CTC Circulating Tumor Cell CTGF Connective Tissue Growth Factor CTHRC1 Collagen Triple Helix Repeat Containing 1 CXCL-12 Chemokine Ligand 12 CXCR4 Chemokine Receptor 4 DKK1 Dickkopf-related Protein 1

Abstract

Bone metastasis is a common complication of advanced breast cancers and is clinically responsible of bone fractures, hypercalcemia and pain for which only palliative therapies are proposed. Breast tumor cells that preferentially invade bone express a set of deregulated genes that enhance bone tropism and facilitate bone marrow engraftment which may lead to the formation of overt osteolytic lesions. Molecular pathways underlining these steps are regulated through the tight control of genes expressed by cancer cells interacting with cells from the bone microenvironment. In this context, microRNAs act as regulators of gene expression and control multiple aspects of bone metastasis, including tumor cell escape from the primary site, dissemination, invasion of the bone marrow and secondary outgrowth.

MicroRNA transcriptomic profiling of osteotropic breast cancer cell lines identified drastic down-regulation of the miR-30 family (miRs-30). In the clinic, low expression of miRs-30 in breast primary tumors is associated with poor distant metastasis-free survival and hormoneinsensitive status. In a model of human bone metastasis in vivo, the forced expression of miRs-30 in a breast cancer cell line that is highly and specifically metastatic to bone inhibited bone metastasis. We demonstrated that miRs-30 inhibit tumor cell invasiveness and stimulate osteoblastogenesis, in vitro, and reduces tumor burden and osteoclast activity, in vivo. Consistent with that, the expression of several genes that promote bone metastasis were inhibited by miRs-30. Among these, expression of connective tissue growth factor (CTGF) was up-regulated in human bone metastasis.

The early steps of bone metastasis were studied in a mouse model using spontaneously metastatic mouse breast cancer cell lines inoculated in the mammary gland. In this model, miRs-30 did not alter tumor growth or metastatic dissemination to bone. However, miRs-30 inhibited cell invasiveness and cancer stem cell-like phenotype of these metastatic cells.

We conclude that miRs-30, by interfering negatively with bone metastasis, represent a potential therapy to repress gene targets that promote bone metastasis.

Résumé

Les métastases osseuses sont des complications fréquentes du cancer du sein, responsables sur le plan clinique d'hypercalcémie, fractures osseuses et douleurs, pour lesquelles, il n'existe que des traitements palliatifs. Les cellules tumorales de carcinomes mammaires qui métastasent au site osseux expriment des gènes qui favorisent le tropisme osseux de ces cellules ainsi que leur ancrage et développement dans la moëlle osseuse. Les mécanismes moléculaires sous-jacents à ces processus sont contrôlés par l'expression génique des cellules tumorales qui interagissent avec le microenvironnement et les cellules osseuses. Dans ce contexte, les microARNs en tant que régulateur endogène de l'expression génique, interfèrent avec les différentes étapes de la formation des métastases osseuses, incluant l'échappement des cellules tumorales de la tumeur primaire, la dissémination et l'invasion du site osseux, ainsi que l'apparition de lésions ostéolytiques.

Les profils transcriptomiques des microARNs de cellules tumorales mammaires à caractère ostéotropique montrent que l'expression de la famille de microARNs-30s (miRs-30) est inhibée dans ces cellules. En clinique, la faible expression des miRs-30 est associée à un mauvais diagnostique de rechute et au statut hormono-résistant. Dans un modèle animal de métastases osseuses, l'expression forcée des miRs-30 dans des cellules tumorales qui métastasent fortement et spécifiquement à l'os, inhibe la formation des métastases osseuses. Nous montrons que les miRs-30 inhibent l'invasion et stimulent l'ostéoblastogenèse, in vitro et réduisent la charge tumorale et l'ostéoclastogenèse, in vivo. En accord avec ces résultats, l'expression de gènes qui stimulent les métastases osseuses est inhibée par les miRs-30. Parmi ces gènes, l'expression du CTGF (connective tissue growth factor) est augmentée dans les métastases osseuses humaines.

Les étapes précoces des métastases osseuses sont étudiées par inoculation de cellules tumorales métastatiques murines dans la glande mammaire de souris. Dans ce modèle, les miRs-30 n'altèrent pas la croissance tumorale et la dissémination métastatique à l'os. Cependant les miRs-30 inhibent l'invasion et le caractère de cellules souches tumorales de ces cellules métastatiques.

Ces résultats suggèrent que les miRs-30, en régulant négativement les métastases osseuses, représentent une thérapie potentielle pour réprimer des gènes cibles qui stimulent les métastases osseuses. Figure 2. Histological section of a mouse tibia, stained with the Masson's trichrome. Figure 3. Osteoblast differentiation. Figure 4. Possible fate of mature osteoblast. Figure 5. Osteoclast-osteoblast interactions in the basic multicellular unit (BMU). Figure 6. Classification of bone metastases. Figure 7. The vicious cycle. Figure 8. MicroRNA biogenesis. Figure 9. MicroRNA genomic locations. Figure 10. Derivation of murine breast cancer cell lines. Figure 11. Metastatic dissemination of breast cancer cell lines after inoculation of these cells in mice mammary gland. Figure 12. Follow-up of bioluminescence imaging from a mouse inoculated with 4T1luc2 in mammary gland. Figure 13. Spontaneous metastatic dissemination of 4T1.2luc2 and 66c14luc2 to bone and lung after cell inoculation into the mice mammary fad pad. Figure 14. Expression of miRs-30 in human and murine breast cancer cell lines. Figure 15. Expression of miRs-30 target genes in 4T1.2luc2 and 66cluc2 expressing miRssponge-30. Figure 16. Expression of miRs-30 target genes in 4T1.2luc2 and 66cluc2 stably transduced with lentiviral microRNA inhibitors. Figure 17. Expression of miRs-30 in 4T1.2luc2 after transient transfection of miR-30 mimics and miR-30 inhibitors. Figure 18. MiRs-30 decreased 4T1.2luc2 invasion whereas trapping miRs-30 increased 4T1.2luc2 invasion. Figure 19. MiRs-30 did not affect 4T1.2luc2 growth in mice mammary fad pad. Figure 20. Expression of miRs-30 in mammospheres obtained by growing 67NR, 66c14luc2 and 4T1.2luc2 without anchorage and without serum.

List of Figures

Figure 21. Expression of cancer stem cell markers in 4T1.2luc2 and 66c14luc2. Figure 22. Expression of Vimentin and Twifilin in 4T1.2luc2 and 66c14luc2. Figure 23. Expression of bone metastasis-related genes in 4T1.2luc2 (A) and 66c14luc2 (B). Table 1. Frequency of site specific metastases according to molecular breast cancer subtype.

List of Tables

Table 2. Arterial blood supply in target organs of cancer metastasis.

Table 3. Comparison of genomic sequences of miR-30 family members between mouse and human.

Table 4. Differences between miR-30 family members in the nucleotide sequence.

INTRODUCTION

Solid primary tumors spread to distant organs with selective affinity, the skeleton being one of the most common organs to be affected by metastatic tumors. Breast cancer is one of the main osteotropic tumors leading to osteolytic lesions characterized by bone destruction that results from enhanced osteoclast activity. These skeletal lesions cause pathological fractures, hypercalcemia and nerve compression and can negatively impact on survival [START_REF] Ell | SnapShot: Bone Metastasis[END_REF]) [START_REF] Weilbaecher | Cancer to bone: a fatal attraction[END_REF]. Therefore there is a need to understand underlying molecular mechanisms that are deleterious to the skeleton in order to improve existing treatments and develop new targeted therapies [START_REF] Coleman | Effects of bone-targeted agents on cancer progression and mortality[END_REF]. The metastatic cascade leading to bone metastasis is initiated in primary tumours where a subset of cancer cells undergoes epithelialmesenchymal transition, acquires mobility to migrate into the surrounding stroma and seeds at distant sites to grow [START_REF] Buijs | Osteotropic cancers: from primary tumor to bone[END_REF]. The specific development of bone metastasis requires the recruitment of circulating tumour cells in the bone marrow, their adaptation to survive in the surrounding microenvironment where they alter the functions of osteoclasts and osteoblasts, and hijack signals coming from the bone matrix. Bone metastasis, being a very inefficient process, is regulated by multiple factors and molecular pathways, mainly through the tight control of genes expressed by interacting cells. The small endogenous noncoding microRNAs (miRs) which have the capacity to control multiple genes are master regulators of gene expression and thus redirect or reprogram biological pathways. Consequently, in cancer, they can act either as promoters or suppressors of tumor development and metastatic progression. Large sets of miRs are underexpressed in tumors compared to normal tissue, whereas miR genes located in fragile chromosomal regions are susceptible to amplification or deletion, often promoting tumorigenesis and metastatic dissemination [START_REF] Krol | The widespread regulation of microRNA biogenesis, function and decay[END_REF]. Here, we asked whether miRs are involved in bone metastasis formation and progression in breast cancer, and whether modulation of their expression might regulate specific steps of bone metastasis.

In the first part of the work, the activity of miRs on bone metastasis has been study in an experimental mouse model of human bone metastasis, after inoculating the breast cancer cells in mice arterial blood. In this, we could investigate the regulation of specific steps of bone tropism, homing, invasion and second outgrowth of breast tumor cells in bone. Analyzing transcriptomic profile of miRs in osteotropic human breast tumor cells identified a drastic down-regulation of the miR-30 family (miRs-30) in these cells. Restoring miRs-30 in an osteotropic breast cancer cell line decreased bone metastasis in vivo predominantly through the down-regulation of genes associated with osteomimicry.

This work is reported in the manuscript under the form of a paper (in preparation of submission for publication), entitled "The Tumor Suppressor miRs-30-5p Family Regulates Human Breast Cancer Cell Colonization in Bone" (pages 59 to 111).

In the second part of the work, the activity of miRs on breast cancer cell dissemination to bone has been studied in an orthotopic tumor xenograft mice model, in which spontaneously metastatic breast cancer cells were inoculated into mammary gland. These results are reported in "Experimental work" (pages 112 to 140).

Finally, we have shown that the transcription factor Twist1 facilitates breast cancer bone metastasis formation through a mechanism dependent of an oncomir, miR10b, which in turn promotes the development of osteolytic lesions. This paper entitled "TWIST1 expression in breast cancer cells facilitates bone metastasis formation" is included in the manuscrit.

PART 1 LITERATURE REVIEW 1. Breast cancer and its complications

Breast cancer is the most common cancer affecting women with 1.67 million new cases in 2012. It is more common in less developed (883,000 cases) than more developed regions (794,000).

Regarding mortality, it ranks as the most frequent in women in less developed regions (324,000 deaths) followed by cancers of the lung (281,000 deaths). In more developed regions, breast cancer is the second leading cause of cancer death (198,000 deaths), after lung cancer (210,000 deaths) (Figure 1) [START_REF] Ferlay | Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012[END_REF]. Breast cancer is a complex and diverse disease and in order to stratify patient risk and outcomes, ongoing research strives for accurate classification of the disease. An important histopatological feature of breast cancer related to patient outcome is the expression of certain receptors, such as oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Based on receptors subtypes and differences in gene expression, molecular subtypes of the cancer have been defined. These include: luminal A, luminal B, HER2-expressing and triple-negative breast cancer (TNBC).

