M Le Professeur

G Rode

La Mme

C Professeure

Burillon

D Bourgeois

Vinciguerra

M X Perrot

A-M Professeure

M Schott

Frédéric Le Professeur

M Fleury

Hamda Le Professeur

Hadid M Ben

Revel M Le Professeur Didier

Philippe Le Professeur

M Chevalier

Vallée Fabrice

Dominique Mme

Marchand

Composantes Sante

At the end of this three-years journey of challenges, obstacles, failures, and success, I can't but express my gratitude to the people that helped me get through the pitfalls and enjoy the ride: I would first like to thank the members of my jury Dr. Thierry Dubois and Dr.Céline Guigon for taking the time to revise my manuscript and evaluate my thesis work . I also would like to thank Dr.Corinne Bousquet, Dr.Hubert Lincet, and Prof. Laurent Morel for accepting to examine my work and be a part of my thesis jury.

I would also like to thank the members of my thesis committee Dr. Jean-Marc Vanacker and Prof. Laurent Morel for their constructive advices and help during the 3 years of my PhD.

Then I would like to thank Dr. Muriel Le Romancer for choosing me four years ago in Lebanon to join her team as a stagiaire, and for making my experience so pleasant and welcoming that I returned as a PhD student after. You gave me a lifetime opportunity of living abroad and training in one of the best research platforms. I also thank you for all your scientific guidance, opinions, and your constructive criticism that helped improve the quality of my PhD.

Most importantly, I would like to deeply thank my PhD supervisor Dr. Chang Zhang for his endless help and support during the 3 years of my PhD. Being a perfect merge between diligence and humanity, you set an example for me of how a real mentor should be. I learned so much working with you many lessons will stay with me my whole life. Scientifically, you taught me how to think and analyze my data objectively, patiently and with much open-mindedness. You always encouraged me to "be brave" and be scientifically curious, to look deep into the reasons and ask why and try to dissect the detailed mechanisms. You also taught me that there is no shame in accepting what we can't fully understand or explain bearing in mind results doesn't always have to be as we expect. On the personal level, you showed me that intelligence alone without discipline and hard work cannot get things done. I learned from you how to be ambitious and focused yet thoughtful and compassionate. The way you listened to me and discussed my ideas instead of telling me what to do helped me become a more confident and independent researcher, made me feel respected and heard. Your patience and perseverance taught me to be more patient myself (a trait I lacked) and not give up even after consecutive failures. For everything you did for me since the day you accepted me as a master 2 intern until today, for every advice and support and push I deeply thank you! I also would like to thank Prof. Mona Diab you for her continuous follow up and care from Lebanon and for your help to find me a scholarship that made PhD dream possible. I want to thank my whole MLR team, every current member and the ones that left us, I surely have learned at least a bunch of lessons from each one of you especially:

The great Dr. Laura Corbo, thank you for all the valuable scientific advices, and more importantly thank you for your compassion and generosity. I will never forget how kind and accommodating you were to me when I was alone at Christmas. Dr.Ali Choucair, I can't thank you enough for saving my life several times when I first arrived to France : you witnessed and helped me get through Bedbugs, knife wounds (Saffa dammi!:p), Water-soaked iphones (rice box:p) and many more I can't remember… I was such a mess and you never failed me every time I asked for an advice or a favor. Thank you for selflessly sharing your knowledge with everyone in the lab and most importantly, thank you for all the paperwork you helped me get through!! Dr.Hanin Lattouf and R'ana Ramouz-Charpentier (Azizam), thank you for being so warm and welcoming and for taking the time to teach me when I first arrived as a master 2 student. You were the first people to show me how to do the basic experiments in the lab and were patient with me every time I messed up and taught me how to learn from every one of them.

Dr.Lucie Malbeteau, thank you for all the interesting scientific discussions and guidance (sho ana w sho enti:p). Thank you for knowing so much about menin (it made me feel less alone), and for always finding some time for me when I need you though you are always busy! "I don't have time":p . Thank you for having the same twisted sense of humor like me and for all the motivating and reassuring words you always told me.

Dr.Romain Teinturier, Thank you for helping me when I first came to the lab, for explaining and discussing the project with me over and over again , and for all the work you have done that made my work afterwards possible.

Dr.Henri-Phillipe Conan, thank you for all your advices and support, for your friendly working personality and your kindness. I will never forget when I first came to the lab and we were eating in the cafeteria, there was not enough places for me to join everyone, and then you directly got up and sat with me on another table so that I don't be alone.

Dr.Coralie Poulard, Thank you for all your scientific suggestions and contributions.

Julien Jacquemetton, thank you for all your technical help and for being our I.T expert! And for saving my life many times by fixing a frozen software or broken computer.

Cecile Languilaire, and Ausra surmielova thank you for all the technical help you offer the team.

Louisane Eve, thank you for your hour help and cooperation in the short time you've been in the lab. Special thanks for your effort and time to help me translate my abstract to French.

I also can't forget the kind and helpful Farida Nasri that made her presence once a week in the lab so effective. Thank you for all the buffers and solutions you prepare for us, you save us lots of time, thank you for all the personal discussions and help and all the "French tutoring"

As for Yakun Luo, Chau Pham, and Lara Noureddine thank you for all your help and for being great colleagues and friends creating a joyful atmosphere to work in: Thank you, Lara, for being so helpful in the short time you have been in the lab (since you spent half of your phd in Lebanon:p), thank you for preparing buffer A(though you always finish it so it's kinda ur job:p), thanks for changing my washes and heating my media to save me time and for tying my lab coat. Most importantly, thanks for the laughter, for talking to yourself too loudly and confusing everyone around you, and for keeping plenty of office supplies on your desk (I might have stolen a couple of sticky notes), and most importantly thank you for having a worse memory than me making me feel better about myself Thank you, Yakun, for every time you allowed me to reveal before you and ordered primers and antibodies for me when I was busy, for being patient with me even after asking you the same question 10 times (I have a bad memory), thank you for the important and inspiring scientific discussions as well as experimental help, it was a pleasure working with an enthusiastic, nice and cooperative person like you.

As for Chau (known previously as Ha!), you might have changed your name but you are still the same hardworking, responsible, smart and witty human I knew since my first year in the lab. Thank you for every time you gave me cells or passed mine, for leaving your work to help me find an antibody or help me with a software (I'm too old for technology), for refreshing my memory about concentrations, volumes, and calculations! For notifying me with bank holidays, and for running to help every time you hear the loud and scared Chau!! And most importantly I want to thank you for being smart enough to know when I need you to crack a joke and when to stay silent or give me a serious quick response.

I also want to thank the Véronique Maguer-Satta's team especially Kevin Geistliche and Nora Jung for the technical help and interesting scientific and personal discussions. It was a pleasure sharing the floor with you, it felt so comfortable and easy to work in such a friendly and nice atmosphere.

To all my colleagues that became friends and family: Lea Jabbour, Mario Flores Violante, Kawtar Arizkane, and Diana Farhat, you made the hard days bearable and the good ones memorable, I can never thank you enough: Lea! thank you for all the help especially recently and for the motivation and hope even when it seemed to be none. Thank you for all the gatherings, food and "fuzed tea"! and I'm sorry again for destroying your kitchen! :( Most importantly, thank you for your understanding and kindness and being a good friend when things get tough… Mario habibiiiiiiiiiii my favorite grandchild! Thank you for making the bench work fun, always having a good response to any situation, a messed-up gel or a messed-up heart! :p thank you for all the jokes, cursing, n'import quoi words in Arabic that doesn't make any sense (mainly insults). I will remember you every time I eat kebbe and shawarma or visit hotel de Ville or touch my belly button or get lost in charpenne :p. Thank you for the best warm hugs (pre-COVID) and for taking care of your old and grumpy grandma! Koukki! my colleague, my best friend, my bundle of joy, my favorite distraction, and my cause of trouble (mashboheen:p)! Thank you for all the emails you wrote for me, the phone calls you made for me and the tons of calories you gave me stuffing my drawer with biscuits and chocolates and "nuages de lait" (though you ate most of them:p) , and thank u for never forgetting to turn off the lights even when im still there ! I can go on about you for pages but there is not enough time and space for that (ya rab ma enta 3aref ba2a!:p) so ill keep it simple , thank you for being one of the reasons I wake up excited to go to the lab and for being a cool breeze of fresh air on a hot summer day! (you kept me sane!) thank you… As for Diana, my person, my support system, and my anchor! There are not enough words to thank you, none of that could have been possible without you, life itself in France couldn't have been possible without you. I can't imagine how my PhD could have been sitting on my desk without getting punched in the back few times or working on a bench without someone stealing my pipettes and TBST, without someone shopping from my drawer and distracting me with not at all important chats and jokes. I also can't imagine how my life could have been facing one challenge after the other and wanting to just give up, without your words and actions pulling me up again and helping me move forward. Watching you closely as a scientist and as a human being made me see life from a different perspective and gave me the courage and determination to search myself and work on it to become the person I am today. Miles away from our country and home we found a home in each other's company, and we formed a strong, unbreakable, and ridiculously hilarious team of two. No matter what happens or where life takes us home will always be where you are…. The best part of my PhD other than "the degree" itself of course was meeting you! So, thank you, Diana Farhat, for touching and changing my life forever! I also want to thank all my friends and big family in Lebanon for emotionally supporting me throughout this hard journey especially my last year of PhD that was extra hard due to the worldwide special circumstances, I want to thank everyone who had faith in me and believed I could do it, special thanks to Asmaa Khalil, Ahmed Hawi, Samer Hijazi, and Rana Lebdy for listening to me nag and complain about it.

Last but not least I want to thank the unseen real soldiers and heroes of my story, the reason I thrive and work hard and want to be better. Mom and Dad, you might have not taught me how to be a good scientist, but you sure taught me how to be a good human. Growing up with you as role models and examples I became familiar with qualities like ambition, sacrifice, dedication, discipline, and hard work, all these qualities shaped my personality and helped me become a better researcher also. You have given me and my siblings a lot and sacrificed a lot more so that we can have opportunities to be better people, and for that I thank you! I dedicate all my hard work and success to you, and I will try my best to always make you proud! I love you! : Table 1:

List of Figures:

List of Tables

Abstract

The MEN1 gene, initially known to be associated with the development of multiple endocrine tumors (MEN1 disease), has been shown over the recent years to play a role in hormone dependent cancers like prostate and breast cancer. Studies have shown that menin, encoded by MEN1, can play a dual role in breast cancer. While MEN1 mutations cause higher risk of breast cancer initiation, menin is also shown to interact with and positively regulate the expression of estrogen receptor alpha (ERα) in ER+ breast cancer. Thus, menin plays a tumor suppressive role in normal mammary cells during tumor initiation procedure, and an oncogenic role in ER+ breast cancer initiated by other factors. However, the underlying mechanisms behind this regulation remain elusive.

To further address this issue, we carried out both clinical and experimental investigations and found that MEN1 silencing led to reduced ERα expression on the transcriptional and protein levels in ERα-positive cell lines. While Dreijerink et al., showed that menin regulates the ESR1 enhancer and promoter via MLL complex, we demonstrated that menin also binds and regulates the proximal ESR1 promoter, probably in an MLL-independent way. Interestingly, by analyzing the expression of menin in a cohort of 354 human breast cancers, our clinical data showed that reduced menin expression was correlated more with the luminal B breast cancer subtype and ER-breast cancer. Consistent with our clinical data, we further demonstrated that menin interacts with GATA3 and FOXA1, co-factors in ESR1 regulation and known marker of the luminal A subtype. Taken together, our data provide relevant clues to the important role of menin in the formation of breast cancer subtypes, likely related to its regulatory role on ERα.

In parallel, we also showed that menin inactivation in BC cells led to reduced cell proliferation in 2 ERα-positive BC cell lines. Searching for molecular explanations, we found that, though MEN1 knockdown activated the mTORC1 pathway, it led to reduced MYC expression on both transcriptional and protein levels in ER+ BC cell lines. We uncovered that menin bound to the MYC promoter and enhancer and regulated the expression of its target genes in these cells. Clinically, we also found that menin expression was negatively correlated with the expression of mTOR and its downstream genes, and more interestingly, with two inhibitory MYC-related genes.

Taken together, my thesis work provides relevant clues to the important role of menin in the formation of breast cancer subtypes, and, for the first time, to the role of menin in the regulation of the mTORC1 and MYC pathways in BC cells.

Résumé

Le gène MEN1 codant pour menin a d'abord été associé au développement de Néoplasies endocriniennes multiples de type 1 (NEM1 syndrome), avant d'être récemment impliqué dans des cancers hormono-dépendants tels que le cancer de la prostate et du sein. Des études ont montré que menin peut jouer un double rôle dans le cancer du sein. En effet, si les mutations germinales du gène MEN1 prédisposent les patientes au développement du cancer du sein, menin interagit avec le récepteur des oestrogènes alpha (ERα) et régule positivement son expression dans le cancer du sein ERα-positives. Ainsi, menin joue un rôle de suppresseur de tumeur dans les cellules saines ou précancéreuses, et un rôle d'oncogène dans les cellules de tumeurs mammaires ER+ initiées par d'autres facteurs. Cependant, les mécanismes sous-jacents de cette régulation restent inconnus.

Dans l'ensemble, mes travaux de thèse fournissent des indices pertinents sur le rôle important de menin dans la formation des sous-types du cancer du sein, et mettent en évidence pour la première fois un rôle de menin dans la régulation des voies mTOR et MYC dans les cellules du cancer du sein. 1) Generalities (incidence):

Cancer is defined as a disease that results from the uncontrolled proliferation of normal cells after acquiring one or several gene mutations forming a tumor mass of cells (NIH Curriculum Supplement). Breast cancer origins from the breast tissues, mainly the mammary ducts and glands (NCI Dictionary of Cancer Term). It is a very common and frequent malignancy among women and one of the major causes of morbidity worldwide. Its prevalence has expanded such that it ranked in the second place after lung cancer in incidence considering all cancers occurring in women (Ferlay et al.,2009). There were over 2 million new cases in the world in 2018, where France ranked 4 th with an incidence Age-standardized rate of 99.1 per 100,000, followed by 93.6 in the UK, and 84.9 in US [START_REF] Bray | Global cancer statistics: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF].

Despite the increasing efforts to raise awareness and encourage early detection all over the world for breast cancer (for example, many countries like France recommend regular mammography tests for women above 50 years), many women still discover this disease late at its advanced stages, which makes it even harder to treat. Late detection often is characterized by the tumor spread and metastasis, in addition to its histological and molecular heterogeneity and complexity.

2) Risk Factors:

There are a lot of risk factors associated with breast cancer development. Taking into consideration that women who live to the age of 85 will have a 1 in 9 lifetime chance of developing breast cancer, many women are at higher risk than others and these are usually advised to take several measures from the simple annual checkups and surveillance to the more complex (if necessary) prophylactic surgery (oophorectomy or mastectomy) and chemoprevention.

a) Age:

Like many diseases, especially cancers, age is one of the most documented associated risk factors for breast cancer as well. Breast cancer incidence is extremely low before age 30 with incidence of less than 25 cases per 100,000, after which it increases linearly to reach 500 cases per 100,000 at the age of 80. Thus, the incidence rate being augmented especially between the age of 55 and 70 [START_REF] Ries | SEER Cancer Statistics Review[END_REF] (Anders et al.,2010).

b) Genetic factors:

About 20% of breast cancer patients show genetic predisposition by having a positive family history of breast cancer, and 5% of patients showed specific germline mutations [START_REF] Easton | Breast and ovarian cancer incidence in BRCA1mutation carriers. Breast Cancer Linkage Consortium[END_REF]. Familial breast cancer was first described over 135 years ago [START_REF] Broca | Traité des tumeurs[END_REF]. Since then, several studies have followed to define levels of risk associated with varying degrees of positive family history. The results have showed that compared to individuals with no family history of breast cancer, the estimated relative risk associated with a first-degree relative who developed breast cancer at 50 years of age or older is 1.8 compared with 3.3 for a first-degree relative who developed breast cancer at an age less than 50 years. The relative risk associated with having a second-degree relative with breast cancer is 1.5, two first-degree relatives (e.g., mother and daughter) is 3.6 (Goldgar et al.,1996) [START_REF] Kelsey | Epidemiology of breast cancer[END_REF]. The BRCA1 and BRCA2 genes are perhaps the most famous genes associated with inherited susceptibility to breast and ovarian cancers [START_REF] Brody | Breast cancer susceptibility genes. BRCA1 and BRCA2[END_REF]. Studies have showed that 55 to 70% of women with BRCA1&BRCA2 mutations eventually develop breast cancer (Nielson et al.,2016). In addition, 13 polymorphisms in 10 additional genes were discovered most of them are responsible for rare genetic syndromes like Li Fraumeni syndrome, Cowden syndrome, ataxia-telangiectasia and MEN1 (multiple endocrine neoplasia syndrome) (please see III 5, page) [START_REF] Brody | Breast cancer susceptibility genes. BRCA1 and BRCA2[END_REF] [START_REF] De | Genes other than BRCA1 and BRCA2 involved in breast cancer susceptibility[END_REF].

c) Reproductive or hormonal factors:

Studies have shown that women who began menstruating before the age of 12 had a relatively higher risk for invasive breast cancer compared to those who began after the age of 15. However, at the other end of the reproductive period, those who did not reach menopause until age 55 or after showed a relatively higher risk compared with those who experienced menopause before the age of 45 [START_REF] Vogel | Breast cancer risk factors and preventive approaches to breast cancer[END_REF]. Based on these data, it was suggested that the risk of breast cancer observed from these gynecologic variables was a simple function of the number of ovulatory menstrual cycles that a woman undergoes during her lifetime (Collaborative Group on Hormonal Factors in Breast Cancer 2012). As women who had both ovaries removed before the age of 40 showed 45% reduction in risk compared with women who undergo a longer period of exposure to estrogen was used to explain the increase in the risk of breast cancer development (Brinton et al.,1983a). Breast cancer risk also increased if a woman was nulliparous or experienced her first live birth at or after the age of 30 compared to a woman with a first live birth at an age less than 20 (Brinton et al.,1983b) (Collaborative Group on Hormonal Factors in Breast Cancer 2002). Having at least one child as well as breast feeding is associated with a decrease in the long-term risk of developing breast cancer compared with risk among the nulliparous, and this protective effect increases with number of children (Collaborative Group on Hormonal Factors in Breast Cancer 2002).

d) Obesity:

Epidemiological studies have also shown that body mass index (BMI) could affect the incidence and outcomes of breast cancer. While in premenopausal women BMI was not that important risk factor, in postmenopausal women, however, it is strongly associated with increased incidence of breast cancer [START_REF] Tretli | Height and weight in relation to breast cancer morbidity and mortality. A prospective study of 570,000 women in Norway[END_REF]. This observation was explained by the fact that postmenopausal women rely on their adipose tissue as an important extragonadal source of bioavailable estrogens and long exposure to estrogen increases the risk of initiation and promotion of breast cancer (Clemens et al.,2001) [START_REF] Verkasalo | Circulating levels of sex hormones and their relation to risk factors for breast cancer: a cross-sectional study in 1092 pre-and postmenopausal women (United Kingdom)[END_REF]. In addition, several studies also suggested that high BMI is associated with increased levels of insulin and insulin like growth factors, which have been associated with increased risk of breast cancer [START_REF] Suga | Molecular significance of excess body weight in postmenopausal breast cancer patients, in relation to expression of insulin-like growth factor I receptor and insulin-like growth factor II genes[END_REF] [START_REF] Goodwin | Fasting insulin and outcome in early-stage breast cancer: results of a prospective cohort study[END_REF].

Other lifestyle and environmental factors could also be mildly associated with breast cancer development like smoking, alcohol consumption, diet, stress, lack of exercise, hormone replacement therapy, and exposure to carcinogens or radiation.

3) Mammary gland anatomy and histology:

The mammary gland is a milk-producing gland regulated by the endocrine system and is a main characteristic of all female mammals, although it's also present in males but however in a rather primitive or nonfunctional form. Mammary gland is composed of a conical disk of glandular tissue covered in fat to give it its characteristic shape. The glandular tissue itself is made up of 15-20 lobes composed of solid cords of ductal cells; each lobe is subdivided into many smaller lobules, separated by broad fibrous suspensory bands (Cooper's ligaments), which connect the skin with the fascia, or sheet of connective tissue, that covers the pectoral muscles beneath the breast. Each lobe is drained by a separate excretory duct which converge beneath the nipple and widen into milk reservoirs, before narrowing again to emerge as pinpoint openings at the summit of the nipple. The areola, a circular disk of roughened pigmented skin surrounding the nipple, contains circular and radiating muscles (to help erect the nipple and facilitate lactation) as well as sebaceous glands to provide lubrication for the nipple during nursing. Under the influence of estrogens from the maturing ovary at puberty, the ductal cells proliferate and form branches. After ovulation, progesterone causes the terminal ductal cells to differentiate into the milk-producing cells, which form acini.

Interspersed with these cells are smooth muscle cells, which can contract and assist in the ejection of milk. The acini are collapsed or filled with desquamated epithelium (epithelium that has been shed), until the stimulus of pregnancy causes proliferation of all the epithelial cells. The actual secretion of milk is induced by prolactin from the pituitary and somatomammotropin from the placenta (Encyclopaedia Britannica 2018). Fig1 Breast cancer was initially histologically divided into several subtypes based on its invasive or not growth features, namely in situ carcinoma and invasive (infiltrating) carcinoma. The carcinomas can be further divided into ductal or lobular depending on its origin, if it rises from the inner wall of the mammary ducts or the mammary glands respectively (Malhotra et al.,2010). The ductal carcinoma in situ (DCIS), being more common than the lobular carcinoma in situ (LCIS), can be further sub-classified into five subtypes: Comedo, Cribiform, Micropapillary, Papillary and Solid. The invasive carcinoma can also be divided into several subtypes such as: infiltrating ductal, invasive lobular, ductal/lobular, mucinous (colloid), and tubular, medullary and papillary carcinomas (Malhotra et al.,2010). With the infiltrating ductal carcinoma (IDC) being the most common subtype accounting for 70-80% of all invasive lesions (Li et al.,2005). IDC is further sub-classified as either well-differentiated (grade 1), moderately differentiated (grade 2) or poorly differentiated (grade 3) [START_REF] Lester | Protocol for the examination of specimens from patients with invasive carcinoma of the breast[END_REF]. Tumor invades into subserosa or non peritonealized pericolic or perirectal tissues (size more than 5cm) T4

Tumor perforates visceral peritoneum and invades other organs

Regional Lymph Node (N) degree of invasion

Nx

Regional lymph node invasion can't be assessed N0 Absence of regional lymph node invasion

N1mi

Presence of micro metastases (less than 2mm)

N1

Metastases to 1 to 3 regional lymph nodes

N2

Metastases to 4 or more regional lymph nodes N3

Metastases to at least 10 regional lymph nodes points. The scores for each of these three criteria are then added together to give an overall final score and corresponding grades (Bloom & Richardson 1975) [START_REF] Genestie | Comparison of the prognostic value of Scarff-Bloom-Richardson and Nottingham histological grades in a series of 825 cases of breast cancer: major importance of the mitotic count as a component of both grading systems[END_REF]. Table 2 Characteristics Score

Distant metastases (M) Absence or presence of metastasis

Tubules formation (%)

Majority or tumoral cells (˃75%) 1 Moderate (10-75%) 2 Rare or absence of tubules (˂10%) 3

Nuclear pleomorphism

Normal cell size and shape, small nucleus [START_REF] Makki | Diversity of Breast Carcinoma: Histological Subtypes and Clinical Relevance[END_REF] (Perou et al.,2000).

There are four main molecular subtypes:

i. Luminal A: is ER positive and or PR positive, HER2 negative and Ki 67 low, causing it to grow slowly and have a significantly good prognosis, being vulnerable to hormone therapy. About 50% of the diagnosed breast cancers are luminal A [START_REF] Rosai | s Surgical Pathology 10e[END_REF].

ii. Luminal B: is ER positive and or PR positive, either HER2 positive or negative, and Ki67 high, having a worse prognosis than luminal A type and variable response to endocrine and chemotherapy [START_REF] Rosai | s Surgical Pathology 10e[END_REF].

iii. HER2 enriched: is ER and PR negative, HER 2 positive (amplification mutations in the coding region of HER2 gene), high Ki-67 expression and common TP53 mutations. They are more aggressive than luminal subtypes but have good response to the therapies that target HER2 receptor [START_REF] Rosai | s Surgical Pathology 10e[END_REF].

iv.

Triple negative / basal-like: characterized by molecular expression of basal cytokeratines like (CK5,6,14,17) and/or EGFR positive, ER negative, PR negative, HER2 negative, high Ki-67 and TP53 mutation. This type has the worst prognosis and frequently seen in BRAC1 mutated patients. It has no response to endocrine therapy or trastuzumab but appears to be sensitive to special kinds of chemotherapy (platinum-based) [START_REF] Rosai | s Surgical Pathology 10e[END_REF]) [START_REF] Shawarby | Molecular classification of breast cancer: An overview with emphasis on ethnic variations and future perspectives[END_REF].

Recently, due to the heterogeneity found among this group of tumors, attempts have made to divide it into seven subtypes: basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal-stem-like (MSL), luminal androgen receptor (LAR), and an unstable cluster (UNS). The Androgen Receptor (LAR) subtype display a luminal phenotype, characterized by high expression of Androgen receptor (AR) and luminal lineage-driving transcription factors (MDAMB453 cell line) [START_REF] Collins | Androgen receptor expression in breast cancer in relation to molecular phenotype: results from the Nurses' Health Study[END_REF] (Lehmann et al.,2011). Breast cancer development is a multi-step process that occurs over an extended period of time, arising from early pre-invasive lesions to invasive metastatic carcinomas. However, not all subtypes present the same malignant phenotypes, such as self-sufficiency in growth signaling, resistance to apoptosis, invasion, and metastasis and abnormal angiogenesis, meaning that the molecular mechanisms behind these phenotypes could vary according to the subtype.

Generally, alterations in signal transduction pathways for mitogenic signals, cell cycle control, DNA repair and epigenetic gene expression modification systems happen in all subtypes but not in the same variation. For example, among the TNBCs, different patterns of molecular alterations exist in terms of RNA expression, somatic mutations and copy number variations, which tend to cluster in genes implicated in specific pathways. The BL1 subtype shows high levels of DNA-damage response and cell-cycle regulation genes like TP53 mutations (highest rate 92%), high gain/amplifications of MYC, CDK6, or CCNE1 and deletions in BRCA2, PTEN, MDM2, and RB1 [START_REF] Bareche | Unravelling triple-negative breast cancer molecular heterogeneity using an integrative multiomic 165 analysis[END_REF]; the BL2 subtype possess enriched growth factor signaling and strong metabolic activity and displays a highly proliferative phenotype.

