorgenesis of mouse prostate cells. We found that prostate-specific (Perou et al., 2000;van de Vijver et al., 2002;Oh et al., 2006) and highly associated with ERα and required for the proper function of most of its target genes (Lacroix et al., 2004;Carroll et al., 2005;Carroll and Brown 2006).

Menin is also known to interact with GATA3 and c-Myb to activate the Th2 cell maturation in primary human peripheral blood T cells (Nakata et al., 2010). In mouse embryonic stem cell (mESC)-derived embryoid bodies, lower Foxa1 and Foxa2 mRNA levels were observed in the absence of Men1 gene expression (Zhang et al. 2011). In addition, menin was found to be able to interact with FOXA2 and to regulate FOXA2 expression in mouse pancreatic cells and derived cell lines, indicating a potential role in MEN1-associated pNET tumorigenesis [START_REF] Bonnavion | Foxa2, a Novel Protein Partner of the Tumour Suppressor Menin, Is Deregulated in Mouse and Human MEN1 Glucagonomas[END_REF]. It is worth mentioning that FOXA1 plays a crucial role in the AR signaling, and possibly in CRPC occurrence (Obinata et al., 2017).

TGF-β and Smad3

Transforming growth factor β (TGF-β) and bone morphogenic protein (BMP)

signaling is transduced to gene transcription via SMA and MAD (SMAD) gene specific transcription factors. Genomic SMAD-binding sites can be both at promoter-proximal and enhancer elements. Menin regulates TGF-β signaling and

TGFβ-induced gene transcription by interacting with SMAD3, a TGF-β downstream effector (Kaji et al. 2001). Menin directly interacts with SMAD3, and antisense menin suppresses TGF-β-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites (Kaji et al. 2001).

When the Men1 gene is deleted in Leydig cells, the effect of TGF-β-induced inhibition of proliferation was reduced [START_REF] Hussein | Deregulation of anti-Mullerian hormone/BMP and transforming growth factor-beta pathways in Leydig cell lesions developed in male heterozygous multiple endocrine neoplasia type 1 mutant mice[END_REF]. Consistently, Canaff and coworkers showed that menin mutation compromises menin's function in promoting TGF-β-induced SMAD3 transcriptional activity [START_REF] Canaff | Impaired transforming growth factor-beta (TGF-beta) transcriptional activity and cell proliferation control of a menin in-frame deletion mutant associated with multiple endocrine neoplasia type 1 (MEN1)[END_REF]. Thus, menin mutation deprives its function in promoting TGF-β signaling-induced repression of cell proliferation, likely contributing to the development of MEN1. On the other hand, it is also reported that TGF-β upregulates menin expression in MLL-AF9-transformed mouse bone marrow cells (Zhang et al. 2011). Moreover, menin expression was downregulated in MLL-AF9-transformed mouse bone marrow cells when TβRII, a vital component in TGF-β signaling pathway, was deleted. Menin expression was also decreased in liver samples from the conditional TβRIIknockout mice after TβRII excision (Zhang et al. 2011).

Runx2 and Smad1/5

Anotherrecognized function of menin is its role in the commitment of multipotential mesenchymal stem cells into the osteoblast lineage and osteoblast differentiation.

However, menin also inhibits the later differentiation of these cells [START_REF] Hendy | Menin and TGF-beta superfamily member signaling via the Smad pathway in pituitary, parathyroid and osteoblast[END_REF]. In the TGF superfamily, bone morphogenetic proteins (BMPs) and TGF-β are crucial regulators of bone formation. The interactions of menin with the BMP-2 signaling pathway and Runx2 might be different depending on the osteoblast differentiation stage [START_REF] Hendy | Menin and TGF-beta superfamily member signaling via the Smad pathway in pituitary, parathyroid and osteoblast[END_REF].

Menin promotes the commitment of multipotential mesenchymal stem cells to the osteoblast lineage through interactions with Smad1 to Smad5 and Runx2, whereas the interaction of menin and Smad3 inhibits later osteoblast differentiation by negatively regulating the BMP-2-Runx2 cascade after the commitment to the osteoblast lineage [START_REF] Sowa | Menin is required for bone morphogenetic protein 2-and transforming growth factor beta-regulated osteoblastic differentiation through interaction with Smads and Runx2[END_REF]. and PTEN were observed in a small percentage of human PanNETs (Jiao et al., 2011;Scarpa et al., 2017), suggesting that menin and Pten may function synergistically to suppress tumorigenesis.

Several studies have focused on the relationship between the PAM (PI3K/AKT/mTOR) and resistance to endocrine therapy in pre-clinical BC models (Boulay et al., 2005), in which the authors showed that Akt can activate the ERα pathway independently of estrogen availability and that the combination of mTOR inhibitors and endocrine therapy can overcome this resistance (Crowder et al., 2009).

In addition, the PAM pathway has also been implicated in trastuzumab resistance in HER2-overexpressing BCs (Nagata et al., 2004). Interestingly, menin interacts with AKT1, downregulates its kinase activity and suppresses both AKT1 induced proliferation and anti-apoptosis in endocrine and non-endocrine cells, mainly by reducing the translocation of AKT1 from the cytoplasm to the plasma membrane during growth factor stimulation (Wang et al., 2011). Another study showed that menin can interact with FOXO1, a downstream effector of AKT, in the hepatocytic cancer cell line HepG2 and in MEFs (Wuescher et al., 2011). A recent study revealed that menin regulates milk protein synthesis through mTOR signaling in normal mammary epithelial cells (Li et al., 2017). According to the authors, menin overexpression caused significant suppression of factors involved in the mTOR pathway, as well as milk protein κ-casein (CSNK). All of the abovementioned data suggest that menin may regulate the PI3K/AKT/mTOR pathway in different tissues and cells, including mammary cells.

Hedgehog signaling

In pancreatic ductal adenocarcinoma, menin expression was suppressed by DNA GRB2 and SOS1 from binding to Ras [START_REF] Wu | Interplay between menin and K-Ras in regulating lung adenocarcinoma[END_REF]; these studies suggest that there exists a potential negative feedback loop between menin and K-Ras, and that menin plays a crucial role in K-Ras-induced lung cancer development.

Cell cycle control & growth factor

Kaji et al. demonstrated that menin could suppress cell proliferation via the transforming growth factor-β (TGF-β) pathway in the rat pituitary cell line by interacting with SMAD3 (Kaji et al., 2001). Agarwal et al. reported that menin is essential for JunD-mediated inhibition of cell proliferation (Agarwal et al., 1999).

Ratineau et al. showed that menin represses cell proliferation in rat intestinal epithelial cells (Ratineau et al., 2004) by inhibiting the expression of Cyclin D1, Cyclin D3, and CDK4. Based on a transcriptomic study of differentially expression genes in mouse Men1 insulinomas, our team demonstrated that Men1 ablation in mouse islet cells greatly affected the expression of factors involved in cell cycle and cell growth control, such as Cyclin A2, B2, and D2 for the former; and IGF2, IGFBP3, and 6 for the latter (Fontanière et al., 2006). Menin can also repress cell proliferation by interacting and inhibiting ASK (S-phase kinase) (Schnepp et al., 2004). In addition, menin was reported to upregulate the expression of Cyclin-dependent kinase inhibitors p18 ink4c and p27 kip1 with the help of the MLL compass-like complex (Fontanière et al., 2006), which adds H3K4me3 marks on their promoters, thus activating gene expression (Karnik et al., 2005;Pei et al., 2009;Stephens et al., 2012). Interestingly, p18 has recently been shown to be a downstream target of GATA3 in luminal BC and to suppress luminal progenitor cell proliferation and tumorigenesis (Milne et al., 2005). P27 is ranked as one of the 18 most significantly mutated genes in luminal A BC, and loss of p27 was associated with poor outcome in BC patients (Schnepp et al., 2006). Schnepp et al. revealed that the infection of cells using menin-expressing adenoviruses could trigger apoptosis in MEFs (Lindsten et al., 2000;Schnepp et al., 2003) by activating an apoptotic pathway that depends on Bax (Milne et al., 2005). They also highlighted that Men1 disruption increased resistance to TNFα-induced apoptosis in Men1 +/-mouse model, further supporting a vital role for menin in regulating apoptosis.

Regulation of menin expression

Regulation of menin expression by prolactin signaling

As menin plays an important and pleiotropic role in regulating multiple functions of islets, it is conceivable that its expression is controlled by numerous signals. During late stage of pregnancy of mice, prolactin is produced, and prolactin binds its cell surface receptor, and phosphorylates and activate STAT1 (Jabbour, et al. 1998;Karnik, et al. 2007;Tourkine, et al. 1995). The activated STAT1 translocates into the nuclear in β cells, and then binds to the promoter of the Men1 gene to suppress Men1 transcription [START_REF] Karnik | Menin controls growth of pancreatic beta-cells in pregnant mice and promotes gestational diabetes mellitus[END_REF]). Notably, controlled expression of the Men1 transgene in the pancreatic islets attenuates the prolactin-induced repression of the Men1 expression in the pancreatic islets, leading to gestational diabetes in the mice [START_REF] Karnik | Menin controls growth of pancreatic beta-cells in pregnant mice and promotes gestational diabetes mellitus[END_REF]). These findings indicate that menin is physiologically regulated to adapt the pancreatic islets to counteract the development of gestational diabetes. More work remains to investigate whether this molecular circuitry is also conserved in human pancreatic islets.

Regulation by FOXO1

Forkhead box O1 (FOXO1) is a member of Forkhead box containing transcription factor family [START_REF] Anderson | Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily[END_REF]. The key feature of this family of transcription factors is that many of them can be phosphorylated by receptor tyrosine kinases (RTKs) by activating the PI3K/AKT pathway [START_REF] Brunet | Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor[END_REF]. AKT can phosphorylate FOXO1 at three different sites Thr24, Ser256 and Ser319. For example, insulin induces phosphorylation of FOXO1 by activating PI3K and AKT axis, and phosphorylated FOXO1 binds to 14-3-3 protein and is isolated in the cytoplasm [START_REF] Tzivion | FoxO transcription factors; Regulation by AKT and 14-3-3 proteins[END_REF]). In addition, phosphorylated FOXO1 in the nucleus can also be exported to the cytoplasm. Thus, the net result of the phosphorylation of FOXO1 is to reduce its nuclear localization and therefore its activity in transcriptional activation [START_REF] Vogt | Triple layer control: phosphorylation, acetylation and ubiquitination of FOXO proteins[END_REF]. Further, it is reported that FOXO1 is crucial for regulating insulin signaling and also cell metabolism [START_REF] Kitamura | The role of FOXO1 in beta-cell failure and type 2 diabetes mellitus[END_REF].

Menin expression is activated by FOXO1 in β-cells. It has been reported that glucose induces activation of AKT and suppresses FOXO1 activity, leading to decreased menin expression and activated β-cell activity (Zhang et al., 2012).

Importantly, the authors also demonstrated that Foxo1 bound to the proximal Men1

promoter and regulated its activity in β-cells (Zhang et al., 2012). Therefore, menin regulation is linked to an important signaling pathway regulating β cell, metabolism and diabetes.

Somatostatin increases menin expression

Somatostatin is a peptide hormone and inhibitor of gastrin expression and secretion [START_REF] Karnik | Inhibition of gastrin gene expression by somatostatin[END_REF]. Menin also inhibits the expression of gastrin. Octreotide is a somatostatin analog that increases mRNA and menin protein levels in the duodenum of mice. While octreotide inhibited PKA enzyme activity, forskolin treatment, which increases cellular cAMP and PKA activity, suppressed menin protein level [START_REF] Mensah-Osman | Somatostatin stimulates menin gene expression by inhibiting protein kinase A[END_REF]. However, whether and how the somatostatin pathway regulates PKA to induce menin expression remains to be investigated. Yaguchi et al found that missense mutant menin protein has much short half-life as compared to wild type menin protein in transfected 293 cells (Yaguchi et al., 2004).

Further studies revealed that the reduced levels of the mutant proteins were a result of rapid menin degradation through ubiquitin-mediated degradation. Several reports further revealed that unstable menin mutants, but not wild type menin, were associated with a heat shock protein Hsp70 and its co-chaperone CHIP, which was shown to function as a ubiquitin E3 ligase towards several substrates presented by Hsp70 and Hsp90 [START_REF] Connell | The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins[END_REF][START_REF] Meacham | The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation[END_REF]. Recently, Wu et al reported that the menin inhibitors (MI), including MI463 and MI503, induced reduction of H3K4m3, and HOXA9 expression was rescued with a proteasome inhibitor that blocks MI-induced menin protein degradation in human leukemia cell lines. Mechanistically, MIs promote the interaction of menin with Hsp70-associated ubiquitin ligase CHIP, resulting in increased menin ubiquitination, leading to increased menin degradation [START_REF] Wu | Disruption of the menin-MLL interaction triggers menin protein degradation via ubiquitin-proteasome pathway[END_REF].

Phosphorylation

The first phosphorylation modification of menin was found in 2006 by Matthewet al [START_REF] Macconaill | Phosphorylation of the menin tumor suppressor protein on serine 543 and serine 583[END_REF]. They showed that menin is phosphorylated on two serine residues, Ser543 and Ser583. However, the functional study of the phosphorylation by mutating both serine residues had no impact on menin's ability to recruit trithorax family complex proteins Ash2L, Rbbp5, and MLL2, nor on cell proliferation. While these two phosphorylation sites are situated between the two NLSs located at the C-terminal part of menin, mutations at both of the serine residues do not affect menin localization into the nucleus [START_REF] Macconaill | Phosphorylation of the menin tumor suppressor protein on serine 543 and serine 583[END_REF]. Further study showed that Ser394 and Ser487 were also identified by mass spectrometry analysis.

Phosphorylation of menin at Ser394 was induced in response to irradiation (IR) or UV treatment, while Ser487 was phosphorylated under normal cell culture conditions.

Nonetheless, HMT activity assay showed both Ser394 and Ser487 mutated menin mutant was still able to immunoprecipitate methylated histone 3 [START_REF] Francis | The menin tumor suppressor protein is phosphorylated in response to DNA damage[END_REF].

Menin and miRNAs

Let-7a

Abnormal microRNA (miRNA) regulation has been attributed to all phases of cancer and affects several of the cancer hallmarks [START_REF] Peng | The role of MicroRNAs in human cancer[END_REF][START_REF] Romano | Small non-coding RNA and cancer[END_REF].

Discovered in C. elegans, let-7 (lethal-7) miRNA family functions as an important regulator of differentiation [START_REF] Reinhart | The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[END_REF][START_REF] Copley | The Lin28b-let-7-Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells[END_REF]. It is categorized as a tumor suppressor because it reduces cancer aggressiveness, chemoresistance, and radioresistance.

Menin does not affect levels of primary-let-7a (pri-let-7a), but increases the levels of mature let-7a [START_REF] Gurung | Menin is required for optimal processing of the microRNA let-7a[END_REF]. Let-7a targets, including insulin receptor (INSR) and insulin receptor substrate 2 (IRS2), pro-proliferative genes that are crucial for insulin-mediated signaling, are up-regulated in Men1-excised cells [START_REF] Gurung | Menin is required for optimal processing of the microRNA let-7a[END_REF]. Inhibition of let-7a using anti-miRNA in wild type cells is sufficient to enhance the expression of IRS2. Depletion of menin inhibits conversion of pri-miRNA to pre-miRNA. These findings unravel a mechanism whereby menin suppresses cell proliferation, at least partly by promoting the biogenesis and processing of certain miRNAs, including let-7a, to insulin signaling and likely endocrine cell proliferation [START_REF] Gurung | Menin is required for optimal processing of the microRNA let-7a[END_REF].

MiR-24-1

The human miR-24 is located at chromosome 19 of the human genome and transcribed as a part of miR-23a-27a-24-2 cluster [START_REF] Chhabra | Cooperative and individualistic functions of the microRNAs in the miR-23a~27a~24-2 cluster and its implication in human diseases[END_REF].

Dysregulation of miR-24 has been reported in various human cancers, such as non small cell lung cancer [START_REF] Franchina | Circulating miR-22, miR-24 and miR-34a as novel predictive biomarkers to pemetrexed-based chemotherapy in advanced non-small cell lung cancer[END_REF], hepatocellular carcinoma (Chen et al., 2016), breast cancer [START_REF] Lu | miRNA-24-3p promotes cell proliferation and inhibits apoptosis in human breast cancer by targeting p27Kip1[END_REF], ec. The miR-24 had different functions in different cancer typies (Wang et al., 2018;[START_REF] Zhang | Up-regulation of miR-24-1-5p is involved in the chemoprevention of colorectal cancer by black raspberry anthocyanins[END_REF]. Interestingly, even in the same type of cancer, miR-24 can play opposite function [START_REF] Duan | Tumor suppressor miR-24 restrains gastric cancer progression by downregulating RegIV[END_REF][START_REF] Zhang | Onco-miR-24 regulates cell growth and apoptosis by targeting BCL2L11 in gastric cancer[END_REF]. Therefore, further studies are needed to confirm its functions and adverse effects.

MiR-24-1 directly binds to the highly conserved 3'UTR region of MEN1 mRNA, and represses menin expression, and the negative feedback loop between miR-24-1 and menin protein is essential for MEN1 tumorigenesis [START_REF] Luzi | The negative feedback-loop between the oncomir Mir-24-1 and menin modulates the Men1 tumorigenesis by mimicking the "Knudson's second hit[END_REF].

Vijayaraghavan et al. also found that miR-24 directly decreases menin expression and impacts downstream cell cycle inhibitors in pancreatic cancer [START_REF] Vijayaraghavan | miR-24 regulates menin in the endocrine pancreas[END_REF]. Moreover, miR-24 inhibition increases menin expression and decreases cholangiocarcinoma cell proliferation [START_REF] Ehrlich | miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation[END_REF]). Our team has been focused on the studies on the role of MEN1 using Men1 mutant mouse models. Our previous works showed that aged heterozygous Men1 mutant mice, in addition to endocrine tumors, developed mammary gland carcinomas in female and prostate cancers in male mutant mice at low frequencies (Bertolino et al., 2003). To further confirm and understand the role of menin in the development of mammary lesions, a conditional mammary-specific Men1 knock-out mouse model was generated by crossing the mice carrying floxed Men1 alleles (Men1 et al., 2021).

The involvement of the

Taken together, all of the data obtained from mouse models suggest a tumor-suppressive role for menin during the initiation and development of murine breast and prostate cancers.

MEN1 gene in human breast cancer

Over the last two decades, several case reports have described breast cancer cases related to MEN1. In 2004, a 44-year-old Japanese woman was diagnosed with MEN1 syndrome, having hyperparathyroidism, primary aldosteronism, and also scirrhous breast carcinoma. The DNA taken from her parathyroid adenoma and breast cancer tissues showed germline MEN1 mutation at codon 451 in exon 10, which resulted in alanine-to-tyrosine substitution (A541T), as well as LOH (Honda et al., 2004).

Another study by Jeong et al. reported a case of a patient with both MEN1-associated tumors and breast cancer. They found a germline MEN1 mutation manifested as a 5-bp duplication in exon 3 (named:196_200dupAGCCC), which resulted in a frameshift mutation. In addition, the tested exon 10 showed a polymorphism at codon 423 with substitution of a cytidine to a thymidine (C423T), causing a change of amino acid (Jeong et al., 2014) The most relevant evidence of the likely involvement of menin in BC arose from the observation that female MEN1 patients were at a higher risk of developing BC (Dreijerink et al., 2014). In this study, Dreijerink et al. referred (Jeong et al., 2014;Papi et al., 2009;Ghataorhe et al., 2007).

However, in a clinical study conducted by Imachi et al. with 65 ERα-positive BC samples treated with tamoxifen for 2-5 years as adjuvant therapies, the authros observed that menin-positive tumors (20 patients) had a worse clinical outcome and were more resistant to tamoxifen than menin-negative tumors (46 patients) (Imachi et al., 2007). They, therefore, proposed that menin could be a predictive factor of resistance to tamoxifen. Furthermore, they found that raloxifene could inhibit the binding of menin to the AF2 domain of ERα and proposed raloxifene as the therapeutic options for menin-positive and ERα-positive BC (Imachi et al., 2011).

Their works suggest an oncogenic role for menin, which raised the controversy as to its precise role in BC. (Grasso et al., 2012). MLL functions as part of a multi-protein complex containing menin (Varier et al., 2011).

Many members of the complex have different levels of aberrations in CRPC (Grasso et al., 2012). Noticeably, Chen et al. analyzed 150 cases for advanced and metastatic human PCa. They observed that the percentage of PTEN and MEN1 co-loss was almost the same as the co-loss of PTEN and PML (Promyelocytic Leukemia), which is around 11% in all cases (Chen et al., 2018). Conversely, Paris et al. reported that the MEN1 locus was amplified in some patients and was predictive of post-operative recurrence (Paris et al., 2004). The similar observation was made Kerstin et al.

(Heselmeyer- Haddad et al., 2014).

More importantly, Malik et al. examined menin expression in a set of human prostate cancer tissue samples (benign: n=38; localized: n=118; metastatic: n=55). By using RNA sequencing (RNA-seq), they observed that MEN1 mRNA expression was associated with disease progression, with significantly elevated levels seen in metastatic prostate cancer compared to those observed in hormone-naive prostate cancer and benign prostate. They also found that menin protein levels were elevated during prostate cancer progression, with notably higher protein levels in metastatic compared to localized disease. Furthermore, the MEN1 mRNA overexpression was predictive of poor patient survival (Malik et al., 2015). Taken together, these data establish that menin is upregulated at both the transcript and protein levels in localized and metastatic prostate cancer and its expression is associated with poor survival. In total, the current data obtained from human studies suggest that the MEN1 gene could play a complex even opposite role in the development of human breast and prostate cancers. (#: co-first author) Please see the paper in Appendices part.

II. My major research themes

II-1. Crosstalk between menin, MYC and AR in AR-dependent prostate cancer cells

Our results demonstrated that menin exerted its oncogenic effects and was critical for MYC transcription and MYC-mediated AR transcription in AR-dependent cells.

Interestingly, we also showed that menin was involved in the regulation of MYC-related LncRNA PCAT1 in AR-dependent PCa cells, through its interaction with EZH2 and their co-binding on the regulatory sequence of PCAT1. The androgen receptor (AR) acts as a vital transcription factor in prostate cancer (PCa) development and progression. Pathway aberrations are extensive in primary and metastatic castration-resistant PCa (mCRPC), where they affect the transactivation activity and transcription of AR [1], although the latter remains elusive. The proto-oncogene MYC has a well-documented role in driving AR oncogenic functions in PCa cells [2], and was recently exposed as a key activator in the regulation of AR transcription [3]. However, the exact mechanisms underlying MYC-mediated AR regulation are lacking, and may be crucial for better understanding PCa development and improving its management. We lately demonstrated that the tumor suppressor menin, encoded by the MEN1 gene, plays an oncosuppressive role in the initiation of prostate cell tumorigenesis and the generation of AR low /CD44 + lesions in the mouse, but exerts oncogenic effects specifically in AR-dependent human PCa cells, both likely through modulating AR transcription [4]. Since menin interacts with numerous transcription and epigenetic factors, we wondered herein whether this newly uncovered function of menin may occur through its interaction with other crucial factors, in particular, MYC.

We initially examined the expression of MYC upon MEN1 knockdown (KD) in PCa cells. We found that MEN1-KD led to a decrease in MYC expression at the transcriptional (Fig. 1a, Fig. S1a) and protein (Fig. 1b-c, Fig. S1b-c) levels in AR-dependent (LNCaP and 22RV1), but not AR-independent (PC3 and DU145), PCa cells. We also detected the interaction between menin and MYC in AR-dependent PCa cells (Fig. S2a-b), previously reported in Hela cells [5]. Recently, several distal regulatory sequences in the MYC locus have been identified and reported to play important role in MYC transcription, including in particular 2 MYC enhancers situated 67 kb upstream and 20 kb downstream of the transcription start site (TSS) of MYC [6,7]. Importantly, ChIP analyses demonstrated that menin bound not only to the MYC promoter, but also to these two enhancers (Fig. 1d, Fig. S2c). Moreover, menin was necessary for major components of the MLL complex (viz KMT2A, KMT2B, ASH2L), to which menin belongs [8], to bind to and activate the MYC promoter and enhancer 2 likely via H3K4me3 marks (Fig. 1e-g, Fig. S2d-g). Consistently, MEN1-KD resulted in decreased expression of known MYC target genes, enhanced cell cycling alterations (Fig. 1h, Fig. S3a-c), and decreased expression of AR variants (Fig. S3d-e), the latter being known to be activated by MYC expression in PCa cells and to contribute to mCRPC development. Notably, the expression of menin and MYC and their interaction, as well as the binding of menin to the MYC promoter and enhancer 2, were augmented upon stimulation with the androgen hormone, dihydrotestosterone (DHT) (Fig. 1i-k, Fig. S4a-c). The above data revealed that menin is a crucial factor positively regulating MYC transcription specifically in AR-dependent PCa cells.

Next, we investigated whether menin was involved in MYC-mediated regulation of AR transcription known to play a critical role in activating AR transcription [3]. ChIP analysis and Luciferase-reporter assays revealed that menin and MYC bound to the same area on the AR promoter, at the location of an E-box site (Fig. 2a-b, Fig. S5a-g).

Deletion of the E-box on the AR promoter significantly reduced the transactivation activity of MYC and that of menin, in Luciferase-reporter assays, whereas this latter activity remained unaffected on other parts of the AR promoter (Fig. S6a-b).

Remarkably, using reChIP analysis, we found that the physical interaction between MYC and menin may be a prerequisite for the binding of menin to the E-box site on the AR promoter (Fig. 2c, Fig. S6c-d). Our data indicate, for the first time, that menin exerts a regulatory role on the MYC binding site on the AR promoter and is essential for the regulation of MYC-mediated AR transcription.

In parallel, we assessed whether menin is involved in the regulation of LnRNA PCAT1, situated 800 kb upstream of the MYC locus and known to play an oncogenic role through increasing the stability of MYC during PCa development [9]. Markedly, MEN1-KD in AR-dependent PCa cell lines led to reduced PCAT1 expression (Fig. 2d, Fig. S7a). Since EZH2 has been reported to directly regulate PCAT1 expression and that the interaction between EZH2 and menin has been described in different contexts [9,10], we investigated whether menin controls PCAT1 transcription through EZH2 in PCa cells. Our data demonstrated that menin was crucial for the expression of EZH2 (Fig. 2e, Fig. S7b) via its physical interaction with EZH2 (Fig. S7c-d) and its binding to the EZH2 promoter (Fig. 2f-g, Fig. S7e-f). Finally, ChIP analysis showed that menin bound to the same binding sites as EZH2 on the regulatory sequences of PCAT1 (Fig. 2h, Fig. S7g), the binding being weakened upon EZH2-KD (Fig. 2i, Fig. S7h). Taken together, the current work revealed that menin is critically involved in the regulation of MYC-related PCAT1, through its interaction with and regulation of EZH2. The function of menin in the activation of PCAT1 expression should further strengthen the activity of the MYC pathway.