Luminal A subtype accounts for 40-55 % of breast cancers, making it the largest tumor gene subtype. It demonstrates ER/PR positivity and HER2 negativity with a low cellular proliferation index (Ki-67 < 14 %). These carcinomas are well-differentiated and they have the lowest systemic relapse rates. The median duration of survival from time of first metastasis is approximately 2.2 years (Chikarmane et al., 2015), this is statistically higher than any other subtype [START_REF] Kennecke | Metastatic Behavior of Breast Cancer Subtypes[END_REF].

Luminal B subtype tumors express ER/PR, can be HER2 positive or negative and often have high cellular proliferation index (Ki-67 > 14 %) (Chikarmane et al.). Patients with ER/PR positivity are more likely to be post-menopausal women with lower-grade tumors at presentation and with worse survival than luminal A patients (1.6 years) [START_REF] Kennecke | Metastatic Behavior of Breast Cancer Subtypes[END_REF].

The HER2-positive tumors constitute 12-30 % of invasive breast cancers and can be ER/PR positive or negative. This subtype is associated with increased proliferation rate, angiogenesis, tumor invasiveness, high nuclear grade and these patients are more likely to have multifocal/multicentric cancers and nodal involvement. It is more often found in pre-menopausal women and was originally associated with poorer clinical outcome (Chikarmane et al.). In 1998, the clinical course for HER2-positive subtype tumors was significantly changed with the introduction of trastuzumab, the monoclonal antibody against extracellular portion of HER2 tyrosine kinase receptor [START_REF] Slamon | Use of Chemotherapy plus a Monoclonal Antibody against HER2 for Metastatic Breast Cancer That Overexpresses HER2[END_REF]. However, without adjuvant trastuzumab treatment these patients have a higher risk of local recurrence than luminal A or B cancers, with a median survival from time of first metastasis of approximately 0.7 years [START_REF] Kennecke | Metastatic Behavior of Breast Cancer Subtypes[END_REF].

TNBC refers to ER/PR and HER2 negativity in immunohistochemistry. It comprises around 13-25 % of breast cancers and presents with higher histological grade and proliferation rate than other subtypes. TNBC is more frequent in young African Americans, and in women with BRCA1 mutation. These tumors are more likely to be present at the physical examination rather than at mammographic screening because they have a rapid growth rate (Chikarmane et al., 2015). Furthermore, patients with TNBC have a poorer prognosis (relapse-free survival and overall survival) when compared to luminal A and B subtypes [START_REF] Lin | Sites of distant recurrence and clinical outcomes in patients with metastatic triple-negative breast cancer[END_REF].

Approximately 30 % of newly diagnosed breast cancer patients will have tumor spread to regional lymph and up to 5 % will already have metastases at presentation (Chikarmane et al.).

Molecular phenotyping of breast cancer on the basis of three receptors mentioned above has become standard of care in the management of breast cancer patients due to the fact that the molecular phenotype of breast cancer determines the timing, pattern and outcome of metastatic disease [START_REF] Kennecke | Metastatic Behavior of Breast Cancer Subtypes[END_REF][START_REF] Perou | Molecular portraits of human breast tumours[END_REF]. patients. Virtually all relapses occurred within the first 5 years among basal-like, TN nonbasal and HER2 groups, whereas luminal subtypes experienced relapses between 5 and 15 years.

Regarding the correlation of breast cancer subtype with site of metastases, it has been found that molecular subtype predicts the secondary events ( The predominant metastatic site for each subtype is marked in green. Numbers reflect the percentage [%] of patients from the specific subtype positive for metastasis in a given organ [START_REF] Kennecke | Metastatic Behavior of Breast Cancer Subtypes[END_REF].

Almost all breast cancer subtype, except for basal-like, predominantly metastasize to bone. This study confirms the previous observation that bone is of particular clinical importance in breast and prostate cancers because of the prevalence of these diseases. At postmortem examination, 70% of patients dying of these cancers have evidence of metastatic bone disease [START_REF] Coleman | Clinical Features of Metastatic Bone Disease and Risk of Skeletal Morbidity[END_REF]. Due to such a high incidence rate of bone metastases in breast cancer patients, it is of urgent need to find effective treatment for bone metastasis. In the next session, the bone physiology, the process of bone metastasis formation and current treatment regiments will be discussed.

Physiology and pathophysiology of bone

Skeleton is responsible for the maintenance of the shape of the body, it shields vital organs from external insults, serves as a scaffold for the muscles allowing their contraction to be translated into bodily movements, resists mechanical load during locomotion and weight bearing, and provides a reservoir of calcium, magnesium, bicarbonate and phosphate. The skeleton is made up of mineralized and unmineralized connective tissue matrix, highly specialized cells, and spaces that include the bone marrow cavity, vascular canals, canaliculi and lacunae. There are two different designs in the architecture of the normal skeleton: cortical and cancellous. Cortical bone accounts for about 80 % of bone mass and forms the solid outer wall of all bones, whereas cancellous, or trabecular, bone rates for 20 % of the total bone mass and consists of threedimensional network of trabeculae located mainly in the metaphysis of long bones such as the femur and inside of flat bones such as ileum or cranium. It is in intimate contact with the hematopoietic marrow [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF].

Like in other tissues, the bone undergoes a process of regeneration or remodeling throughout life.

During development and growth, the skeleton is sculpted in order to achieve its shape and size by the removal of bone from one site and deposition at a different site. Once the skeleton has reached maturity, regeneration continues in the form of a periodic replacement of old bone by new bone at the same location. This process is called remodeling and it is carried out by osteoclasts and osteoblasts, termed basic multicellular units (BMU) [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF].

Osteoblasts and osteoclasts are derived from progenitors originating in the bone marrow and the process of bone remodeling is controlled locally by growth factors and cytokines [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF].

The cellular components of bone and their function

Osteoblasts-bone forming cells

Osteoblasts are cells commonly known for their bone building function. They compose approximately 4-6% of total resident cells in the bone [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF]. As many other cells of the connective tissues (fibroblasts, chondrocytes, myoblasts and adipocytes), osteoblasts arise from a common pluripotent mesenchymal stem cell (MSC).

The expression of Bone Morphogenetic Proteins (BMPs) and Wnt pathways is crucial for the early steps of osteogenesis, where they promote MSCs commitment towards an osteo/chondroprogenitor. The osteo/chondroprogenitor cell is defined when at least the following osteoblast-specific transcription factors are expressed: Runt-related transcription factor 2 (RUNX2), Distal-less homeobox 5 (DLX5) and Osterix (OSX) (Figure 3). The master gene of osteoblast differentiation is RUNX2 [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF]. It has been demonstrated that Runx2-null mice lack of osteoblasts [START_REF] Komori | Targeted Disruption of Cbfa1 Results in a Complete Lack of Bone Formation owing to Maturational Arrest of Osteoblasts[END_REF]. In humans, mutations of RUNX2 cause Cleidocranial Dysplasia (CCD), an autosomal-dominant disease with dramatic abnormalities in bones [START_REF] Lee | Missense mutations abolishing DNA binding of the osteoblastspecific transcription factor OSF2/CBFA1 in cleidocranial dysplasia[END_REF]. RUNX2 up regulates osteoblast-related genes (COL1a1, ALP, BSP, BGLAP) in osteoblasts. Moreover, the overexpression of RUNX2 in human MSCs isolated from adipose tissue triggers commitment towards osteoblasts, by increasing Alkaline Phosphatase (ALP) activity and osteocalcin (OCN) expression [START_REF] Lee | Electroporation-mediated transfer of Runx2 and Osterix genes to enhance osteogenesis of adipose stem cells[END_REF]. At later stage, RUNX2 inhibits osteoblast maturation and causes osteopenia [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF], showing a dual role for RUNX2 in osteoblastogenesis.

Another transcription factor needed to direct MSC into osteoprogenitor differentiation is DLX5.

It promotes osteogenesis under the control of BMPs, is highly expressed in the developing skeleton and is induced during fracture healing [START_REF] Han | Protein kinase A phosphorylates and regulates the osteogenic activity of Dlx5[END_REF].

The last mentioned transcription factor regulating osteoblast differentiation is OSX, also known as SP7. It is a downstream target of RUNX2, whose expression in MSC progenitors is stimulated by BMPs and Insulin-like Growth Factor (IGF) [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF].

Once these main transcription factors have been expressed and a pool of osteoblast progenitors has been established, there is a proliferation phase, during which osteoblast progenitors acquire ALP activity. These cells become now pre-osteoblasts which express relatively high amounts of ALP [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF]. ALP has been long thought to play a role in bone mineralization, however, the precise mechanism of mineralization and the exact role of ALP in this process remain unclear [START_REF] Sharma | Alkaline Phosphatase: An Overview[END_REF]. Pre-osteoblasts also produce Type I Collagen which is a major product of the bone-forming osteoblast. It is initially secreted in the form of a precursor which is further processed extracellularly to mature three-chained Type I Collagen molecules. These then assemble themselves into a collagen fibril. Individual collagen molecules become interconnected by the formation of pyridinoline cross-links which are unique to bone (9).

The late stage of osteoblast differentiation is characterized by a higher expression of the bone matrix proteins OCN, BSP I and II, of Type I Collagen and of proteoglycans, such as decorin and biglycan. This newly formed, non-mineralized bone matrix is called osteoid. Osteoblasts producing osteoid are mature osteoblasts which appear in histological sections as a single row of cuboidal-shaped cells with a round basal nucleus (Figure 3). They also play a role in the early steps of mineralization, which relies on the synthesis of hydroxyapatite crystals within the matrix vesicles, derived from the membrane surface of osteoblasts. The aging osteoblasts have three possible destinies: (I) undergo apoptosis, (II) give way to the osteocytes or (III) become bonelining cells (Figure 4). Osteoblast differentiation is mainly controlled by two signaling pathways, the Wnt pathway and Bone Morphogenetic Proteins patways (BMPs).

Wnt proteins are a family of at least 19 secreted glycoproteins involved in many cell functions.

They signal through a canonical (WNT3a, WNT10b) and a non-canonical pathway (WNT1, WNT5A). The former relies on -catenin stabilization, and is crucial for a correct bone mass achievement. More precisely, in the canonical pathway, Wnt proteins bind to Frizzled (FRZ) and to LRP5/6 receptors, which in turn activate a downstream signaling that inhibits the activity of Glycogen Synthase Kinase 3 beta (GSK3 ), with a consequent prevention of -catenin phosphorylation. When hypophosphorylated, -catenin is more stable, it can translocate into the nucleus where it regulates the transcription of Wnt target genes. Whereas in absence of Wnt, GSK3 is active and phosphorylates -catenin, eventually leading to its ubiquitination, thus turning off Wnt signaling [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF]. Moreover, knock-down of WNT5a and WNT10b show reduced in trabecular bone and bone mass [START_REF] Takada | A histone lysine methyltransferase activated by non-canonical Wnt signalling suppresses PPAR-transactivation[END_REF]. Some molecules specifically interfere with the Wnt pathway, such as the Dickkopf-related protein 1 (DKK1).

Deficiency of DKK1 has been associated with increased bone formation [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF].