JAK/STAT, TNF and NFkB genes that are involved immune cell and cytokine signaling are highly expressed in the IM subtype. Mesenchymal-like TNBC subtypes, M and MSL, display similar expression profiles with the previous subtypes with a unique enrichment in MSL of angiogenesis-and stem cell-associated genes, and low claudin expression. As for the LAR subtype, they display a luminal pattern of gene expression like high levels of FOXA1, GATA3, SPDEF, and XBP1 with elevated mRNA and protein levels of androgen receptor (AR), overlapping in 82% of cases with luminal-A-or luminal-B-intrinsic subtypes. As a result, LAR tumors display high number of mutations in PIK3CA, KMT2C, CDH1, NF1, and AKT1 [START_REF] Bareche | Unravelling triple-negative breast cancer molecular heterogeneity using an integrative multiomic 165 analysis[END_REF]. Studies on HER2+ BC showed in addition to HER2 (also called EGFR2 or ERBB2) mutations, mutations in TP53, PIK3CA, MLL3, MT-ND2, FAT2, NF1 and GATA3 genes (Zongbi et al.,2020) [START_REF] Lesurf | Genomic characterization of HER2-positive breast cancer and response to neoadjuvant trastuzumab and chemotherapy-results from the ACOSOG Z1041 (Alliance) trial[END_REF]. Multiple signaling pathways are also enriched such as: cell cycle, PI3K-Akt signaling, p53 signaling, Wnt signaling, focal adhesion, TGF-beta signaling, ErbB signaling, and immune function [START_REF] Lesurf | Genomic characterization of HER2-positive breast cancer and response to neoadjuvant trastuzumab and chemotherapy-results from the ACOSOG Z1041 (Alliance) trial[END_REF]. In another study conducted on Chinese HER2+ breast cancers, they also showed high number of mutations in CDK12 in addition to TP53(highest) and PI3KCA [START_REF] Li | Genetic mutation profile of Chinese HER2-positive breast cancers and genetic predictors of responses to Neoadjuvant anti-HER2 therapy[END_REF]. As for ER+ BCs molecular mechanisms will be discussed in detail in part III, 5.

II. ERα: functions and role in breast cancer

1) Generalities:

Estrogens are steroid hormones that are derived from cholesterol and exist as estrone, estradiol (E2), and estriol.17β-Estradiol, however, is the most dominant estrogen hormone in the circulation and plays an important role in different tissues like the cardiovascular, musculoskeletal, immune, and central nervous system in addition to its main role in the development and maintenance of reproductive system. Estradiol can also contribute to the initiation and development of target tissue malignancies like breast cancer [START_REF] Gruber | Production and actions of estrogens[END_REF]) [START_REF] Nelson | Estrogen production and action[END_REF].

The effects of E2 are mediated by the estrogen receptors ERα and ERβ [START_REF] Evans | The steroid and thyroid hormone receptor superfamily[END_REF]) [START_REF] Ribeiro | The nuclear hormone receptor gene superfamily[END_REF] which are nuclear receptors that act as transcription factors capable of promoting the transcription of hundreds of target genes, especially those associated with cell differentiation, proliferation and survival (Kos et al.,2001) [START_REF] Harris | Estrogen receptor-beta: recent lessons from in vivo studies[END_REF]. Studies over the years have showed that while ERβ plays a minor role in the uterus, mammary glands, pituitary gland, skeletal muscle, adipose tissue, and bone, it was found to be critical in mediating E2 signaling in the ovary, prostate, lung, cardiovascular and central nervous systems [START_REF] Harris | Estrogen receptor-beta: recent lessons from in vivo studies[END_REF] [START_REF] Hamilton | Estrogen hormone physiology: reproductive findings from estrogen receptor mutant mice[END_REF] (Hewitt et al.,2014) [START_REF] Deroo | Minireview: Estrogen receptor-beta: mechanistic insights from recent studies[END_REF].

Although significant progress has been made towards understanding the mechanism of ERβ signaling in response to E2, ERβ's actions and exact physiological role remain not clear due to limitations of studies [START_REF] Deroo | Minireview: Estrogen receptor-beta: mechanistic insights from recent studies[END_REF] [START_REF] Hamilton | Estrogen hormone physiology: reproductive findings from estrogen receptor mutant mice[END_REF] [START_REF] Haldosén | Estrogen receptor beta in breast cancer[END_REF]) (Marino et al., 2006). In other words, current knowledge indicates that ERα predominates ERβ actions in most tissues and is widely studied in the malignancies of these tissues like mammary gland tumors. ERα and ERβ though encoded by different genes on different chromosomes, share high sequence homology and structural similarities (Marino et al., 2006) [START_REF] Green | Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A[END_REF].

2) ERα (from gene and protein to transduction pathway)

a) Structure of the ESR1 gene

The complete complementary DNA of the estrogen receptor (ERα cDNA) was first cloned and sequenced in 1986 [START_REF] Green | Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A[END_REF]. The gene coding for ERα, ESR1, is of 140 kb in size, localized on chromosome 6q25.1 and composed of 8 exons.

Comparative analysis of the gene between species showed that gene structure is highly conserved (Koike et al.,1987). In breast cancer, studies using exome sequencing have shown that the mutations in ESR1 gene were more chromosomal amplification than point mutations [START_REF] Vincent-Salomon | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF] (Stephens et al., 2012). The NTD or AF1(A/B) domain is involved in both inter-molecular and intra-molecular interactions as well as in the activation of gene transcription in a ligand independent manner, opposite to the LBD or AF2 (E/F) domain which requires the binding of a ligand (Kumar et al.,2011). LBD domain also allows ER to dimerize and to bind to the specific ERE sequence situated on the promoters of its target genes [START_REF] Klinge | Estrogen receptor interaction with estrogen response elements[END_REF]. The estrogen receptor includes 2 more domains: the centrally situated hinge domain (D region) that has a role in receptor dimerization and in binding to chaperone heat-shock proteins (Hsp), containing a nuclear localization signal (NLS) that guides the estrogen-ERα homodimer transfer from the cytoplasm to the nucleus, and the C-terminal F domain where the ER transcriptional activity could be modulated (Kumar et al.,2011). As all nuclear receptors, ER is mainly found in the nucleus with and without a ligand [START_REF] Monje | Differential cellular localization of estrogen receptor alpha in uterine and mammary cells[END_REF] [START_REF] Kocanova | Ligands specify estrogen receptor alpha nuclear localization and degradation[END_REF]. However, in the recent years, ER and its several isoforms have been found to play a certain role in the cytoplasm (Thomas&Gutafsson 2015). ERα isoforms exist due to several splice variants in both normal mammary tissues and breast cancer (Taylor et al.,2010) [START_REF] Leygue | Estrogen receptor variants in normal human mammary tissue[END_REF] [START_REF] Pfeffer | Coexpression of multiple estrogen receptor variant messenger RNAs in normal and neoplastic breast tissues and in MCF-7 cells[END_REF]. However, the levels of expression of these variants are lower in normal than in tumor tissue and increase with breast tumor progression, likely involved in the shift from hormone dependence to independence in breast cancer [START_REF] Leygue | Estrogen receptor variants in normal human mammary tissue[END_REF]. Alternative splicing of pre-mRNA being the most occurring event, as most ESR1 splice variants are of the exon skipping variety [START_REF] Hirata | Isoform/variant mRNAs for sex steroid hormone receptors in humans[END_REF] [START_REF] Poola | Identification of twenty alternatively spliced estrogen receptor alpha mRNAs in breast cancer cell lines and tumors using splice targeted primer approach[END_REF]. Other methods of splice variant generation include exon duplication [START_REF] Pink | A novel 80 kDa human estrogen receptor containing a duplication of exons 6 and 7[END_REF] (Murphy et al.,1996) and insertion of additional coding material [START_REF] Hirata | The novel isoform of the estrogen receptoralpha cDNA (ERalpha isoform S cDNA) in the human testis[END_REF]. In addition, different tissues can utilize different upstream promoters and so form pre-mRNA with a variable length non-coding 5′ untranslated region, also some somatic mutations lead to altered stop codons and/or inclusion of intronic material [START_REF] Flouriot | Differentially expressed messenger RNA isoforms of the human estrogen receptor-alpha gene are generated by alternative splicing and promoter usage[END_REF] [START_REF] Weickert | Variants in the estrogen receptor alpha gene and its mRNA contribute to risk for schizophrenia[END_REF]. There are many names and terms to classify the splice variants: the most frequently used system, with exon deletions numbered and prefixed by a delta (Δ)

sign, e.g., ERαΔ5, ERαΔ7. Partial exon deletions are denoted by a preceding

Asterix, e.g., ERαΔ4 7 8, has a full deletion of exon 4 and partial deletions of exons 7 and 8 (Taylor et al.,2010). Perhaps the most simple nomenclature is ERα splice variant proteins named after their size. ERα 36, a 36 kDa protein, that lacks both transcriptional activation domains (AF-1 and AF-2) but it retains the DNA-binding domain, and partial dimerization and ligand-binding domains of hER-α66 (total ERα), and is localized in both plasma membrane and cytoplasm where it mediates non-genomic ERα signaling in the presence( since it retains its ability to bind E2) and absence of ligand (Wang et al.,2006) (Omarjee et al.,2017) increasing the aggressiveness and the antiestrogen resistance in breast cancer cells [START_REF] Chaudhri | Membrane estrogen signaling enhances tumorigenesis and metastatic potential of breast cancer cells via estrogen receptor-α36 (ERα36)[END_REF] [START_REF] Deng | ER-α36mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells[END_REF]. ERα-46, a 46 kDa protein, that lacks only the AF-1 domain (Flouriot et al.,2000) and is membranously localized such that it mediates the membrane-associated estrogen

d) ERα function and targets:

A. ERα signaling pathways:

i. Direct ligand-dependent genomic pathway of ERα:

The genomic or "classical" estrogen mediated ERα signaling pathway starts when estradiol binds the LBD of ERα in the cytoplasm, causing it to dissociate from the chaperone protein Hsp90 (heat-shock protein 90), dimerize, and translocate to the nucleus where it binds, via DBD, to the estrogen responsive elements (ERE) sequences situated on the promoters of its target genes [START_REF] Klinge | Estrogen receptor interaction with estrogen response elements[END_REF] (Carol et al.,2006). Fig 5

ii.

ERE independent or indirect genomic pathway of ERα:

ERα is also capable of regulating several genes that do not contain ERE in their promoters (35% of estrogen target genes lack ERE-like sequences) by interacting with other transcription factors and their response elements (Marino et al.,2006) [START_REF] Aranda | Nuclear hormone receptors and gene expression[END_REF] [START_REF] Göttlicher | Transcriptional cross-talk, the second mode of steroid hormone receptor action[END_REF], like SP-1 [START_REF] Saville | Ligand-, cell-, and estrogen receptor subtype (alpha/beta)-dependent activation at GCrich (Sp1) promoter elements[END_REF], NF-kB [START_REF] Biswas | Crossroads of estrogen receptor and NF-kappaB signaling[END_REF], c-Jun/Fos (AP1) sites [START_REF] Kushner | Estrogen receptor pathways to AP-1[END_REF], STAT5 [START_REF] Fox | Novel actions of estrogen to promote proliferation: integration of cytoplasmic and nuclear pathways[END_REF] and others. This mechanism is known as the indirect genomic signaling or transcriptional crosstalk that allows ERα to regulate a wide number of genes. Fig 5

iii.

Ligand independent genomic pathway of ERα:

Interestingly, ERα can also be activated in the absence of estrogen or other agonists, through its phosphorylation on specific residues (serine, tyrosine) by several protein kinases (PKA, PKC), growth factors (IGF, EGF), cell cycle regulators, and others [START_REF] Thomas | Estrogen receptor mutations and functional consequences for breast cancer[END_REF]) [START_REF] Nilsson | Mechanisms of estrogen action[END_REF]. This observation showed that the nuclear receptor ERα could also be found in the plasma membrane [START_REF] Pedram | Nature of functional estrogen receptors at the plasma membrane[END_REF] and interfere in cytosol signaling pathways such as phospholipase C (PLC)/protein kinase C (PKCs), Ras/Raf/MAPK, phosphatidyl inositol 3 kinase (PI3K)/AKT, and cAMP/ protein kinase A (PKA) (Marino et al.,2006). Additionally, a membrane bound G protein-coupled estrogen receptor 1 (GPER1) was discovered which promotes estrogen-dependent activation of adenylyl cyclase and epidermal growth factor receptor (EGFR) [START_REF] Yu | The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries[END_REF]. Moreover, several ERα isoforms, like ERα36 and ERα 46, are also found in the plasma membrane of several breast cancer cell lines (Omarjee et al.,2017) [START_REF] Li | Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells[END_REF]. These isoforms are capable of activating proliferative and anti-apoptic pathways like Src, PI3K/AKT, MAPK/ERK pathways in response to estrogen and sometimes without (in case of ERα 36) (Kim& Bender 2009) [START_REF] Rao | Advances in the understanding of the structure and function of ER-α36,a novel variant of human estrogen receptor-alpha[END_REF]. [START_REF] Ma | Mechanisms of aromatase inhibitor resistance[END_REF]. Cells in other tissues in the body, such as bones, liver and the uterus, also have estrogen receptors but the structure of estrogen receptor slightly varies depending on cell type, so the advantage of SERMs compared to aromatase inhibitors is their selectivity which allows them to differentiate the estrogen receptors in breast cells from that of the bone and other tissues increasing the efficiency of the treatment and minimizing the side effects. Three SERMs exist: tamoxifen, raloxifene (Evista), toremifene (Fareston). Tamoxifen being the most commonly used SERM that exist in pill form under the brand name "Nolvadex "and in liquid form under the brand name "Soltamox" [START_REF] Clemons | Tamoxifen ("Nolvadex"): a review[END_REF]. Recently and due to the acquired resistance to the previous endocrine treatments, a third type of therapy was discovered and named as the selective estrogen receptor downregulators (SERD) or anti-estrogens that blocks and damages estrogen receptors by decreasing their expression and activity. Fulvestrant (Faslodex) is the drug of use and is currently approved only for use in post-menopausal women [START_REF] Mcdonnell | Oral Selective Estrogen Receptor Downregulators (SERDs), a Breakthrough Endocrine Therapy for Breast Cancer[END_REF]. Estrogens, through the ER signaling pathway, play important developmental and physiological and pathological roles both in the normal and tumor development.

Studies from animals and humans have demonstrated that ERα is essential for the normal development of female reproductive tract, like the uterus and the ovaries, as well as the proliferation and differentiation of the mammary gland (Korarch et al.,2003). These studies have further showed that ERα is also important for male fertility and is implicated in other non-reproductive organs such as regulation of the neuroendocrine and cardiovascular systems and bone metabolism (Korarch et al.,2003) (Lin et al.,2004). Over the last decade, the research focus has been being

mainly on the role of ERα in breast cancer initiation, progression, and response to treatment [START_REF] Ali | Estrogen receptor alpha in human breast cancer: occurrence and significance[END_REF] [START_REF] Persson | Estrogens in the causation of breast, endometrial and ovarian cancers -evidence and hypotheses from epidemiological findings[END_REF].

D. ERα and its target genes' functions in breast cancer:

There are thousands of ERα target genes. One of the earliest ERα identified target genes was pS2/TFF1 (Brown et al.,1984) (Jakowlew et al.,1984) followed by many genes that were discovered by monitoring the global expression changes upon estradiol induction by SAGE or microarray strategies in BC cells (Charpentier et al.,2000) (Cunliffe et al.,2003) (Frasor et al.,2003) (Inoue et al.,2002) (Seth et al.,2002). ERα target genes are of wide and different functions, such that they can be divided into either: pro-proliferative genes, like cyclin D1 (Altucci et al.,1996), MYC (Dubik et al.,1987) (Dubik &Shiu 1988) and IGF-1 (Umayahara et al.,1994), or antapoptotic factors , like TIT-5, and EIT-6 (Seth et al.,2002), enzymes like the lysosomal proteinase cathepsin D (Elangovan & Moulton 1980); or even nuclear receptors like progesterone receptor (Yu et al., 1981), in addition to many other genes of yet unknown function. Interestingly, these global expression experiments indicated that around half of ERα target genes are down regulated upon induction with estrogen, knowing that many of these down regulated genes are cell cycle inhibitors, pro-apoptotic factors or cytokines and growth factors that inhibit proliferation, explaining the view that estrogen promotes cell survival by down regulating proapoptotic genes.

ER+ positive tumors have the advantage of benefiting from endocrine therapy, which results in positive outcomes. However, in many the cases, resistance to endocrine therapy and metastasis occur instead [START_REF] Group | Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials[END_REF] [START_REF] Ma | Mechanisms of aromatase inhibitor resistance[END_REF]. Resistance to endocrine therapy is a complex process that could be due to many factors, like alterations in growth factor receptor tyrosine kinases (RTKs) such as EGFR and HER2, or insulin-like growth factor receptor (IGF1-R) that increase ER transcriptional activity leading to reduced sensitivity to ER targeted therapies and poor outcomes [START_REF] Osborne | Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer[END_REF] [START_REF] Arpino | HER-2 amplification, HER-1 expression, and tamoxifen response in estrogen receptorpositive metastatic breast cancer: a southwest oncology group study[END_REF]) [START_REF] Song | The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane[END_REF].

Deregulation of the PI3K-AKT-mTOR pathway (PIK3CA being the most frequently mutated gene in ER+ breast cancer) (Ellis&Perou 2013) has also been involved in the process of resistance, in which the use of mTOR inhibitors has recently shown to improve progression-free survival (PFS) for patients failing previous endocrine therapies [START_REF] Sanchez | Preclinical modeling of combined phosphatidylinositol-3-kinase inhibition with endocrine therapy for estrogen receptor-positive breast cancer[END_REF] [START_REF] Baselga | Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer[END_REF]. Dysregulation in cell cycle components is common in ER+ breast cancer and CDK inhibitors are now FDA approved for use in combination with endocrine therapy to treat advanced stage ER+ tumors (Cancer Genome Atlas N. 2012) [START_REF] Thangavel | Therapeutically activating RB: reestablishing cell cycle control in endocrine therapy-resistant breast cancer[END_REF].

In addition to the non-ER-related alterations (tumors expressing wild-type ER) listed above, mutations in the ESR1 gene itself such as ESR1 amplifications or point mutations were found in endocrine therapy resistant breast tumors. The latter mainly happening in the ligand-binding domain (LBD) and leading to constitutive hormoneindependent activation of ER (Zhang et al., 1997) (Basudan et al., 2019). Some mutations cause estrogen hypersensitivity like (K303R, E380Q) [START_REF] Fuqua | A hypersensitive estrogen receptor-alpha mutation in premalignant breast lesions[END_REF] ( [START_REF] Toy | Activating ESR1 Mutations Differentially Affect the Efficacy of ER Antagonists[END_REF] or neutral, retaining hormone dependent activation function (S432L, V534E) [START_REF] Toy | Activating ESR1 Mutations Differentially Affect the Efficacy of ER Antagonists[END_REF]. The most common and well characterized point mutations are the activating mutations occurring at the Y537 and D538 residues which induce changes in protein structure, resulting in reduced ligand affinity. This may be a potential mechanism for their ligand-independent activity even upon antagonist binding and resistance to endocrine therapy [START_REF] Toy | ESR1 ligand-binding domain mutations in hormone-resistant breast cancer[END_REF] [START_REF] Pavlin | A Computational Assay of Estrogen Receptor α Antagonists Reveals the Key Common Structural Traits of Drugs Effectively Fighting Refractory Breast Cancers[END_REF]. Several studies have also shown that these mutations could also recruit coactivators like SRC-1 and SRC-3 in the absence of ligand to further potentiate ER transcription [START_REF] Gates | Proteomic profiling identifies key coactivators utilized by mutant ERα proteins as potential new therapeutic targets[END_REF][START_REF] Merenbakh-Lamin | D538G mutation in estrogen receptor-α: A novel mechanism for acquired endocrine resistance in breast cancer[END_REF]. Moreover, several studies have shown that WT and mutant ERs share classical ER signaling signatures in addition to the mutant-specific transcriptional regulation [START_REF] Jeselsohn | Allele-Specific Chromatin Recruitment and Therapeutic Vulnerabilities of ESR1 Activating Mutations[END_REF]) [START_REF] Bahreini | Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models[END_REF] [START_REF] Martin | Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance[END_REF]. There was a high overlap between ER chromatin binding sites of estrogen-stimulated WT receptor and hormone-deprived mutant receptors, indicating that the exact mechanism of how these constitutive activating mutations regulate transcription should be studied further.

3) Estrogen receptor gene (ESR1) regulation:

As I discussed above, ERα expression and levels in the tumor cells is a limiting factor that predicts the progression of the tumor and response to endocrine therapy. The availability of the receptor in the cell is a result of a fine balance between the synthesis and degradation of the receptor (protein turn over) [START_REF] Nonclercq | Ligand-independent and agonist-mediated degradation of estrogen receptor-alpha in breast carcinoma cells: evidence for distinct degradative pathways[END_REF]. The expression of ERα is regulated by several epigenetic and transcriptional factors present on the ESR1 promoter.

a) The structure of the ESR1 promoter

The human ESR1 gene, whose genomic organization was described in 1986 [START_REF] Ponglikitmongkol | Genomic organization of the human oestrogen receptor gene[END_REF], is 300 kb in size, located on chromosome 6q25.1 locus, and includes a 160 kb 5'-region containing regulatory sequences for ESR1 transcription.

The comparison between ERα cDNA sequence in human and other species have showed a high level of conservation except at the 5′-end (Koike et al.,1987) [START_REF] Krust | The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors[END_REF]) [START_REF] White | Structural organization and expression of the mouse estrogen receptor[END_REF], indicating that ESR1, just like other steroid hormone receptors, can have multiple promoters [START_REF] Hodin | Identification of a thyroid hormone receptor that is pituitaryspecific[END_REF] [START_REF] Kastner | Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B[END_REF]. To date, several exons encoding 5′-untranslated regions (UTRs) of ESR1 mRNAs have been identified, alternative splicing of these exons have resulted in the identification of different ESR1 mRNA transcripts that vary in their 5'UTR length in different breast cancer cell lines [START_REF] Flouriot | Differentially expressed messenger RNA isoforms of the human estrogen receptor-alpha gene are generated by alternative splicing and promoter usage[END_REF] [START_REF] Grandien | Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor-mRNA isoform[END_REF] [START_REF] Thompson | Identification of two estrogen receptor transcripts with novel 5' exons isolated from a MCF7 cDNA library[END_REF] [START_REF] Ponglikitmongkol | Genomic organization of the human oestrogen receptor gene[END_REF]. At least seven ESR1 promoters, situated upstream exon 1, have been identified that can control ESR1 expression differently in different cellular contexts or tissues (Kos et al.,2001).

b) Epigenetic regulation of ESR1:

A lot of studies have demonstrated that the ESR1 promoter is epigenetically regulated by many factors that can either activate or silence the gene transcription thus activating or silencing ERα expression. In 1994, Ottaviano et al., have showed that the lack of ERα expression in ER -breast cancer cell lines was due to hypermethylation of the ESR1 CpG islands (Ottaviano et al.,1994). Then, in the following years other studies showed that the ESR1 promoter was occupied by several complexes with inhibitory components like DNA methyltransferases (DNMTs) and histone deacetylases like HDAC1 and msin3A [START_REF] Ferguson | Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells[END_REF][START_REF] Ellison-Zelski | Repression of ESR1 through actions of estrogen receptor alpha and Sin3A at the proximal promoter[END_REF] which explained the mechanisms behind ERα regulation and loss of expression in ER-tumors. HDAC1 and msin3A were shown to associate together at the +1 transcription start site of the ESR1 promoter, the exact function of this complex is not well understood but it has been shown to correlate with the decrease in certain acetylation marks (AcH3K14) and increase in methylation marks (H4K20me3) thus repressing ERα expression in a hormone dependent manner Fig 8A [START_REF] Ellison-Zelski | Repression of ESR1 through actions of estrogen receptor alpha and Sin3A at the proximal promoter[END_REF]. DNA hypermethylation was also thought to be responsible for the resistance to anti-hormonal therapy [START_REF] Lapidus | Mapping of ER gene CpG island methylation-specific polymerase chain reaction[END_REF] , so the use of 5-aza-2'-cytidine (5-aza-dC) inhibitor that inhibits the action of DNMT1 have restored

ERα expression in the ER-MDAMB231 cell line [START_REF] Ferguson | Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells[END_REF]. Another study has showed that 2 molecular complexes pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1 regulate ESR1 in ERα-positive and ERα-negative breast cancer cells respectively (Macaluso et al.,2003). Fig 8B

c) ESR1 regulation by transcription factors:

Several studies showed that the ESR1 promoter was occupied by several transcription factors, like members of the AP1 [START_REF] Wang | Identification of estrogenresponsive genes by complementary deoxyribonucleic acid microarray and characterization of a novel early estrogen-induced gene: EEIG1[END_REF] and FOX (Forkhead box) family (FOXO3A (Guo & Sonenshein 2007) (FOXM1 [START_REF] Madureira | The Forkhead Box M1 Protein Regulates the Transcription of the Estrogen Receptor Alpha in Breast Cancer Cells[END_REF]. However, perhaps the most described ERα associated transcription factors are GATA3 (Lacroix et al.,2004) and FOXA1 (Bernardo et al.,2010) which activate the transcription of the ESR1 gene and are necessary for its proper functioning (Vijver et al.,2002) (Oh et al.,2006) (Lacroix et al.,2004). Recently, a study has also revealed a regulation of the ESR1 between the proximal (promoter) and distal regulatory sequences (enhancer) by a looplike complex involving GATA3, FOXA1 and the MEN1 (multiple endocrine neoplasia 1) gene product: menin (Dreijerink et al.,2017) Fig 9B . In this study, they showed, that menin binds the ESR1 enhancer region (about 70 kb upstream TSS) at sites that are also bound by FOXA1 and GATA3 and that menin recruits MLL compass-like complex to these sites thus forming a complex between ESR1 enhancer and the promoter region and regulating its expression. Another transcription factors that rather negatively regulate ERα expression are MTA1 (metastasis associated protein 1) which can either activate or suppress ESR1 expression depending on the cellular context (ER+ or ER-) by recruiting a complex to its proximal promoter and exon 1. In the ER+ MCF7 cell line MTA1

associates with TFAP2C to positively regulate ERα, while in the ER-MDAMB231 cell line MTA1 associates with IFI16 to mediate its repressive effects [START_REF] Kang | Differential regulation of estrogen receptor α expression in breast cancer cells by metastasis-associated protein 1[END_REF] Fig

9A.