Finally, to explore the relevance of our findings for future therapeutic strategies, we analyzed large PCa data sets through data mining. Significant positive correlations were observed between MEN1 and MYC mRNA expression in mCRPC, between MEN1 and EZH2 mRNA expression in both primary PCa and mCRPC, and a marginal positive correlation between MEN1 mRNA and PCAT1 expression in primary PCa (Fig. 2j). Lastly, the combined use of siMEN1 and of the MYC/Max dimerization inhibitor 10058-F4 had a synergistic effect on reducing the proliferation of LNCaP and 22Rv1 (Fig. 2k, Fig. S8a-b) cells.

Collectively, we uncovered a critical crosstalk between menin, AR and MYC, involving PCAT1 regulation via EZH2, specifically in AR-dependent PCa cells (Fig. 2l), providing novel clues as to the mechanisms underlying the regulation of AR transcription. Considering AR is not only crucial for the development of PCa, but also for its treatment, we believe that the current finding may provide useful clues to new targets for PCa management.

Cell proliferation assay

Cell proliferation assays were performed as described previously [2]. Briefly, cells were seeded onto 96-well culture plates at 5×10 3 cells for 22Rv1, DU145 or PC3, and at 1×10 4 cells for LNCaP. 24 h later, cells were treated with 20 nM of siMEN1( 1)+ (3) or MYC inhibitor (10058-F4) at different concentrations (10 μM, 15 μM and 20 μM),

or with both 20 nM of siMEN( 1)+( 3) and 10058-F4 at different concentrations ( 15μM and 20 μM). All experiments were performed in triplicate and at least three times.

Real time RT-PCR

RNA was isolated from cells using a NucleoSpin RNA Plus kit (ref. 740984, Macherey-Nagel) and mRNAs levels were assessed by quantitative real time RT-PCR as described previously [2]. Each experiment was repeated independently at least three times. Data were normalized against HPRT and represented as fold change.

Primers used are listed in Supplementary information, Table S1.

Western blotting

Total protein extracts from cells were prepared and analyzed as described previously

Flow cytometry analysis

Cells were treated with siCtrl or siMEN1( 1)+( 3) for 72 h, followed by harvesting and re-suspending in 100 μl of Phosphate-Buffered Saline (PBS) and fixed with 70% ethanol at 4°C overnight, and stained with Propidium Iodide (P1304MP,Thermo Fisher) (50 μg/ml) containing 100 μg/ml RNase A (R6148, Sigma) for 15 min.

Analysis of cell cycling was performed by flow cytometry on a FACScan instrument (Beckman Coulter, Brea, CA, USA).

Promoter and Luciferase reporter assays

Promoter constructs for the assays were generated by cloning the regions of the human AR promoter from -2000 to -1500, -300 to +70, +340 to 800, between the XhoI and Hind III restriction sites of the pGL3-Basic vector (Promega). After cloning and validation by sequencing, the constructs were named AR-Luc-1, AR-Luc-2 and AR-Luc-3. The AR-Luc-2-ΔE construct contains the deletion of the E-box site (CAGGTG), generated by overlapping PCR on the basis of AR-Luc-2. Primers used are listed in Supplementary information, Table S1.

Cells were plated in 6-well plates the day before transfections, then, co-transfected with 1 μg of indicated plasmid and 10 ng Renilla luciferase plasmid pRL-TK (Promega) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Cells were also transfected with 20 nM siRNA using the INTERFERin siRNA transfection reagent (#409-10, Polyplus transfection, France).

After 48 h transfection, cells were lysed and luciferase assays were performed with Promega Luciferase assay kit. The results obtained were normalized against Renilla luciferase activity and expressed relative to the activity of untreated cells transfected with the AR-Luc promoter plasmid. Promoter activity was reported as mean ± SD value.

Proximity ligation assays (PLA), image acquisition and analysis

PLA assays were performed according to manufacturer's protocol as described previously [4]. Duolink In Situ PLA Probe Anti-Goat MINUS (#DUO92006, Sigma Aldrich) was used for menin (Goat anti-menin, A300-106A, Bethyl, TX, USA) and Duolink In Situ PLA Probe Anti-Rabbit PLUS (#DUO92002, Sigma Aldrich) for c-Myc or EZH2 (see above antibodies). Briefly, cells were grown on coverslips in 12-well plates and treated (as explained in figures and legends), then fixed with methanol for 5 min. After saturation in the blocking solution, cells were incubated with different pairs of primary antibodies at 37°C for 1 h. The PLA probes consisting of secondary antibodies conjugated with ligation were then added (30 min at 37°C), prior to the amplification phase, for 100 min at 37°C. Cells were finally counterstained with DAPI (DUO82040, Sigma-Aldrich) for 10 min. Images were acquired on an Eclipse-NiE NIKON microscope. For each sample, at least one hundred cells were counted. Analysis and quantification of these samples were performed using the ImageJ software (Version 1.52, NIH, MD, USA). PLA dots were quantified on 8-bit images using the 'Analyse Particles' command, whereas cells were counted using the cell counter plugin.

Immunofluorescence (IF) staining

For IF staining, cells were grown on glass coverslips, then fixed with methanol for 5 min at room temperature. Following fixation, cells were blocked with Dako buffer (S0809, Agilent) for 1 h, and incubated with primary antibodies overnight at 4°C, then with appropriate secondary antibodies conjugated with Alexa 555 (red) or Alexa 488

Chromatin immunoprecipitation (ChIP) assays

Chromatin immunoprecipitation (ChIP) was performed with the Millipore ChIP Assay Kit . Cells were crosslinked with 1% formaldehyde for 10 min at 37°C.

Chromatin was prepared according to the Millipore protocol and sonicated to an average size of 300-500 bp using a Diagenode Bioruptor. Chromatin fragments were immunoprecipitated at 4°C overnight with tested antibodies or normal rabbit IgG used as negative control, and immune complexes were collected on Protein A agarose beads (ChIP assay kit, Millipore). Chromatin complexes were eluted with elution buffer (1% SDS, 0.1 M NaHCO3) and crosslinking was reversed at 65°C overnight [5]. DNA fragments were purified with the QIAquick PCR purification kit (Qiagen, 28104) and used for quantitative PCR reactions with SsoADV Univer SYBR Green Supermix (Bio-Rad). Each ChIP assay was repeated at least three times independently. Primers used for ChIP-qPCR are listed in Supplementary information, Table S1.

Raw data for mining analysis

The cBioPortal for cancer genomics (http://www.cbioportal.org), a recognized and widely used online portal providing analysis of large-scale cancer genomic datasets [6,7], was employed to analyze mRNA co-expression between MEN1 and MYC, MEN1

and EZH2, MEN1 and PCAT1 in PCa. Data on the correlation between MEN1 and MYC mRNA expression were download from SU2C/PCF Dream team (mCRPC) [8]. mRNA co-expression data for MEN1 and EZH2 were downloaded from Firehose Legacy team and PanCancer Altas team (Primary prostate adenocarcinoma) [9]. Finally, mRNA co-expression data for MEN1 and PCAT1 were downloaded from the publication of SMMU team (Primary prostate adenocarcinoma) [10].

Statistical analysis

All experiments were repeated at least three times to ensure accuracy. Several previous studies have highlighted the involvement of the MEN1 gene in PCa.

In the current study, we focused on its role specifically in AR-independent PCa cells. mRNA expression in primary PCa and mCRPC datasets.

Methods

Background

With over 1.4 million new cases and more than 370, 000 deaths reported worldwide in 2020 [1], prostate cancer (PCa) remains a major cause of cancer-related mortality and morbidity in men worldwide. The majority of PCas express the androgen receptor (AR), and dysregulation of the androgen pathway is key to the development and progression of PCa [2]. Androgen deprivation therapies (ADT) are, therefore, a highly effective frontline treatment for PCa [3]. However, ADT are characterized by the virtually unavoidable emergence of resistance, termed castration-resistant PCas (CRPCs), often metastatic (mCRPC) and with a high mortality rate [1,4]. Genomic characterization of CRPCs has led to their subdivision into two subtypes: (1)

AR-dependent CRPCs, containing alterations in the AR gene, such as amplification, point mutations, and generation of splice variants; and (2) AR-independent CRPCs, in which resistant cells lack AR expression or signaling [5]. Markedly, among these AR-independent CRPCs, some PCas express neither the AR nor markers of neuroendocrine (NE) differentiation ("AR null-NE null", or double negative PCa, DNPC), and their incidence has risen over the past 2 decades from 5% in 1998-2011 to 23% in 2012-2016 [6]. Owing to the heterogeneous nature of the disease, addressing the mechanisms specifically underlying different subtypes of PCa is thus highly relevant.

Studies on DNPC and AR-independent mCRPC have considerably advanced our knowledge in this field over the last decade. By extensively characterizing cellular markers of the related lesions and cell models, cell dedifferentiation and/or altered cell plasticity were proposed to be critical for the development and evolution of these cancers [7]. Among these alterations, epithelial-to-mesenchymal transition (EMT), often in conjunction with stem cell-like changes, is considered to be crucial in the development of AR-negative PCas. The proto-oncogene MYC, a potent transcription factor that controls various biological processes [8][9][10][11][12], was reported to be one of the key drivers of CRPC and neuroendocrine prostate cancer (NEPC) [13][14][15].

Furthermore, several genetic factors and signaling pathways were shown to bypass AR pathways and thus be involved in AR-independent PCa development, such as loss of P53 [16], RB1 [16], PTEN [17], glucocorticoid receptor (GR) [18], FGF [19] and Stat3/5, as well as the metabolic ACSL pathway [20,21], whereas the underlying mechanisms remain elusive.

We have previously observed that male heterozygous Men1 mutant mice developed PCa with a low but significant frequency [22]. However, Malik et al.

reported that the physical interaction between menin and AR was essential for the growth of human PCa cell lines both in culture and in xenografts [23]. Recently, we demonstrated the decisive role played by the menin protein, encoded by the MEN1 gene, in regulating AR transcription in AR-dependent PCa cell lines [24]. Our findings also highlighted disparities in the effects of this protein on the proliferation of PCa cells, since MEN1 silencing had no impact (PC3 cells), or even increased (DU145 cells) the proliferation of AR-independent cells, whereas it led to a marked decrease in cell growth of AR-dependent lines (LNCaP, 22Rv1 and VCaP). The distinct role of the MEN1 gene in these two PCa cell populations, albeit intriguing, is not surprising, considering the different cellular and molecular mechanisms involved in their tumorigenesis. Indeed, the MEN1 gene is largely known as a tumor suppressor in several types of endocrine tissues, since its mutation predisposes patients to multiple endocrine neoplasia type 1 syndrome (MEN1 syndrome, OMIM 131100). However, it is now well established that menin displays oncogenic effects in certain types of leukemia containing fusion MLL caused by chromosome translocation [25].

Dreijerink et al. found that the gene expression profile obtained after MEN1 silencing in normal mammary luminal progenitors was highly distinct from that found in ER-positive breast cancer MCF7 cells, suggesting that menin regulates different gene sets in normal mammary luminal cells versus ER-positive breast cancer cells [26].

Here, we focused on the mechanisms underlying the distinct effects observed upon MEN1 silencing in AR-independent PCa cells. Through different analyses using cultured cells and in vivo experiments, our data unveiled the activation of the JunD and β-catenin pathways upon menin inactivation in AR-independent PCa cells, underscoring menin as an oncosuppressive factor in these cells, in marked contrast to its role in AR-dependent PCa cells.

Materials and methods

Cell culture and treatment

Prostate cancer cell lines were purchased from ATCC. Their authentication was renewed recently and Mycoplasma testing was carried out regularly. LNCaP, 22Rv1 and DU145 were cultured in RPMI medium, and PC3 cells in F-12 medium (Gibco Invitrogen), at 37°C with 5% CO2. Inhibition of menin-MLL interaction was achieved through the use of MI503 (Active Biochem).

Cell proliferation and foci formation assays

Cell proliferation assays were performed as described previously [24]. For foci formation assay, cells were seeded in 6-well culture plates at 5×10 2 cells for LNCaP, and at 2.5 ×10 2 cells for 22Rv1, DU145 or PC3. Cells were transfected with siRNA or treated with MI503, and cultured for 2 weeks. The ensuing colonies were stained with 0.05% crystal violet. The images of the plates were analyzed using Image J software.

Each experiment was conducted in triplicate and statistical analyses were performed using the Prism software.

Scratch wound healing assay

Cell migration capacities were evaluated through wound healing assays. 5×10 3 cells were seeded onto 6-well plates. After 24 h in culture, cells were transfected with 20 nM of siRNA. 72 h after transfection, wounds were created, and wound closures were captured at 0, 6, and 12 h.

Colony formation in soft agar

Soft agar assays were performed as described previously [27]. After 4 weeks of incubation at 37°C, colonies were stained with 0.05% (w/v) crystal violet (Sigma) and colonies were counted using Image J software.

Xenograph tumor growth test

Xenograft has been performed using 8-weeks-old male NOD-Scid mice by surgical implantation under the kidney capsule. 1 × 10 5 PC3-GFP cells have been encapsulated in a collagen matrix as previously described [28]. Five days after implantation, the mice were randomized onto two groups and treated three-times a week during 1 month.

Mouse groups were treated with i.p. injections of menin inhibitor MI503 75 mg/kg (MedChemTronica) (n = 11) or Vehicle solution (DMSO/PEG300, Sigma-Aldrich) (n = 9). One month after, xenografted tissues were collected after necropsy and processed for further analyses. The quantification of the number of PC3-GFP cells in xenografted tissues by qPCR assay was performed as previously described [29]. All experiments were approved by Auvergne Ethics Committee (CEMEAA) and registered according the approval number 17296-2018102216428025 v3.

RNA interference and transfection

Transfection of siRNA was performed using Lipofectamine 2000 (Invitrogen)

according to the manufacturer's instructions, and incubated for 72 h.

RNA extraction, reverse transcription, and real-time PCR

Total RNA from cultured cells was extracted by using the RNeasy-Kits (Qiagen, Valencia, USA) as per manufacturer's instructions. cDNAs were amplified and 123 quantified in an ABI Prism 7500 Sequence Detection System (Applied Biosystems) using the SYBR Green I dye (SsoAdvanced Universal SYBR Green, Bio-Rad). Data were normalized against the in-house control HPRT and represented as fold change.

Primers used are listed in the Supplementary information.

Protein extraction and Western blotting

Subcellular fractions were separated using NE-PER Nuclear and Cytoplasmic

Extraction Reagents (ref. 78833,Thermo Scientific). Western blotting was carried out according to the method described previously [30].

Immunofluorescence (IF) and immunohistochemistry (IHC) staining

For IF staining, cells were grown on glass coverslips, then fixed with methanol for 5 min at room temperature. Following fixation, cells were blocked with Dako buffer (S0809, Agilent) for 1 h, and incubated with primary antibodies overnight at 4°C, then with appropriate secondary antibodies conjugated with Alexa 555 (red) or Alexa 488 (green) (Cell Signaling Technology). Cells were counterstained with DAPI (DUO82040, Sigma-Aldrich) for 10 min and visualized by fluorescence microscopy (Eclipse-NiE NIKON microscope).

Xenografts were collected and fixed in 4% PFA prior to paraffin embedding, sectioning, staining with hematoxylin and eosin, and with immunostaining conducted as described previously [24]. Images were acquired on a ZEISS Axioscope 5 microscope.

Chromatin immunoprecipitation (ChIP) and sequential ChIP (reChIP) assays

Chromatin immunoprecipitation (ChIP) was performed with the Millipore ChIP Assay Kit (17-295) as described previously [24]. For reChIP assays, the first immunoprecipitated chromatin complexes were washed and eluted with 10 mM dithiothreitol at 37°C for 30 min and diluted 50-fold with ChIP dilution buffer. The second immunoprecipitations were then performed [31]. Each ChIP or reChIP assay was repeated at least three times independently. Primers used for ChIP-qPCR are listed in the Supplementary information.

Chromosome Conformation Capture (3C) assay and ChIP-3C

3C was performed as described previously [32,33] with minor modifications described in the Supplementary information. ChIP-3C assays were performed as formerly described [34]. Following 3C assays, we obtain cross-linking in 3C samples under ligation or non-ligation conditions. Then, by combining ChIP (as described above) and 3C assays we obtain ChIP-3C assays. Primers used for ChIP-3C qPCR are listed in the Supplementary information.

Raw data for mining analysis

The cBioPortal for cancer genomics (http://www.cbioportal.org) [35,36] was employed to analyze mRNA co-expression between MEN1 and JunD, MEN1 and CTNNB1 in PCa. Data on the correlation between MEN1 and JunD mRNA expression were download from SU2C/PCF Dream team (mCRPC) [37] and Firehose Legacy team and PanCancer Altas team (Primary prostate adenocarcinoma) [38].

Statistical analysis

All experiments were repeated at least three times to ensure accuracy. The values are expressed as mean ± standard deviation (S.D.) and analyzed by Student t-test. The level of significance: ns, non-significant vs Ctrl, * p < 0.05, ** p < 0.01, *** p < 0.001, for all analyses.

Results

MEN1 silencing promotes the tumorigenic potential and maintains MYC expression in AR-independent PCa cells.

Our previous results, showing that MEN1 silencing had distinct effects on cell proliferation of AR-dependent versus AR-independent PCa cells [24], prompted us to dissect cellular consequences of MEN1 inactivation in AR-independent PCa cells.

Firstly, we performed foci formation and soft agar assays to assess cell tumorigenic potential in MEN1-knockdown (KD) PCa cells. MEN1-KD resulted in a significant increase in colony formation in AR-independent PCa cells (DU145 and PC3) in both tests (Fig. 1a and 1b, S1a), whereas it had the opposite effect in AR-dependent PCa cells (LNCaP and 22Rv1) tested for foci formation (Fig. S1b). The efficiency of MEN1 silencing was confirmed by Western blotting (Fig. S1a andS1c). In soft agar assays, MEN1-KD DU145 and PC3 cells gave rise not only to more foci (Fig. 1c), but also to larger ones, with colonies appearing more irregular in shape, compared with siCtrl-treated cells (Fig. 1b). Furthermore, to determine whether menin influenced cell migration, MEN1-KD DU145 and PC3 cells were subjected to scratch wound-healing assays, with wound closure being monitored at 6 h and 12 h. A significant increasing in DU145 and PC3 cell migration was observed at these time-points following MEN1

silencing with siMEN1(1)+(3) (Fig. 1d-e), whereas this reduced 22Rv1 cell migration (Fig. S1d). Taken together, MEN1-KD in DU145 and PC3 cells promoted the cell growth in an anchorage-independent manner and increased cell migration, suggesting that menin plays a tumor suppressive role in AR-independent PCa cell lines, unlike its oncogenic role in AR-dependent PCa cell lines.

Intriguingly, Wu et al. demonstrated that menin physically interacts with MYC

to enhance the transcription of MYC target genes in liver cancer cells [39]. We observed that, while MEN1-KD reduced the expression of MYC at the transcriptional (Fig. 1f, upper panels) and protein levels (Fig. 1g, upper panel) in LNCaP and 22Rv1 cells, its expression was maintained in the two MEN1-KD AR-independent PCa cells (Fig. 1f and 1g, lower panels), suggesting that the difference in cellular activity between AR-dependent and AR-independent PCa cells could be, at least partially, due to MYC expression. Having shown that MEN1-KD increased the tumorigenic potential of AR-independent PCa cells and that MYC expression was maintained, we wondered whether factors known to regulate MYC expression could contribute to the effects triggered by MEN1 silencing.

Reduced menin expression triggers the nuclear translocation of JunD in

AR-independent PCa cells.

One factor in particular, the proto-oncogene JunD, was recently reported to promote the proliferation of PCa cells through MYC signaling [8]. To better understand this mechanism, we investigated the distinct effects of MEN1-KD in AR-dependent and -independent PCa cells using MI503 as a means of comparison, since MI503 is known to inhibit not only the interaction between menin and MLL, but also that of menin and JunD [40]. Indeed, we previously demonstrated that MI503 treatment inhibited the proliferation of both types of PCa cell lines, unlike siMEN1 treatment [24]. Hence, we initially performed foci formation assays to confirm a significant decrease in colony formation in both AR-dependent and AR-independent PCa cells upon MI503 treatment (Fig. 2a-b). To understand the molecular differences triggered by siMEN1 and MI503, we examined the expression of MYC and JunD. The expression of MEN1, MYC and JunD was lower in all MI503-treated PCa cells at the mRNA (Fig. 2c and Fig. S2a) and protein levels (Fig. 2d). However, following MEN1-KD, only AR-dependent (LNCaP and 22Rv1) cells displayed a decrease in mRNA (Fig. 2e and S2b, upper panel) and protein levels (Fig. 2f and S2c, left panel). Indeed, AR-independent PCa cells remained unaffected by this treatment (Fig. 2e-f and S2b-c, right panel), indicating that MI503 suppresses cell growth by inhibiting menin and JunD expression in PCa cells. Consequently, we confirmed the positive role of JunD on cell proliferation in JunD-KD DU145 and PC3 cells (Fig. S2d). We further evaluated JunD protein expression in MEN1-KD DU145 and PC3 cells, and found that, although MEN1 silencing did not affect the JunD expression at the transcriptional (Fig. 2e and S2b, lower panel) and total protein levels (Fig. 2f and S2c, right panel), it triggered the nuclear translocation of JunD in these AR-independent PCa cells (Fig. 2g-h and S2e), indicative of JunD activation [41].

MEN1 knockdown enhances the binding of JunD to the MYC locus in AR-independent PCa cells.

Wang et al. identified four potential AP-1 binding sites in a 5' MYC enhancer, situated 67kb upstream of the transcription start site (TSS) of MYC, and demonstrated the binding of JunD to the enhancer to regulate MYC transcription in breast cancer cells [42]. We wondered whether the activation of JunD may allow maintaining MYC transcription in MEN1-KD AR-independent PCa cells. Through ChIP-qPCR analyses, we observed that menin bound to the MYC promoter and to its 5' enhancer in DU145 and PC3 cells (Fig. 3a), while JunD bound only to the MYC 5' enhancer (Fig. 3b). We then hypothesized that the increased JunD nuclear translocation triggered by MEN1

silencing may augment its binding to the 5' MYC enhancer, which we confirmed by ChIP analysis upon MEN1-KD in DU145 and PC3 cells (Fig. 3c).

Next, we performed 3C assays to determine whether reduced menin expression modulates the "loop" structure between the distal enhancer region and the proximal promoter region [42]. For the 3C-PCR reaction, we amplified a 330bp DNA fragment ("Detection fragment") to assess intramolecular ligation of the looping between the enhancer and promoter of the MYC locus, using the forward primer (DP1) that anneals upstream of the distal enhancer and the reverse primer (DP2) that anneals downstream from the Fat I site in the MYC promoter (Fig. 3d). As shown in Fig. 3e and Fig. S3a, the detection fragment increased in MEN1-KD cells under the ligation condition, compared to siCtrl cells. As expected, the detection fragment failed to yield products in 3C assays under the non-ligation condition. This analysis clearly demonstrates that reduced menin expression triggers increased loop formation between the MYC enhancer and proximal promoter in DU145 and PC3 cells.

We then performed ChIP-3C [43] to further determine how the loop formed between the MYC enhancer and proximal promoter [44] might change in MEN1-KD AR-independent PCa cells. In the non-ligation condition, menin bound to the enhancer and promoter of the MYC locus (Fig. 3f and S3b), and JunD bound only to the 5' MYC enhancer in DU145 and PC3 cells (Fig. 3g and S3c), while, in the ligation condition, both menin and JunD could be detected on the 5' MYC enhancer and proximal promoter, as well as on the intramolecular ligation region (P3-P6),

suggesting that they bound together to the looping structure. Interestingly, JunD binding increased at the intramolecular ligation region (P3-P6) of the MYC locus in MEN1-KD DU145 and PC3 cells under the ligation condition (Fig. 3g andS3c).

These results provide first evidence that menin is present within the corresponding chromatin loop structure between the MYC enhancer and promoter, and that, importantly, its inactivation leads to increased binding of JunD to the 5' MYC enhancer and the loop structure. Taken together, our analyses strongly suggest that JunD, through its nuclear translocation, replaces menin on the MYC locus to maintain MYC transcription in MEN1-KD AR-independent PCa cells.

MEN1 silencing triggers the nuclear translocation of β-catenin in AR-independent PCa cells.

Our previous works highlighted the activation of β-catenin, a menin-interacting protein, upon Men1 disruption in mouse insulinoma [45,46]. Moreover, β-catenin is a well-known oncogene in prostate cancer cells [47]. We, therefore, also analyzed the expression of β-catenin in MEN1-KD PCa cells, and found that MEN1 silencing resulted in the accumulation of β-catenin in the nucleus of DU145 and PC3 cells, with its membrane and cytoplasmic expression being markedly reduced (Fig. 4a-b and Fig. S4a). Conversely, MEN1-KD significantly downregulated β-catenin total protein and cytoplasmic fraction levels in LNCaP cells and 22Rv1 cells, whereas its expression in nuclear and membrane subcellular fractions remained unaltered (Fig. S4b-d).

Furthermore, we confirmed the positive role of β-catenin in cell proliferation in DU145 and PC3 cells knocked-down for CTNNB1 coding for β-catenin (Fig. S4e).

Collectively, these results indicate that, in MEN1-KD AR-independent PCa cells, the nuclear translocation of β-catenin is drastically increased, a hallmark of the activation of the WNT signaling pathway [48].

MEN1 inactivation increases β-catenin binding to the MYC promoter in AR-independent PCa cells.

It has previously been reported that MYC and β-catenin have a strong cooperative action in different cancers [49][50][51]. We thus investigated the eventual interplay between menin and β-catenin in regulating MYC expression in AR-independent PCa cells. Our ChIP analyses showed that β-catenin bound to the MYC promoter and its 3' enhancer, formerly described in colon cancer cells [50] (Fig. 4c and S5a), while menin bound to the promoter, but not to the MYC 3' enhancer in DU145 and PC3 cells (Fig. 4d andS5b). Furthermore, ChIP-reChIP and/ ChIP analyses showed that MEN1-KD elevated the β-catenin binding to the MYC promoter (Fig. 4e andS5c), but not its binding to the MYC 3' enhancer (Fig S5d) in DU145 and PC3 cells, whereas CCTNB1-KD did not affect the binding of menin to the MYC promoter in DU145 and PC3 cells (Fig. 4f andS5e). All these analyses indicate that nuclear translocation of β-catenin allows maintaining MYC transcription in MEN1-KD AR-independent PCa cells.

JunD and β-catenin are critical for the tumorigenic potential of AR-independent

PCa cells and the expression of EMT markers.

To further investigate the role of JunD and β-catenin in AR-independent PCa cells, we initially performed soft agar assays to assess the anchorage independence in JunD-KD or CTNNB1-KD PCa cells. Both JunD-KD (Fig. 5a) and CTNNB1-KD (Fig. 5b) resulted in a significant decrease in colony formation in DU145 and PC3 cells.

Concomitantly, JunD-KD (Fig. 5c-d) and CTNNB1-KD (Fig. 5e-f) upregulated mRNA and protein levels of the epithelial marker E-cadherin in DU145 and PC3 cells. Consistently, a marked decreased in mRNA and protein expression of Twist 1, a

repressor of E-cadherin gene transcription and a known regulator of EMT [52], was observed. Importantly, the expression of HIF-1α, a key mediator in EMT, inflammation and tumorigenesis under hypoxic conditions [53][54][55], was also reduced in JunD-KD and CTNNB1-KD DU145 and PC3 cells. These findings indicate that JunD and β-catenin are critical for the tumorigenic potential and the expression of EMT markers in AR-independent PCa cells.