Other inhibitors of Wnt pathway acting in a similar way is sclerostin (SOST), which is predominantly produced by osteocytes and the secreted frizzled-related proteins (sFRP), whose members inhibit Wnt pathway by sequestering Wnt from binding to FRZ receptors or by directly binding to FRZ [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF].

The second pathway crucial for osteoblastogenesis is related to Bone Morphogenetic Proteins (BMPs). These proteins (at least 20) belong to the Transforming Growth Factor (TGF) superfamily and they were first described as being involved in bone metabolism as components of the bone matrix able to cause ectopic bone formation when injected subcutaneously or intramuscularly in mice [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF]. Mutations in the genes coding for BMPs in mice clarified the role of these proteins in bone metabolism [START_REF] Kingsley | The mouse short ear skeletal morphogenesis locus is associated with defects in a bone morphogenetic member of the TGF superfamily[END_REF]. Among the different isoforms, BMP-2, 4, 5, 6, and 7 are clearly implicated in osteogenesis, likely due to their ability to stimulate the expression of the transcription factors RUNX2 and OSX. BMP-2 is especially expressed in areas surrounding cartilage condensation, where it has a prominent role for chondrocyte proliferation and maturation, thus enhancing endochondral ossification. Moreover, it is crucial for osteogenesis. It has been shown that a lack of BMP-2 and BMP-4 in osteoblast conditional KO mice led to a severe impairment of osteogenesis [START_REF] Bandyopadhyay | Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis[END_REF]. Finally, BMP-7 has been shown to increase ALP activity in osteoblasts and to trigger bone mineralization [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF].

Osteocytes and bone lining cells

As mentioned in the previous section, mature osteoblast can end up as osteocytes being the most abundant cells in bone. They are characterized by a striking stellate morphology, reminiscent of the dendritic network of the nervous system. Their major feature is multiple extensions of their plasma through narrow canaliculi, connecting them to each other as well as with the boneforming osteoblasts and the lining cells. The cell-cell communication is also achieved by an interstitial fluid that flows through the osteocytes canaliculi. This network is essential in coordinating the response of bone to mechanical and biological signals. Currently, a general consensus exists that osteocytes are involved in translating mechanical stimuli into biochemical signals. Following that, osteocytes orchestrate bone formation and bone resorption acting on osteoblasts and osteoclast differentiation and function.

Regarding the role of osteocyte in osteoblast regulation, it has been shown that osteocyte apoptosis or osteocyte network disruption increased bone formation [START_REF] Moriishi | Osteocyte Network; a Negative Regulatory System for Bone Mass Augmented by the Induction of Rankl in Osteoblasts and Sost in Osteocytes at Unloading[END_REF]. Also, it is well known that SOST is a secreted glycoprotein specifically expressed in osteocytes which inhibits osteoblasts differentiation and function by antagonizing the canonical Wnt pathway.

Consistent with this finding, bone formation is enhanced in Sost-deficient mice [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF]. Osteocyte also regulates osteoclast formation. Tatsumi et al. showed that osteocyte apoptosis results in an increase in RANKL (Rank Ligand) expression in bone, thus enhancing the osteoclast formation and function, leading to increase of the bone resorption [START_REF] Tatsumi | Targeted Ablation of Osteocytes Induces Osteoporosis with Defective Mechanotransduction[END_REF]. These data suggest that osteocytes can act as mechanosensing cells but also are able to regulate bone homeostasis in a coordinated and complex manner.

Another possible destiny for mature osteoblast is becoming the so-called bone lining cells. They are quiescent and flat shaped osteoblasts which cover the bone surface, functionally representing the resting phase of the bone remodelling process. There is not much known about their function.

A likely role of these cells is to prevent the direct interaction between osteoclasts and bone matrix when bone resorption should not occur [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF].

Osteoclasts-bone resorbing cells

Osteoclasts are multinucleated giant cells (50-100 μm in diameter) with abundant mitochondria, numerous lysosomes and free ribosomes. They differentiate from myeloid precursors under the influence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand, being RANKL, supplied by osteoblasts and/or osteocytes (Figure 5) [START_REF] Charles | Osteoclasts: more than "bone eaters[END_REF]. Osteoclasts (OCs) differentiate from OC precursors (OCP) under the influence of M-CSF and RANKL produced by osteoblast (OB) lineage cells including osteocytes. As OCs create a resorption pit, growth factors, including TGF and IGF1, are released from the bone matrix. These growth factors may recruit mesenchymal osteoblast progenitors and promote their differentiation into mature cells that secrete osteoid to fill the area of resorbed bone. Some OBs differentiate further into matrix-embedded osteocytes.

A decoy receptor for RANKL, termed osteoprotegrin (OPG), which is synthetized by the osteoblast lineage, inhibits osteoclast differentiation.

The main function of osteoclasts is degradation of bone. They secrete proteolytic enzymes (e.g., cathepsin K, O, B, L) and acid, which hydrolyze and solubilize the organic and inorganic components of bone, respectively [START_REF] Charles | Osteoclasts: more than "bone eaters[END_REF]. Enzyme and proton secretion is directed into the resorption lacunae where the degradated bone matrix components are endocytosed. Next, these degradated bone matrix components are transcytosed to the membrane area opposite the bone, where they are released [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF]. Subsequent to the osteoclastic resorptive phase, coupling mechanisms promote the recruitment and differentiation of mesenchyme-derived osteoblast progenitors at the resorption lacunae. After these cells mature into osteoblasts, they line the eroded bone surface and secrete the organic component of boneosteoid, which is mineralized over time by the incorporation of hydroxyapatite. As osteoblasts secrete osteoid, some cells are entrapped within the matrix where they eventually become osteocytes (Figure 5 ) [START_REF] Charles | Osteoclasts: more than "bone eaters[END_REF].

Osteoclasts are characterized by high amounts of the phosphohydrolase enzyme tartate-resistant acid phosphatase (TRAPase). This feature is frequently used for the detection of osteoclasts in bone specimens. Osteoclasts bear on their surface several antigens that are also present on hematopoietic cells, such as: CD45, the common leukocyte antigen; ICAM-1, the cellular ligand for certain integrins; integrinα2 and integrinβ1 that constitute the collagen receptor; integrinαv and integrinβ3 which constitute the vitronectin receptor responsible for mediating the attachment of osteoclasts to the bone surface, and finally CD9, a gene encoding for tetraspin family [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF].

Osteoclast function however is not only restricted to bone degradation. Coupling of bone formation and resorption requires cooperation between BMU and other cells residing within bone matrix, such as osteocytes, macrophages and T cells. Each of these cells produces a variety of factors involved in bone remodeling in health, but also in disease. In the next section, the process of bone remodeling in healthy condition will be reviewed.

Bone remodeling

The balance between bone deposition and resorption is crucial for the proper development and maintenance of bone size, shape and integrity. To maintain this balance, molecular communication between osteoblasts and osteoclasts, and also between bone cells and other bone marrow cells is fundamental. Ideas to explain the coordinate regulation of bone resorption and formation center developed on the notion that osteogenic growth factors are released from the bone matrix during bone resorption, and mediate the subsequent recruitment of osteoblasts to the remodeling site [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF]. Matching bone formation and resorption, is referred to as osteoclast-osteoblast "coupling".

An essential step in the process of remodeling is that sites needing to be remodeled must be selected from among the millions possible at any one time in the skeleton. Bone is maintained in a healthy state because remodeling takes place only where it is needed to replace old or damaged bone. To initiate a remodeling cycle, the site of activity must be chosen, regardless of whether remodeling is initiated in response to damage, to change in loading or to remove old bone.

Although the initiating event has usually been described as resorption by osteoclasts, other mechanisms also occur to ensure that this takes place. Such an initiating event might come from osteocytes recognizing that a specific area of bone needs to be replaced, and signaling through their canaliculae to surface cells. Subsequent signals could arise from apoptosis of osteocytes, or even of lining cells themselves, resulting in the release of paracrine factors and chemokines that attract osteoblast and osteoclast precursors and vascular elements.

Hematopoietic precursors for osteoclast formation are provided from nearby marrow precursors, as well as through the capillary blood supply closely associated to bone. As it has been mentioned before, osteoclast formation requires mainly the signals of M-CSF and RANKL which come from osteoblast lineage. Studies on genetically manipulated mice suggested that it is not only early osteoblast precursors but also fully differentiated and matrix-embedded osteocytes that provide RANKL to the osteoclast precursors [START_REF] Xiong | Matrix-embedded cells control osteoclast formation[END_REF]. Moreover, beside osteoblastogenic lineage, lymphocytes T and lymphocytes B have been shown to be high producers of RANKL [START_REF] Capulli | Osteoblast and osteocyte: Games without frontiers[END_REF].

The most completely described intercellular interaction within the BMU is the balance in production of RANKL and its decoy receptor OPG by the osteoblast lineage, and their interaction with RANK on the cell membrane of osteoclast precursors. A number of local factors, produced by osteoblasts that stimulate RANKL production, have been identified such as semaphorin 3B, oncostatin M (OSM), interleukin-6 (IL-6), interleukin-1b (IL-1b), TNFα, family coreceptor subunit, glycoprotein 130 (GP130) [START_REF] Sims | Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit[END_REF].

Coupling of bone formation to resorption

It is essential that the formation component of remodeling needs to replace the exact amount removed by resorption.

Although circulating hormones, including PTH and 1,25-dihydroxyvitamin-D 3 , were considered to be the prime regulators of remodeling, it has been clear for some years that locally generated cytokines are the key influences, influencing bone cell communication and subsequent function in complex ways, and often themselves regulated by the hormones and the nervous system. One of the main examples of osteoclast regulation by osteoblasts is the osteoblast lineage-derived RANKL which signals through its receptor RANK. However, what about the reverse direction? Does the signaling come also from osteoclasts to osteoblasts? Arguments began to be presented in favor of this, and in the past few years a number of candidate 'coupling factors' of osteoclast origin have been proposed.

One of such factors is active TGF 1, released from the bone matrix during bone resorption. It might couple bone formation to resorption by inducing the migration of bone MSCs to sites that have been resorbed, thus making them available for differentiation and bone formation in remodeling [START_REF] Tang | TGF-1-induced migration of bone mesenchymal stem cells couples bone resorption with formation[END_REF]. Complementing that, osteoblast-and matrix-derived insulin growth factor-1 (IGF-1) was found to promote osteoblast differentiation by favoring recruitment of MSCs, in response to osteoclastic bone resorption.

As coupling mechanism, direct interaction between osteoblasts and osteoclasts had been described. Semaphorins and ephrins are axon guidance molecules involved in remodeling and coupling process. Semaphorins include both secreted and membrane-associated signaling molecules that use plexins and neuropilins as receptors. Sema4D that is expressed by osteoclast, but not by osteoblast, is an osteoclast-derived inhibitor of osteoblast differentiation and bone formation, acting as an inhibitor of remodeling [START_REF] Negishi-Koga | Suppression of bone formation by osteoclastic expression of semaphorin 4D[END_REF]. Ephrins and their receptors Eph are axon guidance molecules acting as local mediators of cell function through contact-dependent processes. EphrinB2 has been shown to be expressed by osteoclasts both in vitro and in vivo. Studies in vivo and ex vivo of genetically manipulated mice suggested that osteoclast-derived ephrin B2 can act through its receptor, EphB4, in osteoblasts, to promote osteoblast differentiation, and that reverse signaling by direct contact with osteoblast-derived EphB4 can suppress the formation of osteoclast precursors. This contact-dependent action of osteoclast-derived ephrin B2 on osteoblastic EphB4 is part of coupling mechanism [START_REF] Mundy | Boning up on ephrin signaling[END_REF].