Twist has also been shown to recruit HDAC1 and DNMT3B to the ESR1 promoter and suppress its transcription [START_REF] Vesuna | Twist contributes to hormone resistance in breast cancer by downregulating estrogen receptor-α[END_REF]. NFIB and YBX1 transcription factors physically interact with ER to repress its activity, and drive breast cancer cells toward a less estrogen-dependent cancer phenotype [START_REF] Dermit | Oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells resistant to PI3K inhibition[END_REF].

Studies have shown that ERα loss is a common event in metastasis of the tumors, leading to resistance to endocrine-therapy and thus worse prognosis (Kuukasjarvi et al.,1996) [START_REF] Broom | Changes in estrogen receptor, progesterone receptor and Her-2/neu status with time: discordance rates between primary and metastatic breast cancer[END_REF] diminishment, and loss (Le Romancer et al., 2011). More specifically, studies have revealed that Ser118, Ser167, and Ser305 sites are the most frequently phosphorylated sites of ERα, in which Ser305 phosphorylation activates Ser118 phosphorylation [START_REF] Oladimeji | Synergistic Activation of ERα by Estrogen and Prolactin in Breast Cancer Cells Requires Tyrosyl Phosphorylation of PAK1[END_REF], which in turn contributes to tumor progression, metastasis, and endocrine therapy resistance (Anbalagan et al.,2016) [START_REF] Park | Hypoxia induced phosphorylation of estrogen receptor at serine 118 in the absence of ligand[END_REF]. ERα is also palmitoylated at Cys-447, and studies have demonstrated that the palmitoylation of ERα is essential for its location and function in the plasma membrane [START_REF] Pedram | A conserved mechanism for steroid receptor translocation to the plasma membrane[END_REF]) [START_REF] Acconcia | Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol[END_REF]. After estrogen binding, the palmitoylation of ERα allows the activation of downstream signaling pathways [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF]. At last, although ubiquitination is the primary way of degrading ERα, different studies have showed that the functions of ERα ubiquitination is rather more complicated (Wu et al.,2017) [START_REF] Yang | SMURF1 facilitates estrogen receptor ɑ signaling in breast cancer cells[END_REF] [START_REF] Xue | Regulation of estrogen signaling and breast cancer proliferation by an ubiquitin ligase TRIM56[END_REF]. For example, while ERα ubiquitination promotes tumorigenesis in hepatocellular carcinoma, it results slower growth of breast cancer cells [START_REF] Yang | SMURF1 facilitates estrogen receptor ɑ signaling in breast cancer cells[END_REF] [START_REF] Xue | Regulation of estrogen signaling and breast cancer proliferation by an ubiquitin ligase TRIM56[END_REF].

III. MEN1 syndrome (Multiple endocrine neoplasia type 1) and

MEN1 gene:

1) Generalities:

Multiple endocrine neoplasia syndrome type 1 (MEN1, OMIM#131100) is a hereditary disease, transmitted in an autosomal dominant trait, characterized by the multiple occurrences of neuroendocrine tumors mostly seen in the parathyroid glands, endocrine pancreas and anterior pituitary gland [START_REF] Brandi | Guidelines for diagnosis and therapy of MEN type 1 and type 2[END_REF]. MEN1 syndrome whose prevalence ranges from 3-20/100,000 people and equally in men and women, could also occur sporadically (Francesca et al.,2018). One of the most prevalent manifestations of the disease is the hyper secretion of hormones such as hypergastrinemia, hyperparathyroidism and sometimes hyperinsulinemia.

Many MEN1 tumors can be benign with an only clinical outcome of hypersecretion of hormones, while other MEN1 tumors can be leading to malignancy (Francesca et al.,2005).

Tumors developed by patients show mutations and frequent loss of heterozygosity at the locus of the MEN1 gen, indicating that the MEN1 gene is a tumor suppressor. MEN1 inactivation is also involved less frequently with the development of foregut endocrine carcinoids, adrenal cortical and thymus tumors, as well as non-endocrine tumors, such as angiofibroma and lipomas (Lemos et al.,2008). 2) The MEN1 gene:

The MEN1 gene was identified in 1997 (Chandrasekharappa et al.,1997) (Lemmens et al., 1997), the cytogenetic location of this gene being on chromosome 11q13. It is known for years now that the MEN1 is a tumor suppressor in endocrine tissues but oncogenic in certain types of leukemia [START_REF] Yokoyama | The menin tumor suppressor protein is an essential oncogenic cofactor for MLLassociated leukemogenesis[END_REF]. Recently, the role played by this gene role in mammary and prostate tissues has elicited some debates, because as different studies showed some distinct effects of these gene in these types of tumors.

a) Structure, evolution, and significance:

The human MEN1 gene comprises 10 exons distributed over 7.2 kb in the chromosome 11q13 region and encodes an mRNA of approximately 2.8 kb. The coding sequence starts from exon 2 to 10 forming a total of 1830 bp and encoding the 610-amino acid protein called menin. In humans, another MEN1 transcript of 4.2 kb exist and specifically expressed in thymus and pancreas (Lemmens et al., 1997). The fact that Men1 orthologs exists in species like zebrafish and Drosophila (Manickam et al., 2014) [START_REF] Guru | Characterization of a MEN1 ortholog from Drosophila melanogaster[END_REF] but absent in Saccharomyces cerevisiae and C.elegans indicates that the gene is relatively new or "young" compared to others in terms of gene evolution. The MEN1 is highly conserved between mammals especially human and mice that show high sequence homology [START_REF] Guru | Isolation, genomic organization, and expression analysis of Men1, the murine homolog of the MEN1 gene[END_REF]. This fact was a useful tool in studying the pathology of MEN1 mutations and MEN1 syndrome through the generation of Men1-deficient mouse models (Crabtree et al.,2001) (Bertolino et al.,2003a).

b) MEN1 mutations:

More than a thousand MEN1 sequence abnormalities have been identified to date (1,133 germline and 203 somatic mutations) (171). Loss of heterozygosity (LOH) of MEN1 is a common observation in familial MEN1 patients as well as sporadic tumors (Lemos et al., 2008). The disease follows Knudson's "two hit" model in which the first hit is inherited from a parent in the familial case or sporadically as a consequence of any sudden inactivating mutation in the MEN1 gene during early embryogenesis. These mutations are scattered throughout the entire 1,830-bp coding region and splice sites and can be either frameshift deletions or insertions (41%), nonsense mutations (23%), missense mutations (20%), splice site mutations (9%), in-frame deletions or insertions (6%), and whole or partial gene deletions (1%), leading in all cases to a truncated and/or inactivated protein (Francesca et al., 2006) (Lemos et al., 2008). Unfortunately, despite this wide range of mutations discovered, it was not possible to correlate between each mutation type (genotype) and the phenotype or characteristics seen in MEN1 patients [START_REF] Wautot | Germline mutation profile of MEN1 in multiple endocrine neoplasia type 1: search for correlation between phenotype and the functional domains of the MEN1 protein[END_REF], suggesting that menin needs its different domains to exert its biological functions. Genetic polymorphism in the MEN1 gene have also been reported, a total of 24 different polymorphisms (12 in the coding region, nine in the introns, and three in the untranslated regions). It is important to differentiate between these polymorphisms and mutations, as some studies have reported that between 5% and 10% of MEN1 patients didn't show mutations in the coding region or adjacent splice sites indicating that these patients may have had mutations in non-coding sequences, including the promoter, untranslated regions and introns (Lemos et al., 2008).

3) Mouse models: Table 3 In order to study the normal biological function of the MEN1 gene in different cells and tissues and the mechanisms underlying it involvement in MEN1 disease, different laboratories around the world, including our own, have adopted gene-targeting in the mouse as approach through the generation of Men1 mutant mouse models. In general, these approaches carry out either germline (conventional mutant mice) or cell type-specific (conditional mutant mice)

disruptions of a given gene.

Conventional Men1 mutant models allowed mimicking the genetic events occurring in MEN1 patients, facilitating studies on the natural history of the disease. Results from these mouse models showed that homozygous Men1 mutant embryos (complete menin loss) died in midgestation due to neural tube disclosure, abnormal development of fetal liver and heart (Bertolino et al., 2003b), and cranial and facial developmental defaults (Crabtree et al.,2001).

These findings thus indicated the essential role played by the gene in controlling cell proliferation and differentiation in these tissues during the embryonic mid gestation stage.

Heterozygous Men1 mutant mice, on the other hand, developed multiple endocrine tumors from the age of 9 months onwards, affecting islet cells, parathyroid, pituitary, and adrenal glands, associated with hyperinsulinemia and hyperparathyroidism. In addition, thyroid follicular tumor was also found in some of the mutant mice, although it has been previously considered as a coincidental association in MEN1 patients. all, in MEN1 patients [START_REF] Crabtree | A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors[END_REF] (Bertolino et al., 2003a). 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. Importantly, at a small but substantial proportion of the mutant mice also developed breast and prostate tumors, thus suggesting that the gene could also have relevant function in these two hormone-related tissues.

Conditional Men1 mutant models were generated by crossing mice carrying the floxed Men1 allele Men1 F/F with different transgenic mice expressing Cre recombinase in given cell types or tissues and helped understand the cellular and molecular mechanisms involved in MEN1 disease and. Many models have been created: β-Cell and α-Cell Men1 mutant mice [START_REF] Bertolino | Pancreatic beta-cell-specific ablation of the multiple endocrine neoplasia type 1 (MEN1) gene causes full penetrance of insulinoma development in mice[END_REF]) (Biondi et al.,2004) (Lu et al.,2010) (Shen et al.,2010), Pan-pancreatic Men1 mutant mice (Shen et al.,2009) (Bonnavion et al.,2015), Gastrointestinal epithelium cell Men1 mutant mice (Veniaminova et al. ,2012), Parathyroid Men1 mutant mice (Libutti et al., 2003),

Prolactin-secreting-cell Men1 mutant mice (Biondi et al.,2004) (Seigne et al., 2013),

Craniofacial osteogenic cell and osteoblast Men1 mutant mice (Kanazawa et al.,2015) (Liu et al., 2017) (Lee et al., 2018), and more recently, mammary and prostate gland Men1 mutant mice (Seigne et al., 2013) (Seigne et al., 2010). The last 2 models were significant tools to understand whether and how menin is involved in hormone dependent cancers (see below in part III section 5) MEN1 gene encodes a 610 amino acid protein menin that is highly conserved among species from drosophila to human and shares no homology with other proteins or protein domains [START_REF] Wautot | Germline mutation profile of MEN1 in multiple endocrine neoplasia type 1: search for correlation between phenotype and the functional domains of the MEN1 protein[END_REF]. Menin is ubiquitously expressed and its functions can vary in a tissue specific manner, such that it plays a tumor suppressive role in endocrine organs, whereas it promotes leukemogenesis (Matkar et al., 2013). Menin is mainly a nuclear protein but it is also present in the cytoplasm and even in the cell membrane where it may interact with several cell signaling pathways.

Although menin has been so far only linked to MEN1 syndrome and leukemogenesis, it is important to note that its involvement in several other cancers emerges recently, since it interacts with a variety of proteins from different signaling pathways, causing either transcriptional activation or repression (Matkar et al., 2013).

b) Structure of the protein:

Menin has two classic nuclear localization signals NLS1 and NLS2 [START_REF] Guru | Menin, the product of the MEN1 gene, is a nuclear protein[END_REF] on its C-terminus which are indispensable for its nucleur localization. In the same Cterminus region a novel accessory NLS: NLSa was also identified. These NLSs allow menin to stay in the nucleus, a basis for its binding to the DNA of its target genes [START_REF] La | Tumor suppressor menin: the essential role of nuclear localization signal domains in coordinating gene expression[END_REF]. Menin have two functional nuclear export signals, NES1 that extend from amino acid 33 to 41and NES2 that extend from amino acid 258 to 267 in human sequence of menin. These NESs aid in its translocation between the cytoplasm and nucleus and vice versa (Cao et al., 2009). Menin also contain a SID domain extending from amino acid 371 to 387, with which it interacts with the histone deacetylase (HDAC) corepressor msin3A. Five guanosine tri phosphatase (GTPase) sites: G1 to G5 were found in menin.

Finally, menin also contain the LXXLL motif, extending from amino acid 263 to 267, that is usually present in many transcription factors. This motif allows menin to interact with nuclear receptors like ERα. Fig 11 Because menin lacks homology with other proteins, it was challenging to elucidate its exact molecular and cellular functions. Substantial efforts have been made by many groups to identify menin-interacting proteins, in the hopes of finding clues about how menin biochemically suppresses tumorigenesis. These studies, together with uncovering menin's crystal structure, provided valuable information regarding the biochemical function of menin and its ability to activate or repress gene expression. Menin, that is present at many sites of the chromatin, has been shown to interact with transcription activators or repressors, and cross talks with various signaling pathways and proteins involved in controlling different cellular activities [START_REF] Balogh | Menin and its interacting proteins: elucidation of menin function[END_REF] [START_REF] Poisson | Menin interacting proteins as clues toward the understanding of multiple endocrine neoplasia type 1[END_REF].

A. Menin interacts with epigenetic factors:

i.

Menin and the MLL/COMPASS-like complex:

The first interaction between MLL/COMPASS-like and menin was identified in 2004, indicating that menin's partnership with this complex is an activating one (Yokoyama et al., 2004), then other studies have shown that this partnership could have activating or suppressive roles depending on the tissue.

In acute leukemia the MLL1 (mixed lineage leukemia) gene situated on chromosome 11q23 can undergo chromosomal translocations with one of various partner genes, resulting in the expression of MLL1 fusion proteins (MLL-FPs) [START_REF] Slany | When epigenetics kills: MLL fusion proteins in leukemia[END_REF]) that can induce leukemia through activating the HOX genes [START_REF] Hess | MLL: a histone methyltransferase disrupted in leukemia[END_REF]. Several studies have shown the involvement of menin in the positive regulation of HOX genes. Yokoyama et al have shown that menin binds to different MLL-FP to induce myeloid transformation and the upregulation of genes like HOXA7, HOXA9 and HOXA10 [START_REF] Yokoyama | The menin tumor suppressor protein is an essential oncogenic cofactor for MLLassociated leukemogenesis[END_REF]. The same group then showed that menin links MLL proteins with LEDGF and that this complex recruited histone methyl transferases to the promoter of HOXA9 and Meis1 genes in the mouse hematopoietic cells to regulate their expression (Yokoyama & Cleary 2008). Another oncogenic role of menin/COMPASS-like roles was demonstrated by Thiel et al. They showed that menin recruits both wild-type MLL and oncogenic MLL-AF9 fusion protein to the loci of HOX genes to activate their transcription through both H3K4 and H3K79 methylation [START_REF] Thiel | MLL-AF9-induced leukemogenesis requires coexpression of the wild-type Mll allele[END_REF].

In the endocrine pancreas menin interacts with MLL1 to upregulates the expression of cyclin-dependent kinase (CDK) inhibitors (CDKIs) p18 and p27, thereby reducing beta cell proliferation. In pancreatic MIN6 and MEFs cells, menin was found to bind to the p18 and p27 promoters and recruit the compass like compass like complex that adds tri-methylation to histone H3 at lysine 4 (H3K4me3), a chromatin modification associated with transcriptional activation (Schnepp et al., 2006) (Karnik et al., 2005) (Milne et al., 2005). A decreased expression of P18 and P27 was observed in mouse and human MEN1 insulinomas (Karnik et al., 2005). These results afford the molecular mechanisms indicating that, while menin/COMPASS-like complex plays an oncogenic role in leukemia, it plays a tumor suppressive role in the endocrine pancreas.

Menin's crystal structure could be represented as a curved left hand where the C-terminus resembles the curved fingers, the middle region resembles the palm, and the transglutaminase-like domain resembles the thumb. In addition, a long β hairpin forms the N-terminal domain. The N-terminal region of MLL1 binds the deep central pocket of menin, and the further downstream sequence of MLL1 loops around the N-terminal part of menin, forming an alpha helix that directly contacts the menin N terminus (Huang et al., 2012) [START_REF] Murai | Crystal structure of menin reveals binding site for mixed lineage leukemia (MLL) protein[END_REF]. It is important to note that MLL1 and JunD compete to bind menin in the same pocket of its structure however MLL1 has more affinity (Huang et al., 2012). Based on the crystal structure of menin and MLL1/2, several inhibitors that block efficiently the interaction between the two were produced, including MI-2-2 which has been shown to inhibit HOX genes expression in leukemic cells [START_REF] Shi | Structural insights into inhibition of the bivalent menin-MLL interaction by small molecules in leukemia[END_REF], MI-503 and MI-463 which have also shown some positive results in murine models [START_REF] Borkin | Pharmacologic inhibition of the Menin-MLL interaction blocks progression of MLL leukemia in vivo[END_REF]. ii. PRMT5:

Menin binds the negative regulator PRMT5 of the Hedghog pathway. By using Men1-deficient mouse embryonic fibroblasts (MEF) and primary islets derived from Men1 mutant mice, Gurung et al provided compelling data showing that menin plays a crucial role in inhibiting the Hedgehog pathway through its physical interaction with PRMT5 and its binding to its downstream genes Gli and Gas1 promoters increasing the H4R3me2 inhibitory marks (Gurung et al.,2013). They also showed that, menin plays a tumor suppressive role by inhibiting the shh pathway via PRMT5 in mouse Men1 insulinomas (Gurung et al.,2013).

iii. EZH2:

EZH2 belongs to the polycomb repressive complex (PRC)2 which is known to add the repressive H3K27me3 histone trimethylation marks on their targets'

promoter [START_REF] Margueron | The Polycomb complex PRC2 and its mark in life[END_REF]. Menin was first reported to bind to the pleiotrophin (PTN) promoter together with polycomb repressive complex (PRC)2 in lung cancer cell lines, where it plays a tumor suppressor role by increasing the repressive H3K27me3 marks and thus repressing the expression of this proliferative gene (Gao et al., 2009). Also, in Men1 -/-MEFs, two members of PRC2, EZH2 and SUZ12, were diminished from the PTN promoter, confirming the importance of menin in allowing these factors to bind to the PTN promoter and regulate its expression. Gherardi et al recently showed in vivo and in vitro that menin recruits EZH2 via an indirect mechanism involving Akt-phosphorylation, without physically binding to EZH2, to the Inhbb promoter (gene) and repress its expression by adding H3K27me3 repressive marks (Gherardi et al., 2017). Jun D belongs to the AP1 family of transcription factors and is the first identified menin partner (Agarwal et al., 1999). Jun D has been shown to activate proliferation of menin-deficient MEFs [START_REF] Agarwal | Transcription factor JunD, deprived of menin, switches from growth suppressor to growth promoter[END_REF]. Opposite to menin/MLL interaction (which binds menin in the same pocket as JunD), menin has been shown to play a repressor role through its association with JunD and mediate this effect on different levels. First, menin was indispensable for the recruitment of HDAC1 and msin3A repressors on the promoters of JunD target genes to inhibit their expression (Kim et al., 2003). Second, menin was shown to compete with c-Jun N-terminal kinase (JNK) binding to JunD, which normally phosphorylates JunD leading to its activation (Huang et al., 2012), thus hindering the pro-proliferative activity of JUND. Menin has repressive effects on JunD-mediated transcriptional activation, however the function of menin and JunD interaction in tumorigenesis remains elusive. Agarwal et al hypothesized that, though JunD inactivation has so far not been implicated in the pathogenesis of any tumors, it is possible that tumor suppressor-like properties of JunD are due to the functional synergy between the suppressive effects of menin and JunD. Inversely, when menin is inactivated, JunD may exerts an oncogenic role, since some MEN1 missense mutations were shown to disrupt menin-JunD interaction.

ii. NFkB:

Menin has been shown to inhibit the transcription of NFkB target genes in hepatic cells. In these cells, menin has been shown to physically interact with NFkB and repress its activity by interacting with Sirt 1, a histone deacetylase (HDAC), that deacetylase lysine 310 of p65 subunit of NFkB, thus inhibiting the downstream inflammatory response and cytokine secretion (Heppner et al., et al.,2001) (Gang et al., 2012).

iii. MYC (c-Myc):

MYC belongs to the MYC oncogene family that is called "super-transcription factors" and consists of two other members, N-Myc, and L-Myc [START_REF] Adhikary | Transcriptional regulation and transformation by Myc proteins[END_REF] [START_REF] Pelengaris | c-MYC: more than just a matter of life and death[END_REF] [START_REF] Dang | MYC on the path to cancer[END_REF]. This family regulates the transcription of at least 15% of the entire genome [START_REF] Dang | The c-Myc target gene network[END_REF]. The major downstream effectors of MYC include those involved in DNA repair, ribosome biogenesis, protein translation, cell-cycle progression and metabolism, thus controlling a wide range of biological functions, such as cell proliferation, differentiation, and immune surveillance [START_REF] Dang | The c-Myc target gene network[END_REF] [START_REF] Meyer | Reflecting on 25 years with MYC[END_REF]. MYC mediates its functions by binding to Max that shares a common structural homology (BR/HLH/LZ motif) which is required for DNA-protein interactions [START_REF] Adhikary | Transcriptional regulation and transformation by Myc proteins[END_REF] [START_REF] Pelengaris | c-MYC: more than just a matter of life and death[END_REF] [START_REF] Meyer | Reflecting on 25 years with MYC[END_REF].

The MYC/Max heterodimer then activates transcription by binding to a conserved E-box DNA sequence (CACGTG) located in the transcriptional regulatory region of target genes where it recruits chromatin-modifying complexes, like GCN5 and TIP60 (histone acetyltransferases), TIP48 (ATPbinding protein), and TRRAP (transactivation/transformation-associated protein) [START_REF] Adhikary | Transcriptional regulation and transformation by Myc proteins[END_REF] [START_REF] Pelengaris | c-MYC: more than just a matter of life and death[END_REF] [START_REF] Meyer | Reflecting on 25 years with MYC[END_REF].

Myc family is frequently deregulated in human cancers (>50%). Myc deregulated expression can be induced either by retroviral promoter insertion, chromosomal translocation/amplification, activation of super-enhancers within the MYC gene, and/or mutation of upstream signaling pathways that enhance MYC stability [START_REF] Meyer | Reflecting on 25 years with MYC[END_REF]. Targeting MYC has been challenging for a long time, first due to its "undruggable" protein structure and its nuclear localization. For these reasons alternative approaches that indirectly inhibit MYC oncogenic functions have been achieved such as MYC transcription and/or translation inhibition, and MYC destabilization, or MYC/Max complex disruption like such as the MYC/Max inhibitor 10058-F4 [START_REF] Soucek | Modelling Myc inhibition as a cancer therapy[END_REF]) (Chen et al.,2018) [START_REF] Huang | A small-molecule c-Myc inhibitor, 10058-F4, induces cell-cycle arrest, apoptosis, and myeloid differentiation of human acute myeloid leukemia[END_REF].

MYC is another menin partner that collaborates with menin to both activate or repress the expression of certain genes. However, unlike Menin / MLLmediated H3K4me3 regulation on the promoter of the target gene [START_REF] Jin | c-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis[END_REF], the latest report shows that menin can directly interact with the transactivation domain (TAD) of MYC and then bind to E boxes to enhance the transcription of MYC target genes (Wu et al., 2017). The enhancement of MYC target gene transcription depends on P-TEFb, which is a key factor in promoting the regulation of MYC transcription in HT1080 and HepG2 cells. Menin and MYC can also bind to SKI-interacting protein (SKIP) co-activator at the HIV- et al., 2009). Interestingly, it is reported that menin can interact with MYC to regulate its own expression in HEK293 embryonic kidney cells, where menin and the MLL complex interact with the transcription factor FUSE binding protein 1 (FBP1/FUSBP1) to bind the FUSE site (box) on the MYC promoter and stimulate MYC transcription (Zaman et al., 2014).

1 promoter (Bres
However, the binding of menin on the MYC promoter has not been clearly defined.

C. Menin interferes in several signaling pathways:

As mentioned above, menin is mainly a nuclear protein which allows it to interact with several transcription factors and histone modifiers and play an important role in regulation of gene transcription. However, menin also exists in the cytoplasm and cell membrane, where it interacts with several signaling pathways such as:

i.

Canonical and non-canonical TGF-β pathway:

Menin interacts with SMAD3, the downstream effector of the TGF-β canonical pathway. Menin inactivation inhibited Smad 3 binding to the regulatory transcriptional sites of its target genes, thus leading to abortion of the TGF-β induced inhibitory signal of cell proliferation (Kaji et al., 2001).

Menin also interacts with Runx2 and Smad1/5, the downstream effectors of the TGF-β non canonical pathway BMP (bone morphogenetic pathway) that is responsible for the commitment of the multipotent mesenchymal stem cells into osteoblasts (Sowa et al., 2004). This may explain the abnormalities in facial and cranial bone development in Men1-null mutant embryos (Bertolino et al., 2003b).

ii. Wnt/β-catenin:

Our team together with our collaborators found that menin physically interacts with beta-catenin, and the overexpression of menin reduces nucleus accumulation of beta-catenin and suppresses its transcriptional activity in Men1-null MEFs, whereas nuclear beta-catenin accumulation was found in insulinomas from β-cell-specific Men1 knockout mice (Cao et al., 2009). Jiang et al also found that beta-catenin ablation led to the suppression of tumorigenesis and significantly improved hypoglycemia and the survival rate of Men1-deficient mice. Applying the small molecule inhibitor, PKF115-584, in Men1-deficient mice to antagonize the β-catenin signaling, also suppressed tumor cell proliferation in vitro and in vivo (Jiang et al., 2014). These results

showed that menin negatively regulates the Wnt/β-catenin pathway in pancreatic β-cells, making this pathway a clinical or therapeutic interest to treat MEN1 patients.

iii. PI3K/ PTEN/AKT/MTOR or PAM pathway:

The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathways are often regarded as a single unique pathway, since they are much interconnected. They are involved in several processes that lead to cellular growth and survival especially during stress [START_REF] Datta | Cellular survival: a play in three Akts[END_REF]. Since tumors exist in an intrinsically stressful environment (limited nutrient and oxygen supply, low pH), this pathway plays important role in cancer. Mutations affecting this pathway exist in a number of different solid and hematological tumors (breast, colon, neuroendocrine tumors (NETs), kidney cancer, and some lymphomas). The activation of the PI3K/Akt/mTOR pathway disturbs the control of cell growth and survival, which ultimately leads to a competitive growth advantage, metastatic competence, angiogenesis, and therapy resistance. These facts made this pathway a very good target for anticancer agents and many inhibitors have been designed that inhibit this pathway, some which showed great results in different cancers [START_REF] Hennessy | Exploiting the PI3K/AKT pathway for cancer drug discovery[END_REF] [START_REF] Chen | Inhibition of PI3K/Akt signaling: an emerging paradigm for targeted cancer therapy[END_REF].