The activation of JunD and β-catenin is needed to reverse the oncosuppressive role of menin in AR-independent PCa cells.

We then proceeded to further determine the specific roles of JunD and β-catenin in MEN1-KD AR-independent PCa cells. To this end, we used the foci formation assay to evaluate cell growth upon transfection with siMEN1, siJunD, siCTNNB1, siMEN1+siJunD, siMEN1+siCTNNB1 or siMEN1+siJunD+siCTNNB1 in DU145 (Fig. 6a) and PC3 (Fig. S6a) cells. Knockdown of JunD or CTNNB1 significantly abolished the effect of MEN1-KD, whereas reduced menin expression significantly reversed the effects of JunD-KD or CTNNB1-KD in DU145 and PC3 cells (Fig. 6a and S6a). We obtained similar results on cell proliferation by Incucyte ZOOM analysis (Fig. S6b).

Having shown that both JunD and β-catenin activation are crucial for cell growth in MEN1-KD DU145 and PC3 cells, we hypothesized that MEN1 silencing also alters the expression of genes promoting EMT in AR-independent PCa cell lines. As expected, MEN1-KD in DU145 and PC3 cells led to reduced E-cadherin expression at the mRNA and total protein levels (Fig. 6c-d). Moreover, this silencing led to an increase in the expression of HIF-1α, Vimentin and BMI1 at the transcript and total protein levels in these cells, as well as the nuclear accumulation of HIF-1α, Twist 1 and BMI1 (Fig. 6c-e). These results suggest that menin could be a key factor inhibiting the molecular program favoring EMT in AR-independent PCa cells.

Importantly, we performed cell line-derived xenografts under mouse kidney capsule using PC3-GFP cells to validate our data in vivo (Fig. S7a). Following the transplantation of PC3-GFP cells, mice were treated with either MI503 (n = 11) or vehicle (n = 9) for 1 month. We observed that MEN1 inhibition with MI503 gave rise to significantly accelerated tumor growth, compared to the control treatment (Fig. In parallel, a significant negative correlation between MEN1 and CTNNB1 mRNA expression was seen in two different datasets from TCGA database [37] and mCRPC database [38] (Fig. 7e). These data further support the activated β-catenin due to MI503 treatment observed in PC3-GFP xenografts.

Overall, our data suggest that the activation of both JunD and β-catenin, although they individually play critical roles, is required to produce the effects of menin inactivation in AR-independent PCa cells.

Discussion

In the present study, we uncovered a previously unknown role for menin in preventing AR-independent PCa cells from evolving towards aggravated tumorigenic potential, contrasting drastically with its oncogenic role in AR-dependent PCa cells [24]. More importantly, we demonstrated that MEN1 silencing in AR-independent PCa cells led to nuclear translocation and, therefore, increased binding of JunD and β-catenin to the regulatory sequences of the MYC gene, leading to maintained MYC expression and prominent cellular and molecular alterations.

Although well-known tumorigenic cell lines, MEN1-KD in DU145 and PC3 cells resulted in an even greater increase in their tumorigenic potential, including more marked loss of contact-inhibition, augmented cell anchorage independence and cell migration. Importantly, in vivo xenograft tests in mice further demonstrated that PC3-GFP cells treated with MI503 gave rise to significantly increased tumor growth.

In parallel, we noticed that the changes in cellular behavior were accompanied by remarkable alterations in the expression of EMT makers, including BMI1, Twist1,

HINF1a, E-Cadherin and Vimentin. Our data thus suggest that MEN1 silencing in AR-independent PCa cells affects cell proliferation, cell differentiation and cell migration, resulting in altered cell plasticity and increased tumorigenicity. Both the changes in cell plasticity and abnormal expression of EMT markers are largely documented in PCa, in particular in clinical mCRPC samples [8]. However, we highlighted, for the first time, the involvement of menin in these procedures specifically in AR-independent PCa cells.

JunD was the first menin-interacting partner identified after the identification of the MEN1 gene, with its possible oncogenic role upon menin inactivation being proposed from the beginning [56]. The hypothesis was further strengthened by menin 3D structure analysis, showing that JunD binds to the same menin protein pocket as KMT2A/B [16]. However, the role of JunD in MEN1 tumors has never been clarified.

Interestingly, Wasylishen et al. recently reported that menin plays a tumor suppressive role in mouse Ras-related pancreatic cancer, likely through the activated JunD [57]. It is worth mentioning that our finding is in total agreement with the recent work reporting the role played by JunD in PCa through MYC regulation [8]. Importantly, our data provide new mechanisms showing that the prominent nuclear translocation of JunD triggered by MEN1-KD resulted not only in its increased binding to the regulatory sequence of the MYC locus, thus maintain MYC expression, but also in the altered expression of EMT markers, contributing to the aggravated tumorigenic potential seen in these MEN1-KD AR-independent PCa cells.

In PCa, the activation of β-catenin and the WNT signaling pathway is considered to be among the most commonly occurring molecular alterations involved in its development and progression [58,59]. It has also been suggested that β-catenin could play a critical role in AR-independent CRPC [60]. We and our collaborators have previously demonstrated that menin physically interacts with β-catenin, and that

Men1 deficiency leads to nuclear translocation and activation of the latter in mouse

Men1 insulinoma [61,62]. Interestingly, we uncovered in the current study that, in AR-independent PCa cells, the similar molecular switch can also happen, accompanied by decreased E-Cadherin and increased Vimentin expression, reminiscent of increased EMT. It is worth mentioning that similar changes were observed in mouse Men1 insulinomas and mouse Men1 mammary lesions [63,64].

Furthermore, our ChIP analyses demonstrated an increase in the binding of β-catenin to the MYC promoter, contributing to maintaining MYC expression in these cells.

Importantly, the fact that the nuclear expression of β-catenin increased in MI503-treated xenografted PC3-GFP cells further demonstrates that β-catenin activation is critical for aggravated tumorigenicity triggered by menin inactivation observed in the current work.

Notably, we have depicted the increased nuclear expression of several factors related to cell dedifferentiation, including BMI1, Twist1 and HIF1α. These factors are known to interact with both the JunD and β-catenin pathways and to play relevant roles in cancer progression [65][66][67]. Based on the published data, we could hypothesize that the activation of JunD and β-catenin may lead to, firstly, the activation of HIF1α. Then, the latter could trigger the activation of other factors as previously described [68][69][70][71]. The interplay between menin inactivation and the altered expression of EMT makers revealed in the current study may explain the altered cellular activities observed, and it would be relevant to study whether similar situations could occur during PCa progression, especially in the DNPC subtype of mCRPC.

Our study may also provide insight into novel strategies for AR-independent PCa treatment, if these molecular perturbations could be further confirmed in clinical studies. Considering the data obtained from the current work, especially the above mentioned in vivo test, caution should be taken when using inhibitors of menin/MLL interaction, like MI503, in PCa therapeutic assays, especially in mCRPC cases.

Conclusion

The All experiments were performed in technical and biological triplicates.

Immunofluorescence (IF) staining

For IF staining, cells were grown on glass coverslips, then fixed with methanol for 5 min at room temperature. Following fixation, cells were blocked with Dako buffer (S0809, Agilent) for 1 h, and incubated with primary antibodies overnight at 4°C, then with appropriate secondary antibodies conjugated with Alexa 555 (red) or Alexa 488 (green) (Cell Signaling Technology). Cells were counterstained with DAPI (DUO82040, Sigma-Aldrich) for 10 min and visualized by fluorescence microscopy (Eclipse-NiE NIKON microscope).

Chromosome Conformation Capture (3C) assay

3C assay potentially allows identification of physical interactions between any chromatin segments and is particularly suited to identify chromatin loops formed in genomic regions of up to several hundreds of kilobases in size. 3C was performed as described previously [2,3] with minor modifications described here. Briefly, 2×10 7 cells (DU145 or PC3) were fixed with 1% formaldehyde for 10 min at 37°C. Cells The following primers were used for 3C assays: for the ligated fragment (311bp), DP1: 5′-GTGGTTTCCAGTTATCTAAAC-3′and DP2:

5′-ATAGGGAGGAATGATAGAGGA-3′; for the control fragment (182bp), CP1:

5′-CAAATGCAATGGGAGTTTATT-3′and CP2:

5′-GAGAGTGGAGGAAAGAAGGGTA-3.

ChIP-3C

ChIP-3C assay was performed as described previously [4]. Following the 3C process, we obtain either cross-linked ligation or non-ligation samples. Subsequently, ChIP (see method described above) and 3C assays are combined for ChIP-3C assays.

Primers used in the study (Sequences (5' to 3')

For qPCR

Chapter Ⅲ Discussion and Perspectives

For my thesis work, I have been studying the involvement of the MEN1 gene in the tumorigenesis of prostate cells, using different cellular and mouse models, as well as human PCa samples, with the help from my colleagues and collaborators. The data obtained showed a rather complex and sometimes even opposite role of the MEN1 gene according to cellular and molecular context of PCa. Here, I would like to mainly discuss the questions and issues, raised from these works, that I was not able to, or not fully, address in the above mentioned articles.

From Men1 mouse models and MEN1 disease to PCa 1.1 Men1-knock out (KO) mice: what we my learn about the role of menin in

PCa ?

The first clue showing the possible involvement of the MEN1 in prostate cancer came from our observation that aged male heterozygous Men1 mutant mice developed cancerous lesions at low frequencies, in addition to endocrine tumors.

Men1-disruption accelerates the tumorgenesis

In the first Men1 mouse model established by conventional knockout system, six heterozygous Men1 mutant (Men1 +/-) mice (6/47, 12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions (Seigne et a., 2010).

Based on the above observation, more recently, our team generated a prostate-specific Men1-KO mice model, Men1 Floxed/Floxed -Nkx3.1 CreERT2 , by using a inducible system with tamoxifen. We observed that the tamoxifen treated group found that menin regulates the expression of AR and that of its target genes such as, PSA, FKBP5 and KLK2 in these cells. We further demonstrated that, mechanistically, menin binds to the AR promoter and regulates AR transcription via H3K4me3 histone marker in these cells.

c. Men1-KO induce the cell dedifferentiation of mice prostate cell Men1/Nkx3.1-deficient lesions display a AR low expression and an over 60% of menin-deficient cells expressed high CD44 at a later stage in the prostate cell membrane, accompanied by reduced CK18 and E-cadherin expression. These results suggested that a shift from a luminal to a dedifferentiated epithelial phenotype (Teinturier et al., 2021). Consistently, in my thesis work, we unveiled that menin inhibition activates the JunD pathway and Wnt/β-catenin pathway and their targets, including in particular HIF1α, E-cadherin, Twist 1, Vimentin and BMI1, which should facilitate the cell dedifferentiation and increased tumorigenic potential observed in AR-independent PCa cells. These results further demonstrated that menin involves in the cell dedifferentiation of AR-independent prostate cells, suggesting the oncosuppressive role of the MEN1 gene not only during the initiation of tumorigenesis of normal prostate cell but also in AR-independent PCa cells.

Why there are so few prostate cases reported in MEN1 patients ?

Even compared with the number of breast cancer cases reported in MEN1 patients, the prostate cancer cases found in MEN1 patients are very much limited. I think there are at least two points could afford some answers to this question.

On one hand, the prevalence of MEN1 disease is low, estimated to be approximately 1/10,000 to 1/30,000, whereas the incidence of PCa is very high. On the other hand, PCa is an aged disease, the average age of its occurrence being around 75 yrs [START_REF] Rawla | Epidemiology of Prostate Cancer[END_REF]. Therefore, considering these two factors together, it would be difficult and at least challenging to statistically prove the eventual predisposition of MEN1 mutation to PCa in MEN1 patients, like what has recently been done for breast cancer.

1.3 Any way to conceal the differences or provide more evidence ?

On the one hand, as I mentioned in the Introduction part, the researchers investigated the role of the MEN1 gene in breast cancer patients by independent cohort study. I do think that we still could try to apply the cohort study to ascertain whether PCa could be part of MEN1 pathology. I hope that the coming of more studies on the role played by menin in the future could encourage MEN1 community to address the issue.

On the other hand, we could also make more effort to study the role of MEN1 in sporadic PCa cases, through more updated technology such as RNA-seq, single cell seq, ect. Meanwhile, in this thesis using human PCa cell lines, we demonstrated that menin plays an oncogenic role in AR-dependent, whereas it can play an oncosuppressor role in AR-independent PCa cells. Therefore, we should pay more attention to different subtypes in PCa patients, in order to study the dual role of menin in the future. As I mentioned in the Introduction part and in the second article, the incidence of AR-negative PCa is increasing and the study on the mechanisms underlying AR-independent CRPC is challenging. I believe that dissecting menin's dual role in PCa could be part of the efforts to better understand PCa development and progression, as well as to improve its treatment, especially for mCRPC.

Regulation of AR transcription by menin in PCa cells.

Whether there are other factors participating the regulation of AR by menin in prostate cancer cells?

In this thesis, we proposed a new model showing that menin regulates AR transcription through the MYC pathway, involving PCAT1 and EZH2, in PCa cells. I believe that we still need to know whether other factors may also be involved in this regulation, in order to make this new model clearer and more completed.

Menin inhibition reduces AR and its downstream targets expression in AR-dependent PCa cells. It has been reported that SMAD3, a TGFβ signal transducer, represses androgen receptor-mediated transcription (Hayes et al., 2001). It is known that menin interacts physically with SMAD3 and is also involved in the TGFβ/BMP pathway in leydig cells [START_REF] Kaji | Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type beta signaling[END_REF][START_REF] Hussein | Deregulation of anti-Mullerian hormone/BMP and transforming growth factor-beta pathways in Leydig cell lesions developed in male heterozygous multiple endocrine neoplasia type 1 mutant mice[END_REF]. It would be interesting to investigate whether menin activates AR signaling via inhibiting SMDA3 expression in AR-dependent PCa cells. Menin has been reported to interacts with Forkhead transcription factors such as FOXA1 and FOXA2. In mouse embryonic stem cell (mESC)-derived embryoid bodies, lower Foxa1 and Foxa2 mRNA levels were observed in the absence of Men1 gene expression (Zhang et al. 2011). In addition, our team previously reported that menin interacts with Foxa2 and to regulate Foxa2 expression in mouse pancreatic cell lines [START_REF] Bonnavion | Foxa2, a Novel Protein Partner of the Tumour Suppressor Menin, Is Deregulated in Mouse and Human MEN1 Glucagonomas[END_REF]. In ER positive breast cancer cells, menin is enriched at enhancers that contain FOXA1-binding motifs (Dreijerink et al. 2017). In prostate cancer, Gao et al have also

shown that the FOXA1 protein physically interacts with the AR protein, thereby acting as an AR-collaborating cofactor [START_REF] Gao | The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes[END_REF]. Furthermore, FOXA1 and FOXA2 were shown to induce the expression of AR target genes such as PSA or FKBP5 in PCa cells [START_REF] Yu | Foxa1 and Foxa2 interact with the androgen receptor to regulate prostate and epididymal genes differentially[END_REF]Connelly et al., 2018). Taken together, now the question is whether menin activates AR signaling via activating FOXA1 and/or FOXA2 in PCa cells. The detailed mechanism should be further addressed in the future.

How menin regulates AR splicing variants in prostate cancer cells ?

2.2.1 AR-Vs transcription is regulated by AR-FL signaling

The expression of AR-V7 in cell lines was shown to be dynamic, and can be rapidly suppressed by addition of androgens or induced by inhibition of AR-FL signaling (e.g., through androgen deprivation, anti-androgen enzalutamide, or AR-FL siRNA) [START_REF] Hu | Distinct transcriptional programs mediated by the ligand-dependent full-length androgen receptor and its splice variants in castration-resistant prostate cancer[END_REF], and AR-FL/AR-V expression can be dramatically upregulated upon castration in prostate cancer xenograft models (VCaP and LuCaP35) [START_REF] Watson | Constitutively active androgen receptor splice variants expressed in castration-resistant prostate cancer require full-length androgen receptor[END_REF], or upon treatment with abiraterone in two CRPC xenografts (LuCAP35CR and LuCaP23CR) (Mostaghel et al., 2011). Increased expression of AR-FL/AR-V7 was also observed in other prostate cancer cells (e.g., LAPC4 and DuCaP cells) following long-term culture in the presence of enzalutamide (Hoefer et al., 2016). However, the detailed mechanism is still not clear yet.

Regulation of AR-Vs by alternative splicing

Nadiminty et al showed downregulation of heterogeneous nuclear ribonucleoproteins (hnRNPA) 1 and 2, both regulated by NF-kB, decreased the expression of AR-V7 in CWR22Rv1 and VCaP cells (Nadiminty et al., 2015). The recruitment of hnRNPA1 and hnRNPA2 to AR-Vs including AR-V7 splicing sites were enhanced in enzalutamide resistant CRPC cell lines, and could be inhibited by chemical inhibition of hnRNPA1 (Tummala et al., 2017). Takayama et al also reported that the expression of AR-Vs in prostate cancer was affected by RNA-binding protein PSF. PSF enhances various splicing factors to promote AR expression and splicing (Takayama et al., 2017).

Regulation of AR-Vs by menin

Although the above studies in multiple models have established that transcription of AR-Vs can be regulated by AR gene rearrangement or the AR locus, the detailed mechanistic investigation should be carried out to uncover additional key regulators in PCa. Surprisingly, in my work, MEN1 silencing reduced AR-Vs expression on the mRNA levels, including AR-FL, AR-V1, AR-V3, AR-V567es and AR-V7.

Meanwhile, menin inhibition also induced down-regulation of ARV7 protein expression in VCaP and 22Rv1 cells. These results indicated that menin involves in the regulation of AR-Vs or participates in the AR splicing variants in PCa cell lines.

Basis on these results, many work are still needed to identify the detailed mechanisms. For example, a. From above literature, whether menin participates the regulation of AR-Vs by NF-kB signaling or by RNA binding proteins? b. Is it possible that menin directly regulates AR-Vs expression in PCa cells, through its binding to their promoter? c. Whether MLL complex participates in the AR-Vs regulation by menin in PCa cells. All these points are still needed to be further identified. AR is a key factor for cell proliferation in AR-dependent PCa cells (Zhu et al., 2008).

What

Interestingly, my thesis works provided evidence, for the first time, that menin plays an oncogenic role through its positive regulation of AR transcription. Recent paper reported that MYC regulates the AR expression in AR dependent PCa cells [START_REF] Bai | A positive role of c-Myc in regulating androgen receptor and its splice variants in prostate cancer[END_REF]. Therefore, our initial question was whether and how menin participates in the regulation of AR by MYC in AR-dependent PCa cells. As my data showed, menin and MYC co-bound to the AR promoter to regulate its transcription in these cells. My further analysis, such as reChIP, revealed that menin is essential for the MYC-mediated AR transcription in AR-dependent PCa cells.

Moreover, my work demonstrated that menin regulated MYC transcription, via its binding to the MYC promoter, mediated by H3K4me3. Our data indicate that MYC is a downstream target of menin in AR-dependent PCa cells. It has been reported that MYC target genes related to cell proliferation and ell differentiation, such as cyclin D, cyclin E, CDK4 and β-catenin, ect (Amati et al., 1998;[START_REF] Yochum | A beta-catenin/TCF-coordinated chromatin loop at MYC integrates 5' and 3' Wnt responsive enhancers[END_REF].

Coincidentally, menin inhibition reduced the MYC targets expression and caused cell cycle arrest in AR-dependent PCa cells in my thesis work. More interestingly, it was revealed in my study that other partners of MYC, such as EZH2 and long-noncoding RNA PCAT1, were also involved in the regulation of AR by menin and MYC in AR-dependent PCa cells (Fig. 3.1).

In conclusion, menin plays an oncogenic role through its critical involvement in the regulation of both MYC transcription and MYC-mediated AR transcription in AR-dependent PCa cells. To my knowledge, it is the first time that such mechanisms underlying the involvement of menin as oncogenic factor in PCa cells were so far reported.

Menin suppresses the cell growth and dedifferentiation via inactivating JunD

and β-catenin in AR-independent PCa cells. Meanwhile, menin inhibition induced the expression Hypoxia-inducible factors 1α (HIF1α), further accelerating the carcinogenesis of the AR-independent PCa cells (Fig. 3.2). What would be the role of HIF1α in this context ? HIF1α is a transcription factor regulating many pivotal pathways in normal as well as cancerous cells (Ryan et al., 2000;Elson et al., 2000). It is overexpressed in organs or tissues where oxygen level drops below threshold level (Hockel et al., 2001). High levels of HIF1α point towards angiogenesis, cell proliferation, survival and tumor progression through regulation of growth promoters, oncogenes, glycolytic pathways and pH regulation. A large number of studies support the relation of increased level of HIF-1α with aggressive tumor growth and poor patient prognosis (Baba et al., 2010;Semenza et al., 2013;Chen et al., 2014).

Menin inhibition induces the cell dedifferentiation via activation of JunD and

Wnt/β-catenin pathway in AR-independent PCa cells Epithelial-to-mesenchymal transition (EMT) is a crucial step in metastasis initiation.

Several observations from the reprogramming field also provide evidence that dedifferentiation occurs when EMT factors are expressed (Takahashi and Yamanaka, 2006;Yu et al., 2007;Takahashi et al., 2007;Park et al., 2008). EMT-related gene expression changes may also provide input to the cell differentiation state, such as E-cadherin, occludin, Twist 1, Snail1 and Traffic Jam 1 (TJ1). Interestingly, it has reported that the activation of the Wnt/β-catenin signal pathway is the critical factor that directly controls the process of EMT induced by HIF-1a in PCa cells [START_REF] Jiang | MEN1 mutation analysis in Chinese patients with multiple endocrine neoplasia type 1[END_REF]. In addition, as I mentioned in the 3.1, it has been known that β-catenin is a target of MYC in colorectal cancer cells [START_REF] Yochum | A beta-catenin/TCF-coordinated chromatin loop at MYC integrates 5' and 3' Wnt responsive enhancers[END_REF].

In our analysis, menin inhibition activated the Wnt/β-catenin pathway and its targets, such as HIF1α, E-cadherin, Twist 1, Vimentin and BMI1, leading to cell dedifferentiation in AR-independent PCa cells. (Fig. 3.

2) Consistently, in our previous

Men1 knock out mouse model, we found that early-stage lesions exhibited a decreased expression of AR and its target genes, accompanied by reduced CK18 and E-cadherin expression, suggesting a shift from a luminal to a dedifferentiated epithelial phenotype. Intriguingly, over 60% of menin-deficient cells expressed CD44 at a later stage (Teinturier et al., 2021). These results suggest that menin could be a key factor inhibiting the molecular program favorite for EMT and stemness in AR-independent PCa cells or AR low expression of PCa. Nevertheless, many works are still needed to identify the similar molecular changes in human prostate cancer tissues and to detail the underlying mechanisms.

Mechanisms underlying the suppression of JunD and β-catenin by menin

Menin represses JunD expression

Menin-JunD interaction has been identified by the yeast two-hybrid method in the human brain cDNA library and has been confirmed in vitro and in vivo (Agarwal et al., 1999). Menin represses JunD-activated transcription through a histone deacetylase (HDAC)-dependent mechanism [START_REF] Gobl | Menin represses JunD-activated transcription by a histone deacetylase-dependent mechanism[END_REF] in which mSin3A, a general corepressor, recruits HDAC to menin [START_REF] Kim | a tumor suppressor, represses JunD-mediated transcriptional activity by association with an mSin3A-histone deacetylase complex[END_REF]. Several naturally occurring and clustered MEN1 missense mutations disrupted menin's interaction with JunD.

These observations suggest that menin's tumor suppressing function involves direct binding to JunD and inhibition of JunD-activated transcription (Agarwal et al., 1999).

Our analysis found that menin suppresses JunD expression on the mRNA and protein level in the AR-independent PCa cells. Furthermore, we also showed that menin inhibits the JunD translocation into nuclear to function in AR-independent PCa cells. However, the detailed molecular mechanisms need to be investigated in PCa cells.

A new mechanism was reported whereby menin binds JunD and thus block c-Jun N-terminal kinase (JNK)-mediated phosphorylation of JunD at residues S90, S100 and T117, an event crucial for activating JunD function in transcriptional regulation in HEK-293 cells. Furthermore, menin had no effects on JNK binding and JNK-mediated phosphorylation of c-JUN, a close homologue of JunD that lacks a menin-binding motif (Huang et al. 2012). These results could give us a promising clue to investigate the mechanism that JunD suppression by menin in PCa cells.

Menin represses β-catenin expression

Our team and collaborators previously demonstrated that menin directly interacts with β-catenin and carries β-catenin out of the nucleus through its two functional nuclear export signals (Cao et al., 2009). In Men1-null MEFs and insulinoma tissues from β-cell-specific Men1 knockout mice, β-catenin is accumulated in the nucleus (Cao et al., 2009). Since over expression of menin reduces nuclear accumulation of β-catenin and its transcriptional activity (Cao et al., 2009), menin may repress cell proliferation through suppression of the Wnt/β-catenin signaling, which is pro-proliferative in many cells (Cao et al., 2009). However, in cultured rodent islet tumor cells, menin may repress cell proliferation partly by activating the Wnt/β-catenin signaling [START_REF] Chen | Menin promotes the Wnt signaling pathway in pancreatic endocrine cells[END_REF]. When menin and activated β-catenin is over expressed, the Wnt/β-catenin downstream target gene, Axin2, is significantly enhanced correlating with increased H3K4 trimethylation at the promoter of the Axin2 gene [START_REF] Chen | Menin promotes the Wnt signaling pathway in pancreatic endocrine cells[END_REF]. Indeed, it is already known that β-catenin plays as an oncogenic factor in PCa cells. However, we didn't know the relationship between menin and β-catenin in PCa cells when we started our work. In my thesis work, we found that menin inhibition by siRNA causes up-regulation of CTNNB1 and increased nuclear expression of β-catenin protein in AR-independent PCa cells. Notably, menin also displayed the ability that inhibiting the β-catenin translocation in these cells. Nevertheless, the detailed mechanism that how menin regulates the CTNNB1 transcripts and how menin inhibits β-catenin translocation is still needed to be investigated in AR-independent PCa cells.

3.4 Tumorigenic potential: different usage and the limit of cultured cell approaches

In vitro model for cancer research

In vitro refers to the technique of performing a given procedure in a controlled environment outside of a living organism. For decades, cultured animal and human cells are used as an important tool for cell biology and cancer research. Cells can be grown in attached to specific surfaces forming cell monolayer cultures in two-dimension (2D) system, typically surface platforms are tissue culture-plastic or surfaces with extracellular matrix components such as collagen, fibronectin among others fibrous components. However, monolayer cultures have been subject to criticism because assays still reflect a highly artificial cellular environment that may not correspond to a tissue or organ situation.