Bone metastasis

Bone is one of the most common sites of cancer spread. Although metastatic cells could theoretically invade any organ, clinical experience demonstrates that they have preference for bone and lung [START_REF] Clezardin | Bone metastasis: pathogenesis and therapeutic implications[END_REF]. Why only certain cancers preferentially spread to bone is not fully elucidated, however it is true that bone provides a fertile microenvironment which facilitates colonization of metastatic cancer cells. The outer part of bone is composed of hard calcified matrix which shows relatively low cellularity and metabolic activity. Osteocytes are likely the only cells that are metabolically active and show some biological function. Calcified matrix, however, stores varieties of growth factors which may serve as essential nutrients for cancer cells that begin to house in bone. These growth factors are continuously released into the bone marrow cavity as a consequence of osteoclastic bone resorption during bone remodeling [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF]. This appears to be one of possible explanations for preferential bone colonization.

In this regard, bone is a representative target organ, which is consistent with the "seed and soil" theory, proposed by Paget more than a century ago [START_REF] Paget | THE DISTRIBUTION OF SECONDARY GROWTHS IN CANCER OF THE BREAST[END_REF].

The inner part of bone is made up of multicellular and highly metabolic bone marrow. It is rich in HSCs, which give rise to all blood cell elements and osteoclasts, as well as MSCs which differentiate into osteoblasts, chondrocytes, adipocytes and stromal cells. Especially important is the presence of osteoclasts because it seems that without osteoclastic bone resorption, growth factors stored in calcified matrix are less available to metastatic cancer cells. Tumor cells do not destroy bone by themselves but rather dependent on osteoclasts for the progression of osteolysis.

Therefore, osteoclastic bone resorption is essential for cancer cells to obtain sufficient nutrients and space for growth in the hard calcified tissue. However, cancer cells colonizing the bone will not only interact with osteoclasts, but also with osteoblasts, stromal cells and immune cells found in the bone marrow cavity [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF].

These interactions may lead to development of bone metastases even many years after the surgical removal of the primary tumor, suggesting that the osteotropic cancer cells may have a long period of quiescence before growing as secondary lesions. Growth of tumor cells in bone leads to the dysregulation of normal bone homeostasis, causing bone destruction and, ultimately, severe pains and fractures for which only palliative therapies are proposed. Thus, there is a need to better understand specific stages of tumor cell dissemination and growth in the bone marrow in order to prevent bone metastasis formation.

Classification and cellular features of bone metastases

Bone metastases are classified as osteosclerotic (osteoblastic), osteolytic or mixed lesions (Figure 6) [START_REF] Guise | Basic Mechanisms Responsible for Osteolytic and Osteoblastic Bone Metastases[END_REF].

Figure 6. Classification of bone metastases.

Left panel depicts a normal bone in which osteoclasts and osteoblasts function in a coordinated manner. Osteoclasts remove the old bone matrix which, after reverse phase populated by mononuclear cells (reverse cells) participating to the coupling between osteoclasts and osteoblasts, is replaced by new osteoid released by osteoblasts. Middle panel: in osteosclerotic bone metastases, numerous osteoblasts appear forming new trabeculae that occlude bone marrow. This bone matrix has features of woven bone and is inordinately deposited in the medullary cavity. Right panel: in osteolytic metastases, high number of osteoclasts is formed which resorb the mineralized matrix destroying the tissue (adapted from (Clezardin and Teti, 2007)).

Osteosclerotic metastases are more typical of prostate cancer and are caused by cancerderived factors that stimulate the differentiation and activity of bone forming cells, the osteoblasts, thus leading to increased bone formation. Osteosclerotic lesions appear in radiography as dense areas, often located to the axial skeleton and, particularly, in vertebral bodies and pelvis. Histologically, tumor cells residing in the bone marrow are surrounded by a high number of osteoblasts that form wide trabeculae of woven bone similar to that observed in primary ossification. Tumor-associated woven bone has a poorly organized microstructure, increasing the risk of pathological fractures. On the other hand, osteolytic metastases are common in breast cancer. They are caused by tumor-derived factors that stimulate the activity of boneresorbing cells, the osteoclasts, leading to enhanced bone destruction. Radiographically, osteolytic lesions appear as radiolucent areas, frequently located in the skull and proximal ends of the long bones. Histologically, tumor cells reside in the bone marrow, and are surrounded by a number of osteoclasts, actively degrading bone [START_REF] Chappard | Bone metastasis: histological changes and pathophysiological mechanisms in osteolytic or osteosclerotic localizations. A review[END_REF]. These osteolytic areas frequently fracture.

As it has been noted in the previous section, bone resorption and bone formation are almost always coupled. The coupling of bone resorption and bone formation is altered in cancer, thereby leading to skeletal lesions that are predominantly osteolytic or osteoblastic. However, in many instances, bone metastases may consist of mixed lesions. It is believed that a bone metastasis may evolve from an osteoblastic to an osteolytic pattern through a continuous process of which we only have a static representation at the time of the radiographical or histological assessment [START_REF] Clezardin | Bone metastasis: pathogenesis and therapeutic implications[END_REF].

Bone metastasis formation consists of a series of inter-related steps. Molecular mechanisms involved in each of these steps are gradually being unraveled, and are potential therapeutic targets for the prevention and treatment of bone metastases. Further complexities are introduced by the fact that the bone microenvironment does not only include the cellular architecture of the bone tissue but also bone marrow-derived haematopoietic progenitors and cancer stem cells (CSCs) that altogether constitute a niche supporting the development of metastases. In the next section, these steps of so-called metastatic cascade and determinants for overt bone metastases formation will be debated.

Steps of cancer metastasis to bone

Cancer metastasis to bone consists of multiple sequential steps which include diverse and complex cellular and molecular events. The early steps of metastasis to bone are common to most solid tumors and start with local tissue invasion and tumor cell escape from primary tumors.

After growing in the primary tumor, cancer cells invade surrounding tissue, intravasate, circulate in blood and entry into bone marrow sinus to migrate into bone marrow and colonize the tissue.

Growth at the primary sites, detachment from the primary site, local invasion and intravasation

First essential step for the successive colonization is cancer growth. Production of autocrine growth-promoting factors associated with the activation of receptor tyrosine kinase signaling pathways, oncogene expression and loss of tumor-suppressor gene expression are probably responsible for initiation and promotion of carcinogenesis. More than 30 % of human primary breast cancers exhibit increased expression of HER2/neu receptor tyrosine kinase which is a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family. Targeted expression of neu proto-oncogene in the mammary gland of transgenic mice induces highly metastatic mammary tumors, suggesting that this growth factor influence invasive properties of cells. Apart from growth factors, mutations in tumor-suppressor genes also predispose to cancer.

In aggressively growing tumors, cancer cells appear to be pushed further from existing vasculature due to unregulated cell proliferation and inadequate vascularization. These cancer cells are subject to hypoxia and nutrient depletion which induces angiogenesis [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF]. In the 90s, Weidner at al. proved that there is a correlation between metastasis and angiogenesis. He found that the number and density of microvessels in primary breast tumors were significantly higher in patients with metastases than in patients without metastases [START_REF] Weidner | Tumor Angiogenesis and Metastasis -Correlation in Invasive Breast Carcinoma[END_REF].

Among the angiogenic molecules released by tumors or tumor stromal cells are: basic fibroblastic growth factor (bFGF), TGF-, tumor necrosis factor alpha (TNF-) and VEGF. Their expression correlated with aggressiveness of solid tumors. At certain time point, cancer cells start to detach from the primary site. Cell adhesion molecules (CAMs) play an important role in cancer invasion and metastases. Their downregulation is necessary for detachment from the primary site and initiation of invasion. One of CAMs which is critical in the detachment is E-cadherin. It is a cellsurface glycoprotein involved in calcium-dependent cell-cell adhesion. E-cadherin expression is decreased in breast and prostate cancer with high metastatic potential [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF]. It has been also shown that overexpression of E-cadherin in the human breast cancer cell line MDA-MB-231 profoundly suppresses bone metastases in a nude mouse model of experimental bone metastases [START_REF] Mbalaviele | E-Cadherin Expression in Human Breast Cancer Cells Suppresses the Development of Osteolytic Bone Metastases in an Experimental Metastasis Model[END_REF]. In parallel with the growth at the primary tumor site, cancer cells invade surrounding tissues to enlarge the tumor mass by secreting proteolytic enzymes. The matrix metalloproteinases (MMPs) are a family of Zn2+ -dependent endopeptidases. Increased plasma levels of MMPs have been correlated with invasion and metastasis in patients with breast cancer. Tissue inhibitors of matrix metalloproteinases (TIMPs) on the other hand, function as metastasis-suppressor proteins. The metastatic potential of cancer cells depends on the balance between MMP and TIMP production. For example, overexpression of TIMP-2 in MDA-MB-231 cells inhibits osteolytic bone metastases and prolongs survival of tumor-bearing animals [START_REF] Yoneda | Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with the bisphosphonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2[END_REF]. Subsequently to growth and invasion at the primary site, cancer cells enter the blood stream to start their journey to secondary sites. This process is initiated by the adhesion of cancer cells to the endothelium of interior of lymph vessels. Then, the endothelial cells retract, the basement membrane is exposed and cancer cells bind to extracellular matrix (ECM) molecules of the basement membrane. Two major components of the basement membrane ECM are type IV collagen and laminin [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF].

Migration in the circulation and arrival at the bone

Cancer cells that intravasate into the blood vessels are named CTCs. If they resist to anoïkis or to killing by the immune system, they can extravasate from the sinusoids and reach the bone marrow. For this, they express specific gene signatures that would be predictive of bone metastasis. The preferential attraction and engraftment of CTC to bone is probably the result of several mechanisms. One of them, called hemodynamic, is based on random migration of cancer cells. In 1928, Ewing proposed that the development of metastases in a given organ is dependent on the blood volume flowing into that organ. This theory has been proven in some examples of colon cancer metastasis [START_REF] Body | Tumor bone diseases and osteoporosis in cancer patients[END_REF]. However, it does not explain the high frequency of cancer metastasis to bone, where blood flow is much lower compared with that in other organs which are also preferential sites of cancer spread, such as lung and liver (Table 2) [START_REF] Weiss | Organ vascularity and metastatic frequency[END_REF].

Organ

Blood flow (ml/min/g) Therefore, it has been proposed that breast and prostate cancer cells possess distinct capacities that not only direct them to bone, but also enable them to be attracted to bone then to survive and proliferate and further colonize the tissue.

Osteotropism

This non-random bone seeking is called bone tropism (also osteotropism). Different molecular mechanisms are responsible for this propensity of cancer cells to metastasize to bone.