The PI3K/AKT/mTOR pathway is stimulated by RTK (receptor tyrosine kinase) and cytokine receptor activation. Tyrosine residues are then phosphorylated and provide anchor sites for PI3K translocation to the membrane. Activated PI3K catalyzes the formation of PIP3, which then recruits PDK1 and AKT to the cell membrane [START_REF] Simioni | Targeting the phosphatidylinositol 3-kinase/Akt/mechanistic target of rapamycin signaling pathway in B-lineage acute lymphoblastic leukemia: An update[END_REF] [START_REF] Tuncel | Receptor tyrosine kinase-Ras-PI 3 kinase-Akt signaling network in glioblastoma multiforme[END_REF]) [START_REF] Yu | Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy[END_REF]. AKT is regulated by a variety of hormones, including insulin and growth factors [START_REF] Simioni | Targeting the phosphatidylinositol 3-kinase/Akt/mechanistic target of rapamycin signaling pathway in B-lineage acute lymphoblastic leukemia: An update[END_REF]. PDK1

phosphorylates at Thr308 in the AKT kinase catalytic region [START_REF] Szymonowicz | New Insights into Protein Kinase B/Akt Signaling: Role of Localized Akt Activation and Compartment-Specific Target Proteins for the Cellular Radiation Response[END_REF]. Activated AKT mediates the regulation of cell growth, proliferation, the cell cycle and glucometabolism by further phosphorylating the GSK-3, FoxOs, Bad, Caspase 9, nuclear transcription factor-kappa B (NF-kappa B), mTOR and p21 proteins [START_REF] Risso | Akt/PKB: one kinase, many modifications[END_REF] [START_REF] Kumar | AKT kinase pathway: a leading target in cancer research[END_REF].

mTOR, a serine/threonine protein kinase activated downstream the PI3Kassociated kinase protein family, interprets nutritional signals and regulates cell growth and proliferation [START_REF] Wei | Roles of mTOR Signaling in Tissue Regeneration[END_REF]. mTOR includes mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1, sensitive to rapamycin, composed of mTOR, Raptor and mLST8, mainly regulates cell growth and energy metabolism. mTORC2, not sensitive to rapamycin, composed of mTOR, Rictor, Sin1 and mLST1, is mainly involved in reconstruction of the cytoskeleton and cell survival (Murugan 2019) [START_REF] Kim | mTOR as a central hub of nutrient signalling and cell growth[END_REF]. mTORC1 is a downstream molecule of AKT and is activated by phosphorylated AKT. The downstream effectors of mTORC1 include two signaling pathways: 4EBP1 and S6Ks. 4EBP1 inhibits the assembly of the eukaryotic translation initiation complex eIF4F. High levels of eIF4F are frequently observed in cancer, and could be related with poor prognosis, drug resistance. The eIF4F complex is composed of eIF4A which forms the enzymatic core, eIF4E which is responsible for cap dependent translation of all nuclear encoded mRNA, and eIF4G that act as a scaffold protein to link them together and associates with 43S preinitiation complex and eIF3 (Chu et al., 2016). mTORC1 can phosphorylate 4EBP1 (Rivera-Calderon et al., 2019).

Hypo-phosphorylated 4E-BP1 has a high affinity for eIF4E, while highly phosphorylated 4E-BP1 releases eIF4E, allowing the binding of eIF4G to eIF4E to initiate the translation of relevant mRNAs [START_REF] Mahoney | Insulin signaling controls neurotransmission via the 4eBP-dependent modification of the exocytotic machinery[END_REF]. Studies have also shown that mTORC1 can activate SREBP1 through S6K1 affecting cell lipid production (Yaguchi et al., 2019) and mTOR overactivation enhances lipid anabolism in mouse models of obesity and diabetes [START_REF] Zhang | Global Phosphoproteomic Analysis of Insulin/Akt/mTORC1/S6K Signaling in Rat Hepatocytes[END_REF].

Menin has been shown to interact with AKT1 and downregulate its kinase activity in pancreatic cells. Menin suppresses both AKT1 induced proliferation and anti-apoptosis in endocrine and non-endorcine 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 reported that menin can interact with FOXO1, a downstream effector of Akt, in the hepatocytic cancer cell line HepG2 and MEFs (Wuescher et al., 2011). In the same year, a study showed that MEN1 and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors (Jiao et al., 2011). All these publications being focused mainly on endocrine cells. Interestingly, a recent study demonstrated that menin regulates milk protein synthesis through mTOR signaling in the normal mammary epithelial cells (Honghui et al., 2017). In this study they showed that menin overexpression caused significant suppression of factors involved in the mTOR pathway, as well as milk protein κ-casein (CSNK), which suggests that menin can also regulate the PI3K/akt/mTOR pathway in mammary cells.

PTEN is a tumor suppressor gene mutated in many types of cancer. As a lipid accelerated NE tumorigenesis (Wong et al., 2019). Co-mutations of MEN1 and PTEN have also been observed in a small percentage of human sporadic

PanNETs (Jiao et al., 2011) (Scarpa et al., 2017). All these results suggest that menin can play important roles in the PAM pathway at different levels.

D. Menin interacts with nuclear receptors:

As I mentioned above, menin's protein structure contains a putative nuclear receptor interaction motif (LXXLL) that allows it to bind to nuclear receptors, including ERα, AR, peroxisome proliferator-activated receptor (PPARγ), liver X receptor (LXRα) and vitamin D receptor (VDR) (Dreijerink et al., 2006) (Malik et al., 2015) [START_REF] Cheng | Menin prevents liver steatosis through co-activation of peroxisome proliferator-activated receptor alpha[END_REF]) (Drejink et al., 2009a) [START_REF] Cheng | Tumor suppressor Menin acts as a corepressor of LXRα to inhibit hepatic lipogenesis[END_REF]) (Dreijerink et al., 2009b). Dreijink et al showed that menin acts as a cofactor of ERα in which it interacts with ERα ligand-binding domain in a hormone-dependent fashion to regulate its expression by recruiting MLL-HMT to the ESR1 promoter (Dreijerink et al., 2006). Malik et al have also shown that menin bound MLL (COMPASS like complex) physically interacts with AR and act as a coactivator of AR targets in castration resistant prostate cancer (Malik et al., 2015). Menin was also found to physically interact and regulate the transcriptional activity of PPARγ and LXRα in the hepatocytes [START_REF] Cheng | Menin prevents liver steatosis through co-activation of peroxisome proliferator-activated receptor alpha[END_REF] [START_REF] Cheng | Tumor suppressor Menin acts as a corepressor of LXRα to inhibit hepatic lipogenesis[END_REF] (Dreijerink et al., 2009a). While menin enhances PPARα trans activity and prevent triglyceride accumulation in the liver, it inhibits the transcriptional activity of LXRα and repress hepatic lipogenesis. Accordingly, while menin overexpression results in reduced expression of LXRα target genes (such as lipogenic enzymes), menin on the other side acts synergistically with the nuclear receptor PPARα to control gene expression of fatty acid oxidation in the liver. These results show the importance of menin in maintaining the balance between lipid synthesis and metabolism and protecting the liver from steatosis. Menin can also interact directly with the vitamin D receptor (VDR) and enhance its transcriptional activity. mRNA levels of VDR target genes were also significantly lower in MEN1 parathyroid adenomas compared to normal tissue (Dreijerink et al., 2009b).

d) Menin is inhibited by several signaling pathways:

In addition to the various roles that menin plays in different signaling pathways, menin itself is also regulated by many other factors and signaling pathways, either activated or repressed. For example, menin expression is thought to be stimulated by somatostatin to inhibit the expression of gastrin in the stomach [START_REF] Mensah-Osman | Somatostatin stimulates menin gene expression by inhibiting protein kinase A[END_REF]. Another activating regulation involves TGFβ pathway that menin normally stimulates via its interaction with the downstream effector SMAD3. It has also been shown that TGF-β increases menin's expression in parathyroid, MLL-AF9-transformed leukemia cells and mouse hepatocytes (Sowa et al., 2004), [START_REF] Zhang | Menin expression is regulated by transforming growth factor beta signaling in leukemia cells[END_REF]. Menin can also be repressed, such as in the case with K-ras that down regulates menin expression through recruiting the DNA methyl transferase 1 (DNMT1) on the MEN1 promoter [START_REF] Wu | Interplay between menin and K-Ras in regulating lung adenocarcinoma[END_REF]. It was reported that glucose, via PI3K/Akt pathway, inhibited menin expression in the endocrine pancreas (Zhang et al., 2012). Furthermore, menin was found to be prone to posttranslational modifications such as phosphorylation, ubiquitylation and SUMOylation, and to be post translationally repressed by miR24-1 (Matkar et al., 2013).

All menin interactions mentioned above are summarized by Figure

5) Menin and breast cancer:

a) Molecular studies:

The first molecular clues of the possible involvement of menin in breast cancer came from the observation that menin protein binds physically to ERα. In 2006, Dreijerink et al. revealed that menin could physically interact with several nuclear receptors, including

ERα, playing the role of a cofactor. In the same study, they detailed that menin binds to the AF2 domain of ERα and coactivates the transcription of TFF1, the estrogenresponsive ERα target gene through recruitment of compass-like complex that trimethylating H3K4 on the TFF1 promoter (Dreijerink et al., 2006). Then, in 2009, Imachi et al. confirmed the above wrok in the ERα + MCF7 breast cancer cell line, by demonstrating that menin binds and coactivates ERα in a hormone (estrogen) dependent manner. They also documented that menin could be a predictive factor of tamoxifen resistance. For that, they conducted a clinical study with 65 ER-positive breast cancer samples-all of which had been treated with tamoxifen for 2-5 years as adjuvant therapies. They observed that menin positive tumors had worse clinical outcome and were more resistant to tamoxifen treatment than menin negative tumors in these samples (Imachi et al., 2010). Thus, these results suggested an oncogenic role for menin, raising the controversy about the exact role of menin in breast cancer. A recent study conducted by Dreijerink et al., in 2017 demonstrated that menin regulates the expression of the ESR1 gene (as I described above) through an upstream enhancer, via a looping structure connecting the TSS bound menin&MLL1/2 (adding H3K4 trimethylation marks), to the enhancer bound transcription factors GATA3&FOXA1 (Dreijerink et al., in 2017).

b) Mouse models:

Meanwhile, we have observed that aged heterozygous Men1 mutant mice developed, in addition to endocrine tumors (pancreas, pituitary parathyroid, etc..), mammary gland carcinomas in female and prostate cancers in male mutant mice at low but substantial frequencies (Bertolino et al., 2003a). To further confirm and understand the role played by menin in the development of mammary lesions, we generated a conditional mammary-specific Men1 knock-out mice 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, known to be expressed in luminal mammary epithelial cells. The results showed that female Men1 F/F -WapCre mice developed high incidence of early mammary intraepithelial neoplasia (MIN), precursor lesions of mammary cancer, in comparison with control Men1 +/+ -WapCre mice (Seigne et al., 2013). 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. Also, cell membrane expression of beta-catenin and E-cadherin was nearly absent in the mammary lesions of Men1 F/F -WapCre mice compared with control mice; neither E-cadherin nor beta-catenin was detected in the TS1 cell line derived from the mouse Men1 breast cancer (Seigne et al., 2013). Consistent with the observations made in the mouse, through the analyses of two series of human breast cancers, we demonstrated that a substantial proportion of breast cancers displayed reduced menin expression.

c) Human studies:

The decisive evidence of possible involvement of menin in breast cancer came from the observation that female MEN1 patients were at higher risk to develop breast cancer (Dreijerink et al., 2014). , PTEN, etc.) in breast cancer (Nik-Zainal et al., 2016). In addition, several other case reports have identified MEN1 mutations among breast cancer patients, some being independent of germline mutations in BRCA1 and BRCA2 genes which are usually associated with hereditary breast cancer Zorka et al., 2012) (Jeong et al., 2014), and others being combined with BRCA mutations (Papi et al., 2009) (Ghataorhe et al., 2007).

In
(

d) Other clues menin's role in breast cancer:

As mentioned above, many different factors and signaling pathways are involved in mammary cell carcinogenesis. By investigating the possible link between the formers and menin, one may gain clues into the eventual role played by menin in breast cancer.

Epigenetic factors:

Interestingly, several epigenetic factors reported to be relevant in the carcinogenesis of mammary cells are known menin interacting partners. Histone methylase MLL1

(KMT2A) and MLL4 (KMT2B), menin's most known partners, have been shown to coordinate with ERs (ERα&ERβ) to mediate the estrogen-induced transcriptional activation of HOXB9 gene that is a critical player in mammary gland development and breast cancer (Ansari et al., 2011). The HDAC family, mainly HDAC1, known partners of menin (Kim et al., 2003) [START_REF] Wu | Regulation of cyclin B2 expression and cell cycle G2/m transition by menin[END_REF], has also been implicated in the regulation of ERα expression, mainly silencing the ESR1 gene, and even been proposed that this is how ERα expression might have been lost in ER negative breast cancer (Kawai et al., 2003) (Macaluso et al., 2003). The HMTs PRMT5 and EZH2

are other examples of common partners between menin and the ERα pathway (Gurung et al., 2013) (Gao et al., 2009). EZH2 has been shown to inhibit the transcription of estrogen-responsive genes through its association with the transcriptional corepressor REA (repressor of estrogen receptor activity) (Hwang et al., 2008). Although there is currently no clear evidence of interaction between PRMT5 and ERα, PRMT5 still plays an important role in breast cancer by methylating programmed cell death 4 {PDCD4, a tumor suppressive protein having many anti-proliferative functions} on arginine residue 110 (Powers et al., 2011).

Transcription factors:

JunD, menin's first identified partner, shows higher expression in breast cancers compared to adjacent non-tumor tissues (Kharman-Biz et al., 2013) and binds ERα and facilitate its binding to target genes (Paech et al., 1997). Prostaglandin E2 (PGE2)-induced JunD and JunB have been shown to activate the aromatase promoters I.3/II , while JunD and c-Jun mediated suppression of aromatase promoter I.4, leading to high local levels of estrogen and thus breast cancer progression (Chen et al., 2011). JunD activation has also been shown to reduce the proliferation of Δ 9tetrahydrocannabinol (THC) (cannabinoids) treated breast cancer cells [START_REF] Caffarel | JunD is involved in the antiproliferative effect of Delta9tetrahydrocannabinol on human breast cancer cells[END_REF].

Another important factor is MYC, a well-known estrogen-regulated oncogene (Shang et al., 2000) (Shang et al., 2002), that is overexpressed in approximately 20%-30% of breast cancers (Bièche et al., 1999) and has also been shown to interact with ER to modulate estrogen-mediated signaling (Cheng et al., 2006).

At last, menin has also been recently shown to interact with Gata3 and Foxa1 (Dreijerink et al., 2017) in breast cancer to regulate the ESR1 promoter as we detailed before, Gata3 and Foxa 1 being markers of luminal breast cancer especially luminal A (Perou et al., 2000) (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) (Carrol et al., 2005) (Carrol et al., 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) Signaling pathways and cell cycle regulators:

Menin is involved in the regulation of several factors in the cell cycle and cell growth control. Indeed, the altered expression of a substantial numbers of these factors, including cyclins, CDKs and CDK inhibitors was observed in mouse Men1

insulinomas (Fontaniere et al.,2006). Menin represses cell proliferation in rat intestinal epithelial cells (Ratineau et al., 2004), by inhibiting the expression of cyclin D1, cyclin D3 and cyclin CDK4, and promoting cell cycle transition from G1 to S phase. Menin can also repress cell proliferation by interacting and inhibiting ASK (Sphase kinase), an essential regulatory component of cdc7 protein kinase (Schnepp et al., 2004a). In addition, menin was reported to be able to upregulate the expression of cyclin-dependent kinase inhibitors p18 ink4c and p27 kip1 with the help of the MLL compass like conplex that adds H3K4 trimethylation marks on their promoters activating gene expression (Schnepp et al., 2006) (Karnik et al., 2005) (Milne et al., 2005). Interestingly, p18 has recently been shown to be a downstream target of GATA3 in luminal breast cancer, and to suppress luminal progenitor cell proliferation and tumorigenesis (Pei et al., 2009). P27 has recently been ranked as one of the 18 most significantly mutated genes in luminal A breast cancer and loss of p27 was associated with poor outcome in breast cancer patients (Stephens et al., 2012).

The PAM pathway is another common pathway between menin and breast cancer, as the activation of the PI3K/PTEN/AKT/mTOR pathways has been estimated to be in as frequent as 70% of breast cancers overall (Castaneda et al., 2010). PIK3CA

(subclass of the PI3K family of genes) is the most commonly mutated gene of significance in estrogen receptor (ER) positive breast cancer (Ellis et al., 2013),

occurring in approximately 35% of HR-positive breast cancers, 20%-25% of HER2overexpressing breast cancers, and with a lower frequency (8.3%) in triple-negative breast cancers (Stemke-Hale et al., 2008). PTEN loss in breast cancer is negatively correlated with estrogen receptor (ER) and progesterone receptor (PR) status and associated with the basal-like phenotype (Jones et al., 2013), more aggressive behavior (tumor size, lymph node metastasis, etc..) and worse outcomes (worse DFS&OS) (Li et al., 2017). Several studies have focused on the relation between the PAM (PI3K/Akt/mTOR) and resistance to endocrine therapy in preclinical breast cancer models where they showed that Akt can activate the ERα pathway independent of estrogen availability and that the combination of mTOR inhibitors with endocrine therapy can overcome this resistance (Boulay et al., 2005) (Miller et al., 2010). In addition, the PAM pathway has also been implicated in trastuzumab resistance in HER2-overexpressing breast cancers (Crowder et al., 2009) (Nagata et al.,2004). subtype is ER -positive and/or PR-positive and HER2-negative, displays a reduced expression of proliferation-related genes 3 and is sensitive to endocrine therapy and has an overall favorable prognosis. The luminal B subtype represents around 20% of breast cancers and displays lower expression of ER -related genes, a variable expression of HER2, and a higher expression of proliferation-related genes 4 . This subtype harbors more genomic instability and has a poorer prognosis than the luminal A subtype 5 . The HER2-enriched subtype is ER -negative, PRnegative, HER2-positive and highly sensitive to therapies targeting the HER2 receptor. The triple negative breast cancer (TNBC) subtype is negative for all three receptors 6 and is the most aggressive and has the worst prognosis.

Results

Patients harboring MEN1 mutations are predisposed to multiple endocrine neoplasia type 1 (MEN1) syndrome, which is associated with multi-occurring endocrine tumors 7 , as well as several types of non-endocrine tumors 8 . Numerous studies have revealed that menin is a multifaceted protein involved not only in the development and control of cell growth of endocrine cells, but also in a variety of biological processes, including hematopoiesis and osteogenesis 9-11 . The wide range of biological functions regulated by menin results from its interaction with numerous proteins 12 . These menin-interacting proteins include transcription factors (the components of AP1, NFkB and the TGF signaling pathways) and chromatin modifying proteins (mixed lineage leukemia, MLL, and Sin3A, HDAC) 12,13 . Notably, menin physically interacts with a range of nuclear receptors, including ER and Androgen Receptor (AR), to regulate their corresponding pathways 14-16 .

Over the last few years, evidence has emerged, in vivo, to suggest that menin may play a role in breast cancers 17 . 1) Female heterozygous Men1 knockout mice develop cancers of mammary cells with a low frequency 18 , and conditional mammary gland-specific Men1 disruption leads to the development of mammary intraepithelial neoplasia (MIN) in over 50% of female mutant mice 19 . 2) Importantly, an exhaustive analysis of several cohorts of MEN1 patients revealed a significant predisposition to breast cancer 20 . 3) Menin downregulation was detected in a substantial proportion of human sporadic breast cancer samples 19 , and MEN1 mutations were found, although rarely in sporadic breast cancers, justifying its addition to the list of driver mutations/genes of this pathology 21,22 . Of note, the above-mentioned analyses all highlight the suppressive role of menn in mammary cell tumorigenesis. However, Imachi et al. found that, among 65 ER + breast cancer samples treated with tamoxifen, menin-positive tumors (20 patients) had worse clinical outcome and were more resistant to tamoxifen than menin-negative tumors, suggesting that menin exerts oncogenic effects in these cases 15 . Interestingly, a recent publication, revealing the role of menin in the regulation of the enhancer of the ESR1 gene, suggests distinct functions for menin in primary normal mammary cells and in breast cancers 23 . The authors showed that, although menin possesses a crucial tumor suppressive role in normal mammary cells, it acts as an oncogenic factor in ER + breast cancer cell lines through an enhancer-mediated regulation of ESR1 transcription. Notably, they showed that, by using ZR75-1 BC line which does not express menin, the re-expression of menin led to enhanced ER expression. Given the heterogeneous nature of breast cancers, we sought to further investigate the regulation of ESR1 by menin and assess the putative relationship between ESR1 dysregulation due to menin inactivation and the occurrence of human breast cancer subtypes.

Results

Men1-deficiency in mice leads to ER downregulation in early mammary lesions

We have previously reported the occurrence at a high incidence of mammary intraepithelial neoplasia (MIN) lesions, displaying weak ER expression, in Men1 mammary conditional mutant mice 19 . To further determine the causative role of menin deficiency in reduced ER expression, we carried out double IF analysis of menin and ER expression in normal and young mutant mice with MIN lesions. Three mice per control or mutant group were analyzed. As shown in Fig. 1a, all of the mammary luminal cells in Men1 F/F -WapCre -control mice expressed menin. Conversely, menin expression was lost in 80% of mammary cells in the 10-month-old Men1 F/F -WapCre + mice (Fig. 1a). ER was expressed in approximately 40% of luminal cells expressing menin in the former group (Fig. 1a, upper panel), whereas immunofluorescence revealed that ER expression was lost in Men1-deficient cells in Men1 F/F -WapCre + mice (lower panel). The merged images of menin and ER staining clearly highlight that ER is less expressed specifically in the nuclei of menin-deficient luminal cells (Fig. 1a).

Menin downregulation in human ER + mammary cells leads to reduced ER expression

Next, we further dissected the regulation of ER expression by menin using different approaches in ER + breast cancer cell lines. To achieve this, we first performed MEN1 knockdown (KD) using an SiRNA approach. As shown in Fig. 1 b, MEN1 KD MCF7 and T47D cells displayed reduced ER protein expression by Western blot analysis, unlike the menin-negative ER + line, ZR75-1. Moreover, MEN1 knockdown led to a twofold decrease in ERS1 mRNA levels by RT-qPCR analysis in MCF7 and T47D cell lines (Fig. 1c). We then verified the effects of MEN1 KD on ERS1 mRNA and ER expression levels under estrogen stimulation. Western blot and RT-qPCR analyses showed that MEN1 silencing further abrogated ER expression under E2 stimulation but had no additional effect on ESR1 transcription (Fig. 1d-e). All the above data thus confirm our in vivo analyses, indicating that, menin is essential in maintaining ESR1 transcription and ER expression.

Menin binds to the proximal region of the ERS1 promoter

Dreijerink et al. reported that menin plays a crucial role in the regulation of ESR1 transcription in an enhancer-mediated way 23 . We noticed that, although the study revealed the binding of menin at the transcription start site (TSS) of the ESR1 promoter, no further analyses were reported on this region. We thus carried out ChIP analyses to evaluate the binding of menin to the -2500 bp to +2000 bp around the TSS previously defined (Fig. 2a) 24 , to fully decipher the regulation of ESR1

transcription by menin at the proximal ESR1 promoter region. Menin was significantly enriched in the ESR1 promoter region encompassing the TSS to +2000 bp in MCF7 (Fig. 2b, left panel) and T47D (Fig. 2b, right panel) cells, and more specifically in the promoter area C in MCF7 cells.

Importantly, we confirmed by using luciferase reporter assays that the transcriptional activity of the proximal ESR1 promoter region A/B, and C was markedly reduced when MEN1 was knocked down (Fig. 2c).

Considering the data published by Dreijerink et al 23 , showing that H3K4me3 marks are enriched in the proximal part of the ESR1 promoter, we sought to investigate the involvement of MLL complex, a major actor modifying H3K4me3 marks 13 , in the regulation of the ESR1 promoter. By treating MCF7 and T47D cells with an inhibitor of the menin-MLL interaction, MI503, RT-qPCR analyses unveiled a more than two-fold decrease in ESR1 transcription (Fig. 3a). Western blot analyses in MCF7 and T47D cells using the same inhibitor also revealed a decrease in ER expression at the protein level (Fig. 3b). We then verified the potential alteration of H3K4me3 marks at this region upon inhibition of the MEN1/MLL complex. ChIP analysis with anti-H3K4me3 antibodies showed that, while MI503 treatment led to a markedly reduced binding of menin (Fig. 3c

Lower menin expression is associated with luminal B and basal-like breast cancer subtypes

Our above observation prompted us to perform a thorough investigation of the expression of the expression was significantly associated with ER-negative breast cancers (P = 0.041) and with the HER2-enriched subtype (P = 0.049, Table 1). Moreover, among the 294 ER+/HER2-patients, we observed that low menin expression was associated with the luminal B breast cancer subtype (P = 0.006), larger tumors (P = 0.016), and higher SBR grades (P = 0.005, Table 2).

Interestingly, among the ER+/HER2-cohort, we found that low menin expression was associated with worse distant metastasis-free survival (DMFS), with a 10-year DMFS of 71.5% versus 81.2% in patients with high menin expression, P = 0.053 (Fig. 4a). Furthermore, low menin expression was also associated with a trend for worse disease-free survival (DFS), with a DFS of 65.7% at 10 years versus 75.0% in patients expressing high levels of menin (P = 0.088, Fig. 4b).

Finally, lower expression of menin was also associated with a tendency towards worse OS (10year OS of 77.5% versus 85.2%, P = 0.092, Fig. 4c).

The abovementioned data obtained in human patients, reminiscent of the observations made in Men1-deficient mutant mice, highlighted a relationship between reduced menin expression and weaker ER expression, suggesting that decreased ER expression triggered by Men1-defficiency could be related to the occurrence of luminal B and basal-like breast cancer subtypes.

Menin downregulation alters GATA3 and FOXA1 expression in MCF7 cells

Having demonstrated a clinical correlation between menin inactivation and breast cancer subtypes, we wondered whether the factors important for luminal cell differentiation could be affected by menin. GATA3 is known to be a major factor involved in the regulation of ESR1 expression and is ubiquitously present in luminal A breast cancers 25 . Western blot analysis revealed that GATA3 expression was markedly reduced in MCF7 cells at the protein level after MEN1 KD, although its level of mRNA was not impacted (Fig 5a,b). In parallel, we investigated the expression of FOXA1, which plays an important role in mammary cell differentiation and tumorigenesis, and found that its protein expression was increased upon MEN1 KD in MCF7 cells, whereas no transcriptional alteration could be detected (Fig. 5a,b). Since GATA3 has been reported to interact with menin in lymphocytes 26 , and menin is known to interact with one member of the FOXA family, FOXA2 27 , we performed immunoprecipitation (IP) and PLA analyses, to determine whether menin could interact with GATA3 and FOXA1 in breast cancer cells. The data obtained demonstrated that they indeed interacted with menin in MCF7 cells, as evidenced by IP at the endogenous level (Fig. 5c), by GST pull-down (Fig. 5d) and by PLA (Fig. 5e).