Nevertheless, researchers developed a series of methods such as foci formation assay and soft agar colony formation assay, to better characterize the tumorigenicity of cancer cells, in terms of loss of contact inhibition and anchorage independence. It cannot be denied that they provide a simple and safe way for scientists to perform early tests or basic research on new procedures and treatments. However, sometimes when we apply a single assay to research, the results may not reflect the real situation.

In our analysis, menin inhibition by siRNA did not reduce the cell proliferation of PC3 cells by the Incucyte ZOOM analysis. But, when other assays were applied, I got different results. I found that the MEN1 inactivation not only promotes the cell proliferation, but activates cell migration and cell invasion in AR-independent PCa cells by wound healing assay and soft agar assay, reapectively. Taken together, we should, when possible, use several complementary culture assays in our research work.

In vivo model for cancer research

In vivo refers to experimentation using a whole, living organism as opposed to a partial or dead organism. Animal studies and clinical trials are two forms of in vivo research. In vivo testing is often employed over in vitro because it is better suited for observing the overall effects of an experiment on a living subject. As I mentioned in the Introduction part, Men1-KO mice model were generated by different system. The dual role of menin has been revealed in Men1 gene deficient of mammary gland lesions or prostate lesions.

In this thesis, owing to our collaboration with Dr. S Baron from Université Clermont Auvergne, we adopted a xenograft mouse model using PC3-GFP cells to confirm the results which we obtained in vitro. On the one hand, we found that MI503 treatment

3D culture

A new dimension to cancer research has been achieved by the advent of 3-Dimensional cell culture techniques. This approach increased many folds the ways in which cancer cell lines can be utilized for understanding complex cancer biology.

3D cell culture techniques are now the preferred way of using cancer cell lines to bridge the gap between the "absolute in vitro" and "true in vivo". The aspects of cancer biology that 3D cell culture systems have contributed include morphology, microenvironment, gene and protein expression, invasion/migration/metastasis, angiogenesis, tumour metabolism and drug discovery, testing chemotherapeutic agents, adaptive responses and cancer stem cells. The new technique should give us a new clue to confirm our analysis in vitro studies at least.

New conception of therapeutic strategy to propose based on the findings from

the current work.

4.1 MI503, inhibits the menin and JunD interaction in prostate cancer cells ?

Molecular inhibitor, MI503, blocks the menin/MLL interaction driven leukemia progression in mouse models of MLL leukemia [START_REF] Malik | Targeting the MLL Complex in Castration-Resistant Prostate Cancer[END_REF]. Wu et al further demonstrated that MI503 promoted the interaction of menin with Hsp70-associated ubiquitin ligase CHIP, resulting in increased menin ubiquitination, leading to increased menin degradation [START_REF] Wu | Disruption of the menin-MLL interaction triggers menin protein degradation via ubiquitin-proteasome pathway[END_REF]. Interestingly, Malik et al. reported that MI503 treatment blocked AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice (Malik et al., 2015). Interestingly and importantly, in our hands, MEN1 inactivation by siRNA only inhibited the cell proliferation in AR-dependent PCa cells, but not in the AR-independent PCa cells (Teinturier et al., 2021). This finding inspired us to investigate why MI503 could inhibit the cell proliferation in AR-independent PCa cells.

Huang et al established the crystal structures of human menin in its free form and in complexes with MLL1 (Fig. 3.4a) or with JUND (Fig. 3.4b) heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner (Huang et al., 2012). Their results indicated that MI503 not only inhibits the interaction between menin-MLL, but can also block the interaction between menin-JunD. Notably, Elliott et al demonstrated recently that JunD plays an oncogenic role and is essential for MYC-mediated the cell proliferation in PCa cells (Elliott et al., 2019). Thus, our hypothesis was that MI503 treatment could deregulated menin-JunD interaction and at the same time decrease both menin and JunD expression to suppress cell proliferation in PCa cells, which could explain the difference observed in MI503-treated and siMEN1-treated AR-independent PCa cells. As we expected, MI503 treatment not only reduced the menin expression, but also decreased the JunD and MYC expression on the transcriptional and protein level in both cultured AR-dependent and AR-independent PCa cells. It has been reported

that MI503 decreased menin protein stability by activating ubiquitination pathway [START_REF] Wu | Disruption of the menin-MLL interaction triggers menin protein degradation via ubiquitin-proteasome pathway[END_REF]. Further works are needed to determine whether MI503 affects the JunD protein stability in a similar way.

4.2 Should we use the MI503 to treat the PCa ?

Our analysis should give some clues for this question. We should be cautious when we use the MI503 to treat the PCa. On the one hand, the mechanism of MI503 is not fully understood. As I mentioned in 4.1, MI503 not only reduces the menin expression, but also inhibits the JunD expression. However, the underlying mechanism for the latter is poorly understood for the moment. More efforts should be made to better understand the mechanistic action of MI503. On the other hand, in this thesis, we revealed that menin plays as a tumor suppressor role in AR-independent PCa cells. Hence, MI503 would not be suitable for treating AR-negative or AR-independent PCa. Therefore, it will be useful in the future to distinguish the AR expression in PCa during future clinical trials.

Chapter Ⅳ Conclusion

My thesis works afforded novel evidence suggesting distinct roles of menin in PCa cells, depending on different cellular and molecular contexts. On the one hand, we report here a novel mechanism underlying menin's oncogenic activity in AR dependent prostate cancer (PCa) cells. Our results demonstrate that menin is a critical cofactor for MYC-mediated AR transcription in AR-dependent PCa cells.

Interestingly, we also showed that menin was involved in the regulation of MYC-related LncRNA PCAT1 in AR-dependent PCa cells, through both its positive regulation of EZH2 transcription and its physical interaction with EZH2, as well as their co-binding on the regulatory sequence of PCAT1. As MYC is known to be deregulated in 30-50% of human malignancies with poor prognosis (Chen et al., 2018), our novel discovery may have significant implication for cancer therapy:

targeting both menin and MYC pathways might potentially work for AR-dependent PCa.

On the other hand, our findings provide evidences that menin plays a tumor suppressive role in AR-independent PCa cells. We found that MEN1 inactivation promotes tumorigenic potential, via triggering nuclear translocation of JunD and β-catenin, in AR-independent PCa cells. Mechanistically, upon MEN1 inactivation, both JunD and β-catenin could replace menin to bind to the MYC proximal promoter, and ultimately result in maintaining the MYC expression in AR-independent PCa cells. Moreover, the analysis of culture AR-indepedendent cells and PC3-GFP xenografts in the mouse demonstrated that both JunD and β-catenin are necessary for the altered tumorigenic potential triggered by MEN1 inactivation in AR-independent PCa cells. Interestingly, we also found MEN1 inactivation alters the expression of several molecular markers of EMT and stemness, including E-cadherin, BMI1, Twist1 and HIF1α, in these cells. Nevertheless, many works are still needed to identify the detailed mechanisms underlying the involvement of menin in the above phenomenon in AR-independent PCa cells.

Introduction

Prostate cancer (PCa) is the second most common cancer in men worldwide [1]. The androgen receptor (AR) plays a crucial role not only in the development of PCa, but also in its treatment. Indeed, anti-AR therapy by second-generation antiandrogens is highly effective, though it ultimately results in resistance and may favor the occurrence of an AR-negative PCa subtype [2][3][4]. The latter has risen over last two decades [2], in particular the AR-negative and nonneuroendocrine (AR -NE -) tumor subtype, which has overtly augmented from 5 to 20%.

Aside from the previously known common genetic alterations, such as gain of AR function, loss of NKX3.1, and PTEN, recent works on metastatic castration-resistant PCa (mCRPC) detected recurrent genomic alterations implicating the factors involved in chromatin structure modification [5]. Remarkably, components of the COMPASS-like complexes, KMT2B, KMT2C, and KMT2D, as well as menin encoded by the MEN1 gene and interacting with KMT2A/B, were shown to be mutated [5][6][7]. MEN1 mutations predispose patients to multiple endocrine neoplasia type 1 syndrome (MEN1, disease identifier OMIM131100). Interestingly, it has also been shown that menin interacts with KMT2A, KMT2D, and other partners to promote leukemogenesis [8,9]. Importantly, a recent study revealed a higher breast cancer incidence in female MEN1 patients [10]. The finding is reminiscent of our previous observation that female Men1 mammary gland-specific Men1 knockout mice developed breast precancerous lesions, whereas male heterozygous Men1 mutant mice developed PCa with a low but significant frequency [11]. However, Malik et al. reported that the interaction between menin and AR is required for the growth of human PCa cell lines both in culture and in xenografts [12]. Therefore, an obvious discrepancy exists between data from spontaneous prostate tumors (in human and mouse models) and those from some PCa-derived cell lines (or xenografts), which may reflect opposite functions of menin in prostate cells in different contexts. A dual cell type-specific role for menin has been proposed in breast cancer [13]. We hypothesized that, in PCa, the effects of menin were dependent on the stage and molecular context of disease progression. To test this hypothesis we generated a novel Men1 mutant mouse model, in which Men1 was specifically disrupted in Nkx3.1deficient prostate cells. Intriguingly, a link between MEN1 and AR regulation was uncovered, highlighting the role played by menin in the regulation of AR expression and the AR signaling pathway.

Results

Men1 disruption accelerates tumorigenesis in

Nkx3.1-deficient prostate cells in mice

To decipher the role played by Men1 in the initiation of prostate cell tumorigenesis, we chose to disrupt Men1 in a mouse model in which precancerous prostate lesions develop, in order to either accelerate or rescue tumor development. To this end, Men1 F/F mice [14] were crossed with Nkx3.1Cre ERT2 mice, in which the activity of Cre recombinase controlled by the endogenous Nkx3.1 promoter can be induced in prostate luminal cells upon tamoxifen injection. Men1 F/F -Nkx3.1Cre ERT2-/+ mice thus generated were subjected to tamoxifen treatment (Men1NT) or not (Men1N), at 1.5 months of age, and prostate glands were subjected to pathological examination 1.5, 4.5, and 8.5 months after injection (Fig. 1a). Immunohistochemical (IHC) analysis confirmed Men1 disruption in prostate luminal cells exclusively in Men1NT mice (Fig. 1b,c).

Since Nkx3.1Cre ERT2 mice develop mouse prostatic intraepithelial neoplasia (mPIN) lesions at around 6 months of age at a low frequency and reach full penetrance after 10 months [15,16], we speculated that Men1NT mice would allow us to determine whether additional Men1 disruption rescues or exacerbates the phenotype of Men1N mice. We observed mPIN lesions in dorsal, ventral, lateral, and anterior lobes in all male Men1NT mice (n = 7) 1.5 months after induction of the Cre recombinase, with high-grade mPIN being observed in six of the seven Men1NT mice and low-grade mPIN in the remaining Men1NT mouse, whereas only one mouse displayed lowgrade mPIN among the untreated age-matched Men1N mice (n = 5, Figs. 1d and S1 and Table 1). Having shown that a majority of prostate cells within lesions in Men1NT mice lost menin expression, we then noticed that these mice progressively developed more advanced lesions with prominent pleomorphic changes (Figs. 1e andS2) and microinvasive carcinoma (MIC) at 6 (2/7, n = 7) and 10 months (4/6, n = 6) of age (Fig. 1f, upper panel, Table 1). As experimental controls, no age-matched Men1N mice developed MIC (n = 5 for all groups of age) (Table 1), Men1 F/F mice either treated or not with tamoxifen developed no mPIN lesions within the 10-month experimental time course, and tamoxifen-treated Nkx3.1Cre ERT2-/+ mice developed no MIC lesions at 5 and 10 months of age (n = 5 for both groups, data not shown). Consistently, αSMA staining revealed an attenuated or broken smooth muscle cell layer around the lesions, highlighting alterations in the prostate gland structure during the process of MIC formation (Figs. 2a,upper panel,and S3). Ki67 staining of 6month-old Men1NT mice revealed that mPIN lesions lacking menin expression showed a significantly higher proliferative activity (Figs. 2a,lower panel,and S4). Taken together, our results demonstrated that prostate-specific Men1 disruption in mice leads to the acceleration of tumorigenesis in Nkx3.1 -/+ prostate cells.

Men1/Nkx3.1-deficient lesions display early AR low and late membrane CD44 high expression lineage markers known to be crucial for defining differentiation of prostate cells. Notably, double immunofluorescence (IF) staining using antibodies against menin (red) and AR (green) on prostate glands from 3-and 6-month-old tamoxifenuntreated or -treated double-mutant mice revealed a reduced AR expression in menin-deficient prostate cells (Figs. 2b andS5). IHC analyses also showed that this decrease was accompanied by a decrease in the expression of AR-target genes, such as FKBP5 (Fig. 2c). The strongly reduced AR expression in Men1-deficient lesions prompted us to further investigate the effects of menin downregulation on the expression of AR and its target genes in PCa cell lines. MEN1 knockdown (KD) with different siRNA in LNCaP and 22Rv1 cells (AR + cell lines) resulted in a significant decrease in AR and PSA (prostatespecific antigen) transcriptional expression (Fig. 2d, upper panel). To verify whether the menin-mediated effects described above were linked with the MLL complex, we treated LNCaP cells with MI503, which inhibits the interaction between menin and MLL. As anticipated, MI503 treatment led to a dose-dependent reduction in the transcription of AR and PSA (Fig. 2d, lower panel). Moreover, a strong reduction in the protein expression of AR, PSA, and TMPRSS2 (transmembrane protease, serine 2), another AR-target, in siMEN1-treated LNCaP and 22Rv1 cells was observed by western blot analysis (Fig. 2e). Consistently, AR binding to the promoter of its known target genes, KLK3, FKBP5, and KLK2, was significantly reduced in siMEN1-treated LNCaP, 22Rv1, and VCaP cells (Figs. 2f andS6).

Furthermore, IHC analyses revealed a decrease in CK18 expression in menin-deficient prostate cells, whereas no increase in CK5 expression was observed (Fig. 3a). In addition, Men1-deficient luminal cells expressed no basal marker p63 (Figs. 3b andS7). Consistently, E-cadherin expression, prominently expressed on the membrane of normal epithelial luminal prostate cells, was reduced (Fig. 3c), whereas no vimentin expression could be detected (data not shown), suggesting a partial epithelial-mesenchymal transition. The expression of the neuroendocrine cell marker chromogranin A was absent from menin-deficient cells (Fig. S8), suggesting that these cells were not NE + cells.

A reduced AR expression has recently been linked to the appearance of prostate cell populations expressing molecular markers reminiscent of stem cells [17,18]. We therefore analyzed the expression of several of these markers. IF analyses revealed that, although no CD44 expression could be detected in prostate glands of control mice, or in PIN lesions from double-mutant mice 1.5 and 4.5 months after Cre recombinase induction, surprisingly, prominent CD44 expression was detected in the majority of Men1deficient prostate cells from all Men1NT mice 8.5 months after the induction. Indeed, the percentage of CD44expressing cells reached around 60% of Men1-deficient prostate cells (Fig. 3d). However, other stem cell-related markers, including CD133, α2β1 integrin (CD49b), and CD49f, remained undetected (data not shown).

Menin participates in the regulation of AR expression and of cell proliferation in AR + cell lines

To further study AR regulation by menin revealed by the above mentioned observations made in the mouse model and human PCa cell lines, we sought to define the occupancy of menin on the AR promoter by ChIP analysis. We uncovered a significant menin-enriched region on the proximal part of the AR promoter encompassing the -1500 to +550 bp region flanking its transcription start site, menin-occupancy signal diminishing drastically when cells were pretreated with siMEN1 (Figs. 4a,upper left panel,and S9). Importantly, we found that the same region was highly methylated at H3K4me3 and was co-occupied by one of the COMPASS-like components ASH2L, suggesting that the complex binds to this region (Fig. 4a, lower left panel). Moreover, when the cells were treated with MI503, the ChIP signals obtained with anti-menin and H3K4me3 antibodies were significantly reduced, further confirming the involvement of the menin-MLL complex in this region (Fig. 4a, right panel). Our data thus demonstrated, to our knowledge for the first time, that menin regulates AR transcription, most likely via its binding to the AR promoter region, mediated by the MLL complex.

Considering the crucial role played by AR in the differentiation and proliferation of normal prostate cells and in PCa, we further investigated the effects of menin inactivation on cell growth in 22Rv1,and VCaP) and AR-independent (DU145 and PC3) cell lines, as well as in a PC3 cell line constitutively expressing exogenous AR (PC3-AR). Our analyses showed that MEN1 KD with siMEN1 resulted in a significant decrease in cell proliferation in all three AR-dependent lines, reaching levels similar to those observed using siAR, but, surprisingly, not in the two AR-independent cell lines (Figs. 4b andS10). Even a slight increase in cell proliferation could be noted in DU145 cells: ~1.4-fold at 120 h after siMEN1 treatment. Conversely, we observed a slight but significant decrease in cell proliferation in PC3-AR cells treated with siMEN1. Our data suggest that the previously reported role played by menin in promoting cell proliferation [12] may vary among different PCa cell lines with distinct activities of the AR pathway. Intriguingly, we observed that the treatment of DU145 and PC3 cells with MI503 resulted in a decrease in cell proliferation, unlike siMEN1-treated DU145 and PC3 cells, likely due to an effect unrelated to reduced menin expression (Fig. S11).

MEN1 KD leads to increased CD44 expression in PC3 cells re-expressing AR

A previous work reported reduced CD44 expression in PC3 cells re-expressing AR, suggesting AR expression could inhibit CD44 expression in this context [19]. Since our data revealed the role played by menin in AR regulation, we speculated that ectopic CD44 expression observed in the prostate lesions developed in double Men1/Nkx3.1-deficient mice could be, at least partially, due to the reduced AR expression triggered by Men1 deficiency. To gain further evidence to support our hypothesis, we carried out MEN1 downregulation by siMEN1 in both PC3 cells and a PC3 cell line with reconstituted AR expression (PC3-AR), and subsequently analyzed menin, AR, and CD44 expression. Unlike PC3 cells, MEN1 KD PC3-AR cells displayed both reduced menin and AR, but interestingly, increased CD44 expression (Fig. 4c, left panel). We noticed, as expected, that AR KD using three different siAR, with two targeting coding sequence, resulted in similar CD44 overexpression in PC3-AR cells (Fig. 4c, right panel).

Menin expression diverges in different human PCa

We further analyzed menin expression in 37 human PCa with different clinical features, including 14 low-grade and less advanced cases, 14 high-grade and more advanced cases, and 9 cases with castration resistance (CR). Our data showed that, compared to normal prostate glands, nuclear menin staining varied in the human PCa tested, with a reduction in menin expression not only frequently seen in low-grade PCa (9/14, Fig. 4d and Table 2), but also observed in high-grade (7/14) and CR-staging PCa (4/9). However, we noticed that a strong menin expression could be detected in more than half of the CR samples (5/9). To gain further insight into eventual correlations between menin and AR expression, as well as those between menin and CD44 expression in PCa, data mining analyses were carried out using different publicly available PCa mRNA expression datasets (see "Materials and methods"). Interestingly, a significant and a marginally significant positive correlation between MEN1 and AR mRNA expression were seen in two mCRPC datasets, whereas a significant inverse correlation between MEN1 and AR mRNA expression was found in two primary PCa datasets (Figs. 4e andS12a).

Remarkably, a significant inverse correlation between MEN1 and CD44 mRNA expression was uncovered in two primary PCa datasets and a marginally significant inverse correlation was seen in one of two mCRPC datasets (Figs. 4f andS12b).

Discussion

The current work highlights the oncosuppressive role played by MEN1 in the initiation of prostate cell tumorigenesis involving the dysregulated AR pathway and epithelial luminal cell dedifferentiation. The data provide unprecedented clues as to the role played by menin in regulating AR expression, and consequently, influencing the AR signaling pathway. Specific Men1 disruption in Nkx3.1-deficient prostate cells led to the acceleration of tumorigenesis, both for its initiation and development of advanced lesions. The observation is in line with our previous findings obtained in male Men1 heterozygous mutant mice and with the MEN1 mutations found in sporadic human PCa [5,11]. The current work hence provides further genetic proof of a tumor suppressive role for MEN1 in the initiation of prostate cell tumorigenesis. The characterization of Men1-and Nxk3.1deficient PIN lesions indicates that the affected cells lose their typical luminal cell characteristics and display some dedifferentiation features. Indeed, from the early-stage onward, the lesions developed in Men1NT mice showed downregulation of both AR and its target genes, and a general decrease in CK18 expression, as well as a reduction in E-cadherin expression. Importantly, the characteristics of these lesions are reminiscent of recent findings in human PCa in which undifferentiated PSA low tumors were observed among both advanced primary PCa (GS9/10) and CRPC [20]. Interestingly, Beltran et al. described an ARindifferent cell state as a mechanism of treatment resistance through divergent clonal evolution implying epigenetic modifiers [21]. Our mouse model may provide a useful tool for improving the characterization of AR low PCa, and addressing the mechanisms involved in the development of PCa related to the complex dysregulation of the AR pathway caused by its activation and/or inactivation [22].

Our work provides compelling evidence, to our knowledge for the first time, that menin critically regulates AR transcription, likely through its binding to the proximal AR promoter together with MLL complex. Interestingly, in different PCa cell lines, the effect of AR regulation by menin seems to be correlated with the effects of menin on cell proliferation. Of note, variable response to MEN1 KD in different PCa cell lines was already observed [23]. Our data imply that cellular and pathological consequences of menin inactivation in prostate cells vary according to the activity of the AR pathway. It provides meaningful explanation for the dual role of menin seen in our experiments, on the one hand, as a tumor suppressor in Men1 mutant mice, and on the other hand, as pro-oncogenic factor in AR + cell lines. Although AR is known for its vital function as a transcription factor, how the transcription of its own expression is regulated remains elusive. Further investigations into the regulation of the AR pathway by menin, should shed light on this issue, as well as on the crosstalk between AR and menin in prostate cell tumorigenesis.

Intriguingly, Men1-deficient late-stage lesions massively expressed CD44. However, our data do not seem to suggest that Men1-deficient cells were converted into cancer stem cells. Firstly, the activation of CD44 was detected in the majority of the cells in more advanced prostate lesions, whereas no CD44 + cells were detected in early PIN lesions. Furthermore, the expression of other stem cell markers was not found in the same lesions. Instead, CD44 expression may represent a more advanced dedifferentiation of luminal cells, providing them with more proliferative and invasive capacities, as suggested by Patrawala et al. [24]. It is worth mentioning that the inverse correlation between menin and CD44 expression found in primary human PCa is in line with the observations made in our mouse model. Interestingly, our data demonstrated that the observed ectopic CD44 expression is at least partially related to the reduced AR expression triggered by Men1 disruption. It would be interesting in the future to investigate whether the particular AR low /CD44 + phenotype could be found among human AR low PCa.

Consistent with the above observations made in Men1 mutant mice, reduced menin expression was observed in a substantial proportion of human PCa tested. A more frequent reduction was detected in low-grade PCa arguing in favor of its putative suppressive role in some PCa at the tumor initiation stage. We concomitantly found that a considerable number of CRPC expressed high levels of menin, reminiscent of the proproliferative effects of menin observed in AR-dependent PCa cell lines. Interestingly, the significant positive correlation between MEN1 and AR mRNA expression were found in two mCRPC datasets, reminiscent of our observations made in three ARdependent PCa cell lines, all of them derived from mCRPC. In parallel, the inverse correlation between menin and AR in human primary PCa suggests a different role for menin in these PCa compared with mCRPC. However, we are aware of the heterogeneous nature of primary PCa samples and the possibility that the analysis could be biased by low proportions of AR low PCa present among the samples used. Together with our observations made in different experimental models, our data indicate that menin could play distinct roles in different PCa, likely in relation with the AR pathway and in a spatiotemporal-specific way.

Collectively, the specific Men1 disruption in prostate cells of Men1NT mice resulted in the occurrence of early AR low mPIN and late AR low /CD44 + mPIN and MIC, further suggesting the oncosuppressive role of the MEN1 gene in the initiation of prostate cell tumorigenesis. In parallel, the current work, revealing the regulatory role played by menin on AR and the dysregulation of the AR pathway triggered by MEN1 inactivation in AR-positive PCa cells, may have implications for new PCa treatment strategies.

Materials and methods

Human patient samples

Human paraffin embedded prostate tissues were obtained from Biological Resources Platform at Henri Mondor hospital (CPP no. 16169). The study was conducted in accordance with the guidelines in the Declaration of Helsinki.

Animals

Men1 F/F (on a mixed C57B:6 and 129/SvJ background) were generated, bred, and maintained as previously described [14]. Nkx3.1Cre ERT2-/+ mice were provided by the Jackson Laboratory, and bred with Men1 F/F mice to generate Men1 F/F -Nkx3.1Cre ERT2-/+ mice, as well as Men1 +/+ -Nkx3.1Cre ERT2-/+ mice. Male Men1 F/F -Nkx3.1Cre ERT2-/+ mice were treated by intraperitoneal injection of 100 μl tamoxifen at 10 mg/ml daily for 5 consecutive days at 1.5 months of age after birth, and control mice were injected with oil. Prostatic glands were subjected to pathological examination and other analyses at 1.5, 4.5, and 8.5 months after injection. Animals were maintained in a specific pathogen-free animal facility AniCan at Cancer Research Center of Lyon at Center Léon Bérard, Lyon, France.

Cell culture and treatment

All the PCa cell lines were purchased from ATCC, except the stable cell line PC3-AR constitutively expressing an exogenous AR as described [25]. Their authentication was renewed recently and Mycoplasma testing was carried out regularly. LNCaP, 22Rv1, and DU145 were cultured in RPMI medium, and PC3 and PC3-AR cells in F-12 medium (ThermoFisher Scientific) containing 25 mM glucose and supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 unit/ml penicillin, 100 μg/ml streptomycin, HEPES 10 mM, and sodium pyruvate 1 mM, at 37 °C with 5% CO 2 . Inhibition of menin-MLL interaction was achieved through the use of MI503 (Active Biochem), at the concentration of 0.5, 2.5, 5, and 10 μM.

Cell proliferation assay

Cells were seeded onto 96-well culture plates at 5 × 10 3 cells for 22Rv1, DU145, and PC3, and at 1 × 10 4 cells for LNCaP. Cell morphology, attachment, spreading, and proliferation were monitored every 30 min for 0-120 h by IncuCyte ZOOM Live-Cell Analysis system (Essen BioScience), which was placed in a humidified incubator maintained at 37 °C with 5% CO 2 . After 24 h cells culture, cells were also transfected with 20 nm siRNA against MEN1 and AR using INTERFERin siRNA transfection reagent (#409-10, Polyplus-transfection) according to the manufacturer's protocol. All the experiments were performed in triplicate and at least three times.

Real-time reverse transcriptase-PCR

RNA were extracted using RNeasy-Kits (Qiagen, Valencia, USA). Real-time PCR analyses were carried out on a Step-One RT-System (Applied Biosystem, France) using SYBR-Green (ThermoFisher) and results of each samples normalized to 36B4.

Immunostaining

Prostate tissues were collected and fixed in 4% PFA prior to paraffin embedding, sectioning, and staining with hematoxylin and eosin according to a standard protocol. Briefly, endogenous peroxidases were quenched in 3% H 2 O 2 solution for 30 min at room temperature. Heat-induced epitope retrieval was performed by immersion in antigenunmasking solution (catalog no. H-3300; Vector Laboratories) in a microwave oven for 15 min. After blocking with antibody diluent (Dako), sections were incubated overnight with a primary antibody. For double IF, stains were detected with a Cy3 or Cy5 tyramide amplification kit (Perki-nElmer), according to the manufacturer's instructions, with prior incubation with the appropriate biotinylated secondary antibody or incubation with appropriate Alexa Fluor 488, 555, coupled secondary antibodies (Life Technologies) for 1 h.