One of them, the chemokine receptor CXCR4 controls the metastatic destination of breast cancer cells in organs where its ligand, the chemokine CXCL-12 is produced in high quantity. An organ which is eligible for these criteria is lung, liver and bone marrow. It has been shown that the blockade of CXCR4 reduces the formation of experimental lung and bone metastases caused by CXCR4-expressing breast cancer cells [START_REF] Liang | Inhibition of Breast Cancer Metastasis by Selective Synthetic Polypeptide against CXCR4[END_REF]. However, the inhibition of chemokine receptors only partially blocks metastasis formation, in vivo, suggesting that there are additional factors involved in the bone tropism of cancer cells.

Bone-derived cytokine RANKL triggers the migration of RANK-expressing cancer cells in vitro, and OPG, a natural inhibitor of RANK-RANKL interaction, blocks the bone tropism of these cancer cells in vivo [START_REF] Clezardin | Bone metastasis: pathogenesis and therapeutic implications[END_REF] [START_REF] Clezardin | The role of RANK/RANKL/osteoprotegerin (OPG) triad in cancer-induced bone diseases: physiopathology and clinical implications[END_REF].

There is also evidence from preclinical research showing that integrins mediate metastasis to specific organs. For instance, v 3 integrin overexpression in breast cancer cells enhances bone metastasis incidence in animals, and a nonpeptide v 3 integrin antagonist causes a profound and specific inhibition of bone colonization by v 3-expressing cancer cells, in vivo [START_REF] Zhao | Tumor v 3 Integrin Is a Therapeutic Target for Breast Cancer Bone Metastases[END_REF]. lpha2 1 integrin has been also shown to mediate bone colonization by prostate cancer cells [START_REF] Clezardin | Bone metastasis: pathogenesis and therapeutic implications[END_REF]. It is possible that these integrins act in concert with CXCR4 and RANKL to promote the bone colonization by cancer cells.

Another important chemoattractant for bone is the proto-oncogene c-SRC. In many tumors, c-SRC is upregulated or hyperactivated thus affecting cancer cell properties linked to proliferation, motility and responses to growth factors [START_REF] Clezardin | Bone metastasis: pathogenesis and therapeutic implications[END_REF]. Reduced c-SRC activity in breast cancer cells decreases their malignant phenotype and osteotropism in experimental metastases [START_REF] Rucci | Inhibition of Protein Kinase c-Src Reduces the Incidence of Breast Cancer Metastases and Increases Survival in Mice: Implications for Therapy[END_REF].

Osteomimicry

Osteomimicry is the acquisition of bone cell-like properties by tumor cells, which improve homing, adhesion, proliferation and survival in the bone microenvironment. Osteotropic malignant cells are able to express RUNX2 which is a master regulator of osteoblast differentiation and strong inducer of the expression of bone proteins. For example, bone matrix proteins, such as osteopontin, osteocalcin, osteonectin and bone sialoprotein II, are frequently highly expressed in breast and prostate cancers, which represent tumors with the highest propensity to colonize bone. They are also highly expressed in human breast cancer cell lines that form experimental bone metastases in mice. In our lab, a subclone variant from the human breast cancer cell line, MDA-MB-231 has been obtained by selection pressure in mice. This MDA-B02 cell line, highly and specifically metastasizes to bone and presents an osteoblast phenotype [START_REF] Peyruchaud | Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions[END_REF]. A global transcriptome analysis performed on MDA-BO2 cell line, has confirmed that several genes among those up-or down-regulated relative to the parental cell line, correspond to genes whose expression is associated with the osteoblast differentiation process.

Also, the proteins such as: cadherin 11 (CDH11), cytochrome c oxidase subunit II (COX-2), connective tissue growth factor (CTGF), connexin 43 (CX43) and secreted protein, acidic, cysteine-rich (SPARC), which are overexpressed by MDA-BO2 and known to be up-regulated during osteoblast differentiation, are selectively overexpressed in human breast cancer bone metastases compared to the primary tumor and liver metastases [START_REF] Bellahcène | Transcriptome analysis reveals an osteoblast-like phenotype for human osteotropic breast cancer cells[END_REF].

These data suggest that malignant cells acquire osteomimetic properties, favoring the development of secondary lesion in bone microenvironment. The question remains if these osteomimetic properties refer only to the osteoblast phenotype or if they can be extended to the osteoclast phenotype as well. For instance, it has been shown that cathepsin K, a highly specific osteoclast gene, is overexpressed in human breast cancer cells that metastasize to bone [START_REF] Gall | A Cathepsin K Inhibitor Reduces Breast Cancer-Induced Osteolysis and Skeletal Tumor Burden[END_REF]. Therefore, it seems that a multigenic mimicry program is crucial for a tumor cell to develop in bone, and that this program includes both osteoblastic and osteoclastic genes.

Entry into the bone marrow sinus and migration into the bone marrow cavity

There are four major arteries running into bone: the nutrient artery, the metaphyseal artery, the epiphyseal artery and the cortical capillaries communicating with the marrow arteries.

Cancer cells arriving at bone enter the bone marrow cavity through one of these arterial routes.

Cells penetrating to the bone marrow cavity must have certain capacities to make this step possible. It has been shown in vitro that cancer cells show preferential attachment to the endothelial cells of their in vivo target organs [START_REF] Mccarthy | Heterogeneity of the endothelial cell and its role in organ preference of tumour metastasis[END_REF]. The interactions between the endothelial and cancer cells are mediated by cell adhesion molecules (CAMs). One of them is vascular cell adhesion molecule 1 (VCAM1). When it is aberrantly expressed in breast cancer cells, VCAM-1 mediates distinct tumor stromal interactions facilitating metastasis to bone [START_REF] Lu | VCAM-1 Promotes Osteolytic Expansion of Indolent Bone Micrometastasis of Breast Cancer by Engaging 4 1-Positive Osteoclast Progenitors[END_REF].

Colonization through interactions with bone cells-the vicious cycle

Among factors that regulate bone remodelling are systemic hormones and local factors such as interleukins, cytokines, colony-stimulating factors, eicosanoids, the RANKL/OPG axis and parathyroid hormone-related protein (PTHrP). In addition, many growth factors are synthesized by the osteoblasts and embedded into the bone matrix, mostly as inactive peptides, during the bone formation phase. These factors, which include TFG-, platelet-derived growth factor (PDGF), BMPs, are then released from the matrix during bone resorption and activated both by the low pH created by the osteoclasts to remove the bone mineral, and by a set of proteases present in the microenvironment. Therefore, during bone resorption, the bone/bone marrow microenvironment is enriched in agents that regulate many cellular activities.

According to the "seed and soil" theory, these environmental factors provide a fertile ground in which tumor cells can grow. Moreover, during the formation of bone metastases, the perturbation of the microenvironment is initiated by the tumor cells that produce many factors stimulating the osteoclasts, with the end point of an increased bone resorption. In addition, cancer cells secrete bone morphogenetic and Wnt protein antagonists (noggin, DKK-1) that inhibit osteoblast activity which, in turn, enhance the osteolytic pattern of bone metastases. It is a so called "vicious cycle" [START_REF] Clezardin | Bone metastasis: pathogenesis and therapeutic implications[END_REF]) (Figure 7). Bone metastases are likely to rely on multiple factors: the ability of cancer cells to exhibit specific receptor-ligand interactions that direct the cells to bone, the osteomimicry that leads to tumor development on the bone/bone marrow tissue and the microenvironmental factors which establish a vicious cycle. The stem cell niche maintaining cancer cell dormant until permissive conditions waken them up is also an important player in establishing bone metastases [START_REF] Clezardin | Bone metastasis: pathogenesis and therapeutic implications[END_REF]. Recently, it has been published that the perivascular niche regulates breast tumor dormancy. Specifically, work from Ghajar et al. reveals that stable microvasculature constitutes a dormant niche, whereas sprouting neovasculature sparks micrometastatic outgrowth [START_REF] Ghajar | The perivascular niche regulates breast tumour dormancy[END_REF].

In vivo models of breast cancer bone metastasis

Transplantable tumor model system

In this model, cancer cell lines or tissues can be grown in mice. There are various ways to mimic breast cancer growth and metastasis in tumor transplantation experiments. The primary and secondary metastatic growth is largely defined by the site of injection, together with the cell line specific tropism. Moreover, depending on the species or genetic background of donor and host, xenograft or syngeneic tumor transplantations can be distinguished [START_REF] Fantozzi | Mouse models of breast cancer metastasis[END_REF].

Xenograft tumor transplantations

The majority of in vivo studies of bone metastases have used xenograft models, where human tumor cells are implanted in immunocompromised mice [START_REF] Holen | In vivo models used in studies of bone metastases[END_REF]. The resultant tumors are a mosaic of human cancer cells and murine stromal cells. Cancer cell-stromal cell interactions are important in the biology of cancer progression and metastasis.

For some pathways, it cannot occur across species boundaries. This limited tumor-stromal interaction is one of the major drawbacks in xenograft models. Secondly, in order for human tumors to grow in mice, the murine host must be immunocompromised to prevent immune rejection. Such an approach eliminates the ability to examine the role of the immune system in tumor progression in xenografts. Thirdly, each immunocompromised mouse strain is characterized by specific features that can influence the biology and study of metastasis. For instance, nude mice that are depleted in T cells and have impaired T and B cell function, have been shown to have impaired angiogenesis [START_REF] Khanna | Modeling metastasis in vivo[END_REF]. Human tumor cells are now routinely engineered to express green fluorescent protein (GFP), luciferase or other tags, allowing non-invasive in vivo imaging of tumor development. Depending on multiple factors, such as: the cell line used, the age and strain of the animal, as well as the route of implantation, tumors will develop in bone between 3 and 5 weeks, providing the opportunity to study both the biological aspects of bone metastases and effects of therapy [START_REF] Holen | In vivo models used in studies of bone metastases[END_REF].

Human breast cancer cells for metastasis models can be injected intra-arterialy, intracardially or intraosseouslly. First type of injection results mainly in lung metastasis, whereas intracardiac (i.c.) injection gives rise to a broader target organ spectrum, including bone [START_REF] Khanna | Modeling metastasis in vivo[END_REF]. Intraosseous injection is generally used to investigate late-stage disease, as a large number of tumor cells are introduced directly into bone, bypassing the early steps of homing and colonization [START_REF] Holen | In vivo models used in studies of bone metastases[END_REF].

Intra-arterial injection

We set up a specific breast cancer cell lines with high propensity to metastasize to bone, by isolation of cells from bone metastases (originally introduced by i.c. injection), expanding the cells in culture and re-inoculating them into the left ventricle. Following six passages through bone marrow, a cell line that homes specifically to bone following intra arterial. injection has been generated.

With this approach, the MDA-B02 model has been developed. This is a bone-homing strain of MDA-MB-231 breast cancer cells [START_REF] Peyruchaud | Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity[END_REF], subsequently used to investigate whether therapeutic targeting of tumor v 3 integrin prevents bone metastasis formation [START_REF] Zhao | Tumor v 3 Integrin Is a Therapeutic Target for Breast Cancer Bone Metastases[END_REF].

Intracardiac injection

In this approach, human breast cancer cells are injected into the left ventricle of the heart.

The cells will bypass the pulmonary vasculature resulting in widespread metastasis formation in several skeletal sites (mainly long bones) within 4-5 weeks [START_REF] Holen | In vivo models used in studies of bone metastases[END_REF].