Discussion

The current work provides both clinical and experimental data showing that menin is critically involved in ER expression and that its inactivation in normal mammary cells is correlated with the occurrence of luminal B and basal-like breast cancer subtypes. Our data highlighted cellular and molecular consequences of reduced menin expression in mammary cells, which may affect not only cell proliferation, but also other hallmarks of cancer cells, in particular, cell differentiation.

We previously observed that the mammary lesions developing in mammary cell-specific Men1 mutant mice displayed low ER expression 19 . Our current study further demonstrates that the decrease is an early event, suggesting that menin inactivation favors the occurrence of mammary lesions with weak ER expression. Interestingly, by analyzing this expression in a large cohort of breast cancer patients, we found that reduced menin expression is significantly correlated with both ER -negative and luminal B breast cancer subtypes. Consistently, low levels of menin correlated with larger tumors, more advanced SBR grades and worse prognosis, all of which are major features of these two breast cancer subtypes 4 . Moreover, while searching for luminal cell factors likely interacting with menin, we unveiled that menin binds physically to GATA3 and FOXA1 in mammary cells, and that MEN1 silencing reduces GATA3 expression but upregulates FOXA1 expression. Of note, reduced GATA3 expression is often seen in the luminal B breast cancer subtype but not in luminal A 28 . However, the mechanisms leading to the occurrence of both luminal breast cancer subtypes remain elusive. The current work may provide useful insight and generate interest for further studies. In the meantime, considering the retrospective nature of the study and the heterogeneity of the therapies received by the patients included, the clinical analyses should be confirmed in other cohorts, preferably through prospective studies.

Dreijerink et al first described the capacity of menin to regulate ESR1 transcription by binding to the remote upstream part of regulatory sequences of the ESR1 gene, through an enhancer-mediated looping mechanism, involving GATA3 23 . Moreover, the occupancy of this enhancer sequence by GATA3 has been reported to play an important role in the regulation of ER expression upon estradiol stimulation 25 . Our findings provide complementary information related to the role of menin in ESR1 regulation through its proximal promoter. Intriguingly, our data showed neither altered H3K4me3 methylation on the proximal ESR1 promoter with MI503 treatment, nor affected ESR1 transcription after single MLL1, MLL2, or their combined knockdown with SiRNA. Since MI503 has been demonstrated not only to be capable of inhibiting the interaction between menin and MLL1/MLL2, but also reducing menin expression itself 29 , our data suggest that the factors other than the MLL complex may also participate in this regulation.

It would be interesting in the future to identify the factors or cofactors that interact, positively or negatively, with menin to regulate this gene. In addition, our data seem to support the oncogenic role played by menin in ER + breast cancer cell lines, the proliferation of which is highly ERdependent. Therefore, by combining the data obtained from our experimental and clinical analyses, we consider that menin most likely plays the opposite role in luminal A breast cancer subtype.

Conclusions

The emerging role for the MEN1 gene in mammary cell tumorigenesis appears to be multifaceted.

Our current results provide further data showing that menin may play different, even opposite, roles in the development of different breast cancers, in agreement with the findings reported by Dreijerink et al. Taken together, these results may explain seemingly controversial data reported so far, in particular when comparing data obtained from naturally occurring tumors and those of cultured cancer cells. Furthermore, our findings may also raise awareness to the breast cancer subtypes selected when designing new therapeutic strategies involving the eventual use of menin and MLL inhibitors.

Materials and Methods

Patients

We screened a total of 433 consecutive female patients with breast cancer who underwent surgery

TMA analysis of human breast cancers

Formalin fixed paraffin embedded breast cancers were prepared and processed for immunostaining as previously described 19 . Tissue micro-array (TMA) block preparation, menin nuclear expression assessment using IHC, and statistical analyses were performed as previously described 19 . The percentage of stained cells was multiplied by the intensity of staining to obtain the H score 30 . For sake of correlations and survival analyses, the whole cohort of patients was divided into high menin expression (H score > 100) and low menin expression (H score ≤ 100).

Animal breeding

Men1 F/F -WapCre + and Men1 +/+ -WapCre + mice previously generated in our lab were used 19 . All animal experiments were conducted in accordance with accepted standards of animal care and were approved by the Animal Care and Use Committee of the University Lyon 1.

Cell culture, transfections and luciferase assays

Three breast cancer cell lines expressing ERα, namely MCF7, T47D and ZR75-1 were used in this study. Transient transfections were carried out in phenol red-free medium supplemented with 10%

Luciferase assays

For luciferase assays, MCF7 cells were cultured in 24-well plate. 48h after transfection with 250 ng of the reporter plasmid PrAB or PrC, and 5 ng pRL-TK internal control vector, cell lysates were prepared and analyzed using a dual-luciferase reporter assay system (Promega, Madison, WI), as previously reported 27 . Comparisons between mean values were assessed using the two-tailed Student t-test.

Real-time reverse transcription and qPCR analyses

RNAs 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 (Life-Technology, France) and corresponding primers (Additional file 2). Results of each sample were normalized.

Protein extraction, immunoprecipitation, GST pull-down and immunoblotting

Total protein extracts from cells and immunoprecipitation were prepared and analyzed as described previously 27 . For GST pull-down assays, 1.25 μg purified GST-menin protein or GST control protein was incubated with 1 mg or 2 mg of nuclear cell extracts prepared from MCF7 cells, as previously described 27 . The co-sedimented proteins were detected by Western blot using standard conditions.

Immunostaining

Tissue preparation, immunostaining and statistical analyses were performed as previously described 19 . Briefly, endogenous peroxidases were quenched in 3% H2O2 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 (Additional file 2). For immunofluorescence (IF) staining, signals were detected with a Cy3 or Cy5 tyramide amplification kit (PerkinElmer), with prior incubation with the appropriate biotinylated secondary antibody according to the manufacturer's instructions. Images were acquired on an Eclipse-NiE NIKON microscope, using the NIS-Elements Software.

Proximity ligation assay (PLA), image acquisition and analysis

MCF7 cells were fixed in methanol for 5 min and washed twice in PBS, then treated and analyzed according to the manufacturer's instructions (Duolink II Fluorescence, Olink Bioscience, Sweden). Images were acquired on an Eclipse-NiE NIKON microscope, using the NIS-Elements Software. For each sample, at least one hundred cells were counted. Analysis and quantification of these samples were performed using the Image J software (free access). PLA dots were quantified on 8-bit images using the 'Analyse Particles' command, while cells were counted using the cell counter plugin.

ChIP-qPCR assay

Chromatin for ChIP analysis was prepared from 5 million MCF7 or T47D cells. Briefly, cells were fixed in 1% formaldehyde for 10 min, nuclei were obtained and lysed in 300 μl ice-cold RIPA buffer prior to Chromatin-DNA shearing with a Diogene Bioruptor. ChIP was performed using 5

μg of primary antibodies. Dynabeads® Protein G (10003D, Life Technologies, France) was used to retrieve immunocomplexes according to manufacturers' instructions.

Statistical analyses

For molecular biology experiments, statistical analyses were performed as described in the Fig.

legends; unpaired Student t-tests were used unless otherwise indicated. All analyses were conducted using the Prism5 software (GraphPad, USA); a P value of < 0.05 was considered to be significant. Results are expressed as means +/-standard errors of the means (SEM). For the patient samples, numerical variables were compared using Student's T test while categorical variables compared using Chi-square test. Distant metastasis-free survival (DMFS) was defined as time from diagnosis to the date of distant metastasis or death or last follow-up. Disease-free survival (DFS), defined as the time from diagnosis to death or progression or to date of last follow-up (for censored patients), was also calculated. Survival rates were estimated using the Kaplan-Meier method, and comparisons between menin expression groups were performed using the log-rank test. All statistical tests were two-sided, and the p-value was considered statistically significant if lower than 5%. Statistical analyses were performed using SPSS 20.0 statistics package. and anti-GATA3 antibodies in MCF7 and ZR75-1 cells, the latter expressing no menin. Under: the quantification of PLA analysis. treatment. As such, MYC, overexpressed in approximately 20-30% of BCs 4 , is not only a wellknown estrogen-regulated oncogene 5 , but has also been shown to interact with ER to modulate estrogen-mediated signaling 6 . Identifying the factors influencing the expression and functions of MYC, in order to find ways to control its oncogenic effects, could thus be a promising approach.

The PI3K/PTEN/AKT/mTOR pathways are also considered having close relation with the ER signaling pathway. It has been reported that the activation of the PI3K/PTEN/AKT/mTOR pathways can be found in 70% of BCs 7 . PTEN mutations and inactivation are negatively correlated with ERα and PR status in BC 8,9 , associated with more aggressive behaviors and worse outcome 10 .

The activated Akt is known to be capable of promoting the ERα pathway 11 . Therefore, the combination of mTOR inhibitors and endocrine therapy has been proposed, in order to overcome the resistance to endocrine therapy 12 .

Menin, encoded by the MEN1 gene, is a multifaceted cofactor, interacting physically or functionally with numerous transcription and epigenetic factors, including Smad3, JunD, AKT, MYC, KTM2A/B and Ezh2 13 . Interestingly, it has been identified as an ER interacting partner 14 and a crucial factor that binds to the regulatory sequences of the ESR1 gene and participates in the positive regulation of its transcription in ER+ BC cells 15 . Over the recent years, increasing evidence have been published highlighting the complex and important role of menin in BC. For instance, we have observed that heterozygous Men1 mutant mice develop mammary gland tumors in females and prostate cancers in males at advanced age at low frequencies 16 . Moreover, conditional mammary-specific Men1 knock-out female mice displayed higher amounts of early mammary intraepithelial neoplasia (MIN), that represent precursor cancer lesions 17 . Importantly, a comprehensive analysis of several cohorts of MEN1 patients revealed that female MEN1 patients were at a higher risk of developing BC 18 . In human sporadic BCs, MEN1 mutations, albeit at low frequency, was found as being among driver mutations and reduced menin expression was reported 19 . Interestingly, Dreijerink et al. have demonstrated that menin acts as a tumor suppressor in normal mammary cells, through regulating different sets of genes in primary luminal progenitor cells from normal individuals, compared with those found in ER+ BC cells, but plays an oncogenic factor in the latter cells through its positive regulation of the ESR1 gene 9 . Moreover, Imachi et al.

observed that, with 65 ER+ BC samples treated with tamoxifen for 2-5 years as adjuvant therapies, menin-positive tumors (20 patients) had a worse clinical outcome and were more resistant to tamoxifen than menin-negative tumors (46 patients) 20 . Intriguingly, the AKT/mTOR pathways have been reported to be related to the occurrence of resistance to endocrine therapy, whereas menin is known to have close relation with the former. Wang et al. demonstrated that menin interacts with AKT1, inhibits its kinase activity and AKT1 induced proliferation and anti-apoptosis in endocrine and non-endocrine cells 21 . Moreover, a recent study reported that menin is involved in milk protein synthesis through mTOR signaling in normal mammary epithelial cells 22 , by suppressing the mTOR pathway components and milk protein κ-casein (CSNK). The above data suggest that menin may play an important but complex, even opposite role in mammary cells and in different BC subtypes. In the current study, in order to clarify the role of menin in BC, we investigated the above mentioned MYC and mTOR pathways in human BC samples and derived cell lines.

Materials and Methods

Cell

Protein extraction and Western blot

Protein extracts were prepared by washing the plates twice with 1X PBS, then, 200-1000 μl of complete RIPA buffer (50 mM Tris HCL pH=8; 150 mM NaCl; 0.1% SDS; 0.5% Sodium Deoxycholate, 1% NP40 + Protease Inhibitors (Roche) were added and left for at least 20 mins.

Then, the cell lysate was transferred to 1.5 ml tubes and centrifuged at 13,000 rpm for 10 minutes at 4°C, in order to precipitate genomic DNA and other cellular debris. The supernatant was retained, and the proteins dosed by Bradford assay at 595 nm.

20 μg or 30 μg (depending on experimental conditions) of cell lysate was boiled in 5 μl

Laemli buffer and loaded on 8 or 10 or 12% acrylamide gel depending on protein size. Proteins were then rapidly transferred on a 0.2 μm PVDF membrane (Biorad) by electrotransfer carried out at 25 V for 10 mins. The membranes were then saturated with 0.1% TBS-Tween, 5% milk for 30 min and incubated with the appropriate primary antibody overnight at 4°C. Membranes were washed three times for 10 mins with 1X TBS -0.1% Tween solution and were incubated for 45 mins with an appropriate secondary antibody coupled to HRP (horseradish peroxidase).

Membranes were washed again and were revealed using Roche® Lumi-light Plus Western blotting substrate and Chemiluminescence Film (GE Healthcare). GAPDH was used as an internal control for normalization of the results.

The following primary antibodies were used for western blot: rabbit anti-menin at 1/8000

(

Proximity Ligation Assay (PLA)

PLA assays were performed using Duolink In Situ PLA detection kit (orange) and Probes: Anti-Goat MINUS and Anti-Rabbit PLUS ((#DUO92006, #DUO92002, Sigma-Aldrich), according to the recommendation of the supplier. Briefly, MCF-7 cells were seeded in a 12-well plate containing cover slips for 72h, on the second day cells were transfected with SiCtrl, SiMEN1 (hs1), SieIF4G, or SiMEN1 (hs1) + SieIF4G as described above. The last day, cells were treated with Estrogen for 2h. Cells were fixed with methanol for 2 mins and washed 3 times with 1X PBS, then, blocked with Blocking Solution and incubated at 37°C for 30 min. 40 μl/slip of solution containing primary antibodies of interest and secondary antibodies (anti-Rabbit + anti-Goat or anti-mouse+anti-rabbit) coupled to oligonucleotides were added consecutively with 1 h incubation each at 37° after one time wash with buffer A. Slips were washed twice with Buffer A with shaking for 10 min, ligation reaction was performed for 30 min, and the signal was amplified for 100 min at 37°C . Cover slips were then washed twice with 1X Buffer B for 10 min with shaking, then once with 0.01x Buffer B. Cells were mounted with mounting solution containing DAPI and observed under florescence microscopy. The primary antibodies used for PLA assays in this study were goat anti-menin (A300 106A, bethyl laboratories) at 1/1000, rabbit anti-MYC at 1/500, rabbit anti-ER at 1/500, mouse anti-eIF4E at 1/8000, mouse anti-eIF4A at 1/2000, rabbit anti-eIF4G at 1/2000 (Same antibodies used in Western blot),

Cell proliferation assays:

MCF7, T47D, and MDA453 cells were seeded in white complete medium at 15000 cells per well for MCF7 and MDA453 and 20000 cell per well for T47D in a 48-well plate. 24h after seeding, cells were transfected with SiRNA (same as described before) or treated with 10058-F4 inhibitor (abcam, ab145065) at different concentrations. The plate was then left for 144h in the IncucyteTm device (Essen Bioscience), allowing the observation of cell growth in kinetics. The device is connected to the software IncucyteTm that determines the cell confluence in real-time.

ChIP-qPCR assay

Chromatin immunoprecipitation (ChIP) was performed with the Millipore ChIP Assay Kit according to the recommendations of manufacturer. Cells were crosslinked with 1% formaldehyde for 10 min at 37°C. Chromatin was prepared and sonicated to an average size of 123 300-500 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. 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). Rabbit Anti-menin (same used for W.B) was used to immunoprecipitate menin with a concentration of 2 μg/sample, and equal amount of rabbit IgG was used as control.

Primers used for MYC promoter: TSS: Fw: 5'-ACTCACAGGACAAGGATGCG-3', Rev: 5'-TGCTCCTCCGTAGCAGTACT-3'; +300: Fw: 5'-CCAACAAATGCAATGGGAGT-3'; Rev:

Mining of public gene expression data

RNA-seq data (Illumina HiSeq 2000, version 2 analysis) obtained in human BC samples in The Cancer Genome Atlas (TCGA) project were previously collected from the Genomic Data Commons data portal (https://portal.gdc.cancer.gov). Expression data were available for 1095 tumors as RNA-Seq by Expectation Maximization (RSEM) normalized counts. Clinical data including ER status were available as well, allowing the identification of ER+ (n=807) and ERnegative (ER-), (n=237) tumors. Co-expression between MEN1 and all of the 20531 genes, including MYC, was examined using Pearson correlation coefficient. Genes showing significant correlation coefficient with absolute value greater than 0.2 were taken as correlated with MEN1.

Gene set enrichment analysis (GSEA, http://www.gsea-msigdb.org/) was used to identify Gene Ontology (GO) gene sets enriched in MEN1 positively correlated genes.

In addition, using the cBioPortal (https://www.cbioportal.org/), a recognized and widely used online portal for cancer genomics, other BC data sets could be investigated for MEN1 coexpression. Among the most recent ones, we selected the SMC 2018 study focusing on young Asian patients 23 .

Statistical analyses

Statistical analyses were performed as described in the figure legends; unpaired Student t tests were used unless otherwise indicated. All analyses were done using Prism5 software (GraphPad, USA); a P value of < 0.05 was considered significant. Results are given as means +/-standard errors of the means (SEM).

Results:

MEN1 knockdown decreases cell proliferation in ER+ BC cell lines

It is known now that menin regulates ERα expression in ER+ BC cells and menin is critical for cell growth of ER+ BC cell lines 8,9 . To better study the role of menin in BC cells, we studied the effects of MEN1 KD on the proliferation of two ER+ BC cell lines, MCF7 and T47D, and two ER-BC cell lines, MDA231 and MDA453. As shown in Fig 1a (2 upper panels), the proliferation of MCF7 and T47D cells started to decrease 48h after MEN1 KD and continued to decrease even after 120h of transfection, compared to the untreated and SiCtrl cells. In parallel, our data demonstrated that MEN1 KD had the same effects on the proliferation of MDA231 cells, but no effect at all on that of MDA453 cells (Fig 1a, 2 lower panels). Our data confirmed that MEN1 is critical for cell proliferation in tested ER+ cells, but showed that it is differentially needed in ERcell lines, suggesting the role of menin in the proliferation of mammary cancer cells could be context-specific. In our following work, we, therefore, mainly focused on the role of menin in ER+ BC cells.

MEN1 knockdown activates the mTORC1 pathway in ER+ BC cell lines

To investigate the molecular mechanisms triggered by MEN1silencing in ER+ cells, we first studied its effects on the mTORC1 pathway, which is critical for cell growth and breast cancer development, as well as involved in resistance to endocrine therapy 24 . Hence, we knocked down menin expression in MCF7 and T47D cells treated with insulin (to activate the mTORC1 pathway)

and/or the mTOR inhibitor RAD001. We observed, surprisingly, that MEN1 KD in these cells increased the expression of the active form of mTORC1 components, namely phosphorylated p70, and p85 (T389) of S6K1 and S6RP Ser 235/236, after insulin treatment, which was inhibited by RAD001 treatment (Fig 1b). The analysis was further carried out in MCF7 cells treated with essential amino acids (EAA), another mTOR stimulator, and similar result was obtained (Fig S1a).

MEN1 silencing also led to increased phosphorylated 4EBP1(ser 65) in T47D cells (Fig 1c).

4EBP1 is a repressor of the downstream events of mTOR pathway, and while S6K1 and S6RP

phosphorylations have an activating effect, 4EBP1 phosphorylation has an inactivating one. To further confirm these unexpected results, we used a reverse approach by overexpressing menin in MCF7 cell line. The results showed that menin overexpression led to the decrease in the expression of phosphorylated S6K1 and 4EBP1, reversing the effects seen by MEN1 KD (Fig 1d). We also noticed that there was an increase in the expression of total 4EBP1 protein in MCF7 cells with menin overexpression (Fig 1d).

Previous studies have reported a relation between menin and AKT in other types of cancers 21 , where deregulated AKT was found in sporadic pancreatic endocrine tumors, in which menin is frequently inactivated 25 . For that we thought it was important to check if the role played by menin on the mTORC1 pathway is AKT dependent or not in our breast cancer cell lines. We suggesting that the effect of menin knockdown on mTORC1 activation is AKT-dependent.

MEN1 KD increases the activity of the translation initiation complex

To further dissect the downstream effects of mTORC1 activation triggered by reduced menin expression, we studied the consequences of MEN1 silencing on the translation initiation complex eIF4F (one of the downstream targets of the mTORC1 pathway) composed of 3 factors: eIF4A, the enzymatic core, eIF4E, responsible for cap-dependent translation of all nuclear encoded mRNA, and eIF4G, the scaffold protein that holds the complex together 26 . We first checked whether the expression of these factors was altered after MEN1 KD. The data showed a reduced expression of eIF4A and eIF4E, and a stable expression of eIF4G on mRNA levels, but no significant change for all 3 factors on the protein levels (Fig 2a). We then analyzed the formation of eIF4F by assessing the interaction between eIF4G/eIF4E and eIF4G/eIF4A using Proximity ligation assay (PLA). We found that MEN1 silencing resulted in an increase in the interaction between eIF4G and eIF4E, but not that between eIF4G and eIF4A in MEN1 KD MCF7 and T47D cells ( Inversely, four other genes encoding components related to the eIF4F complex showed inverse correlation with MEN1: EIF4E3, EIF4G2, EIF4G3, and EIF4E that was slightly below the correlation threshold. These results corroborate at the mRNA level the increased formation of the translation initiation complex eIF4G/eIF4E after MEN1 KD shown above. A similar inverse relationship was observed for MTOR (Pearson correlation coefficient = -0.236). These data mining results appeared in agreement with the above experimental data obtained in ER+ BC cell lines.

Menin regulates MYC expression and binds to the MYC promoter and enhancer in ER+ breast cancer cells:

The above results showing the activation of the mTOR pathway could not explain the reduced cell proliferation in MEN1 KD ER+ breast cancer cells. We thus chose to study MYC which is an important estrogen regulated oncogene and is known to play a major role in proliferation, Our data demonstrated that MYC KD had no effect on ER expression, but, interestingly, resulted in decreased MEN1 expression both on transcription and protein levels in MCF7 and T47D cells (Fig S3a,b).

We observed that MEN1 knockdown also showed decrease in protein and mRNA expression of several known MYC target genes, which are either involved in cell cycle control, including Cyclins, Cyclin-dependent kinases (CDKs), p53, and cyclin-dependent kinase inhibitors (CKI) like p21 and p27, or in the apoptosis, like cleavage of PARP and caspase 3, in MCF7 cells (Fig 3d). These data are in total consistency with reduced MYC expression detected in MEN1 KD MCF7 and T47D cells.

Menin is needed for estrogen mediated MYC activation and physically interacts with MYC in BC cells

Since MYC is an estrogen regulated oncogene, whereas all our above findings have been conducted in the absence of estrogen (Charcol stripped media), we thought it would be relevant to investigate whether the regulation of MYC expression is influenced by estrogen stimulation. We In order to better understand the interaction between menin and MYC, previously reported in Hela cells 28 , in BC cells, we analyzed whether there was a physical interaction between menin and MYC in ER+ breast cancer cells used in the current work, and, if yes, whether this interaction was estrogen dependent. Our data showed that menin interacts indeed with MYC in MCF7 cells, Next, we focused on the relationship between MEN1 and MYC-related genes. Interestingly, we found that two genes, MYCBP2 and MYCT1 described as negative regulating factors in the MYC pathway, displayed a significant negative correlation with MEN1 in ER+ tumors (Fig 5c).

Knowing that both of these MYC-related proteins play an inhibitory role in the MYC pathway, the data seem to support the inconsistent relationship between MEN1 and MYC uncovered above.

Based on these findings, we tested whether MEN1 KD could provide or not synergistic

Discussion

Our current work revealed that MEN1 silencing dysregulates mTORC1 and MYC pathways, specifically in ER+ BC cell lines, providing useful clues into molecular mechanisms underlying the oncogenic effects of menin observed in these cells.

We noticed that MEN1 silencing led to reduced cell proliferation in ER+ BC cell lines as previously reported 15,20 , whereas it triggered a distinct cell growth reaction in an ER-BC cell line, indicating the effects exerted by the menin protein are dependent on cellular and molecular context.

Since it is known that ER-BC is a particularly heterogeneous subtype, which has recently been suggested to be further divided into at least 6 subgroups 2 , it is not surprising that menin could play different roles in ER-BC cell lines. In fact, Dreijerink et al. reported that menin did not regulate

ERα target genes in ER-BC cell lines. It would, therefore, be interesting to further dissect the role of menin in responsive or non-responsive ER-BC cells, beyond the ERα pathways, in order to gain insight into the factors and/or pathways participating in the growth and survival of these cells.

Menin is known to physically and functionally interact with the AKT/mTOR pathway in pancreatic endocrine cells, mainly by the retention of AKT in the cytoplasm 21 . However, its role in the mTOR pathway, in particular on the downstream effectors remains elusive. Our data showed that menin expression is crucial for mTORC1 activity in BC ER+ cells, since its inactivation led to mTORC1 activation. Moreover, the latter seemed to mainly result in the activation of capdependent translation, since 4EBP1 expression decreased and its phosphorylated form increased after MEN1 KD, whereas 4EBP1 normally represses the assembly of the eIF4F complex and its phosphorylation allows the components of eIF4F complex to interact and thus initiate mRNA translation. Importantly, our data show that menin inactivation in ER+ BC cells specifically favored the formation of eIF4E and eIF4G complex, which is considered to be the rate-limiting determinant of cap-dependent protein synthesis 27 and one of the characteristic activities of cancer cells, including BC cells 30 . Data mining of gene mRNA expression in ER+ human tumors corroborate the effects of menin inactivation on 4EBP1 and on the formation of the eIF4F complex observed in ER+ BC cells. Notably, our clinical data analysis on TamRad trial also supports the possible activation of the mTOR pathway in menin-low BC ER+ patients, even though the trend observed in the trial needs to be further confirmed by other studies with more cases. We noticed that a recent study reported the reduced ER signaling pathway upon PI3K/AKT activation 11 .

Their data showing increased ERα expression after the treatment with PI3K and/or AKT inhibitor is reminiscent of the data reported in the current study. It is, therefore, plausible that the activation of AKT/mTORC1 triggered by MEN1 silencing contributes to the reduced ERα expression and cell proliferation observed in ER+ BC cells in our works, along with the inhibition of the MYC pathway. Another possibility would be that the activation of the mTORC1 pathway triggered by menin inactivation could lead to oncogenic stress in these cells, as formerly reported 31 .

Interestingly, MEN1 knockdown led to reduced MYC expression in ER+ cells. Our analyses demonstrated, for the first time, that menin is a critical factor regulating MYC transcription in these cells, through its binding not only to the MYC promoter, but also to the MYC 5' enhancer.