ChIP-qPCR assay

Chromatin for ChIP was prepared from five million LNCaP cells. Briefly, cells were fixed in 1% formaldehyde for 10 min, nuclei were obtained and lyzed in 300 μl ice-cold RIPA buffer prior to chromatin-DNA shearing with a Diogene Bioruptor. ChIP was performed using 5 μg of antimenin antibody (A300-105A, Bethyl Laboratories Inc., USA), anti-H3K4me3 (C15410003, Diagenode, Belgium), and anti-ASH2L (D93F6, Cell Signaling). Dynabeads ® Protein G (10003D, Life Technologies, France) was used to retrieve Immunocomplexes. DNA was then extracted with phenol/chloroform/isoamyl alcohol, following proteinase-K reverse crosslinking. Two microliters of IP-DNA or two nanograms of INPUT DNA were used for real-time PCR analysis to quantify coprecipitated chromatin DNA. Relative enrichment of a given promoter region obtained with a specific antibody was compared with Input DNA. The blocks region on Chr1 was used as a negative control for menin, ASH2L, and H3K4me3 ChIPs.

Statistical analyses

Statistical analyses were performed as described in the figure legends; unpaired Student's t tests were used unless otherwise indicated. All analyses were done using Prism 5 software (GraphPad, USA); P value < 0.05 was considered significant. Results are given as means ± standard errors of the means (SEM).

Introduction

The most frequently encountered hormone-dependent cancers are breast and prostate cancers. The prevalence of breast cancer (BC) has increased such that its incidence is ranked second after lung cancer among cancers occurring in women [1], with 18.1 million new cases and 9.6 million cancer deaths in 2018. Similarly, prostate cancer (PCa), with its 174,650 new cases and 31,620 deaths estimated in 2019 in the USA alone [2], continues to represent a major cause of cancer-related mortality and morbidity in men. Hence, their global health burden is enormous, especially in developed countries, where their incidence is increasing [3]. Intriguingly, several lines of evidence have recently suggested that the tumor suppressor gene MEN1, the mutation of which predisposes patients to multiple endocrine neoplasia type 1 (MEN1, OMIM131100), may be involved in the development of these two cancers. In this review, we present the currently available data concerning the seemingly complex and multifaceted implications of MEN1 in these two types of hormone-dependent cancers. We believe that a better understanding of the role played by MEN1 should provide useful insights, not only into the mechanisms underlying the development of these two cancers, but also into their treatment, and may provide new markers for their diagnosis and prognosis.

Background about Breast and Prostate Cancers

Histopathology and Classification

2.1.1. Breast Cancer BC is histologically divided into two subtypes based on its invasive features-in situ carcinoma or invasive (infiltrating) carcinoma. BCs can also be divided into ductal or lobular types, depending on the tissue of origin, whether arising from the inner wall of the mammary ducts or the mammary glands, respectively [4]. More recently, a classification based on molecular markers such as estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) was purposed to facilitate the diagnosis and treatment of the four main subtypes. These include: (i) Luminal A, which represents approximately 40% of diagnosed BCs, is ERα-positive, PR-positive, or both; HER2-negative; Ki-67-low; and is associated with a slow proliferation and a significantly good prognosis, being sensitive to hormonotherapy. (ii) Luminal B is ERα-positive, PR-positive, or both; either HER2-positive or -negative and Ki-67-high; and has a worse prognosis than the luminal A subtype. (iii) The HER2-enriched subtype is ERα-and PR-negative, HER2-positive, and more aggressive than luminal subtypes. (iv) Triple-negative breast cancers (TNBCs) are ERα-negative, PR-negative, and HER2-negative [5], and are the most aggressive subtype, with the worst prognosis. Recent studies have further attempted to divide this classification into six subtypes by including basal-like and androgen receptor (LAR) subtypes, the latter displaying a high level of androgen receptor (AR) expression and an enrichment in AR signaling [6]. Treatments for BC including surgery, radiotherapy, chemotherapy, hormonotherapy, and rapidly developing targeted therapies, depend on the BC subtypes. ERα-positive BC subtypes are the most sensitive to hormonotherapy using either selective estrogen receptor modulators (SERMs) or selective estrogen receptor downregulators (SERD), whereas the treatment for HER2-enriched BCs has been greatly improved owing to therapies targeting the HER2 receptor. Unfortunately, there are very limited therapeutic options for TNBCs, although inhibitors of poly (ADP-ribose) polymerase (PARP) have shown promising results [7].

Prostate Cancer

PCa classifications mainly revolve around the Gleason grading system, based entirely on the histological pattern of carcinoma cells in Hematoxylin and Eosin (H&E)-stained prostate tissue sections [8,9] and the local disease state [10]. Aberrant signaling in the androgen pathway is critical in the development and progression of PCa. Androgen deprivation therapies (ADT) are the frontline treatment for PCa [11,12]. Although highly effective, ADT are characterized by the predictable emergence of resistance, termed castration-resistant PCas (CRPCs) [13,14], with a high mortality rate [15]. Genomic characterizations of CRPCs have led to the subdivision of CRPCs into two subtypes:

(1) AR-dependent CRPCs, containing alterations in the AR gene, such as amplification, point mutations, and generation of splice variants; and (2) AR-independent CRPCs, in which resistant cells or metastatic CRPC (mCRPC) lack AR expression or signaling. The latter subtype has recently been reported to be associated with cellular plasticity and neuroendocrine (NE) molecular features. Importantly, there are mCRPCs that neither express the AR nor markers of NE differentiation ("AR null-NE null") [16,17]-their incidence has risen over the past 2 decades from 5% in 1998-2011 to 23% in 2012-2016 [18]. Neuroendocrine prostate cancer (NEPC) displays a more complex spectrum of phenotypes, ranging from anaplastic carcinomas to pure small-cell carcinomas (SCCs) [18,19]. Several studies [20][21][22] have shown that 10-20% of lethal PCa display SCC features with a very poor prognosis [23,24].

Estrogen Receptor-Alpha and Androgen Receptor

Estrogen Receptor-Alpha (ERα)

Structure

ERα belongs to the steroid-stimulated nuclear receptors, which are transcriptional factors involved in regulating the transcription of hundreds of target genes [25]. The gene encoding ERα is called ESR1. This gene is highly conserved, localized on chromosome 6q25.1, and composed of 8 exons on a 140 kb genomic locus (Figure 1, upper panel) [26]. ERα consists of 595 amino acids, with two transactivation domains AF1 and AF2 located in the N-terminal domain (NTD) and domain E, respectively. The NTD is involved in both inter-molecular and intra-molecular interactions, as well as in the regulation of gene transcription, while the DNA-binding domain (DBD) allows ERα to dimerize and to bind to specific estrogen response element (ERE) sequences on DNA. The hinge domain (D region) containing the nuclear localization sequence (NLS) plays a role in receptor dimerization and in binding to chaperone heat shock proteins (Hsp). The ligand-binding domain (LBD, E/F region, C-terminal) comprises the E 2 -binding domain and works synergistically with the NTD in the regulation of gene transcription (Figure 1, upper panel) [27]. At least 2 isoforms of Erα have been identified: Erα-46, lacking the AF1 domain [28]; and ERα-36, devoid of both transcriptional activation domains (AF1 and AF2) and localized in both the plasma membrane and cytoplasm, where it mediates non-genomic ERα signaling [29,30]. ESR1 mutations, such as ESR1 amplifications or point mutations, were found in endocrine-therapy-resistant breast tumors, and occur predominantly in the LBD, leading to constitutive hormone-independent activation of ERα [31,32].

ESR1 Gene Regulation

Several studies have demonstrated that the ESR1 promoter is positively or negatively regulated by epigenetic factors. In 1994, Ottaviano et al. showed that the lack of ERα expression in ERα-negative BC cell lines was due to the hypermethylation of ESR1 CpG islands [33]. It was subsequently shown that the ESR1 promoter was occupied by several complexes with inhibitory components such as DNA methyltransferases (DNMTs) and histone modifiers such as HDAC1 and msin3A [34,35].

Many studies have also reported that the ESR1 promoter was occupied by several transcription factors, including members of the AP1 [36] and Forkhead box (FOX) family (FOXO3A [37], FOXM1 [38]), as well as metastasis-associated protein 1 (MTA1) and Twist [39]. The most extensively described ERα-associated transcription factors are GATA3 [40] and FOXA1 [41], which activate the transcription of the ESR1 gene and are necessary for its proper functioning [40,42,43]. Recently, a study also revealed a regulation of the ESR1 distal promoter by a loop-like complex involving GATA3, FOXA1, and menin [44]. In this study, they showed that menin binds to the ESR1 enhancer region at sites that are also bound by FOXA1 and GATA3, and recruits the mixed lineage leukemia (MLL) compass-like complex containing MLL1/2, menin, ASH2L, RBBP5, and WDR5 [45] to these sites, thus forming a complex and regulating its expression.

Gene Targets and Gene Functions

Estrogens, through the ERα signaling pathway, play important and various developmental, physiological, and pathological roles. ERα is essential for the normal development of the female reproductive tract, including the uterus and the ovaries, as well as the proliferation and differentiation of mammary glands [46]. Furthermore, ERα plays a role in male fertility and in other non-reproductive organs, such as the neuroendocrine and cardiovascular systems and bone metabolism [46,47]. Estrogens can bind to ERα in the cytoplasm and causes their release from bound chaperones, dimerization, and their nuclear translocation, where they bind to ERE and regulate transcription of downstream ERα genes, triggering the "genomic signaling pathway". ERα can also indirectly bind to promoters via protein-protein interactions, activating a variety of transcription factors, such as the activator protein (AP)-1 or the nuclear factor-κB (NF-κB) [48]. Finally, estrogens can bind to ERα in the plasma membrane, thus inducing the "non-genomic pathway".

Among the thousands of ERα target genes, one of the earliest identified was pS2/TFF1 [49,50], followed by many genes that were discovered by monitoring the global expression changes upon estradiol induction [51][52][53][54][55]. ERα target genes display a wide variety of functions, such that they can be divided into (i) pro-proliferative genes, such as Cyclin D1 [56], cMyc [57,58], and IGF-1 [59]; (ii) anti-apoptotic factors, such as TIT-5 and EIT-6 [55]; (iii) enzymes, such as the lysosomal proteinase cathepsin D [60]; (iv) and nuclear receptors, such as progesterone receptor [61], in addition to many other genes of as yet unknown function. Interestingly, these global expression experiments indicated that approximately half of ERα target genes are downregulated upon estrogen induction, reinforcing the view that estrogen promotes cell survival by downregulating pro-apoptotic genes.

Androgen Receptor (AR)

Structure

The AR gene is located on chromosome X (Xq11-12) and consists of 8 exons coding a protein of about 110 kDa (Figure 1, lower panel). The full-length AR has four domains, namely from the N-terminal, the NTD, the DBD, the hinge domain, and the LBD [62,63]. The NTD includes the transcriptional regulatory domain AF1, while the LBD includes AF2. Over 20 splice variants of the AR have been reported in the last 2 decades [64]. Most of them are lacking the C-terminal region containing the LBD [65,66] and are, therefore, functionally active independently of the presence of androgens. Among them, AR-V1 and AR-V7 are the most abundant variants [65]. Somatic AR mutations may occur selectively in response to androgen deprivation [67]. A review of 27 clinical studies revealed that AR mutations in androgen-dependent tumors ranged from 2 to 25%, while the incidence in CRPC tumors was slightly higher at 10-40% [67,68]. Furthermore, the AR LBD was described as a mutational hotspot, placing the incidence of its point mutations in CRPC at ~15-20% [69,70].

AR Gene Regulation

A better understanding of the regulation of AR transcription is crucial for studying prostate cell tumorigenesis. SP1, a zinc finger transcription factor, binds to GC-rich motifs of the AR promoter and activates the transcription of AR, whereas the associated antagonistic transcription factor pur-α can bind to the same region and inhibit AR transcription [71]. More recently, Deng et al. demonstrated that PRMT5 promotes prostate cancer cell growth by epigenetically activating the transcription of AR in prostate cancer cells. PRMT5 binds to the proximal promoter region of the AR gene and mainly contributes to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter upon its interaction with Sp1, forming a complex with Brg1, an ATP-dependent chromatin remodeler [START_REF] Deng | Protein Arginine Methyltransferase 5 Functions as an Epigenetic Activator of the Androgen Receptor to Promote Prostate Cancer Cell Growth[END_REF]. In addition, Grad and colleagues found that AR is regulated by AR itself in osteoblast-like U2OS cells. Indeed, two androgen response elements (AREs) were identified in exons 4 and 5 of the AR gene that were responsible for the androgen-mediated upregulation of AR mRNA [START_REF] Grad | The Androgen Receptor (AR) Amino-Terminus Imposes Androgen-Specific Regulation of AR Gene Expression via an Exonic Enhancer[END_REF].

Gene Targets and Functions AR, playing a key role in both normal prostate development and prostate cancer, is a hormonal transcription factor. Upon binding to androgens, testosterone, or dihydrotestosterone (DHT), the AR localizes to the nucleus [START_REF] Shafi | Androgen Receptors in Hormone-Dependent and Castration-Resistant Prostate Cancer[END_REF][START_REF] Davey | Androgen Receptor Structure, Function and Biology: From Bench to Bedside[END_REF]. There, the receptor dimers bind to AREs in the promoter regions of target genes, such as prostate-specific antigen (PSA) and transmembrane protease serine 2 (TMPRSS2), to regulate transcription [START_REF] Tan | Androgen Receptor: Structure, Role in Prostate Cancer and Drug Discovery[END_REF]. Similarly to other transcription factors, AR-enhanced transcription depends on the recruitment of RNA polymerase II to its target gene promoter. Some dynamic changes in the state of covalent histone modifications, relying on methyltransferase activities, are related to androgen-stimulated transcription. Fu et al. demonstrated direct interactions between p300, CBP, P/CAF, and AR. Moreover, several signaling pathways are known to enhance AR activity [START_REF] Fu | p300 and p300/cAMP-response element-binding protein-associated factor acetylate the androgen receptor at sites governing hormone-dependent transactivation[END_REF], including the EGF, IGF, IL6, Wnt, Ras-Raf-MAP kinase, PI3K/AKT, and MAPK/ERK pathways [START_REF] Davey | Androgen Receptor Structure, Function and Biology: From Bench to Bedside[END_REF][START_REF] Bakin | Attenuation of Ras Signaling Restores Androgen Sensitivity to Hormone-Refractory C4-2 Prostate Cancer Cells[END_REF][START_REF] Bakin | Constitutive Activation of the Ras/Mitogen-Activated Protein Kinase Signaling Pathway Promotes Androgen Hypersensitivity in LNCaP Prostate Cancer Cells[END_REF][START_REF] Gregory | Epidermal Growth Factor Increases Coactivation of the Androgen Receptor in Recurrent Prostate Cancer[END_REF][START_REF] Wu | Interaction of IGF Signaling and the Androgen Receptor in Prostate Cancer Progression[END_REF][START_REF] Schweizer | The Androgen Receptor Can Signal through Wnt/Beta-Catenin in Prostate Cancer Cells as an Adaptation Mechanism to Castration Levels of Androgens[END_REF][START_REF] Leung | Non-Genomic Actions of the Androgen Receptor in Prostate Cancer[END_REF]. Mounir et al. reported that PMRT5 display inhibitory effects on the transactivation of differentiated genes by AR via AR methylation [START_REF] Mounir | ERG Signaling in Prostate Cancer Is Driven through PRMT5-Dependent Methylation of the Androgen Receptor[END_REF].

For prostate cancer cells, studies have shown that AR is a critical regulator of the G1-S transition in AR-dependent cell cycle progression. Indeed, Xu et al. demonstrated that androgen induces Cyclin D expression via mTOR-dependent enhancement of translation [START_REF] Xu | Androgens induce prostate cancer cell proliferation through mammalian target of rapamycin activation and post-transcriptional increases in cyclin D proteins[END_REF]. The p21 cip has been validated as a direct AR target [START_REF] Lu | Androgen Regulation of the Cyclin-Dependent Kinase Inhibitor P21 Gene through an Androgen Response Element in the Proximal Promoter[END_REF], consistent with the findings revealing that p21 cip expression is enhanced in tumors and is correlated with a higher proliferative index and Gleason grade [START_REF] Aaltomaa | Prognostic Value and Expression of P21(Waf1/Cip1) Protein in Prostate Cancer[END_REF][START_REF] Baretton | Proliferation-and Apoptosis-Associated Factors in Advanced Prostatic Carcinomas before and after Androgen Deprivation Therapy: Prognostic Significance of P21/WAF1/CIP1 Expression[END_REF]. Furthermore, Knudsen et al. showed that androgen depletion induces p27 Kip1 , which likely contributes to the observed reduction in CDK2 activity [START_REF] Knudsen | Multiple G1 Regulatory Elements Control the Androgen-Dependent Proliferation of Prostatic Carcinoma Cells[END_REF].

Rokhlin et al. found that androgen and AR signaling could directly regulate p53 to suppress apoptosis. Mechanistically, androgen suppresses TNF-α/Fas-induced apoptosis through the inhibition of p53 expression and caspase-2 activation [90]. Interestingly, Frezza et al. reported that a significant decrease in AR expression leads to an increase in caspase-3 activity in LNCaP and PC-3AR cells, suggesting that AR might suppress caspase-3 expression [START_REF] Frezza | Modulation of the Tumor Cell Death Pathway by Androgen Receptor in Response to Cytotoxic Stimuli[END_REF]. Liao et al. also showed that knockdown of AR via siRNA leads to apoptotic death in PCa cells [START_REF] Liao | Small-Interfering RNA-Induced Androgen Receptor Silencing Leads to Apoptotic Cell Death in Prostate Cancer[END_REF]. Blockade of AR degradation and ectopic expression of Bcl-2 or selected caspase inhibitors can suppress this pro-apoptotic activity [START_REF] Godfrey | Proteasomal Degradation Unleashes the Pro-Death Activity of Androgen Receptor[END_REF].

Zhao et al. demonstrated that AR can act as a transcriptional repressor to directly inhibit gene expression. This repression is mediated by the binding of AR to AREs, and is facilitated by EZH2-mediated repressive chromatin remodeling [START_REF] Zhao | Cooperation between Polycomb and Androgen Receptor during Oncogenic Transformation[END_REF]. More recently and interestingly, Song et al. revealed that AR upregulated EZH2 expression by binding to the EZH2 promoter and stimulating its transcriptional activity in hepatocellular carcinoma (HCC) cells. EZH2 overexpression increased H3K27me3 levels, thereby silencing the expression of Wnt signal inhibitors, resulting in the activation of Wnt/β-Catenin signaling and subsequent induction of cell proliferation and tumorigenesis [START_REF] Song | Androgen Receptor Drives Hepatocellular Carcinogenesis by Activating Enhancer of Zeste Homolog 2-Mediated Wnt/β-Catenin Signaling[END_REF].

The Involvement of the MEN1 Gene in Breast and Prostate Cancers

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary syndrome characterized by the multiple occurrence of endocrine tumors of the parathyroid, pancreas, and anterior pituitary. The large tissue spectrum of the disease, affecting a dozen different endocrine cell lineages [START_REF] Thakker | Multiple Endocrine Neoplasia Type 1 (MEN1) and Type 4 (MEN4)[END_REF], indicates that the predisposition gene, MEN1, possesses a relevant role in all of the endocrine tissues affected. The MEN1 gene, the mutation of which predisposes patients to MEN1 syndrome, was first identified in 1997 [START_REF] Lemmens | Identification of the Multiple Endocrine Neoplasia Type 1 (MEN1) Gene. The European Consortium on MEN1[END_REF][START_REF] Chandrasekharappa | Positional Cloning of the Gene for Multiple Endocrine Neoplasia-Type 1[END_REF], and functional studies have since further improved our understanding of the gene. In particular, both genetic and biochemical experiments suggest that the MEN1 gene has a large spectrum of expression and that the menin protein encoded by the gene plays multifaceted biological functions in a broad range of different tissues and cells, likely through physical and functional interactions with its numerous protein partners. Menin primarily has a nuclear localization, although it can also be located in the cytoplasm and membranes [START_REF] Wautot | Expression Analysis of Endogenous Menin, the Product of the Multiple Endocrine Neoplasia Type 1 Gene, in Cell Lines and Human Tissues[END_REF]. Menin may act as an adaptor protein involved in the regulation of gene expression via its physical interaction with several transcription factors, such as JunD, Smad1/3/5, β-Catenin, MafA/B, Foxa2, and P53, as well as epigenetic factors, including KMT2A/2B, Sin3A, and EZH2 [44,[100][101][102][103][104][105][106][107]. The interactions between menin and several nuclear receptors were recently unveiled (see below). Importantly, various analyses demonstrated that menin is involved in different cellular activities controlled by many signaling transduction pathways, in particular cell proliferation, cell cycle, and cell death. Finally, the experiments using various in vivo models have also revealed that the biological functions of menin extend far beyond endocrine cells to hematopoiesis, adipogenesis, myogenesis, fibrogenesis, or even osteogenesis [108-110].

Molecular Studies

The first clues as to the possible involvement of menin in BC came from the observation that the menin protein binds physically to ERα. In 2006, Dreijerink et al. revealed that menin, owing to an evolutionarily conserved amino acid sequence LXXLL, could physically interact with several nuclear receptors, such as the vitamin D receptor, RXR, and ERα, and played the role of a cofactor. In the same study, they showed that menin binds to the AF2 domain of ERα and coactivates the transcription of TFF1, an estrogen-responsive ERα target gene, through the recruitment of the compass-like complex trimethylating H3K4me3 on the TFF1 promoter [111]. In 2009, Imachi et al. confirmed the previous results by showing that menin coactivates ERα in an estrogen-dependent manner in the ERα-positive MCF7 BC cell line [112]. A recent study conducted by Dreijerink et al. demonstrated that menin regulates the expression of the ESR1 gene (as described above) through an upstream enhancer via a looping mechanism that connects the TSS bound menin&MLL1/2 to the enhancer-bound transcription factors GATA3 and FOXA1 [44].

Almost a decade after discovering the interaction between menin and ERα, menin was identified as an important cofactor for AR signaling due to its physical interaction with AR-NTD and the recruitment of the MLL histone methyltransferase complex to AR target genes [113]. Inhibition of menin-MLL interaction with a small-molecule inhibitor (MI) impaired AR signaling and inhibited the growth of castration-resistant tumors in xenograft experiments in mice [113]. Hence, these results suggest that menin can facilitate oncogene activation through AR signaling in PCa (Figure 2).

Mouse Models

Mammary Gland Lesions in Mouse Men1 Models

Our team is at the forefront of studies on the role of MEN1 using Men1 mutant mouse models. We have observed that aged heterozygous Men1 mutant mice, in addition to endocrine tumors, developed mammary gland carcinomas in female and prostate cancers in male mutant mice at low frequencies [114]. To further confirm and understand the role of menin in the development of mammary lesions, we generated a conditional mammary-specific Men1 knock-out mouse model by crossing the mice carrying floxed Men1 alleles (Men1 F/F ) with WapCre transgenic mice expressing Cre recombinase under the control of the whey acidic protein (Wap) promoter, which is known to be expressed in luminal mammary epithelial cells. Our results demonstrated that female Men1 F/F -WapCre mice developed substantially higher amounts of early mammary intraepithelial neoplasia (MIN), which are precursor lesions, in comparison with control Men1 +/+ -WapCre mice. Interestingly, we found that ERα expression and the number of ERα-positive cells were clearly reduced in MIN lesions of mutant mice compared with normal mammary glands. In addition, cell membrane expression of β-Catenin and E-Cadherin was almost absent in the mammary lesions of Men1 F/F -WapCre mice compared with control mice; neither β-Catenin nor E-cadherin were detected in the TS1 cell line derived from a mouse Men1 BC [115].

Prostate Lesions in Mouse Men1 Models

By following a cohort of 47 male heterozygous Men1 mutant mice (Men1 +/-) and 23 male wild-type (Men1 +/+ ), age-matched littermate mice from 18 to 26 months of age, our group found that six Men1 +/-mice (6/47, 12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. No prostate carcinoma was found in age-matched Men1 +/+ littermates (0/23). In addition, these carcinomas exhibited loss of the non-target Men1 allele (LOH), therefore supporting a tumor suppressor role for the Men1 gene in prostate glands. Moreover, the AR and p27 expression decreased in tumor lesions, likely facilitating prostate cell tumorigenesis due to Men1 inactivation [116].

Taken together, all of the data obtained from mouse models suggest a tumor-suppressive role for menin during the initiation and development of murine breast and prostate cancers. 3.3.1. MEN1 in Human Breast Cancer Over the last two decades, several case reports have described breast cancer cases related to MEN1. In 2004, a 44-year-old Japanese woman was diagnosed with MEN1 syndrome, having hyperparathyroidism, primary aldosteronism, and also scirrhous breast carcinoma. The DNA taken from her parathyroid adenoma and breast cancer tissues showed germline MEN1 mutation at codon 451 in exon 10, which resulted in alanine-to-tyrosine substitution (A541T), as well as LOH [117]. Another study by Jeong et al. reported a case of a patient with both MEN1-associated tumors and breast cancer. They found a germline MEN1 mutation manifested as a 5-bp duplication in exon 3, named c.196_200dupAGCCC), which resulted in a frameshift mutation. In addition, the tested exon 10 showed a polymorphism at codon 423 with substitution of a cytidine to a thymidine (C423T), causing a change of amino acid [118]. More recently, a 41-year old patient with no familial history of breast cancer but with a mother with primary hyperparathyroidism (PHP) was found carrying a variant p.C421R/p.426R in the MEN1 gene. The patient's histopathological study revealed hormone receptor negativity, as well as HER-2 and p53 negativity. A family study showed positive findings for MEN1 in a sister, two maternal nephews, and one of the patient's daughters, with no record of breast cancer development in any of these people [119] Evidence of the likely involvement of menin in BC arose from the observation that female MEN1 patients were at a higher risk of developing BC [120]. In this study, Dreijerink et al. referred to the Dutch longitudinal MEN1 database to assess the incidence of BC in MEN1 patients, and found that out of 190 female patients, the relative risk of invasive BC was 2.83 (p < 0.001) and the mean (±SD) age at diagnosis of essentially luminal-type BC was 48 ± 8.8 years, compared with an age range of 60 to 65 years in the general population. This feature is often observed in the patients harboring a genetic predisposition. The authors validated their results using 3 other independent MEN1 patient cohorts from the United States (p = 0.11), Tasmania (p = 0.22), and France (p = 0.03), which provided similar values for relative risk as those obtained in the Dutch cohort, with an average age at diagnosis of 51 years. Furthermore, 8 out of 10 BC samples obtained from Dutch MEN1 patients displayed more than 50% reduction of menin expression in the nucleus, and subsequent analysis showed loss of heterozygosity at the MEN1 locus in 3 of 9 tumors. Overall, these observations strongly suggest that MEN1 mutations could be involved in human breast tumorigenesis as a tumor suppressor.