The main disadvantage is that it does not represent the initial steps of the metastatic cascade, where tumor cells leave a primary site, enter the circulation and subsequently recolonize a distant site [START_REF] Holen | In vivo models used in studies of bone metastases[END_REF]. Phadke et al. used the i.c. injection model to perform a detailed and informative study of the kinetics of breast cancer cell trafficking in bone.

GFP-expressing MDA-MB-435 cells were injected into the left ventricle of female nude mice and animals were sacrificed at time points ranging from 1 h and up to 6 weeks. This allowed them to precisely localize breast cancer cells from the initial colonization of bone to advanced disease.

One hour post-injection, tumor cells can be detected in the highly vascularized methaphysis of the femur and they are gradually cleared over the next 72 h. One week after injection, small tumor cell foci were detected mainly in the distal methaphysis, and a few of these developed into larger tumors associated with loss of the majority of the trabecular bone during the following 2-3 weeks. Moreover, increasing numbers of tumor cells were associated with a substantial decrease in osteoblast number through induction of apoptosis. There was also a decrease in the number of osteoclasts and the osteoblast:osteoclast ratio was reduced from around 40:1 at baseline to 4:1 by 4 weeks, as bone turnover balance shifted towards increased resorption. This resulted in a rapid depletion of bone forming cells by an unknown mechanism, likely to prevent repair of tumorinduced osteolytic lesions. From the point of view of human disease these results suggest that therapeutic strategies to protect the osteoblast from the detrimental effects of tumor cells should be developed, for use in combination with anti-resorptive agents [START_REF] Phadke | Kinetics of Metastatic Breast Cancer Cell Trafficking in Bone[END_REF].

Intraosseous implantation

Implanting cancer cells directly into bone can also be used to model bone metastasis, although, it is generally used to investigate late-stage disease. Most common site of intraosseous injection is intratibial (i.t.) injection. Holes are drilled in the tibiae of anesthetized animals, the bone marrow flushed out and replaced by a suspension of tumor cells. This will inevitably cause damage to the bone around the injection site, complicating analysis of tumor-induced changes of bone structure. Bone tumors are generated within 3-4 weeks, depending on cell lines.

Syngeneic models

Syngeneic models, unlike the xenograft models, provide the possibility to study the initial steps of metastasis by injecting murine tumor cells into the mammary fat pad of immunocompetent BALB/c mice.

4T1 is a commonly used murine breast cancer cell line that is inoculated into the mouse mammary gland to study tumor growth and cancer cell dissemination. This model allows to study growth of initial mammary tumor in an anatomically correct site, meaning mammary fat pad, followed by metastatic spread to a range of distant organs, thereby mimicking human breast cancer. The main advantage is that the transplanted tissues, the tumor microenvironment and the host are from the same species, therefore, it can be used to study the role of the immune system in tumor progression in bone, as well as in response to anti-cancer therapy. However, syngeneic models do have some important limitations. The murine tumor cells differ from human breast cancer cells in the genetic makeup and potentially in their growth characteristics and responses to therapy. In addition, the primary mammary tumors grow quickly once established, leaving a limited window of opportunity for studies of the metastases unless the primary tumor is removed [START_REF] Holen | In vivo models used in studies of bone metastases[END_REF]. Moreover, these model systems lack many of the important features of human tumors. For instance, they are usually derived from homozygously imbred mice and therefore lack the genetic complexity of human tumors [START_REF] Khanna | Modeling metastasis in vivo[END_REF]. Most importantly, syngeneic models are not bone-metastasis specific, and a large number of animals are required in order to generate sufficiently large groups with skeletal involvement.

There is also considerable variability in time for metastasis appearance in animals, presenting particular challenges in terms of when to initiate treatment and the timing of therapeutic interventions [START_REF] Holen | In vivo models used in studies of bone metastases[END_REF].

Historically, subcutaneous (s.c.) models were used due to rapid primary tumor growth. It was however, uncommon to observe spontaneous metastasis to distant organs. Application of "seed and soil" hypothesis resulted in the use of orthotopic transplantation of tumor cells into mice.

Orthotopic inoculation refers to delivery of cancer cells to the anatomic location or tissue from which a tumor was derived. It is a model in which the primary tumor is formed followed by the development of metastasis [START_REF] Khanna | Modeling metastasis in vivo[END_REF]. Orthotopic model in part resembles the multiple stages involved in malignant breast cancer development in patients. Therefore, it has the advantage over other models in which the direct introduction of cancer cells into the blood circulation should be considered rather as an assay of organ colonization and not a complete metastatic process.

Transplantable models have yielded many important insights into the potential molecular mechanisms of metastasis, however a number of important caveats remain. Neither injection of the tumor cells into the circulation, nor the orthotopic implantation is free of drawbacks.

Tumorigenesis is not simply the result of proliferative activation of a mutated cell, but rather is a complex interaction between neoplastic tissue and the stromal and environment in which it arises. Injection of the tumor cells does not necessarily recapitulate all of the interactions and microenvironmental components that may play important roles in tumor dissemination. Important issue is also mechanical disruption of the target tissue during the implantation process which may permit escape of tumor cells into the circulatory system at the time of implantation, thus seeding distant sites at the onset of the experiment rather than subsequent to tumor growth, as is the intent. A further common caveat of transplantable models is their reliance on cultured cells.

During the selection process and/or subsequent cell culture, the resulting cell lines and clones have been passaged in vitro. Thus, the cells are adapted to growth on a two-dimensional rather than a normal three-dimensional matrix platform, in an artificial or foreign milieu. The adaptations that permit perpetual growth in tissue culture may have significant impact on the pathways and mechanisms by which spontaneous metastases arise. While these experimental systems may imply some of the mechanisms and biological processes that function in tumor dissemination, we have to be aware that they only partially represent those steps in the metastatic process [START_REF] Khanna | Modeling metastasis in vivo[END_REF].

Genetically engineered mouse models of metastasis

To complement transplantable models, investigators need access to autochthonously arising tumors capable of completing the entire metastatic process. Here, genetically engineered models have the application. They encompass a variety of tissue types, with differing degrees of penetrance and different latencies. While it is possible that the constitutive activation or loss of a gene in these models may not completely replicate native metastasis, the tumors do arise in their normal context, more closely replicating the clinical setting and in a system possessing a functional immune system. They also permit the investigation of important determinants of cancer that are difficult or impossible to address using cell culture and transplant systems including the influence of genetic heterogeneity on tumor phenotype [START_REF] Khanna | Modeling metastasis in vivo[END_REF].

Human-to-human models

These models have been developed in order to capture the species-specific interactions involved in homing of breast cancer cells to bone. Pieces of human bone are implanted subcutaneously (s.c.) in immunocompromised animals and subsequently, human breast cancer cells are injected i.a., in the mammary fat pad or directly into the bone marrow cavity.

The main advantage of these models is that the tumor cells have an access to the human bone microenvironment, although the bone fragments used are either fetal or from patients undergoing hip replacements, not from patients with bone metastases. In order to generate consistent bone metastasis, cancer cells must be introduced directly into bone, whereas tumors implanted in the mammary fat pad generate low frequency of bone colonization. These results are in agreement with the observation that not all breast cancer cells home to bone even when the right microenvironment is available. It might be due to a requirement that the human bone actually contains the appropriate niches. Also, there may be problems with a graft versus graft immune response, as the human bone and the human tumor cells are of different origin [START_REF] Holen | In vivo models used in studies of bone metastases[END_REF]. Nevertheless, human-to-human models are becoming important as we seek to understand the pivotal role of the bone microenvironment in the development of bone metastases.

MicroRNAs

MiRs, a class of small noncoding RNAs are cell endogenous regulators of gene expression.

Within 10 years of research, we have gone from discovering the existence of miRs in mammals to exploring their therapeutic applications in numerous diseases, including cancer.

Biosynthesis of microRNAs

MiRs are evolutionary conserved, small non-coding, single-stranded RNA molecules of around 19-23 nucleotides in length. These miRs are predicated to regulate more than 50% of the human protein coding genes. Mature miRs are generated via a two-step processing pathway (Figure 8) [START_REF] Lee | MicroRNA maturation: stepwise processing and subcellular localization[END_REF].

The miR genes are first transcribed by RNA polymerase II into primary transcripts (pri-miRs) in the nucleus. Initial cleavage is catalyzed by Drosha, a RNase III nuclease existing in a multiprotein complex called the microprocessor, (Drosha-DGCR8 (Pasha)), which produces pri-miRs of around 60 nucleotides in length [START_REF] Denli | Processing of primary microRNAs by the Microprocessor complex[END_REF][START_REF] Gregory | The Microprocessor complex mediates the genesis of microRNAs[END_REF]. The Numerous studies have uncovered highly specific miR profiles during development or tumorigenesis. Their functions as important regulators of differentiation, proliferation, apoptosis or metabolism are extensively studied.

MiRs discovery and first experiments

The discovery by Lee, Feinbaum and Victor Ambros, on the role of the lin-4 and lin-14 genes in temporal control of development in the model organism C. elegans has busted an era of intense miR studies [START_REF] Lee | The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[END_REF].

They showed that lin-4 gene does not encode a protein product, but instead gives rise to a 61-nucleotide precursor gene that matured to a more abundant 22-nucleotide. Also, it has been found that Lin-14 levels are inversely proportional to those of lin-4 RNA and that it is complementary to the 3' untranslated region (3'UTR) of the lin-14 gene. This led to the hypothesis that lin-4 regulated LIN-14, in part, through Watson-Crick base pairing, revealing the first miR and mRNA target interaction (van Rooij, 2011). For 7 years, lin-4 was considered an anomaly, until the discovery of a second C. elegans miR, called let-7, which repressed lin-41, lin-14, lin-28, lin-42, and daf-12 expression during development [START_REF] Reinhart | The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[END_REF]. Let-7 homologs were found in many vertebrate species including humans, which lead to the finding that miRs are evolutionarily conserved across many species and are often ubiquitously expressed [START_REF] Van Rooij | The Art of MicroRNA Research[END_REF].

In 2002, shortly after the expression of mammalian miRs was recognized, Calin et al showed a correlation between the loss of miR-15 and -16 and the occurrence of B-cell leukemia [START_REF] Calin | Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia[END_REF]. In 2006, the first cardiac miR-profiling study appeared, linking dysregulation of many different miRs to cardiac remodeling in both mice and humans. Today, microarray analysis and deep-sequencing approaches enable researchers to correlate the dysregulation of miRs to the progression of many different diseases in both animals and humans. When such a disease-specific miR expression signature was first characterized, researchers started investigating whether these expressional changes were causally related to disease. Although many in vitro studies indicated prominent and defined functions for miRs in different aspects of cell biology, the first genetic evidence for the importance of miRs in mammals came when it was shown that homozygous deletion of Dicer disrupted prenatal development of the murine embryo through its role in miR biogenesis [START_REF] Yang | Dicer Is Required for Embryonic Angiogenesis during Mouse Development[END_REF].

MiR-mediated regulation of bone metastasis formation

Bone metastasis is a multi-step process involving a series of molecular events that might already be genetically determined in primary tumours. The misexpression of miRNAs in tumourinitiating cells might up-or down-regulate master genes that drive invasion, extravasation and finally bone seeding of cancer cells. Besides these regulatory mechanisms within tumour cells themselves, miRs interfere with tumour-stroma cross-talks within the bone [START_REF] Ell | Tumor-induced osteoclast miRNA changes as regulators and biomarkers of osteolytic bone metastasis[END_REF]. In this way, stroma-tumour interactions may be mediated by miRNA transfer through cell-to-cell interactions or exosomes to influence osteoclastogenesis and metastasis-related bone destruction.