Furthermore, we observed that MEN1 silencing in these cells triggered altered expression of factors known to be MYC targets and responsible for cell growth, cell cycle and cell death. The novel role of menin uncovered in the current work in regulating MYC transcription and the MYC pathway is, therefore, consistent with molecular alterations observed in these cells. Our finding is also in agreement with the recently emerged notion that menin could play a dual role in the tumorigenesis of BC cells. The fact that menin is essential for ESR1 and MYC transcription revealed in the previous study 18 and the current work on ER+ BC cells makes it a powerful oncogenic factor in these cells, by regulating two main actors. Imach et al previously reported that menin expression in BC patients is clinically correlated with resistance to tamoxifen 20 . It would be relevant to verify whether the role of menin in regulating MYC expression could be one of the molecular bases conferring such resistance. In parallel, we observed that, in ER-BC cells, the role of menin in regulating MYC and cell proliferation varies among different cell lines tested, suggesting more diverse molecular elements may participate in the carcinogenesis of these cancers.

In MEN1 KD ER-BC cells showing reduced MYC transcription and reduced cell proliferation without altered MYC protein expression, as in MDA-MB-231, menin may also be relevant to its post-transcriptional regulation via factors, such as MYCBP1 and MYCT1, and/or to the expression of other genes that are critical for the growth of these cells. Future works on ER-BC cells could provide useful answers to explain these findings. We noticed that the positive correlation between menin and MYC expression in some BC samples. Moreover, even in the absence of MYC-MEN1 co-expression, a clear inverse relationship was detected between MEN1 and MYCT1 or MYCBP2 expression in ER+ as well as ER-tumors, suggesting the involvement of menin in posttranscriptional regulation of MYC. Considering the heterogeneous nature of BC 32 , it would be relevant to further dissect and validate the molecular crosstalk unveiled in the current work between menin and its related pathways, including MYC and mTORC1, in different BC subtypes.

DECLARATIONS SECTION

Consent for publication

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

(

Discussion

The first clue of menin's involvement in breast cancer came from our observations in female aged heterozygous Men1 mutant mice that developed, in addition to endocrine tumors, mammary gland carcinomas at low but noticeable frequency (Bertolino et al., 2003a). Few years later, we generated a mammary specific Men1 knock-out mouse model, showing that ERα expression and the number of ERα-positive cells were clearly reduced in mammary intraepithelial neoplasia (MIN), precursor lesions of mammary cancer lesions of mutant mice, compared with normal mammary glands (Seigne et al.,2013). More recently, female MEN1 patients found to be at a higher risk to develop breast cancer (Dreijerink et al.,2014) and Men1 mutations were also detected (though at very low frequency) in sporadic breast cancer (Nik-Zainal et al.,2016). Interestingly, an international clinical panel on MEN1 disease researched a consensus last year, proposing to the community that breast cancer is from now on considered as among the tumor panel developed in female MEN1 patients.

However, we are still far from knowing exactly the role of menin in BC development, since both the data from basic research and clinical observations are divers and even contradictory. Nevertheless, the recent works carried out in different laboratories in the field, including in our lab, afford some new clues into better understanding of the complex and multifaceted role played by menin in BC.

I. What we know now about the role of menin in breast cancer

Consistent to the above-mentioned works, our clinical studies carried out during my thesis work showed that lower menin expression was significantly associated with ER-and luminal B subtype breast cancers displaying either no, for the former, or weaker ER expression for the latter, whereas high menin expression was associated with luminal A ER+ breast cancers.

These in vivo analyses in total consistency with the regulatory role of menin in ESR1 transcription revealed previously and more detailed in this study is. These data also reinforced what we reported previously based on our observations obtained in mammary-specific Men1deficient mouse model, suggesting the tumor suppressor role of menin during the initiation procedure of tumorigenesis of mammary cells, since both ER-and luminal B subtype breast cancers are more aggressive than luminal A subtype breast cancers.

On the contrary, in ER+ luminal A subtype breast cancer, menin expression may promote cell proliferation by activating ERα expression and ER target genes (Dreijerink et al., 2006). In the matter of fact, Imachi et al found that menin binds and coactivates ERα in a hormone (estrogen) dependent manner and that menin could be a predictive factor of tamoxifen resistance (Imachi et al.,2010). These data highlight a complex and even opposite role played by the Men1 gene in the carcinogenesis of mammary cells. A hypothesis of "dual for menin"

proposed

II. Menin regulates ESR1 transcription in ER+ BCs

We, as well as others, have showed that menin positively regulates ERα expression as MEN1 knockdown leads to decreased ERα expression on both the protein and transcriptional level. To better understand how menin regulate the ESR1 proximal promoter and why MI503

treatment decreased ERα expression and menin binding but not H3K4me3, we investigated the eventual participation of JunD, menin's first identified partners (Agarwal et al.,1999), by using MI503, which inhibits MLL1/2 binding to menin's pocket, the same pocket where JunD binds to menin. Our results, however, showed no effect of JunD knockdown on ERα expression or cell proliferation (data not shown). We also checked other epigenetic factors like HDAC1 and found no effects of its knockdown on ERα expression (data not shown).

Bearing in mind that menin can interact with a large number of epigenetic factors (like HMTs and HATs), and transcription factors that could bind to the ESR1 promoter and due to the limitation of our study, we expect that other factors could be involved in this regulation, and further detailed studies like ChIP-seq or RHIME techniques could be more suitable to identify the factors involved in the regulation of ESR1 transcription.

III. How can MEN1-KD activate mTOR pathway and yet cause cell death?

As we showed in our first part of the work, menin regulates, as a cofactor, ESR1 transcription in ER+ breast cancer cells. Bearing in mind the importance of ERα for mammary cell growth and survival, we thought that it's important to study the biological effect of menin in these types of cells. We found that MEN1-KD decreased cell proliferation in both ER+ cell lines, whereas its effects in ER-cell lines were rather heterogenous. The latter can be explained by the fact that ER-breast cancer is heterogenous and can be subdivided into at least 7 further subtypes (Lehmann et al.,2011) and nothing is known about menin's role in these types of cancers. It was, therefore, not surprising for us to observe different outcomes of MEN1-KD, due to possible different roles played by in the different subtypes of ER-breast cancers.

We focused on ER+ breast cancer cell lines and sought to know how menin MEN1-KD decreased cell proliferation in these cells and the molecular mechanisms behind it. Since AKT/mTOR pathway is highly activated in breast cancer (Castaneda et al., 2010), and that menin has been shown to interact with partners of this pathway in other types of cancer (Wang et al.,2011) (Wuescher et al., 2011), we studied the relation between menin and AKT/mTOR pathway in our breast cancer cells. Surprisingly, our results showed that menin inactivation activates the mTOR pathway and activates the downstream translation initiation complex. We also investigated other mTOR downstream targets like those involved in lipid metabolism, aerobic glycolysis, and pentose phosphate pathway (data not shown) but did not find any obvious change after MEN1-KD. Interestingly, these results may explain why breast cancer patients with low menin expression had better survival rates when treated with tamoxifen plus the mTOR inhibitor everolimus (RAD001) compared with those treated with tamoxifen only.

We noticed that, in 2016, Dermit et al observed that PI3K inhibitor (PI3Ki)-resistant cells developed proliferative defects under the actions of PI3K/mTORC1 in an AKT independent manner, due to accumulated reactive oxygen species (ROS), and hypoxia-inducible factor-1α

(HIF1α)-produced lactate [START_REF] Dermit | Oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells resistant to PI3K inhibition[END_REF]. This metabolic phenotype made resistant cells more sensitive to hydrogen peroxide and nutrient starvation thus hindering their division.

Unfortunately, because of the lack of time, it was not possible for me to carry out further investigation to know whether this is also the case in my cell model.

IV. Menin and MYC, another major partnership underlying menin's oncogenic effect?

We looked at other menin partners that play important role in cell proliferation and death in BC cells, in particular MYC. MYC, an estrogen-regulated oncogene involved in cell proliferation, apoptosis, differentiation, metabolism, is overexpressed in 20%-30% of breast cancers and interacts with ERα in ER+ positive cell lines (Bièche et al., 1999) (Cheng et al., 2006). Menin was reported to interact with MYC in different tissues to regulate MYC target genes, whereas its regulatory role on MYC transcription has not been so far reported.

In our lab, a preliminary menin ChIP-seq analysis in MCF7 cells has showed that menin binds to the MYC promoter (Data not shown). Our further analyses in the current study confirmed that menin positively regulates MYC transcription and its target genes especially those involved in cell cycle and apoptosis. We also detailed, for the first time, that menin not only binds to several areas of the MYC proximal promoter but also to an enhancer previously identified (Wang et al.,2011). We checked if menin and MYC could regulate together ERα expression, but MYC knockdown had no effect on ERα expression, whereas menin knockdown decreased both ERα and MYC expression. Furthermore, our data demonstrated that menin is crucial for the activation of MYC expression, both on transcription and protein levels, under estrogen stimulation. The data highlighted the importance of menin, as an oncogenic factor, in the tumorigenesis procedure mediated by the ERα pathway in ER+ BC cells.

Interestingly, we also found that MYC knockdown led to decreased menin expression at both protein and transcriptional level. The data suggest that menin and MYC may form a positive feedback regulation, playing a crucial role in promoting the proliferation of ER+ BC cells.

This part of the project would be interesting to follow, since very limited studies focus on the regulation of the MEN1 gene and nothing is known so far about on the regulation of menin expression by MYC. It would be interesting to investigate more deeply on the crosstalk between menin/ERα/MYC in ER+ BC cells.

In this study, we were also able to reveal some clinical potential related to the importance of menin as a regulator of MYC expression, one of the most important proliferative genes.

Although menin inactivation alone decreases cell proliferation of ER+ BC cells, combined menin and MYC inactivation gives better results in cultured cell works. The data may provide relevant clue into new therapeutic strategy, since menin expression in ER+ BC cells reported to be more resistant to hormone-therapy. Also, data mining analyses demonstrated certain positive correlations between MEN1 and MYC mRNA expression in breast cancer,

Conclusion and Perspectives:

Menin, a multifunctional scaffold protein, can have different roles in a tissue and molecular specific manner. In cancers, menin have been first identified as a tumor suppressor in endocrine tumors, and later on as an oncogenic factor in several types of leukemia. However, its involvement in hormone-dependent cancers like prostate and breast seems to be more complex. Menin, as a We have also shown that menin inhibition activates the mTORC1 pathway and its downstream translation initiation complex. We have also checked if menin had an influence on other downstream effectors of mTORC1 but didn't go deep into these investigations. Consequently, it could be interesting to investigate if menin regulates pathways like lipid metabolism, glycolysis, pentose phosphate, and autophagy all being controlled by the mTOR pathway.

cofactor
Our works also revealed that menin physically interacts and regulates MYC transcription in ER+ BC cells, through binding to its proximal and enhancer promoter. Menin not only regulate MYC protein and transcriptional expression, but also regulates several MYC target genes mainly those implicated in cell cycle and apoptosis. For this reason and taking into consideration that menin knockdown inhibits cell proliferation in ER+ BC cell lines, it would be interesting to investigate more thoroughly whether menin regulates MYC target genes directly or through MYC itself, and if menin's role on the proximal and enhancer could be different and/or effected by estrogen. In addition, it would be also relevant to dissect the positive feedback regulation between MYC and menin observed in our work, which could be relevant to investigate more deeply the crosstalk between menin, ERα and MYC in ER+ BC cells. We also found that menin could regulate MYC differently in ER+ and ER-BC cells, however we didn't investigate more deeply how menin regulate MYC expression in ER-BC cells, making this an interesting field for other people to explore. Lastly, taking into consideration the clinical complexity and aggressiveness of TNBCs, and that many ER+ BCs eventually acquire resistance to endocrine therapy, menin/myc relation could serve as a potential strategy for novel targeted therapy.

List of publications:

During

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, andS3). 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

We then sought to better characterize the prostate lesions that developed in Men1NT mice by analyzing several major cell 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 AR-dependent (LNCaP, 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, Endocrines 2020,1,doi:10.3390/endocrines1020007 www.mdpi.com/journal/endocrines 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,[START_REF] Dubik | Transcriptional Regulation of C-Myc Oncogene Expression by Estrogen in Hormone-Responsive Human Breast Cancer Cells[END_REF], and IGF-1 [START_REF] Umayahara | Estrogen Regulation of the Insulin-like Growth Factor I Gene Transcription Involves an AP-1 Enhancer[END_REF]; (ii) anti-apoptotic factors, such as TIT-5 and EIT-6 [55]; (iii) enzymes, such as the lysosomal proteinase cathepsin D [START_REF] Elangovan | Progesterone and Estrogen Control of Rates of Synthesis of Uterine Cathepsin D[END_REF]; (iv) and nuclear receptors, such as progesterone receptor [START_REF] Yu | Effects of 17 Beta-Estradiol on Progesterone Receptors and the Uptake of Thymidine in Human Breast Cancer Cell Line CAMA-1[END_REF], 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 [START_REF] Jenster | Domains of the Human Androgen Receptor Involved in Steroid Binding, Transcriptional Activation, and Subcellular Localization[END_REF][START_REF] Fujita | Role of Androgen Receptor in Prostate Cancer: A Review[END_REF]. 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 [START_REF] Bryce | Androgen Receptor Splice Variant 7 in Castration-Resistant Prostate Cancer: Clinical Considerations[END_REF]. Most of them are lacking the C-terminal region containing the LBD [START_REF] Hu | Ligand-Independent Androgen Receptor Variants Derived from Splicing of Cryptic Exons Signify Hormone-Refractory Prostate Cancer[END_REF][START_REF] Van Der Steen | Posttranslational Modification of the Androgen Receptor in Prostate Cancer[END_REF] and are, therefore, functionally active independently of the presence of androgens. Among them, AR-V1 and AR-V7 are the most abundant variants [START_REF] Hu | Ligand-Independent Androgen Receptor Variants Derived from Splicing of Cryptic Exons Signify Hormone-Refractory Prostate Cancer[END_REF]. Somatic AR mutations may occur selectively in response to androgen deprivation [START_REF] Hoang | Androgen Receptor-Dependent and -Independent Mechanisms Driving Prostate Cancer Progression: Opportunities for Therapeutic Targeting from Multiple Angles[END_REF]. 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% [START_REF] Hoang | Androgen Receptor-Dependent and -Independent Mechanisms Driving Prostate Cancer Progression: Opportunities for Therapeutic Targeting from Multiple Angles[END_REF][START_REF] Koochekpour | Androgen Receptor Signaling and Mutations in Prostate Cancer[END_REF]. Furthermore, the AR LBD was described as a mutational hotspot, placing the incidence of its point mutations in CRPC at ~15-20% [START_REF] Grasso | The Mutational Landscape of Lethal Castration-Resistant Prostate Cancer[END_REF][START_REF] Robinson | Integrative Clinical Genomics of Advanced Prostate Cancer[END_REF].

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 [START_REF] Hay | An Sp1 Modulated Regulatory Region Unique to Higher Primates Regulates Human Androgen Receptor Promoter Activity in Prostate Cancer Cells[END_REF]. 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 [START_REF] Rokhlin | Androgen Regulates Apoptosis Induced by TNFR Family Ligands via Multiple Signaling Pathways in LNCaP[END_REF]. 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][109][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. Neuroendocrine tumours form a rare group whose incidence is constantly increasing and the prevalence is relatively high because of its relative slow tumour progression, according to the statistics collected in the States. 1 They may occur as familial syndromes that are caused by genetic mutations. Multiple endocrine neoplasia syndrome type 1 (MEN1) is one of these hereditary neuroendocrine tumour syndromes, transmitted with an autosomal dominant trait 2 .Patients affected by the disease develop multiple endocrine tumors, including the parathyroid glands, endocrine pancreas and anterior pituitary gland. MEN1 mutation is among the genetic abnormalities clearly involved, making this gene a crucial key to address studies of tumorigenesis of endocrine cells. In tumors developed by patients, there is frequently a loss of heterozygosity at the locus of this gene, consistent with the notion that the MEN1 gene is a tumor suppressor. It is important to indicate that MEN1 inactivation is also involved in sporadic neuroendocrine tumours affecting endocrine pancreas, parathyroid, anterior pituitary and adrenal glands, as well as in angiofibromas and lipomas 3 .

Lessons learnt from

The MEN1 gene was identified gene in 1997 4,5 . Since then, along with MEN1 studies using diverse approaches and models, different laboratories around the world, including our own, have engaged in the generation and characterisation of Men1 mutant mouse models. Later on, more laboratories were involved in the use of these models to dissect MEN1 functions. As expected, numerous studies carried out in using Men1 mutant mouse models have allowed not only studying the events related to MEN pathology, but also, dissecting cellular and molecular mechanisms triggered by MEN1 ablation. In this review, we present the currently available data on the downstream molecular events due to MEN1 disruption revealed by studies exploring Men1 mutant mice.

II. Men1 mutant mouse models

Gene targeting in the mouse allows currently to either disrupting a given gene on germline level (conventional mutant mice) or inactivating the gene in a specific cell type (conditional mutant mice). Conventional Men1 mutant models recapitulate genetic events happened in MEN1 patients, hence facilitating the study of the natural history of MEN1 disease. Several published works unanimously pointed out that homozygous Men1 mutant embryos died at mid-gestation stage, whereas heterozygous Men1 mutant mice developed multiple endocrine tumors starting from around 12month-old of age, affecting the islet, parathyroid, pituitary and adrenal glands and associated with hyperinsulinemia. Conditional Men1 mutant models were generated by crossing the mice carrying the floxed Men1 allele, Men1 F/F , with selected Cre mice functioning in given cell types or tissues, including different islet cells, parathyroid, pituitary and mammary cells. In addition, inducible Men1 mutant models were also generated and provided interesting tools for fine analyses of temporal molecular and physio-pathological consequences of Men1 inactivation. All these mouse models played a vital role in dissecting cellular and molecular mechanisms involved in MEN1 syndrome development (Table 1). rather than synergistic effects of these pathways on tumorigenesis 27 . Pei et al. reported that p18, but not p27, functionally collaborates with Men1 in suppressing lung tumorigenesis, likely due to loss of the control on stem cell expansion 28 .

Agarwal et al. has reported that JunD, the first identified menin partner 24 , activates proliferation of menin-deficient MEFs 29 . It appears that, in the absence of menin, c-Jun N-terminal kinase (JNK) phosphorylates JunD, leading the activation of JunD-induced pro-proliferative gene expression 27,30 . The activation of JunD after menin inactivation could also result from the loss of biding to the co-repressor mammalian sin3 protein (mSin3A) and its associated HDAC 31 . However, the role played by JunD in Men1-related tumours remains so far elusive.

The analyses carried out in our laboratory revealed the physical interaction between menin and the Wnt pathway effector -Catenin, and the nuclear translocation of the latter in menin-deficient islet cells 10,32 . Furthermore, Cao et al have shown that menin plays an essential role in controlling -Catenin cyto-nuclear shuttling. Importantly, Jiang et al have provided genetic evidence showing that -Catenin ablation could restore Men1 insulinoma phenotype 33 , highlighting the importance of -Catenin in Men1 insulinoma development. Finally, -Catenin inhibitor was shown to successfully suppress insulinoma development in Men1 F/F -RipCre + mice 33 .

Wang, Y. et al investigated whether menin can regulate the AKT signaling using immortalized meninnull MEFs stably infected or not with menin-expressing retroviruses 34 . They found that menin inhibited AKT1-mediated cell proliferation likely by reducing the translocation of Akt1 from the cytoplasm to the plasma membrane. They also demonstrated that p-AKT (S473) expression was detected in Men1 mouse insulinomas. Interestingly, it is reported that menin deficiency in CD8 T cells affected the mTOR complex 1 (mTORC1) signaling, resulting in increased glycolysis and glutaminolysis, highlighting its role in regulating the AKT/mTOR pathway in these cells 35 .

By using Men1-deficient mouse embryonic fibroblasts (MEF) and primary islets derived from Men1 mutant mice, Gurung et al have provided compelling data showing that menin plays a crucial role in inhibiting the Hedgehog pathway through its physical interaction with PRMT5 and its binding to both the Gli and Gas promoters 36 . The data suggest that menin inactivation may lead to the activation of the Hedgehog pathway known to be enhanced in human PanNET.

Mammary precancerous lesions were found in more than half of WapCre-Men1 F/F female mice where the Men1 gene was disrupted in luminal cells 18 . Further analyses demonstrated that ER expression was reduced in menin deficient mammary cells. Similarly, the quantitation of epithelial cell nuclei displaying either no or very low staining of AR expression showed a significant increase in the number of cells with altered AR expression in prostate carcinomas from heterozygous Men1 mutant mice, compared with prostate glands from Men1 +/+ 37 . These observations are in consistence with the molecular analyses showing the physical and functional interaction of menin with ER and AR 38,39 .

III.2. Cell cycle control & Growth factors

Several studies of the altered gene expression in mouse Men1 insulinomas demonstrated early deregulated expression of the genes involved in cell cycle control, such as cyclin A2, B2, D2, CDK2, CDK4, p18 and p27, and growth factors of the insulin pathway, such as Igf1, Igf2, Igfbp3, and Igfbp6 40,41, 42,43 ,44 . Moreover, the analysis of the methylation state of the DMR2 regulatory regions of the Igf2 gene revealed a hyper-methylation of the four CpG sites whose methylation leads to the increased Igf2 expression 40,45,46,47 . Interestingly, Zhuang et al recently showed that neuron-specific Men1 ablation in the mouse caused decreased CDk5 expression and synaptic dysfunction. Their detailed investigation demonstrated that menin binds the p35 promoter, the main activator of CDK5, and activates its expression via the MLL complex 48 .

III.3 Cell adhesion and angiogenesis

The alteration of several cell adhesion markers were documented in mouse Men1 insulinomas 45 . Interestingly, we were also able to demonstrate an evident decrease in membrane -Catenin and Ecadherin expression in the mammary lesions developed in Men1 F/F -WapCre + model 18 . Early appearance of increase in angiogenesis was observed in different Men1 insulinoma models, using respectively CD31 10 , lectin, VEGF 15 , reminiscent of what observed in MEN1 PanNET.

III.4 Pancreatic endocrine transcription factors

Our works revealed that the expression of Maf family members, in particular MafA and MafB both having important functions in islet cells, is dysregulated in islet tumors in murine models. More precisely, we observed an ectopic expression of MafB, which is normally expressed only in mousecells, in insulinomas. Our analyses demonstrated that MafB expression is negatively regulated by menin and its overexpression can promote the ability of β-cells to form foci on soft agar and the level of cyclin D2 expression 49 . On the contrary, MafA expression is decreased in mouse and human MEN1 insulinomas 50 . The analyses demonstrated that menin binds to the MafA promoter and activates its transcription, which is essential for keeping intact -cell program. Therefore, a finely controlled expression of MafA and MafB seems crucial for normal -cell proliferation and differentiation.

The expression of another islet transcriptional factor Hlxb9 was found increased in mouse islet tumors, the former being a menin-interacting factor 51 . The authors also reported that combined menin knockdown and Hlxb9 overexpression partially rescued the apoptosis induced by Hlxb9 overexpression alone.

We were able to demonstrate the existence of physical and functional interaction between menin and Foxa2. In addition, Foxa2 is overexpressed very early in menin deficient α-cells in Men1 F/F -GluCre + mice and in a substantial proportion of human glucagonomas. This is consistent with our observation that the overexpression of Foxa2 in cultured α-cells (αTC1-9) led to an increase in the proliferation of these cells 52 .

III.5 Epigenetic regulation

The decreased expression of P18 and P27, as well as Mll was observed in mouse and human MEN1 insulinomas 53 . The analyses showed that menin positively regulated the transcription of these gens by recruitment of Mll to the p27Kip1 and p18Ink4c promoters and coding regions. Thus, loss of menin resulted in reduced expression of p27Kip1 and p18Ink4c, and deregulated cell growth.

Several studies have demonstrated that menin stably associates with the N-terminus of MLL and is essential for initiation and maintenance of leukemogenic transformation by MLL fusions 54,55,56 .Thiel et al showed that menin recruits both wild-type MLL and oncogenic MLL-AF9 fusion protein to the loci of HOX genes to activate their transcription through both H3K4 and H3K79 methylation. They also showed that that menin binds the Ezh2 promoter, a polycomb component, and activates its expression, leading to the repression of C/EBPα target genes and of cell differentiation 57 . In parallel, Yokoyama et al. showed that conditional Men1 disruption in the mouse hematopoietic system impaired the self-renewal capacity of hematopoietic stem cells, associated with altered MLL and Ledgfmediated Hoxa9 expression 56 .

Menin loss resulted in increased ActivinB expression in -cell-specific Men1 mutant mice, which occurs along with the loss of insulin expression 58 . The work further revealed that ActivinB expression was mediated through a direct modulation of H3K27me3 marks on the Inhbb locus in Men1-deficient cell lines 59 . More importantly, the data demonstrated that menin binds to the Inhbb promoter where it favours the recruitment of Ezh2 via an indirect mechanism involving Akt-phosphorylation. The data may provide insights onto the molecular mechanisms underlying the occurrence of less differentiated PanNET, likely including non-functional PanNETs.

Feng, Z.J. reported that menin could bind to the PTN promoter together with polycomb repressive complex (PRC)2, increase the repressive chromatin mark H3K27me3, thus repress the expression of the PTN gene. The latter encodes a pro-cell migration receptor, pleotro-phin, in lung cells 60 .

Cao et al reported that menin expression is reduced in the liver of aging mice and that hepatocytespecific Men1 ablation induced liver steatosis in aging mice, due to loss of menin-mediated SIRT1 recruitment to control hepatic CD36 expression and triglyceride accumulation through histone deacetylation 61 .

IV. Summary

The generation and characterisation of Men1 mutant mouse models have greatly accelerated studies to pinpoint cellular and molecular mechanisms underlying MEN1 disease development, from cells of origin of tumorigenesis related to MEN1 syndrome to different factors and /or cell signaling pathways involved in the procedure due to menin inactivation. These advances would help us to better understand the tumour spectrum and tumour progression seen in MEN1 patients. Needless to say, acquired knowledge should also be valuable for finding prognostic biomarkers and potential therapeutic targets for MEN1 disease.