Human Studies

Concomitantly to our work carried out in mice, we also observed that a substantial proportion of human sporadic BCs displayed reduced menin expression, as observed through the analyses of two series of human BCs [115]. More recently, a study in which the whole-genome sequences of 560 BCs were analyzed highlighted sporadic MEN1 mutations, albeit at low frequency, as being among driver mutations (such as BRCA1, TP53, PIK3CA, MYC, CCND1, PTEN) in BC [121]. In addition, several other case reports identified MEN1 mutations among sporadic BC patients, independent or not of germline mutations in BRCA1 and BRCA2 genes that are usually associated with hereditary BC [118,[122][123][124].

However, in a clinical study conducted by Imachi et al. with 65 ERα-positive BC samples treated with tamoxifen for 2-5 years as adjuvant therapies, they observed that menin-positive tumors (20 patients) had a worse clinical outcome and were more resistant to tamoxifen than menin-negative tumors (46 patients) [112]. They, therefore, proposed that menin could be a predictive factor of resistance to tamoxifen. Furthermore, they found that raloxifene could inhibit the binding of menin to the AF2 domain of ERα and proposed raloxifene as the therapeutic options for menin-positive and ERα-positive BC [125]. Their works suggest an oncogenic role for menin, which raised the controversy as to its precise role in BC. In total, the current data obtained from human studies suggest that the MEN1 gene could play a complex even opposite role in the development of human breast and prostate cancers.

Further Clues for the Role of Menin in Breast and Prostate Cancers

As we mentioned above, many different factors and signaling pathways are involved in mammary and prostate cell tumorigenesis. By investigating the possible molecular links between the former and menin, we speculated that we might gain further insight into the possible role played by menin in these cancers (Figure 3).

Epigenetic Factors

Interestingly, several epigenetic factors reported to be involved in mammary cell tumorigenesis are known to be partners of menin. Histone methylase MLL1 (KMT2A) and MLL4 (KMT2B), which are the most characterized partners of menin, were shown to act synergistically with ERs (ERα and ERβ) to mediate the estrogen-induced transcriptional activation of the HOXB9 gene, which is critical for mammary gland development and BC [134]. Menin was also shown to upregulate several members of the same family (mainly HOXA9 gene) in leukemia by associating with the compass-like complex and lens-epithelium-derived growth factor (LEDGF) [45,135]. The HDAC family, which contains known partners of menin [54,55], is implicated in the regulation of ERα expression, mainly by silencing the ESR1 gene. It was proposed that HDAC may be responsible for loss of ERα expression in ER-negative BC [34,35]. EZH2 and PRMT5 are two shared partners of menin [100,136,137] and the ERα pathway. Indeed, EZH2 inhibits the transcription of estrogen-responsive genes through its association with the transcriptional corepressor repressor of estrogen receptor activity (REA) [138]. Although there is no direct evidence of the interaction between PRMT5 and ERα, PRMT5 plays an important role in BC by methylating programmed cell death 4 (PDCD4), a tumor suppressive protein with anti-proliferative functions on arginine residue 110 [139].

Transcription Factors

JunD, a member of the AP-1 family that interacts physically with menin [140,141], has a higher level of expression in BCs [142]. JunD and menin co-expression was found in the mouse submandibular gland, an AR-responsive tissue, with their expression pattern and localization changing with cell differentiation status [143]. Moreover, JunD physically binds to ERα and facilitates its binding to target genes [36]. Intriguingly, it has been shown that prostaglandin E 2 (PGE 2 ) induces JunD and JunB expression, resulting in the activation of the aromatase promoters I.3/II, while JunD and c-Jun mediate the suppression of the aromatase promoter I.4, leading to high levels of local estrogen, and thus to BC progression [144]. JunD is crucial for cell proliferation in PCa cells, as it controls cell cycle regulatory genes [145,146]. Their analyses further suggest that the essential role played by JunD in prostate cancer cell proliferation is mediated by MYC signaling [147]. Furthermore, Mehraein-Ghomi et al. highlighted JunD as an AR co-activator, as it triggers the oxidative stress pathway in prostate cancer cells by regulating the SSAT promoter, which produces large amounts of metabolic reactive oxygen species (ROS) [148].

Another important factor is cMyc, a well-known estrogen-regulated oncogene [149,150], which is overexpressed in approximately [20][21][22][23][24][25][26][27][28][29][30] and has also been shown to interact with ERα to modulate estrogen-mediated signaling [152]. The cMyc overexpression in PCa has been a well-recognized phenomenon since 1986, when Fleming et al. showed a significantly higher level of its expression in adenocarcinoma of the prostate than in benign prostate hyperplasia by Northern blotting [153]. Furthermore, Sato et al. reported that cMyc amplification is strongly associated with higher histopathological grades and Gleason scores, as well as with earlier disease progression and cancer-associated death [154]. It is now known that cMyc is a partner of menin and that they collaborate to either activate or repress the expression of certain genes. The most recent report shows that menin can directly interact with the transactivation domain (TAD) of cMyc, and that they in turn bind to E boxes to enhance the transcription of cMyc target genes [155]. Interestingly, menin can interact with the cMyc promoter to regulate its transcription in HEK293 cells [156].

Finally, menin was recently shown to interact with GATA3 and FOXA1 [44] in BC to regulate the ESR1 promoter (see details above), both of which are markers of luminal BC, especially for the luminal A subtype [42,43,146], and which are highly associated with ERα and are required for the proper function of most of its target genes [40,157,158]. Menin interacts with GATA3 to activate Th2 cell maturation in primary human peripheral blood T cells [159] and to physically interact with a member of the FOXA family, namely FOXA2 [103]. It is worth mentioning that FOXA1 plays a crucial role in the AR signaling, and possibly in CRPC occurrence [160].

Signal Transduction Pathways

Menin is known to interfere with different signaling pathways that play important roles in breast and prostate cancers. This mutation is present in approximately 35% of HR-positive BCs, 20-25% of HER2-overexpressing BCs, and with a lower frequency (8.3%) in TNBCs [163].

PTEN is one of the most commonly deleted and mutated genes in human breast and prostate cancers. Loss of PTEN in BC is negatively correlated with ERα and PR status, and is associated with the basal-like phenotype [164,165], with more aggressive behaviors (tumor size, lymph node metastasis, etc.), and with worse outcome (disease-free survival DFS and overall survival OS) [166]. Accumulating evidence has highlighted an association between loss of PTEN and the development of CRPC, likely due to AR phosphorylation [167,168]. Moreover, loss of PTEN and AR expression has been clinically correlated with increased mortality in CRPC patients [169]. More recently, Wong and colleagues generated mouse models with insulin-specific biallelic inactivation of Men1 and Pten in β-cells, and showed that concomitant loss of Pten and Men1 accelerated islet cell tumorigenesis. Co-mutations of MEN1 and PTEN were observed in a small percentage of human PanNETs [170,171], suggesting that menin and Pten may function synergistically to suppress tumorigenesis.

Several studies have focused on the relationship between the PAM (PI3K/Akt/mTOR) and resistance to endocrine therapy in pre-clinical BC models [172], in which the authors showed that Akt can activate the ERα pathway independently of estrogen availability and that the combination of mTOR inhibitors and endocrine therapy can overcome this resistance [173,174]. In addition, the PAM pathway has also been implicated in trastuzumab resistance in HER2-overexpressing BCs [175]. Interestingly, menin interacts with AKT1, downregulates its kinase activity and suppresses both AKT1 induced proliferation and anti-apoptosis in endocrine and non-endocrine cells, mainly by reducing the translocation of AKT1 from the cytoplasm to the plasma membrane during growth factor stimulation [176]. Another study showed that menin can interact with FOXO1, a downstream effector of Akt, in the hepatocytic cancer cell line HepG2 and in MEFs [177]. In the same year, a study also showed that MEN1 and genes from the mTOR pathway are frequently altered in pancreatic neuroendocrine tumors [170]. A recent study revealed that menin regulates milk protein synthesis through mTOR signaling in normal mammary epithelial cells [178]. According to the authors, menin overexpression caused significant suppression of factors involved in the mTOR pathway, as well as milk protein κ-casein (CSNK). All of the abovementioned data suggest that menin may regulate the PI3K/Akt/mTOR pathway in mammary cells. showed that menin represses cell proliferation in rat intestinal epithelial cells [180] by inhibiting the expression of Cyclin D1, Cyclin D3, and CDK4. Based on a transcriptomic study of differentially expression genes, our team demonstrated that Men1 ablation in mouse islet cells greatly affected the expression of factors involved in cell cycle and cell growth control, such as Cyclin A2, B2, and D2 for the former; and IGF2, IGFBP3, and 6 for the latter [181]. Menin can also repress cell proliferation by interacting and inhibiting ASK (S-phase kinase) [182]. In addition, menin was reported to upregulate the expression of Cyclin-dependent kinase inhibitors p18 ink4c and p27 kip1 with the help of the MLL compass-like complex, which adds H3K4 trimethylation marks on their promoters, thus activating gene expression [183][184][185]. Interestingly, p18 has recently been shown to be a downstream target of GATA3 in luminal BC and to suppress luminal progenitor cell proliferation and tumorigenesis [186]. P27 is ranked as one of the 18 most significantly mutated genes in luminal A BC, and loss of p27 was associated with poor outcome in BC patients [187].

Schnepp et al. revealed that the infection of cells using menin-expressing adenoviruses could trigger apoptosis in MEFs [188,189] by activating an apoptotic pathway that depends on Bax [186]. They also highlighted that Men1 disruption in vivo increased resistance to TNFα-induced apoptosis, further supporting a vital role for menin in regulating apoptosis.

Wnt Signaling

It is well known that the Wnt pathway plays a crucial role in the development of breast and prostate cancers, in particular at late stages [190]. We observed that in Men1-deficient mice insulinomas, β-Catenin expression switched from a membrane expression to a cytoplasmic or even nuclear expression [187]. Along with our collaborators, we also showed that menin physically interacts with β-Catenin, and menin overexpression reduced the nuclear accumulation of β-Catenin and suppressed its transcriptional activity in Men1-null MEFs

Finishing Words

The Dual Role of Menin

The abovementioned data provide clues on the complex and sometimes paradoxical role of the MEN1 gene in mammary and prostate cell tumorigenesis (Figures 2 and3). Dreijerink et al. proposed a hypothesis on the dual role of menin in BC, which may shed light on these discrepancies and the surrounding confusion [44]. They proposed that menin could act as a tumor suppressor in normal luminal mammary epithelial cells and as an oncogene in sporadic ER-positive BCs, the key point being its essential role in the regulation of the ESR1 gene mediated by the MLL-menin complex via H3K4me3 sites. Therefore, when MEN1 is mutated or inactivated in normal mammary and prostate cells, it could result in dysregulated ERα and AR pathways, leading to aberrant cell proliferation and differentiation, and to tumor development with the participation of other oncogenic alterations. Conversely, in ER-positive BC and AR-positive prostate cancer cells, menin could act as a co-activator of these two nuclear receptors, playing a crucial role in promoting cell proliferation by the latter.

Remaining Questions

The currently available data and the abovementioned molecular clues suggest that menin may play a multifaceted but non-negligible role in the tumorigenesis of both mammary and prostate cells. However, concerning the detailed mechanisms underlying its involvement, many questions remain. Among them, one may wonder about the molecular pathophysiological consequences of MEN1 inactivation in these two tissues during the initiation of tumorigenesis. In addition, since menin interacts and regulates the ERα and AR pathways, does menin play different roles in HR-positive than in HR-negative cancers? Last but not least, as menin acts as a scaffold protein, what are the other factors, in particular interacting partners, involved in the process?

To further understand the involvement of menin in these two cancers, there is an urgent need to generate adequate cell, tissue, and animal models in order to better investigate the distinct roles played by menin during the initiation of carcinogenesis on the one hand, and during cancer progression on the other hand. Concurrently, strengthening MEN1 mutation detection and menin expression analysis for breast and prostate cancer samples collected from young and aged patients or in different subtypes would be informative. The availability of more relevant models and crucial data from clinical samples, together with the rapidly improved tools in molecular study, should be of great help in obtaining rightful answers for the abovementioned questions.

Summary

Even though the role of menin in the development of neuroendocrine cancers is well known, its role in human breast and prostate cancers is slowly emerging. Based on the literature presented above, we speculate that future research could unveil further crosstalk between menin and the ERα and AR pathways. Finally, a better understanding of the mechanisms underlying its role in the mammary and prostate cell tumorigenesis could also make menin a potential therapeutic target for the treatment of these cancers, as well as a new marker for their diagnosis and prognosis.

Introduction

The multiple endocrine neoplasia type 1 (MEN1) gene, mutations of which are observed in patients with MEN1 syndrome, [1] was identified in 1997, although its precise biological function initially remained unknown. [2,3] This led to debates among scientists around the world, including about the normal biological functions of the gene in different cells and tissues, the cellular and molecular consequences of its inactivation, and whether the MEN1 gene also plays a role in other tissues and cells? Needless to say, addressing these issues was deemed highly relevant for improving the general understanding of diseases related to its inactivation and for seeking adequate therapeutic strategies for treating MEN1.

Since then, concomitantly to MEN1 studies using diverse approaches and models, different laboratories around the world, including our own, have adopted gene-targeting approaches in murine models to study the MEN1 gene, and have been engaged in the generation and characterization of Men1 mutant mouse models. This approach has coincided with the broad-scale and worldwide development of experimental mouse models for a variety of contexts, as evidenced by the fact that every single gene within the entire mouse genome has now been targeted. Of the mutant mouse strains available, currently exceeding 20,000, no less than 4500 models recapitulate human diseases. Extensive investigations based on these mouse models, especially using continually developing systems biology approaches, is rapidly changing our knowledge on and our strategies to deal with human diseases. Indeed, with the establishment of various Men1 mutant mouse models, an increasing number of laboratories are involved in the use of these models to dissect MEN1 functions. Here, we exclusively present data on the generation and characterization of Men1 mutant mouse models in connection with the MEN1 syndrome.

Men1 mutant mouse models

Current technological advances in gene-targeting approaches enable scientists to perform either germ-line (conventional mutant mice) or cell type-specific (conditional mutant mice) disruptions of a given gene.

Conventional Men1 mutant models

This type of Men1 mutant mouse model mimics genetic events occurring in MEN1 patients, hence facilitating studies on the natural history of the disease.

The first conventional Men1 mutant mouse model was reported in 2001. [4] Authors demonstrated that homozygous Men1 mutant embryos died mid-gestation, whereas heterozygous Men1 mutant mice developed multiple endocrine tumors from the age of 9 months onwards, affecting islet cells, parathyroid, pituitary, and adrenal glands, all associated with hyperinsulinemia. Interestingly, thyroid tumors, considered to be a coincidental finding in MEN1 patients were detected in mutant mice, with loss of menin expression, suggesting that the MEN1 disease may encompass thyroid tumors.

In 2003, a second conventional Men1 mutant model was established following our collaboration with the laboratory of Dr Zhao-qi Wang. [5] Examination of over 150 heterozygous Men1 mutant mice, divided into 3 age groups, 8 to 12 months, 13 to 18 months, and >18 months, for the emergence of tumors, revealed that around 1 year after birth, some heterozygous Men1 mice developed multiple endocrine tumors observed in the MEN1 pathology, concomitantly to hyperinsulinemia and increased levels of serum parathyroid hormone. Importantly, we were able to document the presence of gastrinomas, the most common functional pancreatic neuroendocrine tumor (PanNET) in MEN1 patients, and glucagonomas. Intriguingly, Men1 mutant mice also developed gonadal tumors with complete menin loss in both male and female mice, which are not reported in MEN1 patients, except for 1 Leydig tumor case. [6] The significance of this observation with regards to the human MEN1 pathology remains to be determined.

Two additional conventional Men1 mutant models were later established by the teams of Dr N. Steward and Prof Raj Takker. [7,8] The observations derived from these 2 models largely confirmed data reported by the previous models, with one of the models also reporting the occurrence of adrenocorticotropinomas with hypercorticosteronemia. [8] The analyses carried out on homozygous Men1 mutant embryos revealed that complete Men1 ablation led to neural tube disclosure and abnormal development of fetal liver and heart. [9] Cranial and facial developmental defaults were also observed. [4] These findings thus indicate the essential role played by the gene in controlling cell proliferation and differentiation in these tissues during the embryonic midgestation stage. Of note, comparison of conventional Men1 mutant models set-up in different genetic backgrounds highlighted significant differences in terms of timing and severity of developmental phenotypes. [10] Conditional Men1 mutant models Cell type-or tissue-specific Men1 mutant mouse models were generated by crossing mice carrying the floxed Men1 allele, Men1 F/F , with different Cre recombinant mice for given cell types or tissues. These models played a vital role in dissecting cellular and molecular mechanisms involved in the development of the MEN1 disease.

b-Cell Men1 mutant mice. Two conditional Men1 knockout mice specifically targeting pancreatic b cells were reported in 2003, using different RipCre mice to generate Men1 F/F -RipCre + mutant mice. [4,11] A similar model was described in 2004. [12] Histological analyses revealed that, as early as 2 months of age, hyperplastic islets composed of Men1-deficient b cells appeared in some of the Men1 F/F -RipCre + mice. At 4 months, the number of conditional Men1 mice with hyperplastic and/or dysplastic islets increased significantly, and at 6 months, insulinoma was detected in a substantial proportion of these mice. At 10 months, all Men1 F/F -RipCre + mice developed multiple insulinomas, accompanied with hypoglycemia and hyperinsulinemia. In addition, we and others observed that mouse Men1 insulinomas displayed certain characteristics of tumor progression, including dedifferentiation, loss of cell-cell adhesion and angiogenesis. [11,12] a-Cell Men1 mutant mice. The incidence of glucagonoma is low in MEN1 patients, whereas a-cell pre-tumorous lesions are rather common. [13] To better understand the role played by a cells in MEN1 PanNET development, we carried out a project aiming at specifically disrupting the gene in a cells using GluCre + mice. [14] Characterization of Men1 F/F -GluCre + mice enabled us to fully document the appearance of a-cell proliferation, a-cell pretumorous lesions, the lesions expressing both glucagon and insulin, and the development of both glucagonomas and insulinomas as early as 7 months of age. The data provided evidence, for the first time, of the involvement of cell transdifferentiation in the histogenesis of PanNET. The second a-cellspecific Men1 mutant mouse model was reported shortly after. [15] Intriguingly, only insulinomas, but no glucagonomas, were observed in the model, presumably due to a cross communication between these 2 islet cell populations according to the authors.

Pan-pancreatic Men1 mutant mice. Shen et al [16] generated a Men1 mouse mutant model targeting pancreatic progenitors using Pdx1Cre mice. Indeed, they observed insulinomas in aged mutant mice, whereas no neoplastic lesions were detected in exocrine tissues, albeit Men1 disruption was documented in the latter. Neither glucagonoma, nor pancreatic gastrinoma were observed, and data concerning menin inactivation in non-b-cell linages was not provided. More recently, we generated a novel Men1 pan-pancreatic progenitor mutant mouse model using pTF1Cre mice, which allowed us to follow Men1 pancreatic gastrinoma development. [17] In these mutant mice, we did observe the occurrence of neoplastic lesions not only from b cells, but also a-, g-, and pancreatic polypeptide cells (unpublished data).

Gastrointestinal epithelium cell Men1 mutant mice. Veniaminova et al [18] initially reported that Men1 deletion in gastrointestinal epithelium cells using Villin-Cre resulted in antral G cell hyperplasia and a hyperproliferative epithelium with hypergastrinemia, but no gastrinomas. Later, the same mutant mice were placed on a somatostatin null genetic background, leading to the development of gastric carcinoids (GCs) the occurrence of which could be accelerated by suppressing acid secretion. [19] The authors reported that the GCs developing in this mouse model were reminiscent of human gastrointestinal neuroendocrine tumors, and accompanied by altered p27kip1 subcellular localization and stability.

Parathyroid Men1 mutant mice. A mouse model in which the Men1 gene was specifically disrupted in parathyroid glands was generated by crossing the mice carrying Men1 F/F allele with PthCre mice. [20] Mutant mice displayed parathyroid lesions with neoplastic changes and systemic hypercalcemia.

Mammary gland Men1 mutant mice. Some of the female mutant Men1 heterozygous mice developed breast cancers, with a small but substantial frequency. [5] In an attempt to determine the role played by the gene in controlling mammary cell proliferation, we performed Men1 disruption specifically in murine mammary glands by generating Men1 F/F -WapCre + mutant mice. [21] This led to mammary intraepithelial neoplasia with a high incidence (>50%) in mutant mice aged 12 months. We concomitantly performed analyses of menin expression in sporadic breast cancers and found that, among 124 analyzable cancers, 95 (78%) showed a decrease in menin expression compared with normal controls. The data were corroborated by a recent report revealing the significant predisposition of female MEN1 patients to breast cancer development. [22] Prolactin-secreting-cell Men1 mutant mice. Due to the leakage of RipCre [12] and WapCre [21] in the pituitary, highly hemorrhagic pituitary adenomas developed in the Men1 mutant mice generated using these 2 Cre mice, at the age of 12 months. The tumor cells were shown to be prolactin-secreting cells, with a 2-fold increase in the level of serum prolactin in mutant mice compared to control mice, indicating prolactinomas. Subsequent analyses demonstrated that Men1 ablation in prolactin-secreting cells triggered prolactinoma development in mice, leading to the premature death of these mice.

Craniofacial osteogenic cell and osteoblast Men1 mutant mice. Recently, 2 laboratories published their studies on the function of Men1 in bone. Kanazawa et al carried out Men1 ablation in mature osteoblasts using osteocalcin-Cre mice. They found that the mutant mice displayed a significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness, whereas no tumor developed. Interestingly, in another study, [23] the Men1 gene was disrupted in craniofacial lineages of osteogenic cells using Runx2Cre mice. [24] The mutant mice developed multiple ossifying fibromas (OF) in the mandible with 100% penetrance, generating the first genetic mouse model of OF. [25] The study provided not only interesting clues for better understanding the occurrence of gingival papules in MEN1 patients, but also for dissecting molecular mechanisms of OF development, such as dysregulated Cdkn1a expression due to Men1 inactivation.

Inducible Men1 mutant models

Schnepp et al analyzed the effects of Men1 ablation in an inducible Men1 mutant mouse model by crossing mice carrying floxed Men1 alleles and mice with a tamoxifen-inducible Cre transgene. They observed a slight increase in islet volume and a higher proliferation of islet cells shortly after the induction of Cre activity (2 weeks to 1 month). [26] Interestingly, by either using the same mouse model or an inducible adult b-cell-specific PdxCreER line, the same laboratory reported that acute and temporally controlled Men1 disruption improved pre-existing hyperglycemia in both streptozotocin-treated mice or mice fed with high fat diet, and glucose intolerance in genetic db/db diabetic mice. [27] Based on a similar approach, using a mouse line with tamoxifen-inducible Cre-ER driven by the rat insulin promoter, Line et al examined histological changes in Men1 F/F -RIP2Cre ER mice 2 to 5.5 months after the induction of Cre activity by tamoxifen. They revealed that all of the tamoxifentreated mice developed PanNETs originating from b cells. [28] Defining the cell of origin of PanNETs using Men1 mutant mouse models

The cells at the origin of the development of PanNETs have been the object of a long-standing debate. Previously, multihormonality was observed in at least 35% to 50% of islet tumors. [29] Several pioneering studies provided interesting finely documented observations. One study proposed that the histogenesis of these tumors arose from the differentiation of ductal pancreatic progenitor cells. [30] Another study surprisingly demonstrated that early pancreatic lesions observed in MEN1 patients mainly affected a cells, whereas almost all of the advanced islet tumors observed in these patients were insulin-secreting or nonfunctional types. [13] The histogenesis of these tumors remains to be elucidated, since it is crucial for the improvement of patient management and the treatment of the disease.

In order to determine whether a cells could be at the origin of PanNETs in MEN1, we carried out Men1 disruption specifically in a cells (see above). The data obtained from our analyses indicate that a-cell-specific Men1 disruption first leads to proliferation of these cells and to the development of glucagonomas. Simultaneously, Men1-deficient a cells transdifferentiate into insulin-secreting cells, leading to the occurrence of insulinoma. The tumor phenotype observed in mutant mice reflects one of the most important features of islet tumors described in humans, multihormonality. Data obtained from this model revealed, for the first time, the involvement of transdifferentiation of a cells in the PanNET development and provide us with crucial clues to better understand the histogenesis of these tumors. In addition, they will make it possible to better understand the biology of a cells and the control of their cell proliferation and differentiation for other diseases affecting islet cells. [14] The cells of origin of pancreatic gastrinomas, a rather common PanNET in MEN1 patients, remain an enigma, since no gastrinexpressing cells are found in the normal adult pancreas. We addressed the issue by further characterizing previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. In effect, we provided evidence that pancreatic gastrin-expressing cells, found from embryonic day 12.5 until postnatal day 7, are derived from pancreatic Ptf1a-and neurogenin 3-expressing progenitors, the majority of which coexpress glucagon, and the remaining coexpress insulin. Based on these observations and fine analyses of both Men1 progenitor mutant mice, and Men1 aor b-cell mutant mice, we demonstrated that pancreatic gastrinomas related to Men1 inactivation originated from pancreatic endocrine cells themselves. [17] It is worth mentioning that the hypothesis concerning the ductal origin of PanNETs has so far not been supported or confirmed.

Summary

Huge efforts have been made to generate and characterize Men1 mutant mouse models by different laboratories. It is worth noting that conventional Men1 mutant models have succeeded in genuinely recapitulating clinical features of MEN1 syndrome, whereas conditional Men1 mutant mouse models have enabled scientists to carry out mechanistic studies to unravel cellular and molecular mechanisms underlying the development of the disease, from deciphering the cells of origin of PanNETs to the identification of different factors and/or cell signaling pathways involved in tumorigenesis upon menin inactivation. Interestingly, many of the studies using Men1 mutant mouse models concomitantly revealed the importance of dysregulated cell differentiation and cell proliferation, as well as the factors involved in the corresponding alterations, although, unfortunately, they are beyond the scope of this review. These advances have improved our understanding of the spectrum of tumors and their evolution in MEN1 patients.

The knowledge acquired may also be valuable for designing treatments by finding potential MEN1 targets, as demonstrated by Jiang et al. [31] We believe that a wider use of Men1 mutant mice for drug discovery is thus imminent.