Involvement of miRs in the early stage of metastatic dissemination

Molecular pathways underlining bone metastasis formation are regulated by multiple factors, through the tight control of genes expressed by cancer cells interacting stroma and with cells from the bone microenvironment. In this context, miRNAs can act as master regulators of gene expression to control multiple aspects of bone metastasis formation, including cancer cell escape from the primary tumour site, cancer cell dissemination to bone, invasion of the bone marrow, as well as secondary outgrowth and tumour-stroma cell interactions [START_REF] Croset | Tumour-derived miRNAs and bone metastasis[END_REF]. A small subset of cancer cells in breast primary tumours express specific gene signatures that would be predictive of bone metastasis [START_REF] Smid | Genes associated with breast cancer metastatic to bone[END_REF]) [START_REF] Kang | A multigenic program mediating breast cancer metastasis to bone[END_REF]. In this respect, the identification of miRNA and gene signatures expressed in primary carcinomas and their matching bone metastases provide information on molecular mechanisms driving bone metastasis. It is very likely that before patients are diagnosed with cancer, dormant disseminated tumour cells (DTC) are already present in the bone marrow (Aguirre-Ghiso, 2007). Given this information, dysregulated miRNAs that are associated with the early onset of bone metastasis might be a useful clinical biomarker of the minimal residual disease in patients with cancer. Tavazoie et al.

identified human miRs that affect breast cancer metastasis to lung and bone. MiR profiling of the MDA-MB-231 cell line was compared to its derivatives that are highly metastatic to bone and lung. Hierarchical clustering defined a set of miRs (miR-335, -126, -206, -122a, -199*a, and -489) whose expression was decreased in the two metastatic derivatives. Restoration of miR-335, miR-126, or miR-206 expression in LM2 and BoM2 cells reduced their colonizing activity in both lung and bone when they were inoculated to mice by the intravenous route or orthotopically [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. MiR-126 suppresses tumorigenesis and metastasis, in part, through an inhibition of tumor cell proliferation whereas miR-335 and miR-206 do not affect primary tumor outgrowth, but reduce migratory and invasive capacities of these tumor cells. MiR-335 suppresses breast cancer cell invasion and metastasis by targeting the transcription factor SOX4 and tenascin C, proteins involved in cell adhesion. Additionally, expression levels of miR-335 and miR-126 are low in breast tumors from patients who relapse, further indicating that these miRs act as metastasis suppressors in breast cancer.

Studies performed on human primary prostate tumors and matched bone metastasis showed that expression levels of miR-145, -143, -33a, -100, and -508-5p were the most highly downregulated in bone metastasis specimens. The expression levels of several miRs previously reported as metastasis suppressors (miR-125b, 30e, c, -99a,-20b,c, let7) were also deregulated to a lesser extent. Ectopic expression of miR-143 and miR-145 in the aggressive and androgeninsensitive PC-3 prostate cancer cell line reduced the migratory and invasive capacities of these tumor cells in vitro and their propensity to metastasize to bone in vivo. Neither miR-143 nor miR-145 interfer with the tumorigenic and metastatic properties of the hormone-responsive and weakly-metastatic LNCaP prostate cancer cell line. Upon upregulation of these two miRs, the expression of the mesenchymal markers fibronectin and vimentin (VIM) decreased, whereas Ecadherin expression increased, in PC-3, but not in LNCaP cells. Further, oncogenic protein HEF1

(human enhancer of filamentation, also known as NEDD9) was shown to partially mediate miR-145 suppression of epithelial-mesenchymal transition (EMT) and invasion [START_REF] Peng | Identification of miRs-143 and -145 that Is Associated with Bone Metastasis of Prostate Cancer and Involved in the Regulation of EMT[END_REF]. In a retrospective investigation of 12 patients without bone metastasis and 10 patients with bone metastasis the low expression levels of these two miRs were negatively correlated to bone metastasis, the level of free prostate-specific antigen (PSA) and the Gleason score. Therefore, miR-143 and miR-145 can be potential candidates for biomarkers in discriminating different stages of prostate carcinomas and predicting bone metastasis.

Regarding metastasis-promoting miRs, Huang et al. found that miR-373 and miR-520c, two miRs that share the same seed sequence, promoted cancer cell migration and invasion in vitro and in vivo and that several cell lines required miR-373 expression in order to be invasive.

In vivo studies on SCID mice were conducted by tail-vein injection with either MCF-7 cells expressing miR-373 or miR-520c (miR-MCF-7) or a mutant of miR-373 (Mut-MCF-7) or a control vector (Ctrl-MCF-7). Metastatic nodules appeared in the skull, spine and lungs in the mice inoculated with miR-MCF-7 but not in the mice inoculated with Ctrl-MCF-7 or Mut-MCF-7. Histological analysis showed osteolytic metastases infiltrating into the medullary bone spaces and skull [START_REF] Huang | The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis[END_REF]. Evidence of metastasis-inducing potential of miRs was reported by Weinberg's laboratory when they identified miR-10b as being highly expressed in metastatic breast tumors and promoting tumor cell migration and invasion. 50 % of the metastasis-positive patients had elevated miR-10b levels in their primary tumors when compared to metastasis-free patients. This was in agreement with the observation that miR-10b is highly expressed in metastatic breast cancer cell lines. When miR-10b was overexpressed in otherwise non-metastatic breast tumor cell lines, it initiated strong invasion of orthotopically implanted mammary tumors in animals and promoted dissemination of tumor cells in the lungs. The transcription factor TWIST directly induced miR-10b expression which, in turn, inhibited the expression of the transcription factor homeobox D10 (HOXD10) [START_REF] Ma | Tumour invasion and metastasis initiated by microRNA-10b in breast cancer[END_REF]. Interestingly, Twist1 is expressed in disseminated breast cancer cells that persist in the bone marrow after chemotherapy [START_REF] Watson | Isolation and Molecular Profiling of Bone Marrow Micrometastases Identifies TWIST1 as a Marker of Early Tumor Relapse in Breast Cancer Patients[END_REF]. In an experimental model of human breast cancer bone metastasis, Twist1 facilitates bone metastasis formation through a miR-10b-dependent mechanism [START_REF] Croset | TWIST1 expression in breast cancer cells facilitates bone metastasis formation[END_REF]. Moreover, miR-10b could be a circulating marker of bone metastasis in regard of its high level in serum of breast cancer patients with bone metastasis [START_REF] Zhao | Serum overexpression of microRNA-10b in patients with bone metastatic primary breast cancer[END_REF].The potential of miR-10b as target for anti-metastatic therapy has been therefore studied in an animal model of breast cancer metastasis. MiR-10b antagomir was delivered to rapidly growing tumor cells preventing metastasis dissemination to secondary organs without marks of cytotoxicity [START_REF] Ma | Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model[END_REF].

Involvement of miRs in tumor cell homing and colonization of bone marrow

MiR-203 have an anti-metastatic role in prostate cancer progression and bone metastasis [START_REF] Saini | Regulatory Role of mir-203 in Prostate Cancer Progression and Metastasis[END_REF] miR expression analysis in a set of normal and malignant breast tissues. They reported that the miR-30s were expressed at higher levels in hormone-dependent (ER+/PR+) and well differentiated breast tumors, compared with that observed in hormone-independent tumors [START_REF] Iorio | MicroRNA gene expression deregulation in human breast cancer[END_REF]. Moreover, they have been shown to act as tumor suppressors in anaplasic thyroid carcinoma [START_REF] Visone | Specific microRNAs are downregulated in human thyroid anaplastic carcinomas[END_REF], lung (Kumarswamy et al., 2012a), prostate [START_REF] Kao | miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT[END_REF] and breast [START_REF] Zhang | MicroRNA-30a suppresses breast tumor growth and metastasis by targeting metadherin[END_REF] cancers, mostly by regulating EMT. MiR-30s might play a role during metastatic dissemination of breast carcinomas, as their expression levels was lower in lymph node metastases versus primary tumors [START_REF] Baffa | MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets[END_REF]. TGF-is released from bone in the microenvironment during bone resorption, and it stimulates cancer cells to produce pro-osteolytic factors such as IL-11. Pollari et al. identified three miRs,as potent inhibitors of TGF--induced IL-11 secretion. In addition, gene expression analysis showed that miR-204 and miR-379 downregulated a set of genes involved in TGF-signaling [START_REF] Pollari | Identification of MicroRNAs Inhibiting TGF--Induced IL-11 Production in Bone Metastatic Breast Cancer Cells[END_REF]. MiR-33a is a tumor suppressor that target PTHrP, a potent stimulator of osteoclastic bone resorption, in lung cancer. MiR-33a levels are inversely correlated with PTHrP expression between human normal bronchial cell lines and lung cancer cell lines. When miR-33a expression was restored in lung cancer cells, the production of PTHrP, IL-8 (another stimulator of osteoclast-mediated bone resorption) and OPG decreased. Thus, low miR-33a expression contributes to cancer-mediated bone destruction and may even predict a poor prognosis for lung cancer patients [START_REF] Kuo | MicroRNA-33a functions as a bone metastasis suppressor in lung cancer by targeting parathyroid hormone related protein[END_REF]. Rho-associated kinase (ROCK) signaling plays an important role in regulating cell morphology, adhesion, and motility. ROCK is involved in the enhanced ability of cancer cells to detach and become motile and is highly expressed in primary breast [START_REF] Hassan | miR-218 Directs a Wnt Signaling Circuit to Promote Differentiation of Osteoblasts and Osteomimicry of Metastatic Cancer Cells[END_REF]. Although miR-218 regulates positively the molecular mechanisms underlying bone metastasis formation it appears as tumor suppressor in other types of cancers [START_REF] Tie | MiR-218 Inhibits Invasion and Metastasis of Gastric Cancer by Targeting the Robo1 Receptor[END_REF], suggesting that its biological activity depends on the cellular microenvironment.

Evidence that the bone is able to produce miRs that modulate the growth of DTCs has been reported [START_REF] Ono | Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells[END_REF]. There are also reports that cancer cells can modify the microenvironment to become more favorable for metastatic tumor initiation. Conditioned media from highly metastatic cell lines are able to induce osteoclast differentiation [START_REF] Ell | Tumor-induced osteoclast miRNA changes as regulators and biomarkers of osteolytic bone metastasis[END_REF].

Indeed, exposure of differentiating osteoclasts to tumor-conditioned media activates a specific miRNA signature in osteoclasts that is partly induced by the secretion of slCAM1 by bonemetastatic cells. This suggests that miRNAs from the tumor cells may also alter the bone microenvironment, disrupting bone turnover.

MiRs activity in interfering with bone colonization relies on mRNA targets whose expression is altered in bone metastasis. Uncovering the genes under the control of these miRs and how they integrate into the bone metastatic gene interaction network will further help us understand the disease and hopefully lead to new targeted therapies. Identifying these targets and transferring their bone metastasis-suppressive activity from bench into clinical settings for predictive biomarkers and/or treatment of bone lesions is essential. The already existing miRbased therapeutic approach to interfere cancer is an attractive one. However, it still remains puzzling, mainly due to the large numbers of transcripts sometimes with opposing functional consequences that could be targeted by a single miR. Therefore, the efficiency and safe delivery of agents such as miR mimics or antagomirs to a growing metastatic tumor or to alreadyestablished metastasis need to be further studied.