Faculté

  Institut des Sciences Pharmaceutiques et Biologiques Institut des Sciences et Techniques de la Réadaptation Département de formation et Centre de Recherche en Biologie Humaine Techniques des Activités Physiques et Sportives Observatoire des Sciences de l'Univers de Lyon Polytech Lyon Ecole Supérieure de Chimie Physique Electronique Institut Universitaire de Technologie de Lyon 1 Ecole Supérieure du Professorat et de l'Education Institut de Science Financière et d'Assurances Directeur : M. F. DE MARCHI Directeur : M. le Professeur F. THEVENARD Directeur : Mme C. FELIX Directeur : M. Hassan HAMMOURI Directeur : M. le Professeur S. AKKOUCHE Directeur : M. le Professeur G. TOMANOV Directeur : M. le Professeur H. BEN HADID Directeur : M. le Professeur J-C PLENET Directeur : M. Y. VANPOULLE Directeur : M. B. GUIDERDONI Directeur : M. le Professeur E. PERRIN Directeur : M. G. PIGNAULT Directeur : M. le Professeur C. VITON Directeur : M. le Professeur A. MOUGNIOTTE Directeur : M. N. LEBOISNE

Figure 1 :

 1 Figure 1: Mammary gland anatomy .........................................................................................

Figure 2 :

 2 Figure 2: Histological classification of breast cancer .............................................................

Figure 3 :

 3 Figure 3: Molecular classification of breast cancer and their characteristics ......................

Figure 4 :

 4 Figure 4: Schematic representation of ESR1 gene and ERα and its isoforms. .....................

Figure 5 :

 5 Figure 5: ERα signaling pathways. ...........................................................................................

Figure 6 :

 6 Figure 6: Hormonotherapy: mechanisms of action: ................................................................

Figure 7 :

 7 Figure 7: ESR1 genomic promoter organization. ....................................................................

Figure 8 :

 8 Figure 8: Epigenetic regulation of ESR1. .................................................................................

Figure 9 :

 9 Figure 9: ESR1 regulation by transcription factors. ...............................................................

Figure 10 :

 10 Figure 10: Clinical manifestations of MEN1 syndrome and their penetrance estimated at years of age. .................................................................................................................................

Figure 11 :

 11 Figure 11: Menin protein structure...........................................................................................

Figure 12 :

 12 Figure 12: Menin and the MLL complex ..................................................................................

Figure 13 :

 13 Figure 13: Menin a scaffold protein that regulate gene transcription. ..................................

Table 3 :

 3 TNM classification ....................................................................................................... Table 2: SBR classification ......................................................................................................... Summary of different Men1 mutant mouse models .................................................

DFS 2 FBP1

 2 eukaryotic initiation factor-4A eIF4E: Eukaryotic translation initiation factor 4E eIF4F: Eukaryotic initiation factor 4F eIF4G: Eukaryotic translation initiation factor 4G ERE: estrogen responsive elements ERK: extracellular signal-regulated kinase ERα: estrogen receptor α ERβ: estrogen receptor β EZH2: Enhancer of zeste homolog pointed domain-containing Ets transcription factor SREBP1: Sterol regulatory element-binding transcription factor 1 STAT: signal transducer and activator of transcription proteins TFAP2C: Transcription factor AP-

Figure 1 :

 1 Figure 1: Mammary gland anatomy Adapted from austincc.edu

Fig 2 Figure 2 :

 22 Figure 2: Histological classification of breast cancerAdapted fromMalhotra et al., 2010

Figure 3 :

 3 Figure 3: Molecular classification of breast cancer and their characteristics

b)

  ERα structure and localization: ER consists of 595 amino acids, composed of two domains of functional activity AF1 & AF2 located in the N-terminal domain (NTD) and C-terminal Ligand binding domain (LBD) respectively. These 2 domains are separated by a connecting DNA binding domain (DBD) and work together to mediate the transcriptional activity of the protein.

Fig 4 c)

 4 ERα isoforms:

4 Figure 4 :

 44 Figure 4: Schematic representation of ESR1 gene and ERα and its isoforms: (A) ESR1 structure composed of 8 exons (grey boxes) separated by introns (black lines), the vertical lines show the coding sequences leading to the functional domains of ERα protein. (B) Different ERα domains, A/B region composing the AF1 domain, C region or DNA binding domain, D region which serves as hinge region, and region E which forms the AF2 domain. Underneath are the 2 ERα isoforms showing the missing regions compared to thefull-length ERα.

  genomic pathway of estrogen signaling was discovered, owing to the observation of a quick response to estrogen treatment, as fast as 15 seconds compared to what was usually known of a 2-hour time lag after the treatment.

Fig 5 Figure 5 :

 55 Figure 5: ERα signaling pathways: A: Classical ligand-dependent genomic pathway, B: Nonclassical ERE independent genomic pathway, C: Ligand independent genomic pathway, D: Non genomic pathway.

Fig 6 Figure 6 :

 66 Figure 6: Hormonotherapy: mechanisms of action:

Fig 7 Figure 7 :

 77 Figure 7: ESR1 genomic promoter organization based on Ros et al.,2001 nomenclature: The promoter region of the ESR1 gene contains 9 non-coding exons (boxes), annotated from A to F-T1, T2, and 7 alternative promoters each having its own TSS (arrows), Exons E1 and T2 expression depends on the promoters E2 and T1, respectively. Exons A to D constitute the proximal part of the ERα promoter. Expression of exons T1 and T2 is restricted to the testicles while exons E1 and E2 in the liver. The numbers below represent the distance in (bp) from the transcription start site (+1), and the numbers above represent the distance in (kb) between exons. All exons represented in this scheme undergo alternative splicing due to a splice acceptor site located at position +163 bp from +1 transcription start site.

Figure 8 :

 8 Figure 8: Epigenetic regulation of ESR1: (A): In response to estrogen, msin3A and ERα are recruited to ESR1 proximal promoter mainly on the +1 position from transcription start site to repress ERα expression. (B) (upper panel):In ER+ cells pRb2/p130 recruits HDAC1, SUV39H1 and p300/CBP to ESR1 proximal promoter and increase acetylation marks opening the chromatin and activating its expression. In ERlines however, (lower panel), DNMT1 is recruited instead of p300/CBP decreasing acetylation marks and increasing methylation marks (CpG islands) on the promoter, leading to more compact chromatin and thus repressing ERα expression.

Figure 9 :

 9 Figure 9: ESR1 regulation by transcription factors: (A): differential regulation of ESR1 by MTA1 in ER+ and ER-breast cancer cells. (B): Schematic representation (as proposed by Dreijerink et al.,) of the regulation of ESR1 through formation of a loop between its distal (enhancer) promoter bound by GATA3 and FOXA1 and its proximal bound by menin and the MLL complex that activates gene transcription by increasing the H3K4me3 marks.

Fig 10 Figure 10 :

 1010 Figure 10: Clinical manifestations of MEN1 syndrome and their penetrance estimated at 40 years of age.

Figure 11 :

 11 Figure 11: Menin protein structure

Fig 12 Figure 12 :

 1212 Figure 12: Menin and the MLL complex: (A) In the endocrine pancreas menin interacts with MLL1/2 and play a tumor suppressive role by positively regulating the expression of cyclin inhibitors p18 and p27. (B) in leukemic cells, however, menin plays an oncogenic role by recruiting both wild type and fused MLL proteins (MLL-FP) to increase the expression of Hox genes. Inspired by Matkar et al.,2013

  Taking into consideration that menin loss in β-cell-specific Men1 mutant mice developing insulinomas resulted in increased Activin B encoded by the Inhbb gene, these results uncovered detailed mechanisms underlying the interaction

  phosphatase and negative regulator of the PI3K/AKT/mTOR pathway, PTEN controls several cellular processes, including proliferation, growth, metabolism, migration, and survival. Recently, Wong et al generated MPR (Men1 flox/flox Pten flox/flox RIP-Cre) and MPM (Men1 flox/flox Pten flox/flox MIP-Cre) mouse models with insulin-specific biallelic inactivation of Men1 and Pten in β-cells. Their results showed that concomitant loss of Pten and Men1

Figure 15 :

 15 Figure 15: Menin a scaffold protein that regulates gene transcription: 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.

Figure 14 :

 14 Figure 14: Menin a scaffold protein that regulates gene transcription: 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.

Figure 13 :

 13 Figure 13: Menin a scaffold protein that regulates gene transcription: 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.

  this study, Dreijerink et al. referred to the Dutch longitudinal MEN1 database to assess the incidence of breast cancer in MEN1 patients, and they found that out of 190 female patients, the relative risk of invasive breast cancer was 2.83 (P<0.001) and the mean (±SD) age at diagnosis of mostly luminal-type breast cancer was about 48 years ±8.8 years as compared with an age of 60 to 65 years in the general population, a feature being often observed in the patients carrying genetic predisposition. They validated their results using 3 independent cohorts from United States, Tasmania, and France which gave almost the same incidence risk values as those of the Dutch and the average age at diagnosis was 51 years. Furthermore, 8 out of 10 breast-cancer samples obtained from MEN1 Dutch 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. All these observations suggested strongly that MEN1 mutations could be involved in human breast carcinogenesis. More recently in 2016, a study that has analyzed the whole-genome sequences of 560 breast cancers has shown sporadic MEN1 mutations (though at very low frequency) to be one of the many other driver mutations (like BRCA1, TP53, PIK3CA, MYC, CCND1

:I. Article 1 :

 1 Reduced menin expression leads to decreased ERα expression and is correlated with the occurrence of human luminal B and ERnegative breast cancer subtypes My contribution: The study was initiated by my colleague Romain Teinturier (former PhD student in our lab), especially on the regulation of ERα expression by menin and menin's binding to the ESR1 promoter. I took part in this study by confirming his results, then, further elaborating the work especially for the MLL1/2, GATA3 and FOXA1 part. I conducted the related experiments, analysed and interpreted the data, prepared the figures and contributed to the manuscript writing. Status: Submitted in the journal of Breast Cancer Research and Treatment II. Article 2: Menin downregulation triggers the deregulated mTORC1 pathway and MYC transcription in ER-positive BC cell lines My contribution: Based on some preliminary data from my colleague Romain Teinturier who found that menin inhibition caused altered protein expression of members of the Akt/mTOR family, I went on further and investigated more in detail the relation between menin and the mTORC1 pathway, especially the translation initiation complex, downstream targets of mTORC1, in ER+ breast cancer cell lines. Given the contradiction between the molecular alteration and cell growth behavior of these cells, I developed the new axis on the role of menin in regulating the MYC pathway breast cancer. I conducted the experiments, analysed and interpreted the data, prepared the figures and contributed to manuscript writing. Status: Submitted in the journal of Breast Cancer Research.Results: Immunofluorescence staining revealed that early mammary neoplasia in Men1 mutant mice displayed weak ER expression. Furthermore, MEN1 silencing led to both reduced ESR1 mRNA and ER protein expression in MCF7 and T47D cells. To further dissect the regulation of ESR1 transcription by menin, we next examined whether and in which way menin could regulate the proximal ESR1 promoter, which has not been fully explored. Using ChIP analysis and reporter gene assays covering -2500 bp to +2000 bp of the TSS position, we showed that the activity of the proximal ESR1 promoter was markedly reduced upon menin downregulation independently of H3K4me3 status. Importantly, by analyzing the expression of menin in 354 human breast cancers, we found that a lower expression was associated with ER-negative breast cancers (p = 0.041).Moreover, among the 294 ER -positive breast cancer samples, reduced menin expression was not only associated with larger tumors (p = 0.01) and higher SBR grades (p = 0.005), but also with the luminal B breast cancer subtype (p = 0.006). Consistent with our clinical data, we demonstratedIntroductionBreast cancers are among the most common malignancies worldwide and remain the leading cause of cancer-related mortality in women1 . Previous receptor expression analyses enabled their classification into 4 major clinical subtypes, including luminal A, luminal B, HER2-enriched and triple negative 2 . The luminal A subtype encompasses approximately 44% of breast cancers. This

  left panel), H3K4me3 methylation was not altered upon menin/MLL inhibition in the tested region (Fig. 3c right panel). Furthermore, RT-qPCR analyses also showed that neither SiMLL1, nor SiMLL2, nor their combination affected ESR1 transcription (Fig. 3d left panel) and ER expression (Fig. 3d right panel). Taken together, our data provide evidence that menin regulates the proximal ESR1 promoter, likely via mechanisms involving other factors part from the compass-like complex.

  and (neo)/adjuvant therapy at the Centre Léon Bérard (CLB) between January 2001 and December 2003 (Additional file 1). Patients with complete data and with adequate samples assessable for menin by IHC were 354, among which 294 patients had ER+/HER2-tumors. The study was conducted in accordance with the guidelines in the Declaration of Helsinki and the use of all patient tissues was approved by local IRB and carried out according to French laws and regulations.

  charcoal-stripped serum(Biowest), in order to remove steroid hormones (steroid depletion). Cells were transfected with 20 nM SiRNA of, respectively, control SiRNA (5 nmoles, Eurogentec), two different SiRNA targeting human MEN1 transcript (SiMEN1 hs1 (HSS106462) and hs2 (HSS181079), ThermoFisher Sci.), SiRNA targeting human MLL1 (SiKMT2A: siRNA 107890 ThermoFisher Sci.), SiRNA targeting human MLL2 (SiKMT2B: siRNA s18833 ThermoFisher Sci.), using Jetprime® transfection reagent (Polyplus) for 72h according to the manufacturer's instructions. Inhibition of the menin-MLL interaction was achieved by MI503 (Active Biochem) at different concentrations. Prior to performing treatment with E2 and MI503, cells were grown in phenol red-free medium supplemented with 10% charcoal-stripped serum (Biowest), in order to remove steroid hormones (steroid depletion). Cells were then treated for 3 hours with E2 (Sigma) 10 -8 M and MI503 for 48h. The treatment was repeated after 24h due to the degradation of the inhibitor with time. Please also see Additional file 2 -Supplemental Materials & Methods.Construction of luciferase constructsWe used genomic DNA extracts from MCF7 cells to generate regions of the human ESR1 promoter, PrAB (genomic location: Chr6q25.1; 152127793-152129027) and PrC (genomic location: Chr6q25.1; 152124474-152127509) (Additional file 2). The resulting fragments of the proximal ESR1 promoter were cloned into the pGL3 Basic vector (Promega, Madison WI).

LegendsFig. 1

 1 Fig. 1 Reduced menin expression leads to decrease in ER expression. a, Co-

Fig. 2

 2 Fig. 2 Recruitment and effects of menin on the proximal ESR1 promoter. a, Schematic

Fig. 4

 4 Fig. 4 Comparison of Kaplan-Meier estimates for a, distant metastasis-free survival (DMFS), b, disease-free survival (DFS) and c, overall survival (OS) in the ER + /HER2 -cohort of breast

Fig. 5 Fig. 3

 53 Fig. 5 Menin interacts with GATA3 and FOXA1 and influences their expression. a, Western

Fig. 1 Fig. 2

 12 Fig. 1 Reduced menin expression leads to decrease in ERα expression. a, Co-immunofluorescence against menin and ERα on mammary gland sections from Men1 F/F WapCre -mice (upper panel) and Men1 F/F WapCre + mice (lower panel) at 10 months of age. Arrows in the inset illustrate that menin expression is correlated with ER expression. b, Western blot analyses using antibodies against menin and ER in MCF7, T47D and ZR75-1 cells treated with SiRNA control (SiCtrl) or SiRNA targeting the MEN1 gene (SiMEN1 hs1). c, Quantitative RT-qPCR analyses of MEN1 and ESR1 gene expression in MCF7 and T47D cells treated with SiCtrl or two different SiMEN1 (hs1 or hs2). d, Western blot analyses using antibodies against menin and ER in MCF7 and T47D treated with SiRNA control (SiCtrl) or SiMEN1 hs1, then subjected to estradiol (E2) stimulation at a concentration of 10 nM. e, Quantitative RT-qPCR analyses of the MEN1 and ESR1 gene expression in MCF7 and T47D cells treated with SiCtrl or SiMEN1 hs1, then subjected to estradiol (E2) stimulation at a concentration of 10 nM. All data are expressed as mean +/-SEM, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, ****. 105

Fig. 3

 3 Fig. 3 The regulation of the proximal ESR1 promoter by menin does not entirely rely on the MLL complex. a, Quantitative RT-qPCR analyses of ESR1 gene expression in MCF7 and T47D cells treated or not with the inhibitor of menin-MLL interaction, MI503 at a concentration of 2 μM. b, Western blot analyses of ER expression in MCF7 and T47D cells untreated (UT) or treated with MI503 at concentrations of 1, 2.5 and 5 μM. c, ChIP-qPCR analysis of the binding of the anti-menin antibody (left panel), or anti-H3K4me3 antibody (right panel) to the -2500 bp / +2000 bp area flanking the transcription start site (TSS) on the ESR1 gene in MCF7 cells treated or not with MI503 at a concentration of 2 μM. d, Quantitative RT-qPCR (left panel) and Western blot (right panel) analyses of the expression of MLL1 (KMT2A), MLL2 (KMT2B) and ESR1 genes in MCF7 cells treated with SiCtrl, SiMLL1, SiMLL2, or combined SiMLL1 and SiMLL2.

Table 1 :Table 2 :

 12 Correlation between menin expression and the clinico-pathologic factors of from 354 breast cancer patients. *Correlation by Pearson's Chi square test unless otherwise specified † Difference between means by Student's T test. Correlation between menin expression and the clinico-pathologic factors of 294 ER+/HER2-breast cancer patients. *Correlation by Fisher's exact test.

Fig. 4

 4 Fig. 4 Comparison of Kaplan-Meier estimates for a, distant metastasis-free survival (DMFS), b, disease-free survival (DFS) and c, overall survival (OS) in the ER + /HER2 -cohort of breast cancer patients included in the TMA of breast cancer patients, showing the survival curves of tumors with high (> 100) (green curve) or low menin nuclear staining (≤ 100) (blue curve).

Fig. 5

 5 Fig. 5 Menin interacts with GATA3 and FOXA1 and influences their expression. a, Western blot analyses using antibodies against menin, GATA3 and FOXA1 in MCF7 cells treated with SiCtrl or SiMEN1 hs1. b, Quantitative RT-qPCR analyses of the GATA3 and FOXA1 transcription in MCF7 cells treated with SiCtrl or SiMEN1 hs1 c, Co-immunoprecipitation analyses were carried out by incubating nuclear lysates of MCF7 cells with either anti-IgG, or anti-GATA3 or FOXA1 antibodies and subjected to Western blot analyses. d, GST pull-down using GST-full-length (FL) menin and nuclear fraction of protein lysates of MCF7 cells, detected by Western blot using the anti-GATA3 or FOXA1 antibodies. Coomassie blue stained gel showing levels of recombinant GST proteins used in GST pull-down assay. e, Upper: PLA analysis with anti-menin and anti-GATA3 antibodies in MCF7 and ZR75-1 cells, the latter expressing no menin. Under: the quantification of PLA analysis. 111

  culture, estrogen treatment and SiRNA transfections MCF7, ZR75-1 and T47D, derived from human invasive breast ductal carcinomas, are ER+ breast cancer cell lines displaying luminal mammary epithelium features. MCF7 and ZR75-1 cells were cultured in DMEM (Gibco Thermofisher Scientific) containing 25 mM glucose supplemented with 10% (v/v) foetal calf serum (FCS), 2 mM L-Glutamine, 100 unit/ml Penicillin, 100 μg/ml streptomycin, 1X MEM non-essential amino acids solution (all being supplied by Gibco by life technologies), 100 mM β-mercaptoethanol, at 37°C with 5% CO2. T47D cells cultured in RPMI medium (Gibco by life technologies) containing 25 mM glucose and supplemented with 10% (v/v) FCS, 2 mM L-Glutamine, 100 unit/ml Penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 1 mM Sodium Pyruvate, at 37°C with 5% CO2. MDA-MB-231 and MDA-MB-453 cell lines are human metastatic ER-breast cancer cell lines, which were cultured in the same medium as MCF7 and ZR75-1 except non-essential amino acids. MCF7 and ZR75-1 cells were seeded at 250,000 cells/well and T47D cells were seeded at 300,000 cells per well in 6-well plates and cultured for 24h. Then, cells were cultured in white complete phenol-red free medium containing 10% charcoal stripped serum (Biowest) and treated with 10 nM E2 (β-estradiol, E2758-1G, SIGMA) for 30 mins,1h, 2h, 4h, 8h, 24h (depending on the experiment) or transfected with 20 nM SiRNA of either control SiRNA(5 nmoles, Eurogentec) or SiRNA against the targeted gene using Jetprime® transfection reagent (polyplus) for 72h. Two different SiRNA targeting human MEN1 transcript (SiMEN1 hs1 (HSS106462) and hs2 (HSS181079), ThermoFisher Sci.), two SiRNA against MYC (SiMYC1 (SASI_HS01_00222676) and SiMYC2 (SASI_HS01_00222677), Sigma-Aldrich), One SiRNA targeting eIF4G (eIF4G SiRNA (h): sc-35286, SANTA CRUZ BIOTECHNOLOGY, INC) were used.

  Bethyl laboratories, A300 105A), rabbit Anti-ERα 60c at 1/2000 (Millipore, 04-820), mouse Anti GAPDH at 1/25000 (Santa cruz, sc-47734), rabbit Anti MYC at 1/1000 (cell signaling, 9402), rabbit Anti-CDK2 at 1/1000 (Santa Cruz, sc-163) ,rabbit Anti-CDK4 at 1/1000 (Santa Cruz, sc-260) , mouse Anti-cyclin E at 1/1000 (Santa Cruz, sc-377100) , mouse Anti-cyclin D at 1/1000 ( Santa Cruz, sc-450) ,rabbit Anti-p53 at 1/1000 (cell signaling , 9282) , rabbit Anti-caspase 3 at 1/1000 (cell signaling 8G10), rabbit Anti-PARP at 1/1000 (cell signaling, 46D11) , rabbit Anti-eIF4G at 1/1000 (cell signaling, C45A4), mouse Anti-eIF4E at 1/1000 (Santa Cruz, A-10), mouse Anti-eIF4A at 1/1000 (Santa Cruz,H-5), rabbit Anti-phospho 4EBP1 ser 65 at 1/1000 (Cell Signaling, S65), rabbit Anti-Phospho p70 S6K1 at 1/1000 (Cell signaling, 49D7), rabbit Anti-AKT total at 1/1000 (Cell signaling), rabbit Anti-Phospho 4EBP1 Thr37/46 at 1/1000 (cell signaling, 236B4), rabbit Anti-Phospho AKT Ser 473 at 1/1000 (Cell signaing,D9E), rabbit Anti-Phospho 4EBP1 total at 1/1000 (Cell signaling, 53H11), rabbit Anti-Phospho p60 S6K1 (T389) at 1/1000 (Cell signaling, 9205S), rabbit Anti-Phospho S6RP Ser 235/236 at 1/1000 (Cell signaling, 2211S) , rabbit Anti-Phospho p85 S6K1 (T389) at 1/1000 (Cell signaling, 9205S), rabbit Anti-S6K1 total at 1/1000 (Cell signaling).Real-time reverse transcriptase and qPCR analysesRNAs were extracted using RNeasy-Kits (Macherey-Nagel). Real-time PCR analyses were carried out on a Step-One RT-System (Applied-Biosystem, France) using SYBR-Green (Life-Technology, France) and results of each sample were normalized with the 28S house keeping gene used here as a control. Primers used were as following: 28s Fw: 5'-CGATCCATCATCCGCAATG-3', Rev: 5'-AGCCAAGCTCAGCGCAAC-3'; MEN1 Fw: 5'-CATCCAGGACTACAACTACT -3', MEN1 Rev: 5'-ATGACATCATTGGCTACTTC -3';

  thus analyzed AKT expression after menin knockdown in MCF7 cells and found that MEN1 KD increased slightly the phosphorylated AKT without altering the total protein level (Fig 1e), and the use of AKT inhibitor inhibited the activation of S6K1 and S6RP triggered by MEN1 KD (Fig S1b),

  Fig 2b), compared to the menin negative ZR75 cell line with same treatment (Fig S2a). In addition, mining MEN1 gene co-expression in BC TCGA data provided a correlation coefficient for each of the genes available in the data set. Gene set enrichment analysis identified the GO term Translational initiation as enriched in genes positively correlated with MEN1 in ER+ tumors (Fig 2c). As shown in Fig 2d and S2b, among the so-annotated genes, EIF4EBP1 encoding 4EBP1, as well as EIF4BP3 were positively correlated with MEN1, in agreement with the increased expression of 4EBP1 protein observed in MCF7 cells with menin overexpression.

  metabolism, and apoptosis of different cancer cells, including BC cells, and analyzed the effects of MEN1 silencing on MYC expression. The results showed that menin inactivation decreased MYC expression at both the mRNA (Fig 3a, left panels) and protein levels (Fig 3a, right panels) in MEN1 KD MCF7 and T47D cells. However, our data showed heterogeneous results in 2 tested ER-cell lines, with reduced MYC transcription but increased MYC protein levels in MDA231 cells, and no effect at all in MDA453 cells (Fig 3b). To understand how menin regulates MYCexpression on transcriptional level, we sought to investigate the binding of menin to both the MYC proximal promoter and the MYC 5' enhancer previously reported 27 by ChIP analysis. We found that menin bound indeed the MYC 5' enhancer and the proximal MYC promoter, including the areas involving transcription start site (TSS) and +300bp sites of the MYC promoter in MCF7 cells(Fig 3c). We also analyzed the influence of MYC expression on the expression of ER and menin.

  thus first treated MCF7 cells with β-estradiol (E2) at different time points(Fig S4). We found that while the expression of ERα protein was decreased after E2 stimulation, the expression of MYC was significantly increased at both protein and mRNA levels, starting 1h after treatment, whereas menin expression did not change (Fig4a). Interestingly, MEN1 silencing resulted in decreased MYC expression under E2 stimulation in MCF7 cells, suggesting that menin expression is critical for estrogen mediated MYC activation) (Fig4b).

  but their interaction was reduced upon E2 stimulation (Fig 4c left panel), We also noticed that E2 treatment in MCF7 cells led to increased interaction between menin and ER (Fig 4c right panel), suggesting menin's role in estrogen-mediated MYC activation functions likely through its physical interaction with ER .Co-expression of menin and MYC in some breast cancers and their combined inhibition has better effects on cell proliferation reductionData mining analyses were then carried out using different publicly available BC mRNA expression datasets to further investigate co-expression between menin and MYC in BC. Although no positive correlation was detected between MEN1 and MYC mRNA expression in TCGA BC samples in all tumors and in ER+ tumors, an interesting significant positive correlation was seen in all tumors from the SMC 2018 dataset, as well as in ER+ tumors only(Fig 5a). With regards to the TCGA data, a positive correlation was observed for ER-tumors (Pearson correlation coefficient = 0.256) (Fig5b).

  effects with MYC inhibition to reduce cell proliferation. Although separate inhibition of MYC and menin using SiRNA or 10058-F4 (MYC/MAX inhibitor) decreased cell proliferation in MCF7 cells, the combined use of MEN1 KD and SiRNA and MYC inhibitor 10058-F4 did not decreased further cell proliferation in MCF7 line, suggesting that the effects of menin depends mainly on MYC expression (Fig 5d).