Fig. 1 . 1

 11 Fig. 1.1 The anatomical relations of the prostate gland. Obtiained from:

Fig. 1 . 2

 12 Fig. 1.2 Zonal anatomy of the prostate (Patek, 2018).

Fig. 1 . 3

 13 Fig. 1.3 Schematic depiction of the cell types within a human prostatic duct.

Fig. 1 . 4

 14 Fig. 1.4 Histopathology of normal prostate tissue and BPH. (A) Normal prostate tissue and (B) BPH characterised by epithelial and stromal. Obtained from Association AU. Pathology for Urologists:

Fig. 1 . 5

 15 Fig. 1.5 Histopathology of high-grade prostatic intraepithelial neoplasia. (A) Haematoxylin and eosin (H&E) stained section of HGPIN with luminal epithelial cell proliferation and prominent nuclei and nucleoli (B) Prostate biopsy section stained for high molecular weight keratin, a basal cell marker, showing HGPIN with disruption of the basal cell layer. Obtained from[START_REF] Bostwick | High-grade prostatic intraepithelial neoplasia[END_REF]

Fig. 1 . 6

 16 Fig. 1.6 Gleason patterns and the Group Grade system. Adapt from: Chen and Zhou Q, 2016.

a

  protein about 110 kDa. AR has four regions: from the N-terminal, an NH2 terminal transactivation domain (NTD) encoded by exon 1, a DNA-binding domain (DBD) encoded by exons 2-3, a hinge region encoded by exon 4, and a ligand binding domain (LBD) encoded by exons 5-8 (Fig. 1.7).

Fig. 1 . 7

 17 Fig.1.7 Structure of androgen receptor (AR). a. Full-length AR is composed of 8 exons. b. NTD: N-terminal domain, DBD: DNA-binding domain, HR: hinge region, LBD: ligand-binding domain, AF-1: activation function-1, AF-2: activation function-2, CE: cryptic exon. Adapt from: Fujita and Nonomura, 2019.

Fig. 1 . 8

 18 Fig. 1.8 The androgen-AR signaling pathway (Ligand-dependent). Testosterone diffuses into the cells and gets converted into dihydrotestosterone (DHT) via the action 5-α-reductase (5-a-R).

Fig. 1 . 9

 19 Fig. 1.9 Common splice variants of the AR. Adapt from: Lallous et al., 2013.

Fig. 1 .

 1 Fig.1.10 Schematic illustration of the mechanisms of action of drugs used in castrationresistant prostate cancer. Abiraterone is a CYP17 inhibitor that blocks androgen synthesis not only in the testis but also in the adrenal gland and in tumor tissue. Bicalutamide and enzalutamide are AR antagonists that bind the AR ligand site, thus preventing ligands to bind to the AR. Cabazitaxel and docetaxel are taxanes that stabilizes microtubule which results in blocking of cell division, thereby inducing cell death, taxanes also inhibits nuclear translocation of the AR. Alpharadin (Radium 233 radioisotope) targets new bone growth in and around bone metastases and induces double-strand DNA breaks through alpha radiation over a short distance, thereby inducing cell death. Adapted from Emma Jernberg, 2013.

  shRNA knockdown, they confirmed MYC regulation of expression and activity of AR-FL and AR-Vs in cell lines (LNCaP, 22Rv1 and VCaP) and a patient-derived xenograft model. Mechanistically, MYC promotes AR transcription and enhances the stability of AR-FL and AR-Vs proteins without altering AR RNA splicing. In addition, analysis of gene-expression data from 159 metastatic CRPC samples and 2142

Fig. 1 .

 1 Fig. 1.11 Schematic model of regulation of PCAT1 at SNP rs7463708. The prostate cancer risk-associated SNP rs7463708 is associated with increased expression of PCAT1 expression via modulating ONECUT2 and AR binding at adistal enhancer. PCAT1 recruits AR and LSD1 to the GNMT and DHCR24 enhancers to activate transcription of these genes.Abbreviations: PCAT1, prostate cancer-associated transcript 1; SNP, singlenucleotide polymorphism; ONECUT2, one cut homeobox 2; AR, androgen receptor; LSD1, lysine-specific demethylase 1; GNMT, glycine N-methyl-transferase; DHCR24, 24-dehydrocholesterolreductase. Adapt from:Guo et al., 2016.

  Wnt signaling is the stabilization and nuclear localization of β-catenin. Phosphorylation of β-catenin normally targets β-catenin for degradation. Its inhibition results in cytoplasmic β-catenin stabilization and accumulation which facilitates its translocation into the nucleus. Nuclear β-catenin acts as a transcriptional coactivator and interacts with transcriptions factors such as T-cell factor (TCF) and lymphoid enhancer factor (LEF) and leads to increased transcription of target genes, such as MMP7, MYC, c-Jun, Fra and other members of the c-Fos family. In addition, Wnt signaling can also regulate other noncononical pathways such as the JNK pathway independent of β-catenin leading to changes in cell polarity, movement and survival

Fig. 1 .

 1 Fig. 1.13 The PI3K/AKT/mTOR pathway and cross talk with the AR signaling and RAS/RAF/MEK pathways. AR, androgen receptor; ARE, androgen-responsive element; DHT, dihydrotestosterone; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; ERK, extracellular signal-related kinase; FKBP5, FK506-binding protein 5; MEK, mitogen-activated protein/ERK kinase; PHLPP, PH and leucine-rich repeat phosphatase; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; RAS, rat sarcoma oncogene; Rheb, RAS homolog enriched in the brain; S6K, S6 kinase; TSC, tuberous sclerosis protein; TORC, target of rapamycin complex; +/-, Akt activity can both enhance and suppress AR signaling. Possible mechanisms of AKT-mediated regulation of AR activity include direct phosphorylation of AR(Wen et al., 2000; Lin et al., 2001).

4. 3 . 3

 33 Preliminary study for AR-negative PCa therapy Furthermore, Bluemn et al confirmed that gene sets reflecting the activity of FGF signaling, MAPK signaling, and MEK/ERK signaling are significantly enriched in AR-negative CRPC tumors by RNA sequencing. Across all of the CRPC metastases, AR activity was inversely associated with FGF8/9 expression and FGFR activity.They also found that the downstream mediator of FGF/MAPK signaling ID1 (inhibitor of differentiation 1) was upregulated in androgen independent LNCaP cells in comparison to wild-type LNCaP cells. Stimulation of wild-type LNCaP cells with FGF8 in androgen-deprived conditions led to an increase in ID1 mRNA and protein levels, indicating that FGF8 activation is sufficient to trigger ID1 expression. While ID1 knockdown did not impair wild type LNCaP cell growth, it significantly impaired androgen independent LNCaP cell growth, ID1 transcripts were diminished in mouse xenograft models after treatment with an FGFR inhibitor. Together, these data support the hypothesis that ID1 mediates the FGFR dependent survival of AR-negative

  locus in Men1-KO cell lines. More importantly, they demonstrated that menin bound to the Inhbb promoter where it favours the recruitment of EZH2 likely via an indirect mechanism involving AKT phosphorylation (Gherardi et al 2017). 5.4.2 Menin interacts with numerous transcriptional factors By investigating the possible molecular links between different transcription factors and menin, we speculated that we might gain further insight into the possible role played by menin in MEN1 tumors and other cancers (Fig. 1.14). 5.4.2.1 JunD JunD, a basic leucine-zipper (bZIP) transcription factor, is a member of Jun family (c-Jun, which is the cellular counterpart of the v-Jun oncogene, JunB and JunD), forming the dimeric transcription factor complex AP-1 with Fos members (c-Fos, FosB, Fra1 and Fra2). These bZIP transcription factor family members dimerize at their C-terminal leucine zipper domains in synergy with their adjacent basic domain binding to DNA. They regulated transcription on promoters by binding to a consensus TRE (12-O-teyradecanoylphorbol-13-acetate [TPA] responsive element) sites.

Fig. 1 .

 1 Fig. 1.14 Mechanistic clues underlying the involvement of menin in tumorigenesis. Menin interacts with numerous menin-interacting factors, consequently participating in the regulation of many target genes and interfering with different signaling pathways strongly implicated in breast and prostate cancers. EF: epigenetic factors; TF: transcriptional factors.

  Men1-deficient mice developed accelerated mPIN and later displayed microinvasion adenocarcinoma. More importantly, Men1-KO led to markedly downregulation of AR in early-stage lesions. Interestingly, we also found the expression of AR was decreased in MEN1 silencing LNCaP, 22Rv1 and VCaP cells. Moreover, we demonstrated menin bound to the proximal AR promoter and positively regulated AR transcription via the H3K4me3 histone mark in PCa cells. These findings highlight the regulation of the AR promoter by menin and the crosstalk between menin and the AR pathway(Teinturier et al., 2021).

5. 4 . 3

 43 Menin regulates various signaling pathways The wingless-related integration site (Wnt)/β-catenin signaling has important roles not only in development, but also in cancer biology. Menin was shown to be crucial for regulating canonical Wnt/β-catenin signaling in cultured rodent islet tumor cells by interaction with β-catenin[START_REF] Chen | Menin promotes the Wnt signaling pathway in pancreatic endocrine cells[END_REF]. In Men1-null MEFs and insulinomas from β-cell-specific Men1-knockout mice, β-catenin accumulates in the nucleus, but overexpression of menin reduces nuclear accumulation of β-catenin and suppresses its transcriptional activity(Cao et al. 2009). Wnt signaling stimulates pancreatic islet β cell proliferation, possibly by increasing expression of paired-like homeodomain 2 (Pitx2)[START_REF] Rulifson | Wnt signaling regulates pancreatic beta cell proliferation[END_REF]. When menin and activated β-catenin are overexpressed in islet tumor cells, the Wnt/β-catenin downstream target gene, Axin2, is significantly enhanced, correlating with increased H3K4me3 at the promoter of the Axin2 gene[START_REF] Chen | Menin promotes the Wnt signaling pathway in pancreatic endocrine cells[END_REF]. These opposite results for the role of menin on the Wnt signaling pathway and cell proliferation may depend on distinct context of cells. Inhibition of Wnt signaling resulted in reduced proliferation of Men1-deficient rodent β-cells. Accordingly, combining a β cell-specific mouse knockout model of the Men1 gene with knockout of the Ctnnb1 gene (encoding β-catenin) resulted in lower tumor numbers and sizes in mice defective in Men1 and Ctnnb1 vs Men1 alone (Jiang et al. 2014), accompanied with the inhibition of the expression of pro-proliferative genes and improved hyperinsulinemia and hypoglycemia. Antagonizing β-catenin signaling by the small molecule inhibitor PKF115-584 in Men1-deficient mice also suppresses tumor cell proliferation in vitro and in vivo (Jiang et al. 2014). These results suggest that activated Wnt signaling after loss of MEN1 gene expression could be a targetable mechanism in MEN1-related tumors. 5.4.3.2 PI3K/AKT/PTEN/mTOR signaling More recently, Wong and colleagues generated mouse models with insulin-specific biallelic inactivation of Men1 and Pten in β-cells, and showed that their concomitant loss accelerated islet cell tumorigenesis. Co-mutations of MEN1

methyltransferase 1 (

 1 DNMT1) downstream of the Hedgehog signaling pathway, and menin overexpression strongly antagonized the positive effect of Hedgehog signaling on pancreatic cancer cell proliferation[START_REF] Cheng | Interplay between menin and Dnmt1 reversibly regulates pancreatic cancer cell growth downstream of the Hedgehog signaling pathway[END_REF].Gurung et al. study indicated that menin antagonizes Hedgehog signaling, partly via increasing PRMT5-mediated repressive H4R3me2s at the Gas1 promoter in neuroendocrine cells(Gurung et al. 2013b). Men1-null cells supplemented with menin mutants cannot inhibit Gas1 mRNA levels, but wild type can do(Gurung et al. 2013b). Moreover, menin and PRMT5 also suppress Hedgehog signaling at a second step besides Gas1, by inhibiting the expression of Hedgehog downstream effector Gli1. These findings indicate that loss of menin-mediated repressive histone methylation of H4R3me2 sites at the Gas1 and Gli1 promoters and resulting upregulation of Hedgehog signaling play a role, at least in part, in the pathogenesis of MEN1 syndrome. 5.4.3.4 Menin inactivation triggered pancreatic islet α cell to β cell transdifferentiation Menin is also a crucial factor maintaining the cell differentiation of pancreatic islet alpha-and β-cells. Mice with alpha-cell-specific knockout of the Men1 gene developed insulin-secreting β-cell tumors or insulinomas 6 months following Men1 disruption (Lu et al. 2010). Genetic cell lineage tracing analysis showed that insulinoma cells were directly derived from transdifferentiating glucagon-expressing cells (Lu et al. 2010), associated with altered expression of transcription factors critical for islet cell differentiation, including MAF-B and FOXA2. It remains to further investigate as to what pathway(s) is(are) perturbed or the epigenetic program altered in the Men1-deficient alpha cells, resulting in alpha-cell to β-cell trans-differentiation. 5.4.3.5 Menin and K-Ras signaling regulate each other Menin suppresses both proliferation and migration of lung adenocarcinoma cells partly via inhibiting PTN and its receptor RPTP β/ζ signaling (Gao et al. 2009, Feng et al. 2010). However, menin also is repressed by K-Ras through DNMT1-dependent DNA demethylation of the promoter of the MEN1 gene in lung adenocarcinoma cells and inversely menin reduces the level of active Ras-GTP at least partly by preventing

5. 5 . 4

 54 Regulation of menin by posttranslational modifications 5.5.4.1 Ubiquitination

  MEN1 gene in hormone-related cancers 5.7.1 Hormone-related cancers observed in Men1 mouse models 5.7.1.1 Mammary gland lesions in mouse Men1 models

PrefaceI.

  My initial PhD research theme I started my PhD research works with my participation in characterizing a mouse model where the Men1 and Nkx3.1 genes were conditionally disrupted in prostate luminal cells. The major findings achieved in this work are: Men1 disruption accelerates tumorigenesis of prostate cells in Nkx3.1-deficient mice. Men1/Nkx3.1-deficient lesions display low AR and high CD44 expression, accompanied by reduced CK18 and E-cadherin expression. Mechanistically, we demonstrated that menin bound to the AR promoter and regulated AR transcription via H3K4me3 in human PCa cells. More interestingly, we also found menin could play an opposite role in AR-dependent versus AR-independent PCa cells. My major contribution to this work was to dissecting the molecular mechanisms underlying the regulation of AR transcription by menin. In particular, the data I have obtained allowed us to uncover that the MEN1 gene plays distinct roles in controlling cell proliferation in AR-dependent versus AR-independent PCa cells. Consequently, I've focused my major PhD research them to investigate the cellular and molecular mechanisms underlying the phenomenon. Publication: Teinturier R#, Luo Y#, Decaussin-Petrucci M, Vlaeminck-Guillem V, Vacherot F, Firlej V, Bonnavion R, Abou Ziki R, Gherardi S, Goddard I, Gadot N, Bertolino P, Le Romancer M, Zhang CX. Men1 disruption in Nkx3.1-deficient mice results in ARlow/CD44+ microinvasive carcinoma development with the dysregulated AR pathway. Oncogene. 2021;40(6):1118-1127. doi: 10.1038/s41388-020-01589-1.

Prepared manuscript :II- 2 .

 manuscript2 Yakun Luo, Virginie Vlaeminck-Guillem, Romain Teinturier, Razan Abou Ziki, Philippe Bertolino, Muriel Le Romancer and Chang Xian Zhang. Menin is essential for activating the MYC locus and MYC-mediated androgen receptor transcription in AR-dependent prostate cancer cells. Cancer Communication. (Under review) Activation of JunD and B-catenin triggered by MEN1 silencing in AR-independent PCa cells.Our data provide evidence that menin plays a tumor suppressor role in AR-independent PCa cells. We found that MEN1 inactivation promoted the tomorigenic potential of these cells via triggering nuclear translocation of JunD and β-catenin. Mechanistically, both JunD and β-catenin could replace menin to bind to the regulatory sequences of the MYC locus, ultimately resulting in maintaining MYC expression. Interestingly, we also found that MEN1 inactivation altered the expression of several molecular markers of EMT and stemness, including E-cadherin, BMI1, Twist1 and HIF1α. Finally, our analyses using cultured AR-independent PCa cells and PC3-GFP xenografts in the mouse demonstrated that both JunD and β-catenin are necessary for the altered tumorigenic potential triggered by MEN1 inactivation. Prepared manuscript: Yakun Luo, Virginie Vlaeminck-Guillem, Silvère Baron, Chang Xian Zhang and Muriel Le Romancer. MEN1 silencing aggravates tumorigenic potential of AR-independent prostate cancer cells through nuclear translocation and activation of JunD and β-catenin. Journal of Experimental & Clinical Cancer Research (Under review)

Fig. 1

 1 Figure legends:

Fig. 2 Fig. 1 Fig. 2

 212 Fig. 2 Menin is crucial for MYC-mediated AR transcription and EZH2-regulated PCAT expression in AR-dependent PCa cells. a-i Shown are the experiments conducted exclusively in AR-dependent LNCaP cells. a ChIP-qPCR of the binding of menin to the AR promoter. b Relative luciferase activity after transfection of siCtrl or siMEN1(1)+(3)-treated cells with AR-Luc-2 or AR-Luc-2-ΔE. c ChIP-reChIP assay assessing the effect of siMEN1(1)+(3) on menin and MYC recruitment to the E-box

Fig. S2 a

 S2 Fig. S2 Menin recruits the MLL complex and activates MYC transcription in AR-dependent PCa cells.

Fig.

  Fig. S3 MEN1-KD reduces the expression of downstream MYC target genes and AR splice variants in AR-dependent PCa cells.

Fig. S4 a

 S4 Fig. S4 The regulation of MYC by menin is androgen-dependent in PCa cells.

Fig. S5

 S5 Fig. S5 Menin and MYC co-regulate AR transcription in PCa cells.

Fig. S6

 S6 Fig. S6 Menin is crucial for the MYC-mediated regulation of the AR promoter through the E-box site in AR-dependent PCa cells.

Fig. S7

 S7 Fig. S7 Menin participates in the regulation of PCAT1 through EZH2.

  Fig. S1

[ 3 ]

 3 . The cellular nuclear fractions were separated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ref. 78833, Thermo Scientific). The following primary antibodies were used: Rabbit anti-menin (1/4000) (A300-105A, Bethyl), Rabbit anti-c-Myc (1/2000) (#9402, Cell Signaling Tech.), Rabbit anti-AR (1/2500) (A303-965A, Bethyl), Rabbit anti-ARV7 (1/2000) (#19672, Cell Signaling Tech.), Mouse anti-CDK2 (1/1000) (sc-6248, Santa Cruz), Mouse anti-CDK4 (1/1000) (sc-56277, Santa Cruz), Mouse anti-Cyclin A (1/500) (sc-271645, Santa Cruz), Mouse anti-Cyclin D1 (1/1000) (sc-20044, Santa Cruz), Mouse anti-Cyclin E (1/1000) (sc-377100, Santa Cruz), Rabbit anti-EZH2 (1/2000) (#5246, Cell Signaling Tech.), Rabbit anti-ASH2L (1/2000) (#5019, Cell Signaling Tech.). Secondary antibodies included HRP Goat Anti-mouse (1/10000) (ref.111-035-003) and HRP Goat Anti-rabbit (1/10000) (ref. 111-035-003), and were purchased from Jackson immunoresearch. Mouse anti-GAPDH (1/1000) (sc-47724, Santa Cruz) and anti-Histone H3 (# 4499, Cell Signaling Tech.) antibodies were used as loading controls.

(

  green) (Cell Signaling Technology). Cells were counterstained with DAPI (DUO82040, Sigma-Aldrich) for 10 min and visualized by fluorescence microscopy (Eclipse-NiE NIKON microscope). The following primary antibodies were used for immunostaining: Rabbit anti-menin (1/2000) (A300-105A, Bethyl), Rabbit anti-c-Myc (1/2000) (#9402, Cell Signaling Tech.)

F

  studies highlighted the increased frequency of AR-low or -negative prostate cancers (PCas) and the importance of AR-independent mechanisms in driving metastatic castration-resistant PCa (mCRPC) development and progression.

:

  Cell tumorigenic features were evaluated by proliferation assay, foci formation, colony formation in soft agar, wound healing assay and xenograft experiments in mice. Quantitative RT-PCR, Western blot and immunostaining were performed to determine the expression of different factors in human PCa lines. Different ChIP-qPCR-based assays were carried out to dissect the action of JunD and β-catenin. Results: We found that MEN1 silencing in AR-independent cell lines, DU145 and PC3, resulted in an increase in anchorage independence and cell migration, accompanied by sustained MYC expression. By searching for factors known to positively regulate MYC expression and play a relevant role in PCa development and progression, we uncovered that MEN1-KD triggered the nuclear translocation of JunD and β-catenin. ChIP and 3C analyses further demonstrated that MEN1-KD led to, on the one hand, augmented binding of JunD to the MYC 5' enhancer and increased formation of loop structure, and on the other hand, increased binding of β-catenin to the MYC promoter. Moreover, the expression of several molecular markers of EMT, including E-cadherin, BMI1, Twist1 and HIF1α, was altered in MEN1-KD DU145 and PC3 cells. In addition, analyses using cultured cells and PC3-GFP xenografts in mice demonstrated that JunD and β-catenin are necessary for the altered tumorigenic potential triggered by MEN1 inactivation in AR-independent PCa cells. Finally, we observed a significant negative clinical correlation between MEN1 and CTNNB1

  7a-b). Morphologically, MI503-treated PC3-GFP cells app eared more variable in size and more invasive towards surrounding mouse tissues, with disorientated alignment (Fig.7a, c). In agreement with our in vitro observation, qPCR and immunostaining analyses of PC3-GFP xenografts confirmed reduced expression of menin and JunD in xenografted cells, but revealed increased CTNNB1 transcription and overt nuclear expression of β-catenin in MI503-treated-PC3-GFP cells (Fig.7c-d, S7b-d), suggesting that inhibition of both menin and JunD could still lead to aggravated tumorigenic potential of xenografted PC3-GFP cells, due to activated β-catenin.

  present work unveiled the activation of the JunD and β-catenin pathways upon menin inactivation specifically in AR-independent cells. Of note, the oncosuppressive role of menin in AR-independent PCa cells is closely associated with maintained MYC expression and altered expression of EMT markers (Fig 7f), which may pave the way for new strategies for PCa treatment. interpretation; M.L.R and C.X.Z conceived and supervised the study and manuscript preparation, and obtained funding.

Fig. 1 MEN1

 1 Figure legends:

Fig. 2

 2 Fig. 2 MEN1 silencing triggers the nuclear translocation of JunD in AR-independent PCa cells. Representative images of foci formation assays (a) and their quantification (b) using LNCaP, 22Rv1, DU145 and PC3 cells treated or not with MI503 (2.5 μM). qRT-PCR analysis of JunD and MYC mRNA expression (c) and Western blot analysis showing JunD and MYC expression (d) in 22Rv1 and DU145 cells treated or not with MI503 (2.5 μM). qRT-PCR analysis of JunD mRNA expression (e) and Western blot analysis of JunD protein expression (f) in MEN1-KD 22Rv1 and DU145 cells. g IF staining showing menin and JunD in DU145 cells treated with siCtrl or siMEN1(1)+(3). Scale bar = 25 μm. h Western blot analysis of JunD expression in different subcellular fractions in DU145 and PC3 cells treated with siCtrl or siMEN1(1)+(3).

Fig. 3

 3 Fig. 3 MEN1 knockdown leads to increased JunD binding and loop structure formation in the MYC locus in AR-independent PCa cells. a ChIP-qPCR analysis using anti-menin to evaluate the binding of menin to the MYC 5' enhancer (-67kb) and promoter in DU145 and PC3 cells treated with siCtrl or siMEN1(1)+(3). Chr1 served as a negative control. b ChIP-qPCR analysis using anti-JunD to detect the binding of JunD to the MYC 5' enhancer and promoter in DU145 and PC3 cells treated with siCtrl or siJunD. c ChIP-qPCR analysis to assess the effect of MEN1-KD on the level of JunD recruitment to the MYC 5' enhancer in DU145 and PC3 cells. d Primer locations for qPCR of 3C and ChIP-3C analyses. e 3C detection results (DP1-DP2 fragment) and control (CP1-CP2) by PCR in siCtrl or siMEN1(1)+(3)-transfected DU145 cells upon ligation or non-ligation. ChIP-3C qPCR analysis detecting menin ChIP-qPCR analysis showing menin binding (f) and JunD binding (g) to the MYC 5' enhancer, MYC promoter and the looping fragment (F3-F6) in DU145 cells transfected with siCtrl or siMEN1(1)+(3) under ligation (right panel) or non-ligation (left panel) conditions, P1-P2 and P7-P8 were used as negative controls.

Fig. 4

 4 Fig. 4 MEN1 silencing results in nuclear translocation of β-catenin and an increase in its binding to the MYC promoter in AR-independent PCa cells. a Western blot analysis of β-catenin expression in different subcellular fractions in siCtrl or siMEN1(1)+(3)-treated DU145 and PC3 cells as indicated. b Double IF staining showing menin and β-catenin in siCtrl or siMEN1(1)+(3)-treated DU145 cells. Scale bar = 25 μm. ChIP-qPCR analysis assessing β-catenin (c) or menin (d) binding to the MYC promoter and the MYC 3'enhancer in siCtrl or siMEN1(1)+(3)-treated DU145 cells. reChIP analysis evaluating the effect of MEN1-KD (e) and CTNNB1-KD (f) on menin (left panel) and β-catenin (right panel) co-occupancy on the MYC promoter in DU145 cells.

Fig. 5

 5 Fig. 5 JunD and β-catenin are involved in the regulation of EMT marker expression in AR-independent PCa cells. Representative images of soft agar colony formation assay

Fig. 6

 6 Fig. 6 The expression of both JunD and β-catenin is critical for the tumorigenic potential of AR-independent PCa cells. a Representative images of foci formation of DU145 cells upon transfection with siMEN1, siJunD, siCTNNB1, siMEN1+siJunD, siMEN1+ siCTNNB1 or siMEN1+siJunD+siCTNNB1. The quantification of the data are shown in the right and lower panel. b qRT-PCR analysis showing mRNA expression of HIF1A, E-cadherin, Vimentin, Twist 1 and BMI1 in DU145 and PC3 cells transfected with siCtrl or siMEN1(1)+(3). c Western blot analysis showing total protein expression of menin, HIF-1α, E-cadherin, Vimentin, Twist 1 and BMI1 in DU145 and PC3 cells transfected with siCtrl or siMEN1(1)+(3). d Western blot analysis showing menin, HIF-1α, Vimentin, Twist 1 and BMI1 expression in nuclear and cytoplasmic subcellular fractions in siCtrl or siMEN1(1)+(3)-treated DU145 and PC3 cells.

Fig. 7 a

 7 Fig. 7 MI503-treated PC3-GFP xenografts display increased tumor growth with nuclear overexpression of β-catenin. a Representative images of HE stained xenografts treated with DMSO (upper) and MI503 (lower). Scale bar = 200 μm. b qRT-PCR analysis evaluating the number of PC3-GFP cells transplanted in mouse kidney treated (n = 11) or not (n = 9) with MI503. Representative images of IHC staining (c, Scale bar = 100 μm) for menin, JunD and β-catenin or IF staining (d, Scale bar = 50 μm) for JunD and β-catenin in xenografts in the Ctrl group (DMSO

Fig. S2

 S2 Fig. S2 MEN1 silencing triggers the nuclear translocation of JunD in AR-independent PCa cells, but not in AR-dependent cells.

Figa

  Fig. S3 MEN1 silencing elevates JunD binding to the MYC locus in AR-independent PCa cells.

Fig.

  Fig. S5 β-catenin replace menin to bind to the MYC promoter in MEN1-KD AR-independent PCa cells.