Fig 1 .b

 1 Fig 1. Menin downregulation leads to mTORC1 activation in ER+ cell lines. a Cell

Fig 2 .

 2 Fig 2. MEN1 silencing results in activation of eIF4F formation. a Quantitative RT-PCR (left

Fig 3 .

 3 Fig 3. Menin regulates MYC expression and binds to the MYC promoter in ER+ BC cells.

Fig 4 .

 4 Fig 4. Menin is needed for estrogen-mediated MYC activation and physically interacts with

Fig 5 .

 5 Fig 5. Correlation between MEN1 and MYC mRNA expression in breast cancer and effects

Fig 1 .

 1 Fig 1. Menin downregulation leads to mTORC1 activation in ER+ cell lines. a Cell proliferation curves of MCF7, T47D (2 upper panels), and that of MDAMB231 and MDAMB453 cell lines (2 lower panels) transfected with SiCtrl or SiMEN1 hs1 at 24h after cell culture.Western blot showing menin expression in the above cells treated with different siRNAs as indicated. Data shown are mean (± SEM) of technical replicates from one representative experiment out of three. b Western blot analysis showing the protein expression of phosphorylated and total S6K1 and S6RP in MCF7 (left panel) and T47D (right panel) cells transfected with SiCtrl or SiMEN1 hs1 and treated with insulin and/or the mTOR inhibitor RAD001 as indicated. c Western blot analysis detecting the phosphorylated and total 4EBP1 in T47D cells transfected with SiCtrl or SiMEN1 hs1 and treated with insulin and/or the mTOR inhibitor RAD001 as indicated. d Western blot analysis showing the protein expression of S6K1, S6RP, and 4EBP1 in MCF7 cells transfected with the control vector PCI-Neo or the vector PCI-MEN1 overexpressing menin and treated with insulin and/or RAD001 as indicated. e Western blot analysis showing the protein expression of phosphorylated (Ser 473) and total AKT in MCF7 cells transfected with SiCtrl or SiMEN1 hs1 and treated with insulin and/or the AKT inhibitor MK2206 as indicated. 144

Fig 2 .

 2 Fig 2. MEN1 silencing results in activation of eIF4F formation. a Quantitative RT-PCR (left panel) and Western blot (right panel) showing the mRNA and protein expression respectively of eIF4E, eIF4A, eIF4G in MCF7 cells transfected with SiCtrl or two different SiMEN1 hs1 or hs3. Data shown, normalized against GAPDH, are presented as means (± SEM) of technical replicates from one representative experiment out of three. b Proximity ligation assay (PLA) showing the interaction between either eIF4G/eIF4E or eIF4G/eIF4A in MCF7 (Upper panel) and T47D (lower panel) cell lines transfected with SiCtrl, SiMEN1 hs1, SieIF4G and SieIF4G+MEN1 hs1, respectively. Quantification of PLA signals, represented by dots/cell, for each condition is shown on the right panels. c GO term Translational initiation gene set was identified among MEN1 correlated genes with Gene Set Enrichment Analysis. d Clinical correlation from TCGA data between MEN1 and mTOR, MEN1 and 4EBP1, MEN1 and EIF4E, MEN1 and EIF4G2 in ER+ BC. 145

Fig 3 .

 3 Fig 3. Menin regulates MYC expression and binds to the MYC promoter in ER+ BC cells. Quantitative RT-PCR (left panels) and Western blot (right panels) showing MYC expression in ER+ MCF7 and T47D cell lines (a) and ER-MDAMB231 and MDAMB453 cell lines (b) transfected with SiCtrl or two different SiMEN1 hs1 or hs3. Quantitative RT-PCR data shown, normalized against GAPDH, are presented as means (± SEM) of technical replicates from one representative experiment out of three. c Schematic representation of the primers used ChIP-quantitative PCR analysis (left panel). ChIP-qPCR analyses analysis showing menin binding (normalized with input) on different regions of the MYC promoter (right panel). Results were compared to IgG and an unrelated region on chromosome 1 (Chr1) used as controls for ChIP. Cells were also transfected with SiMEN1 hs1 to confirm the specificity of binding. d Quantitative RT-PCR (left panel) and Western blot (2 right panels) showing the mRNA and protein expression of different MYC target genes in MCF7 cells transfected with SiCtrl or two different SiMEN1 hs1 or hs3. 146

Fig 4 .

 4 Fig 4. Menin is needed for estrogen-mediated MYC activation and physically interacts with MYC in BC cells. a Quantitative RT-PCR (left panel) and Western blot (right panel) showing the protein and mRNA expression of menin, ERα, and MYC in MCF7 cells after estrogen treatment for 1 or 2h. b Left panel: Quantitative RT-PCR showing the expression of menin, ERα, and MYC in MCF7 cells transfected with SiCtrl or SiMEN1 hs1 or hs3 in the absence or presence of 1h estrogen stimulation; Right panel: Western blot showing the expression of menin, ERα, and MYC in MCF7 cells transfected with SiCtrl or SiMEN1 hs1 or hs3 in the absence or presence of 2h estrogen stimulation. c Upper panel: PLA showing the physical interaction between menin and MYC (left panel) or menin and ERα (right panel) in MCF7 cells before and after estrogen treatment. SiMEN1 hs1 and SiMYC were used as controls; Lower panel: Quantification of PLA signals measured in terms of numbers of dots/cell.

Fig 5 .

 5 Fig 5. Correlation between MEN1 and MYC mRNA expression in breast cancer and effects of cell proliferation inhibition with combined inactivation of MEN1 and MYC. a Left panel: Data mining analysis from the SMC 2018 dataset showing the positive correlation between MEN1 and MYC mRNA expression in BC including all subtypes. Right panel: same analysis from the SMC 2018 dataset in ER+ BC subtypes. b Correlation of mRNA expression between MEN1 and MYC observed in ER+ (left panel) and ER-(right panel) tumors from TCGA dataset. c Inverse correlation between the expression of MEN1 and 2 inhibitory factors in the MYC pathway, MYCB2 and MYCT1, in ER+ BC from the TCGA dataset. d Proliferation curve of MCF7 cells transfected with either SiMEN1 hs1 or SiMYC or both (left panel), and transfected with SiMEN1 hs1 or treated with 10058 F4 (MYC/MAX inhibitor) or both (middle panel). Western blot showing MYC protein expression in MCF7 cells treated with DMSO or 20 and 50 μM of 10058 F4 inhibitor (right panel).

  by dual role Dreijerink et al., in 2017 may help to solve these discrepancies and the eventual confusions thus elicited. They proposed that menin could play as a tumor suppressor during the procedure of the initiation of tumorigenesis of normal luminal mammary epithelial cells, but as an oncogenic role in sporadic ER+ breast cancer initiated by other factors, the key point being the essential role of menin in the regulation of the ESR1 gene mediated by MLL/menin complex via H3K4me3 sites. The data obtained from my thesis works support fully such a hypothesis. Therefore, when the MEN1 gene is mutated or inactivated in normal mammary cells, it results in decreased ERα expression, leading to aberrant cell proliferation and differentiation, and tumor development, whereas in ER+ breast cancer triggered by other factors, menin could act as a coactivator of ERα, playing a crucial role in promoting the cell proliferation by the latter.

  After the fine analyses carried out by Dreijerink et al mainly on distal regulatory sequences of the ESR1 gene, our current works has been focalized on the proximal ESR1 promoter. We have demonstrated by ChIP analysis and luciferase assay that menin binds to a large area of the proximal promoter of ESR1 and regulates its activity, the area extending from -2500bp to +2000bp of ESR1 transcription start sites (TSS). Dreijerink et al showed that menin regulates ERα through binding to ESR1 enhancer region through a loop-structure bound by a complex involving MLL1/2, GATA3 and FOXA1. Intriguingly, our results showed that, though treatment with menin/MLL inhibitor MI503 decreased the expression of ERα and menin binding to the ESR1 proximal promoter, no changes were detected in the H3K4me3 marks.Moreover, the combined MLL1 and MLL2 knockdown had no effect on ERα expression in MCF7 cells, thus raising the questions about the exact role of MLL in the regulation of ESR1 transcription by menin. Taking Dreijerink et al's observations into account, we also investigated the relation between menin, GATA3 and FOXA1. We found that menin does physically interact with GATA3 and FOXA1 in our cellular model, but only regulate their expression at the protein not the transcriptional level. All these results suggest that the complex described by Drejerink may not be enough to fully explain the molecular mechanisms underlying the regulation of ESR1 transcription by menin.

Article 1 :Article 2 :Article 3 :Article 4 :Article 5 :

 12345 my 3-years PhD, I had the opportunity to contribute to other work done by my colleague Yakun Luo, who works on menin's role in prostate cancer. I helped with the results analysis and discussion as well as provided technical help and support. Men1 disruption in Nkx3.1-deficient mice results in AR low/CD44 + microinvasive carcinoma development with the dysregulated AR pathway (published in the journal Oncogene) Menin is essential for activating the MYC locus and MYC-mediated androgen receptor transcription in AR-dependent prostate cancer cells (submitted to the journal Oncogene) I also had the chance to read and integrate different studies done in our field, mainly menin. Together with my colleague and Dr. Chang, we were able to summarize this work in three review articles: Involvement of the MEN1 Gene in Hormone-Related Cancers: Clues from Molecular Studies, Mouse Models, and Patient Investigations (published in the journal Endocrines) Research progress of MEN1 gene mutation mouse model: exploring the molecular mechanism of MEN1 inactivation in tumor development (published in in Journal of Pancreatology) Generation and characterization of Men1 mutant mouse models for studying MEN1 disease (published in Chinese Journal of Digestion)

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.

3. 3 . 2 .

 32 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].

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].

  Men1 mutant mouse models: molecular mechanisms underlying the role played by the MEN1 gene in tumorigenesis I. Introduction:

  Oncogene, 22(23), 3511-3517. https://doi.org/10.1038/sj.onc.1206578Macaluso, M., Cinti, C., Russo, G., Russo, A., & Giordano, A. (2003). pRb2/p130-E2F4/Oncogene, 22(23), 3511-3517. https://doi.org/10.1038/sj.onc.1206578Madureira, P. A., Varshochi, R., Constantinidou, D., Francis, R. E., Coombes, R. C., Yao, K. M., & Lam, E. W. (2006). The Forkhead box M1 protein regulates the transcription of the estrogen

Table 1

 1

Extent of resection (R) Absence or presence of residual tumor

	Mx	Distant metastasis can't be assessed
	M0	No distant metastasis
	M1	Distant metastases
	Rx	Presence of residual tumor can't be
		assessed
	R0	No residual tumor
	R1	Microscopic residual tumor
	R2	Macroscopic residual tumor

Table 1 : TNM classification

 1

	c) SBR classification:
	SBR (Scarff-Bloom-Richardson) classification is based on the histological and
	cytological characteristics of a tumor in which tumors are graded by adding up scores
	for tubule formation (percent of the tumor forms normal duct structures) nuclear
	pleomorphism (irregular breast duct epithelial cells) and mitotic count (the number of
	dividing cells seen in 10x high power microscope field), each of which is given 1 to 3

Table 2 : SBR classification

 2

	d) Molecular classification:
	More recently in attempts to better reflect the genetic heterogeneity of breast cancer and
	improve prediction of tumor behavior and therapy, classification into molecular subtypes
	depending on molecular markers such as the estrogen receptor (ER), progesterone
	receptor (PR), and human epidermal growth factor receptor 2 HER2 was purposed to
	facilitate diagnosis and treatment, though it should still be used in association with on
	other characteristics such as the tumor size, grade, and lymph node invasion

  Hormonotherapy: ERα targeting in breast cancer treatmentBreast cancer treatments usually include surgery, radiotherapy, chemotherapy, and endocrine or hormonotherapy. ER+ breast cancer types are the most benefited from the latter, because they depend on ERα signaling for survival as it regulates several genes required for proliferation and metastasis. Different types of endocrine therapies exist that can block ERα's actions at different levels. Selective estrogen receptor modulators (SERM) have been used for over than 30 years now and have shown great efficiency in treating ER+ breast cancer in premenopausal women.Unlike aromatase inhibitors that block the synthesis of estrogen from testosterone,

B.

SERMs' mechanism of action involves blocking the ER action in the cancer cells by binding, instead of estrogen, to the receptor, SERMs have proven their efficiency in treating postmenopausal women.

(Maximove et al.,2013)

Table 3 : Summary of different Men1 mutant mouse models

 3

	56

  menin protein in a cohort of breast cancer patients having surgery at the Centre Léon Bérard (CLB) hospital from 2001 to 2003. For sake of correlations and survival analyses, the whole cohort of patients was divided into high menin expression (H score > 100) and low menin expression (H score ≤ 100). Accordingly, among the whole cohort of 354 patients, 151 (42.7%) had low menin

	expression, while 203 (57.3%) had high expression. Among the 294 patients with ER+/HER2-
	tumors, 116 patients (39.5%) had a low nuclear menin expression and 178 patients (60.5%) had a
	high expression. In this cohort of 354 patients, we found that lower nuclear menin (H score ≤ 100)

  26. Nakata, Y. et al. that plays a pivotal role in human T helper type 2 cell development c-Myb , Menin , GATA-3 , and MLL form a dynamic transcription complex that plays a pivotal role in human T helper type 2 cell development. 116, 1280-1290 (2013). 27. Bonnavion, R. et al. Foxa2, a novel protein partner of the tumour suppressor menin, is deregulated in mouse and human MEN1 glucagonomas. The Journal of Pathology 1-12 (2017) doi:10.1002/path.4885. 28. Mair, B. et al. Gain-and Loss-of-Function Mutations in the Breast Cancer Gene GATA3

Result in Differential Drug Sensitivity. PLoS Genetics 12, 1-26 (2016) 29. Wu, Y. et al. Disruption of the menin-MLL interaction triggers menin protein degradation via ubiquitin-proteasome pathway. Am J Cancer Res 9, 1682-1694 (2019).Choucair, A. et al. The arginine methyltransferase PRMT1 regulates IGF-1 signaling in breast cancer. Oncogene 38, 4015-4027 (2019). 30. Choucair, A. et al. The arginine methyltransferase PRMT1 regulates IGF-1 signaling in breast cancer. Oncogene 38, 4015-4027 (2019).

Table 1 Correlation between menin expression and the clinico-pathological factors of breast cancer patients

 1 . *Correlation by Pearson's Chi square test unless otherwise specified † Difference between means analyzed using the Student t-test.

Table 2 Correlation between menin expression and the clinico-pathological factors of ER + /HER2 -breast cancer patients

 2 . *Correlation by Fisher's exact test.

  30) Dermit, M.; Casado, P.; Rajeeve, V.; Wilkes, E. H.; Foxler, D. E.; Campbell, H.;

	Critchlow, S.; Sharp, T. V.; Gribben, J. G.; Unwin, R.; Cutillas, P. R. Oxidative Stress
	Downstream of MTORC1 but Not AKT Causes a Proliferative Defect in Cancer Cells
	Resistant to PI3K Inhibition. Oncogene 2017, 36 (19), 2762-2774.
	https://doi.org/10.1038/onc.2016.435.
	(31) Jackson, H. W.; Fischer, J. R.; Zanotelli, V. R. T.; Ali, H. R.; Mechera, R.; Soysal, S. D.;
	Moch, H.; Muenst, S.; Varga, Z.; Weber, W. P.; Bodenmiller, B. The Single-Cell Pathology
	Landscape of Breast Cancer. Nature 2020, 578 (7796), 615-620.
	https://doi.org/10.1038/s41586-019-1876-x.

  and regulator of ERα and ESR1 respectively, is now considered playing a dual role depending on the stage and type of the tumor, meaning that in the early stages or initiation of breast

cancer, menin plays a tumor suppressive role, while in already developed ER+ breast cancer, menin plays an oncogenic role. Both molecular and clinical data acquired during my thesis works provide relevant clues supporting this notion. I noticed, though Dreijerink et al showed that menin recruits MLL/compass-like complex to the distal (enhancer) ESR1 promoter and regulate its expression, we didn't find any changes in H3K4me3 on the proximal promoter after treatment with menin/MLL inhibitor. Thus, this area of the promoter remains to be an interesting field of research to study. How menin exactly regulates ESR1 on this site? What other factors could be involved? We have tried the candidate gene approaches (already tested few factors like HDAC1 and JunD). But given the fact that menin interacts with a wide number of epigenetic and transcription factors, this approach could be long and inefficient. For this reason, applying techniques like RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) on this area of the promoter could give later on better, faster, and more detailed description of all the factors and mechanisms involved in this regulation.

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.

	Patient	Stage	Gleason score	Menin expression
	013CHI	pT2c	6 (3 + 3)	Low
	015FRE	pT2c	7 (3 + 4)	Absent
	024TAP	pT2c	6 (3 + 3)	Low
	027LHO	pT2c	7 (3 + 4)	Absent
	031GEO	pT2c	7 (3 + 4)	Low
	043F0N	pT2c	6 (3 + 3)	Low
	044COU	pT2c	7 (3 + 4)	Low/Normal
	054BRY	pT2c	7 (3 + 4)	Low
	055SER	pT2c	7 (3 + 4)	Low
	082MAD	pT2c	7 (3 + 4)	Low
	005FRA	pT4	8 (4 + 4)	Absent
	016BEN	pT3b	9 (4 + 5)	Absent
	022SAN	pT3a	8 (4 + 4)	Low/Normal
	028NAH	pT3a	8 (4 + 4)	Absent
	029BAF	pT4	9 (4 + 5)	Low
	034LIT	pT3a	8 (4 + 4)	Low
	049SAR	pT3b	8 (4 + 4)	Absent
	052PEI	pT3a	8 (4 + 4)	Low
	063GIR	pT3a	8 (4 + 4)	Low
	065GUI	pT3a	9 (4 + 5)	Low
	179RIB	HR	N/A	Low
	181MAN	HR	N/A	Absent
	184GEH	HR	N/A	Normal
	186MBA	HR	N/A	Absent
	187FLE	HR	N/A	High/Normal
	189DIO	HR	N/A	High/Normal
	193CAS	HR	N/A	Normal/low
	211CEV	HR	N/A	Normal/Low
	212JAC	HR	N/A	Normal
	213DRO	HR	N/A	Normal
	Quantitation of menin intensity staining based on a high, normal, and
	low expression has been assessed in the sample with normal prostate
	and different tumor grades according to Gleason's pattern scale.
	Quantitation of menin intensity staining by IF analysis based on a
	high, normal and low expression has been assess in the sample with
	normal prostate and different tumor grades according to Gleason's
	pattern scale.

Afin de mieux comprendre ces mécanismes, nous avons mené des recherches cliniques et expérimentales et avons constaté que l'inhibition de l'expression de MEN1 entraînait une diminution de l'expression de ERα aux niveaux transcriptionnels et protéiques dans les lignées cellulaires ERα-positives. Alors que Dreijerink et al. ont montré que menin régule le promoteur et l'enhancer ESR1 via le complexe MLL, nous avons découvert que menin se lie et régule le promoteur ESR1 proximal, vraisemblablement indépendamment du MLL. Il est intéressant de noter qu'en analysant l'expression de menin dans une cohorte de 354 cancers du sein humains, nos données cliniques ont montré que la réduction de l'expression de menin était davantage corrélée au sous-type de cancer du sein luminal B et au sous-type de ERα-négatif. Ces données cliniques sont en cohérence avec nos données expérimentales, puisque nous avons aussi démontré que menin interagit avec GATA3 et FOXA1, des cofacteurs de la régulation de ESR1 et des marqueurs connus du sous-type luminal A. Dans l'ensemble, nos données mettent en évidence sur le rôle important de menin dans la formation des sous-types de cancer du sein, probablement lié à son rôle de régulateur de l'ERα.En parallèle, nous avons également montré que l'inactivation de menin dans les cellules du cancer du sein entraînait une réduction de la prolifération cellulaire dans deux lignées ERα-positives. D'un point de vu moléculaire, nous avons découvert que, bien que l'inhibition de l'expression de MEN1 active la voie mTOR, elle entraîne une réduction de l'expression de MYC aux niveaux transcriptionnel et protéique dans les lignées cellulaires du cancer du sein ERα-positives. Nous avons également constaté que menin se lie au promoteur et à l'enhancer MYC, et régule ainsi l'expression de ses gènes cibles dans ces cellules. Sur le plan clinique, nous avons également observé que l'expression de menin est négativement corrélée avec mTOR et ses gènes en aval, mais également avec deux gènes inhibiteurs de MYC.

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

Acknowledgments

Acknowledgements

We are grateful to Brigitte MANSHIP for her assistance in proofreading the manuscript.

Acknowledgements

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

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.

Acknowledgement & Funding

We would like to thank Dr. M Le Romancer for fully supporting our research activities, and Dr. P Bertolino for his regular scientific input. This 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.

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). R.T was the recipients of a PhD-fellowship from

Funding

This study was supported by 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. R.A.Z was the recipient of a PhD-fellowship from the Association "G04MEDIA S.A.R.L", Lebanon. 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.

Data availability statement

The data that support the findings of this study are available from the corresponding author, [CXZ for basic research and IT for clinical data analyses], upon reasonable request.

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.

Author contribution statement

RT and RAZ conducted the experiments, analysed and interpreted the data, prepared figures and manuscript; LK participated in the design of the clinical study, performed the statistical analysis and participated in manuscript preparation; YL participated in the molecular and cellular studies and interpreted the data; LM participated in the molecular and cellular studies and interpreted the data; SG participated in the design of the molecular study and established methods; LC participated in the design of the molecular study and data interpretation, and helped to draft the manuscript; PB provided technical and material support, and participated in data interpretation and manuscript preparation; TB conceived the design of the clinical study and participated in clinical data collection and data interpretation; IT conceived the design of the clinical study, participated in clinical data collection, data interpretation and manuscript preparation, and provided critical revision of the manuscript; M.L.R and C.X.Z conceived and supervised the study and manuscript preparation, and obtained funding.

Conflict of interest statements

Nothing to disclose

Consent for publication

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

Ethics approval and consent to participate

The specimens of patients with breast cancer were conducted according to French laws and

Competing of Interests

The authors declare that they have no competing interest. supporting that the interplay between menin and MYC could be existing in a substantial proportion of BC.

Author Contributions

The current work allowed us to uncover an important partnership between menin and MYC in ER+ BC cells. It'll be relevant to know, in the future, what would be the role of menin/MYC in ER-BC cells, and whether menin could also be involved, as an oncogenic factor, in other cancers where MYC plays a decisive role.

Supplementary information

The online version of this article (https:// doi.org/10.1038/s41388-020-01589-1) contains supplementary material, which is available to authorized users.

Author contributions RT and YL conducted the experiments, analyzed and interpreted the data, and prepared figures and manuscript; MD-P, VF, and FV supervised the development of the tissue banks, and performed pathological analyses and data interpretation; VV-G, RAZ and RB provided technical and material support and participated in data interpretation; NG and IG provided material and methodological support and participated in data interpretation; PB and MLR participated in study design and provided critical revision of the manuscript; and CXZ conceived and supervised the study and manuscript preparation and obtained funding.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

Ethical approval

The specimens of patients with PCa were conducted according to French laws and regulations. All animal experiments were performed in accordance with the animal care guidelines of the European Union and were validated by the local Animal Ethic Evaluation Committee (CECCAPP: C2EA-15 agreed by the French Ministry of High School and Research, Autorisation de projet CLB-2012-053).

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author Contributions: R.A.Z. and Y.L. prepared manuscript and figures. M.L.R. and V.V.-G. participated in study design and provided critical revision of the manuscript. C.X.Z. conceived and supervised the manuscript preparation and obtained funding. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.

Abbreviations

ADT Androgen deprivation therapies AP-1

Activator protein

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.

Abstract

MEN1 mutation results in the predisposition to multiple endocrine neoplasia type 1 (MEN1) syndrome affecting various endocrine and non-endocrine cells. The generation of murine models using gene targeting approach has afforded remarkable tools for studying the MEN1 gene and its related diseases. In particular, these studies led to unravelling the cellular and molecular events occurred after MEN1 inactivation, revealing, therefore, detailed mechanisms underlying the development of the disease. In this review, we focus on the currently available data concerning the molecular functions of the MEN1 gene uncovered using Men1 mutant mouse models in connection with MEN1 syndrome.

Different models Tempero-spacial spectrum of Men1 disruption

Conditional β-Cell Men1 mutant mice (Men1 F/F -RipCre + ) Bertolino P et al.,2003 10 ;Crabtree et al.,2003 11 ;Biondi CA et al.,2004 12 α-Cell Men1 mutant mice (Men1 F/F -GluCre + ) Lu J et al., 2010 13 ; Shen HC et al., 2010 14 Pan-pancreatic Men1 mutant mice (Men1 F/F -Pdx1Cre or -pTF1Cre) Shen HC et al., 2009 15 ; Bonnavion R et al.,2015 16 Parathyroid Men1 mutant mice (Men1 F/F -PthCre) Libutti SK et al., 2003 17 Mammary gland Men1 mutant mice (Men1 F/F -WapCre + )

Seigne C et al.,2013 18 Craniofacial osteogenic cell and osteoblast Men1 mutant mice (Men1 F/F -osteocalcin-Cre and -Runx2Cre) Kanazawa I et al., 2015 19 Liu P et al.,2017 20 Inducible Crossing Men1 F/F mice with transgenic mice carrying tamoxifen-inducible CreER Schnepp RW et al., 2006 21 inducible β-cell-specific PdxCreER Yang Y et al., 2010 22 Inducible CreER driven by the rat insulin promoter (Men1 F/F -RIP2Cre ER )

Lines KE et al.,2017 23

III. Molecular mechanisms triggered by Men1 inactivation

The MEN1 gene codes for a protein named menin 24 that interacts physically with many protein partners, including in particular those involved in transcriptional regulation 25 . It is believed, therefore, that menin may exert various biological functions through its partners to influence different cell signaling pathways in a cell context dependent manner. Different Men1 mutant mouse models and derived Men1-null cells provide relevant tools for dissecting complex events triggered by MEN1 inactivation under distinct cellular and molecular contexts.

III.1 Synergistic effects of Men1 disruption with other cancer genes and pathways

Heterozygous Men1 mutant mice have been used to investigate eventual synergistic effects between the Men1 gene and other cancer genes involved in endocrine tumour development. The double heterozygotes Men1 and pRb mutant mice displayed a combined tumour spectrum observed in each of the individual heterozygote's mutant mice, without decrease in age of onset 26 . Similar approach was used by the same lab to determine whether complete or partial Trp53 inactivation could alter the tumour phenotype seen in Men1 mutant mice. Their data demonstrated that simultaneous heterozygous deletion of Men1 in mice with either heterozygous or homozygous Trp53 disruption did not result in formation of tumours at any new sites. The works implies that there are independent