Fig. S6 Both

 S6 Fig. S6 Both JunD and β-catenin are needed for increased tumorigenic potential triggered by menin inactivation in AR-independent PCa cells.

Figa

  Fig. S7 MI503-treated Xenografted PC3-GFP cells displayed accelerated tumor growth with increased nuclear β-catenin expression.

  Fig.S3

  were lysed in 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP-40 and protease inhibitor cocktail. Nuclei were pelleted and chromatin was digested overnight with 2000 U Fat I (NEB, Category Number R0650) and ligated with 4000 U T4 DNA Ligase (NEB) for 4 h at 16°C followed by 30 min at room temperature. 3C samples (ligation or non-ligation) were reversed cross-linked by proteinase K treatment at 65°C for 4 h and purified by phenol/chloroform extraction (Sigma). Typically, 2.5 μg of DNA was recovered at this step, and 100 ng was used for each 3C PCR amplification with specific pairs of primers and resolved on a 2.0% (w/v) agarose gel.

3 . 1

 31 are the mechanisms underlying the regulation of the cell growth and differentiation by menin in PCa cells ? Menin promotes the cell proliferation through MYC-mediated AR transcription in AR-dependent PCa cells.

3. 2 . 1

 21 Fig. 3.1 Molecular mechanism that MEN1 inactivation suppresses the cell proliferation of AR-dependent PCa cells.

Fig. 3 . 2

 32 Fig. 3.2 Molecular mechanism that MEN1 inactivation facilitates the cell growth and dedifferentiation of AR-independent PCa cells.

  reduces the menin and JunD expression and triggers the β-catenin translocation in PC3-GFP cells. These results are consistent with our in vitro studies. On the other hand, the proliferation of MI503-treated xenografted PC3-GFP cells was increased and cells appeared more variable in cell size, disorientatedly aligned and more invasive towards surrounding mouse tissues. Conversely, the MI503 treatment significantly inhibits the cell proliferation of PC3 cells in vitro. These results indicated that: a. for the technique part, sometimes, one could get different even opposite results in vitro and in vivo. I think this is not surprising, because the in vitro study happens in an artificial cellular environment that may not correspond to a real situation in tissue or organ. Meanwhile, cell line derived xenograft is not a "real" in vivo study. The PC3-GFP cells still come from in vitro cell culture. It would be interesting to carry out patient derived xenografts (PDX) to confirm our current data. For the function of MI503, our work or previous paper has already found that MI503 not only decrease menin expression, but that of JunD in vitro and in xenograft model. It would also be interesting to study whether MI503 could inhibit other factors interacting with menin or down-regulate other factors in in vivo environment.

Fig 3 . 4

 34 Fig 3.4 Structural studies of the menin -MLL1 and menin-JUND interaction. a. Crystal structure of the menin-MLL1 complex. b. Crystal structure of the menin-JUNDMBM complex. Menin is coloured as in Fig. 3.1a and JUNDMBM is shown as a purple stick model. Obtained from: Huang et al., 2012, Nature.

4. 3

 3 Could we use the combine MI503 with β-catenin inhibitors to treat the PCa ? It still depends on the the AR level in PCa patients. If the AR expression is positive, combined MI503 and β-catenin inhibitor may have the advantage to antagonise the cancer cell growth. Conversely, if the AR expression is negative, it is still too early to carry out the assay. Although according to our results, menin inhibition triggers the β-catenin activation in AR-independent PCa cells, we still don't know whether other partners of menin participates the increased tumorigenic potential of AR-indepedent PCa cells due to menin inhibition. Further studies are needed, in order to dissect the cellular and molecular events triggered by MEN1 inactivation in these cells.

Fig. 1

 1 Fig. 1 Characterization of the Men1 F/F -Nkx3.1Cre ERT2+/-mouse phenotype. a Schematic overview of the approach used for the generation of Men1 F/F -Nkx3.1Cre ERT2+/-mice. b Depletion of menin in prostate glands and development of prostatic intraepithelial neoplasia (PIN) lesions in Men1 F/F -Nkx3.1Cre ERT2+/-mice. Immunohistochemical analysis using an anti-menin antibody was performed in prostate sections of Men1 F/F -Nkx3.1Cre ERT2+/-treated with tamoxifen (Men1NT), and in untreated control mice (Men1N), at 3, 6, and 10 months of age. Right columns for each condition, a twofold magnification showing Men1 expression in the normal prostate gland or neoplastic cells is included. Scale bar = 25 μm. c Quantification of luminal cells positive and negative for menin expression in each group of age following tamoxifen treatment (n = 5). d Men1 inactivation accelerates tumorigenesis of prostate cells. Prostate glands were collected from the control and mutant mice at 3 months of age.

Fig. 2

 2 Fig. 2 Men1 disruption in Nkx3.1-deficient prostate cells results in deregulated AR expression and the AR pathway. a Upper: αSMA immunofluorescence (IF) performed in prostate specimens of Men1NT mice at 10 months of age, arrows indicate discontinuous αSMA staining in surrounding stromal compartment adjacent to menindeficient luminal cells. Scale bar = 50 μm. Middle: IF staining against Ki67 on prostate sections from Men1N and Men1NT at 6 months of age. Scale bar = 25 μm. Lower: Quantification of Ki67 staining. Data are presented as means±SEM, **P < 0.01; ***P < 0.001. b Double IF against androgen receptor (AR) and menin in Men1 F/F -Nkx3.1 (Men1N) and Men1 F/F -Nkx3.1 + tamoxifen (Men1NT) mouse prostate tissues at 3 (left panel) and 6 (upper right panel) months of age. The left and right histograms represent respectively the percentage of cells with or without AR expression in menin-positive luminal cells from Men1N mice and menin-negative luminal cells from Men1NT mice (n = 5). Scale bar = 25 μm. c Immunohistochemical (IHC) detection of

Fig. 3

 3 Fig. 3 Men1 inactivation in Nkx3.1-deficient prostate cells led to cell dedifferentiation. a H&E staining (left column) and IHC against menin, CK18, and CK5 on Men1N and Men1NT mouse prostate sections at 6 months of age. b Double IF staining against menin and p63 with DAPI counterstaining on prostate sections from Men1N (upper panel) and Men1NT (lower panel) at 10 months of age, showing no evidence of p63 expression in menin-negative luminal cells. c Triple IF staining against menin, E-cadherin (E-cadh), and α-SMA with DAPI counterstaining on prostate sections from Men1N (upper

Fig. 4

 4 Fig. 4 Menin binds to the proximal AR promoter and is crucial to cell differentiation and proliferation of AR-dependent cells. a Upper graph: ChIP analysis was used to define the binding of menin and menin-MLL complex to the AR promoter. Quantitative PCR analyses of menin and H3K4me3 binding on the -2000 bp/+550 bp area flanking the AR gene transcription start site (TSS) in LNCaP cells (left panel) and in LNCaP cells treated with DMSO or MI503 at 5 μM (right panel). b Cell growth of LNCaP, 22Rv1, VCaP, DU145, PC3, and PC3-AR cells treated with siRNA ctrl or siMEN1 (1 + 3). Data shown are mean (±SEM) of technical replicates from one representative experiment out of three. c Western blot analysis of menin, AR,

Figure 1 .

 1 Figure 1. Schematic representation of the structure of the ESR1 gene and the AR gene and their proteins. Different protein domains are indicated, including the N-terminal domain (NTD), the DNA binding domain (DBD), the hinge domain (D for ERα protein and HR for AR protein), and the ligand binding domain (LBD).

Figure 2 .

 2 Figure 2. The menin protein interacts physically and functionally with ERα and AR, and is involved in the regulation of ESR1 transcription and the transactivation of the target genes of both ERα and AR.

Figure 3 .

 3 Figure 3. Mechanistic clues underlying the involvement of menin in mammary and prostate cell tumorigenesis. Menin interacts with numerous menin-interacting factors, consequently participating in the regulation of many target genes and interfering with different signaling pathways strongly implicated in breast and prostate cancers. EF: epigenetic factors; TF: transcriptional factors.

4. 3 . 1 .

 31 The PI3K/PTEN/AKT/mTOR Pathways Activation of the PI3K/PTEN/AKT/mTOR pathways occurs in 70% of BCs overall [161]. PIK3CA (a subclass of the PI3K family of genes) is the most commonly mutated gene in ER-positive BCs [162].

4. 3 . 2 .

 32 Cell Cycle, Growth, and Death Control Kaji et al. demonstrated that menin could suppress cell proliferation via the transforming growth factor-β (TGF-β) pathway in the rat pituitary cell line by interacting with Smad3 [179]. Agarwal et al. reported that menin is essential for JunD-mediated inhibition of cell proliferation [140]. Ratineau et al.

  [104]. Jiang et al. further demonstrated that β-Catenin ablation leads to the suppression of tumorigenesis and significantly improved hypoglycemia and the survival rate of Men1-deficient mice [105]. Applying the small molecule inhibitor, PKF115-584, in Men1-deficient mice to antagonize β-Catenin signaling suppressed tumor cell proliferation in vitro and in vivo [105]. Kim et al. reported that menin promotes ubiquitin-mediated degradation of β-Catenin and menin overexpression downregulates the transcriptional activity of β-Catenin and target gene expression, as well as the proliferation of human renal carcinoma cells with an activated β-Catenin pathway [191].

Table 1 .1 Criteria for diagnosis of prostatic adenocarcinoma.

 1

Table 1 .3 The TNM classification system in Prostate Cancer.

 1

Table 1 .4 Age specific PSA values.

 1

Table 1 .5 Risk stratification of men with localised prostate cancer

 1

. NICE. Prostate Cancer: Diagnosis and Treatment. Clinical Guideline. 2014.

Table 1 .6 AR variants/isoforms expressed in tissues and cell lines

 1

. Adapt from: Pelekanou et al., 2013.

4. Other mechanisms underlying prostate cancer initiation and progression 4.1 Genes involved in the carcinogenesis of prostate cells

	4.1.1 NKX3.1
	NKX3.1 is a prostate-specific tumor suppressor located on chromosome 8p, whose
	loss or reduction represents a key initiating event in prostate cancer (Cancer Genome
	Atlas Research, 2015; Baca et al., 2013). Inactivation of the NKX3.1 homeobox gene,
	expressed in normal prostate luminal cells, represents a frequent and critical event in
	prostate cancer initiation, likely involving multiple mechanisms (Abate-Shen et al.,

Table 1 .7 MEN1 pathology(tumors and hyperplasia)

 1

  Men1 F/F mice(Bertolino et al., 2003) were crossed with Nkx3.1Cre ERT2 mice, in which the activity of Cre recombinase controlled by the endogenous Nkx3.1 promoter can be induced in prostate luminal cells upon tamoxifen injection. Men1 F/F -Nkx3.1CreERT2-/+

	mice thus generated were subjected to tamoxifen treatment (Men1NT) or not (Men1N)
	F/F ) at 1.5 months of age, and prostate glands were subjected to pathological examination
	with WapCre transgenic mice expressing Cre recombinase under the control of the 1.5, 4.5, and 8.5 months after tamoxifen injection. Having shown that a majority of
	whey acidic protein (Wap) promoter, which is known to be expressed in luminal prostate cells within lesions in Men1NT mice lost menin expression, we observed that
	mammary epithelial cells. Female Men1 F/F -WapCre mice developed substantially these mice progressively developed more advanced lesions with prominent
	higher amounts of early mammary intraepithelial neoplasia (MIN), which are pleomorphic changes and microinvasive carcinoma (MIC) at 6 (2/7, 28%) and 10

precursor lesions, in comparison with control Men1 +/+ -WapCre mice. Interestingly, ERα expression and the number of ERα-positive cells were clearly reduced in MIN lesions of mutant mice compared with normal mammary glands. In addition, cell membrane expression of β-Catenin and E-Cadherin was almost absent in the mammary lesions of Men1 F/F -WapCre mice compared with control mice; neither β-Catenin nor E-cadherin were detected in the TS1 cell line derived from a mouse Men1 BC

(Seigne et al., 2013)

.

5.7.1.2 Prostate lesions in mouse Men1 models

By following a cohort of 47 male heterozygous Men1 mutant mice (Men1 +/-) and 23 male wild-type (Men1 +/+ ) age-matched littermate mice from 18 to 26 months of age, Seigne et al. found that six Men1 +/-mice (6/47, 12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. No prostate carcinoma was found in age-matched Men1

+/+ littermates (0/23). In addition, these carcinomas exhibited loss of the non-target Men1 allele (LOH), therefore supporting a tumor suppressor role for the Men1 gene in prostate glands. Moreover, the AR and p27 expression was decreased in tumor lesions, likely facilitating prostate cell tumorigenesis due to Men1 inactivation (Seigne et al., 2010). More recently, our team generated a conditional Men1-KO mouse model to decipher the role played by Men1 in the initiation of prostate cell tumorigenesis. months (4/7, 57%) of age. As experimental controls, no age-matched Men1N mice developed MIC (n = 5), Men1 F/F mice either treated or not with tamoxifen developed no mPIN lesions within the 10-month experimental time course, and tamoxifen-treated Nkx3.1CreERT2 -/+ mice developed no MIC lesions at 5 and 10 months of age (n = 5). Our results indicated that prostate-specific Men1 disruption in mice leads to the acceleration of tumorigenesis in Nkx3.1 -/+ prostate cells (Teinturier

  . More recently, a 41-year old patient with no familial history

	of breast cancer but with a mother with primary hyperparathyroidism (PHP) was
	found carrying a variant p.C421R/p.426R in the MEN1 gene. The patient's
	histopathological study revealed hormone receptor negativity, as well as HER-2 and
	p53 negativity. A family study showed positive findings for MEN1 in a sister, two
	maternal nephews, and one of the patient's daughters, with no record of breast cancer
	development in any of these people (Herranz-Antolín et al., 2018).

  2.Grad JM et al. Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation ofAR messenger RNA. Mol Endocrinol. 1999;13(11):1896-911. doi:

	10.1210/mend.13.11.0369.
	3.Nadiminty N, et al. MicroRNA let-7c suppresses androgen receptor expression and
	activity via regulation of Myc expression in prostate cancer cells. J Biol Chem.
	2012;287(2):1527-37.doi: 10.1074/jbc.M111.278705.
	4. Teinturier R, et al. Men1 disruption in Nkx3.1-deficient mice results in
	ARlow/CD44+ microinvasive carcinoma development with the dysregulated AR
	pathway. Oncogene. 2020. doi: 10.1038/s41388-020-01589-1.
	opposite effects on transcription. Nature. 2012;482(7386):542-6. doi:
	10.1038/nature10806.
	9. Prensner JR, et al. Transcriptome sequencing across a prostate cancer cohort
	identifies PCAT-1, an unannotated lincRNA implicated in disease progression. Nat
	Biotechnol. 2011;29(8):742-9. doi: 10.1038/nbt.1914.

5. Brès V, et al. SKIP interacts with c-Myc and Menin to promote HIV-1 Tat transactivation. Mol Cell. 2009;36(1):75-87. doi: 10.1016/j.molcel.2009.08.015. 6. Wang C, et al. Estrogen induces c-myc gene expression via an upstream enhancer activated by the estrogen receptor and the AP-1 transcription factor. Mol Endocrinol. 2011;25(9):1527-38. doi: 10.1210/me.2011-1037. 7. Gao L, et al. Androgen receptor promotes ligand-independent prostate cancer progression through c-Myc upregulation. PLoS One. 2013 May 21;8(5):e63563. doi: 10.1371/journal.pone.0063563. 8.Huang J, et al. The same pocket in menin binds both MLL and JUND but has

Table S1 . Primers for this study

 S1

	Application	Primers'name	Sequence	Reference
	RT-qPCR	MEN1

The values are expressed as mean ± standard deviation (S.D.) and analyzed by Student t-test. The level of significance: ns, non-significant, * p <0.05, ** p < 0.01, *** p < 0.001.

  developed earlier and more frequently PIN lesions, and progressively developed more advanced lesions with micro invasive carcinoma (MIC) at 6 (2/7, 28.6%) and 10 months (4/6, 66.7%) of age. In addition, these carcinomas exhibited loss of the non-target Men1 allele (LOH), therefore supporting a tumor suppressor role for the CreERT2 ) is more specific and efficient to disrupt the Men1 in the prostate luminal cell. And finally, since the NKx3.1 gene is known as a prostatic tumor suppressor gene, the combined disruption of both Men1 and Nkx3.1 could accelerate the tumorgigenesis. Thus, these are the reasons for which the 2 nd mice model is quicker than 1 st mice to develop the PCa lesions. Indeed, pathological examination revealed that Men1-disruption affected the AR expression in Men1-KO mice prostate cells. We found that PCa from Men1 mouse models did display heterogeneous and reduced AR expression . These results were the first indication that menin could regulate AR expression in mice prostate cell. To confirm the observation, we investigated in AR dependent human PCa cells, and

	Men1 gene in prostate glands.
	Taken together, our two Men1-KO mouse models demonstrated that Men1
	disruption accelerates the tumorgenesis of mouse prostate cells, indicating that menin
	plays a tumor suppressor role in normal prostate glands in the mouse.

1.1.2 Several points remain to be elucidated a. Difference in age of mice developing the prostate carcinoma in these two types of mouse model

For the first mouse model, heterozygous Men1 mutant (Men1 +/-) male mice were monitored for cancer development and examined by histology analysis between 18 and 26 months. We noticed that, in the 2 nd mouse model, prostate glands were subjected to pathological examination at 1.5, 4.5 and 8.5 months after tamoxifen injection. As we all know, in the first model, the occurrence of LOH is crucial for PCa development, whereas in the conditional model, the gene disruption is largely complete. Also, for the 2 nd mice model, the inducible system (tamoxifen-Nkx3.1 b. Down-regulation of AR in Men1-KO mice prostate cell

Table 1

 1 Men1 inactivation accelerates tumorigenesis ofNkx3.1 +/-prostate cells.

	Genotype	Pathological classification	3 months	6 months	10 months
	Men1N	Normal	(4/5) 80%	(0/5) 0%	(0/5) 0%
	Men1 F/F -Nkx3.1Cre ERT2-/+	mPIN	(1/5) 20%	(5/5) 100%	(5/5) 100%
	Untreated	MIC	(0/5) 0%	(0/5) 0%	(0/5) 0%
	MEN1NT	Normal	(0/7) 0%	(0/7) 0%	(0/7) 0%
	Men1 F/F -Nkx3.1Cre ERT2-/+	mPIN	(7/7) 100%	(7/7) 100%	(7/7) 100%
	Tamoxifen treated	MIC	(0/7) 0%	(2/7) 28%	(4/7) 57%

Table 2

 2 Menin expression is decreased in numerous prostate tissue sections of PCa patients.

  3.3.2. MEN1 in Human Prostate CancerPerakakis et al. reported two cases of PCa seen in a MEN1 family with atypical tumor spectrum[126]. The DNA sequencing analysis revealed a novel mutation-Ser38Cys (TCC > TGC) in exon 2, located in a region of menin that is responsible for interaction with the transcription factor JunD. The latter has recently been associated with prostate cancer.Only limited sporadic MEN1 mutations have so far been reported in human sporadic PCa[127]. Manson-Bahr et al. found that missense mutations of the MEN1 gene were detected in 2 of 8 formalin-fixed prostate needle biopsy materials[125]. Interestingly, Grasso et al. analyzed 58 human CRPC samples by aCGH and found that 17.2% of all samples (10 of 58) harbored mutations in the MLL complex, including the MEN1 gene[69]. MLL functions as part of a multi-protein complex containing menin[128]. Many members of the complex have different levels of aberrations in CRPC[69]. Noticeably,Chen et al. analyzed 150 cases for advanced and metastatic human PCa. They observed that the percentage of PTEN and MEN1 co-loss was almost the same as the co-loss of PTEN and PML (Promyelocytic Leukemia), which is around 11% in all cases [129]. Conversely, Paris et al. reported that the MEN1 locus was amplified in some patients and was predictive of post-operative recurrence [130]. The similar observation was made Kerstin et al. [131]. Moreover, MEN1 knockdown resulted in a decrease in cell proliferation in DU145 cells [132,133], but curiously not in the PC3 cell line [132].

Men1 disruption in Nkx3.1-deficient mice results in. . .

Endocrines 2020, 1, 58-81; doi:10.3390/endocrines1020007www.mdpi.com/journal/endocrines

A Acknowledgements

I would like to extend my thanks and gratitude to my supervisor Dr. Chang Xian ZHANG for his unwavering support, encouragement and invaluable advice throughout this project.

Without his input, I could not have recruited so successfully. His experience, understanding, and patience have added considerably to my graduate experience.

I would like to thank Dr. Muriel Le Romancer for allowing me the opportunity to undertake this project. I am very grateful for this amazing opportunity and for the financial and technical support she has provided me throughout my period of study.

I would also like to thank the members of my advisory committee, Dr. Virginie Vlaeminck-Guillem and Dr. Silvère Baron, for their experience, advice and support throughout my project. Thanks to Dr. Philippe Bertolino and Dr. Romain Teinturier, who

ACKNOWLEDGEMENTS

We are grateful to Brigitte MANSHIP for her assistance in editing and proofreading the manuscript, Zhi Chong Wu for his scientific and technical input.

Acknowledgements

We are grateful to Brigitte MANSHIP for her assistance in editing and proofreading the manuscript, Zhi Chong Wu for his scientific and technical input.

The primary antibodies used for IF, IHC and ChIP analyses: rabbit anti-menin (1/2,000) (A300-105A, Bethyl), rabbit anti-β-catenin (1/2,000) (#8480S, Cell Signaling Tech.), rabbit anti-JunD (1/2,000)(#720035, Invitrogen), rabbit anti-MYC (1/2,000) (#9402, Cell Signaling Tech.), and goat anti-GFP (1/2,000)(ab5450, Abcam).

Acknowledgements The authors are grateful to Emilie Servoz for the maintenance and dissection of the mouse colonies, and Dr. Brigitte Manship for her assistance in editing and proofreading the manuscript. The authors would also like to thank VV-G for providing the prostate cell lines.

Funding This study was supported by Epigenetics & Cancer Program (ASC14092CSA), the Fondation de l'Association pour la Recherche contre le Cancer (SFI20101201530), the Ligue Inter-régionale contre le Cancer (R19040CC), the Association Le Cancer du Sein Parlons-en, and the Région Auvergne, Rhône-Alpes, (SICORRA22425). RT and RB were the recipients of a PhD fellowship from French Ministry of Higher Education and Research, and of a 4th year doctoral fellowship from Société Française d'Endocrinologie and the Fondation ARC, France, respectively. YL was the recipient of a PhD fellowship from China Scholarship Council. RAZ was the recipient of a PhD fellowship from Association "G04MEDIA S.A.R.L," Lebanon.

Acknowledgments:

We are grateful to Brigitte Manship for her assistance in editing and proofreading the manuscript.

Acknowledgments

We would like to thank Dr M Le Romancer for fully supporting our research activities, and Dr P Bertolino for his regular scientific input. We are grateful to Dr Brigitte MANSHIP for her assistance in proofreading the manuscript.

FUNDING

This study was supported by Epigenetics & Cancer Program (ASC14092CSA), the Fondation de l'Association pour la Recherche contre le Cancer (SFI20101201530), the Ligue Inter-régionale contre le Cancer (R19040CC), and the Association : « Le Cancer du sein, parlons-en ». R.T was the recipients of a PhD-fellowship from Ministère de l'Enseignement Supérieur et de la Recherche, France. Y.L was the recipient of a PhD-fellowship from the China Scholarship Council. R.A.Z was the recipient of a PhD-fellowship from the Association "G04MEDIA S.A.R.L", Lebanon. Funding This study was supported by the Ligue Inter-Régionale contre le Cancer (R19040CC), and the Association: « Le Cancer du sein, parlons-en ». Y.L was the recipient of a PhD-fellowship from the China Scholarship Council.

Funding: This study was supported by the Fondation de l'Association pour la Recherche contre le Cancer (SFI20101201530), the Ligue Inter-Régionale contre le Cancer (R19040CC), and the Région Auvergne, Rhône-Alpes (SICORRA22425). R.A.Z was the recipient of a PhD-fellowship from Association "G04MEDIA S.A.R.L", Lebanon. Y.L. was the recipient of a PhD-fellowship from China Scholarship Council.

Availability of data and material

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.

Availability of data and material

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.

Continue above

Article 1. Menin is essential for activating the MYC locus and

MYC-mediated androgen receptor transcription in AR-dependent prostate cancer cells

Short Report

Menin is essential for activating the MYC locus and MYC-mediated androgen receptor transcription in AR-dependent prostate cancer cells

Running title Crosstalk between menin, AR and MYC in AR-dependent PCa cells

Yakun Luo 1 , Virginie Vlaeminck-Guillem 1,2 , Romain Teinturier

DECLARATIONS

Consent for publication

All of the authors approved the contents and the data in this manuscript and agreed for its submission for publication.

Competing of Interests

The authors declare that they have no competing interest.

AUTHORS' CONTRIBUTIONS

Continued above

Conclusions: Our current work highlights an unrecognized oncosuppressive role for menin specifically in AR-independent PCa cells, through the activation of JunD and β-catenin pathways.

Keywords: (6) Prostate cancer, AR-independent cells, MEN1, MYC, JunD, β-catenin

Declarations

Ethics approval and consent to participate

All animal experiments were performed in accordance with the animal care guidelines of the European Union and were validated by the local Animal Ethics Evaluation Committee in agreement with the French Ministry of High School and Research. All experiments were approved by Auvergne Ethics Committee (CEMEAA) and registered according the approval number 17296-2018102216428025 v3.

Consent for publication

All of the authors approved the contents and the data in this manuscript and agreed for its submission for publication.

Competing of Interests

The authors declare that they have no competing interest.

SUPPLEMENTARY INFOMATION

Supplementary Materials and Methods

Cell culture and treatment

LNCaP, 22RV1 and DU145 were cultured in RPMI medium, and PC3 cells in F-12 medium (Gibco Invitrogen) containing 25 mM glucose and supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM L-Glutamine, 100 unit/mL Penicillin, 100 μg/mL streptomycin, HEPES 10mM, Sodium Pyruvate 1 mM, at 37°C with 5% CO2.

Inhibition of the menin-MLL interaction was achieved through the use of MI-503 (Active Biochem), at 1 μM, 2.5 μM, and 5 μM.

RNA interference and transfection

MEN1 siRNA sequences, that are siMEN1(1) (MEN1HSS106462) and siMEN1(3) (MEN1HSS181079), were designed by Invitrogen. JunD siRNA (siJunD(1), s7664; siJunD(2), s7665) and CTNNB1 siRNA (siCTNNB1(1), #146154; siCTNNB1(2), #146154) were purchased from ThermoFisher Scientific. A non-targeting control pool (siCtrl) from Sigma-MERCK was used as negative control for each RNA interference experiment. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and incubated for 72 h.

Cell proliferation assay

Cell proliferation assays were performed as described previously [1]. Briefly, cells were seeded onto 96-well culture plates at 5×10 3

Author contributions

None.

Financial support

The study was supported by the Fondation de l'Association pour la Recherche contre le Cancer (PJA 20151203335), SCUSI 2017 program from la Région Auvergne Rhône-Alpes and the grant from Ligue contre le Cancer inter-régionale 2018.

Conflicts of interest

The authors declare no conflicts of interest.