MicroRNA-30 family

Genomic arrangement of miR-30 family

In most multi-cellular organisms there are hundreds of miR genes. MiRs can be located within exons, or introns of coding or non-coding transcripts. Some miR genes are intergenic and not subject to splicing. MiRs can also be co-transcribed as part of one primary transcript in cluster, which can be intronic or exonic (Figure 9) [START_REF] Finnegan | MicroRNA biogenesis: regulating the regulators[END_REF].

Figure 9. MiR genomic locations.

MiR can be located within exons (A) or introns (B) of coding or non-coding transcripts. Some miR genes are intergenic and not subject to splicing (C). Multiple miRNAs constitute miR clusters co-transcribed as part of one primary transcript, which can be intronic (D) or exonic (not shown) as part of a coding RNA or a non-coding RNA Blue represents non-transcribed DNA, pink represents exons, and white represents introns. Adapted from [START_REF] Finnegan | MicroRNA biogenesis: regulating the regulators[END_REF].

54

The Regarding the similarity between the members of the family, they are conserved between human and mouse. Not a single base is changed between these two organisms (Table 3).

Table 3. Comparison of genomic sequences of miR-30 family members between mouse and human.

(based on http://www.mirbase.org/ )

There is one base difference between the member a and e or d. Thus, between d and e this difference is of 2 nucleotides. On the other hand, members b and c differ in 7 or 6 nucleotides respectively when compared to member a. The member b is of the same length, even though, there are two deletions in its sequence. Member c is one base longer (Table 4).

Table 4. Differences between miR-30 family members in the nucleotide sequence

Nucleotides that are different are marked in red. Deletions are marked with a hyphen.

Based on http://www.mirbase.org/ Typically, a family of miRs includes all miRs, regardless of genomic location, that share 2-7 nucleotides, called the seed sequence [START_REF] Bartel | MicroRNAs: Target Recognition and Regulatory Functions[END_REF]. In miR-30 family, all members share the same seed sequence 5' UGUAAAC 3'. By this sequence, miR-30 members bind to target 3'UTR region of the mRNA based on the antisense complementarity. If the complementarity is perfect, the target mRNA is cleaved and the miR acts as a siRNA. If the complementarity is partial, the translation of the target mRNA is repressed [START_REF] Nelson | The microRNA world: small is mighty[END_REF].

The biological role of miR-30 family

MiR-30 has been reported to stimulate adipogenesis [START_REF] Zaragosi | Small RNA sequencing reveals miR-642a-3p as a novel adipocytespecific microRNA and miR-30 as a key regulator of human adipogenesis[END_REF], B-cells differentiation [START_REF] Lin | Follicular dendritic cell-induced microRNA-mediated upregulation of PRDM1 and downregulation of BCL-6 in non-Hodgkin's B-cell lymphomas[END_REF], pronephros development [START_REF] Agrawal | The miR-30 miRNA family regulates Xenopus pronephros development and targets the transcription factor Xlim1/Lhx1[END_REF], endothelial cell behavior [START_REF] Bridge | The microRNA-30 family targets DLL4 to modulate endothelial cell behavior during angiogenesis[END_REF], structural changes in myocardium [START_REF] Duisters | miR-133 and miR-30 Regulate Connective Tissue Growth Factor Implications for a Role of MicroRNAs in Myocardial Matrix Remodeling[END_REF] and cellular senescence [START_REF] Martinez | miR-29 and miR-30 regulate B-Myb expression during cellular senescence[END_REF]. MiR-30s can suppress apoptosis in the heart [START_REF] Li | miR-30 Regulates Mitochondrial Fission through Targeting p53 and the Dynamin-Related Protein-1 Pathway[END_REF], osteoblast differentiation [START_REF] Wu | miR-30 Family Members Negatively Regulate Osteoblast Differentiation[END_REF] and EMT [START_REF] Braun | Downregulation of microRNAs directs the EMT and invasive potential of anaplastic thyroid carcinomas[END_REF][START_REF] Joglekar | The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells[END_REF][START_REF] Zhang | miR-30 inhibits TGF-1-induced epithelial-to-mesenchymal transition in hepatocyte by targeting Snail1[END_REF], a process which correlates with more invasive potential of cancer cells.

Regarding cancer disease, in the majority of reports miR-30s are described as tumor suppressors. Reduction of miR-30s has been correlated with more invasive properties of cancer cells. Downregulation of the family in aggressive BT-ICs (breast tumor initiating cells) results in the maintenance of self-renewal and apoptosis inhibition in these cells [START_REF] Yu | Mir-30 reduction Mayntains selfrenewal and inhibits apoptosis in breast tumor-initiating cells[END_REF]. In this view, miR-30a reduction may correlate with the expansion of putative BT-ICs. In addition, miR-30d overexpression suppresses cell proliferation and induces apoptosis in renal carcinoma cells [START_REF] Wu | MiR-30d induces apoptosis and is regulated by the Akt/FOXO pathway in renal cell carcinoma[END_REF]. By targeting Wnt/ -catenin/BCL9 (B-cell CLL/lymphoma 9) pathway, miR-30s reduce tumor burden and metastatic potential in vivo in murine models of human multiple myeloma [START_REF] Zhao | miR-30-5p Functions as a Tumor Suppressor and Novel Therapeutic Tool by Targeting the Oncogenic Wnt/ -Catenin/BCL9 Pathway[END_REF]. It has been also shown that miR-30c influences the chemotherapy resistance of breast cancer cells. In fact, by targeting twinfilin 1 (TWF1) and IL-11, a secondary target of TWF1, miR-30c reduces chemotherapy resistance of these cells [START_REF] Bockhorn | MicroRNA-30c inhibits human breast tumour chemotherapy resistance by regulating TWF1 and IL-11[END_REF].

MiR-30c expression correlates with endocrine therapy resistance in advanced ER-positive breast cancer [START_REF] Rodríguez-González | MicroRNA-30c expression level is an independent predictor of clinical benefit of endocrine therapy in advanced estrogen receptor positive breast cancer[END_REF]. In this study, miR-30c has been suggested as a predictor of endocrine therapy in ER+ breast cancer. Interestingly, it has been reported that miR-30 family members are all downregulated in both ER and PR negative tumors, suggesting that expression of these miRs is regulated by these hormones [START_REF] Iorio | MicroRNA gene expression deregulation in human breast cancer[END_REF]. Indeed, miR-30 have been recently shown to be dowregulated by progestins [START_REF] Cochrane | Progestin regulated miRNAs that mediate progesterone receptor action in breast cancer[END_REF].

Despite a well-recognized role of tumor suppressor in epithelial cancer and activity to oppose EMT and to maintain epithelial phenotype, miRs-30 has been reported as oncomir. A miR analysis of human melanoma, a highly invasive cancer, found that miR-30b/30d upregulation correlates positively with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment [START_REF] Gaziel-Sovran | miR-30b/30d regulation of GalNAc transferases enhances invasion and immunosuppression during metastasis[END_REF]. MiR-30c actually promotes the invasive phenotype of MDA-MB-231 by a mechanism dependent on NOV gene [START_REF] Dobson | hsa-mir-30c promotes the invasive phenotype of metastatic breast cancer cells by targeting NOV/CCN3[END_REF]. Taking into account the high expression of miRs-30, it is important to investigate the impact of their dysregulation in specific tissue, pathology and cell lines. Depending of gene expression in the considered tissues, miRs-30 would have different regulating activity. As a consequence of their different location in genome, they may be differently regulated, especially in cancer, and this will impact on their own regulative property.

Objective of the project

The early steps of breast cancer metastasis to bone are common to most solid tumors and start with local tissue invasion and tumor cell escape from primary tumors. Following tumor cell dissemination into the blood and/or lymphatic system, cancer cells that express a specific gene signature disseminate to bone and invade the bone marrow. If they adapt to the local microenvironment and exit the dormancy step, they may further colonize the tissue. Each of the molecular pathways underlining these steps is regulated by multiple factors, through the tight control of genes and their master cell endogenous regulators, the miRs.

The objective of this work is to find out if tumour-derived miRs act as endogenous regulators of bone metastasis, in breast cancer. We have first studied miR activity in the steps of bone tropism, bone homing and engraftment of cancer cells and subsequent osteolytic lesions of bone. We used a model of human bone metastasis in vivo, in which a tumor cell line that highly and specifically metastasizes to bone is injected in caudal artery. We have also studied the potential activity of miRs-30 in breast tumorigenesis and dissemination of cancer cells, the early steps of metastasis in a syngeneic orthotopic mouse model, based on mammary gland injection of breast cancer murine cells.

Overall, we looked for miRs that could be prognosis and/or diagnosis markers of bone metastasis and might represent potential therapeutic targets of the disease.

MiRs-30 activity in breast tumorigenesis and tumor cell dissemination to bone.

In the first part of the work, we have shown that the miRNA-30 family (miRs-30) regulates the expression of a set of genes which are mediators of bone metastasis formation, at the steps of bone tropism, homing, colonization and macro-metastasis development. Through the carcinomainduced down-regulation of Dicer expression, large sets of miRNAs are underexpressed in breast human tumors compared to normal tissues and this interference with miRNA processing enhances experimental tumorigenesis [START_REF] Valastyan | Metastasis suppression: a role of the Dice(r)[END_REF]. Therefore, the decrease of miRs-30 levels, which are highly expressed in epithelial tissues, in the breast primary tumor may affect breast tumorigenesis and the subsequent metastatic dissemination [START_REF] Joglekar | The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells[END_REF]. Based on the analysis of a genome-wide miRNA expression in a set of normal and tumor breast tissues the pioneer work of Iorio et al. [START_REF] Iorio | MicroRNA gene expression deregulation in human breast cancer[END_REF] demonstrates the existence of a breast cancer-specific miRNA signature. The authors reported that miRs-30, the levels of which did not differ in tumor versus normal tissue, was expressed at higher levels in hormone dependent and well differentiated breast tumors versus hormone independent tumors. MiRs-30 are tumor suppressor in lung (KuMarchwamy et al., 2012b), prostate [START_REF] Kao | miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT[END_REF], breast [START_REF] Zhang | MicroRNA-30a suppresses breast tumor growth and metastasis by targeting metadherin[END_REF] and anaplasic thyroid [START_REF] Visone | Specific microRNAs are downregulated in human thyroid anaplastic carcinomas[END_REF] cancers mostly by regulating EMT and related transcription factors such as SNAIL1 and ZEB2. Their lower expression levels in lymph node metastases versus primary tumors suggest a role for miRs 30 during metastatic dissemination of breast carcinomas [START_REF] Baffa | MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets[END_REF]. Moreover, miRs-30 were reduced in putative breast tumor-initiating cells (BT-ICs) inducing the up-regulation of ubiquitin-conjugating enzyme 9 (Ubc9) and ITGβ3 [START_REF] Yu | Mir-30 reduction Mayntains selfrenewal and inhibits apoptosis in breast tumor-initiating cells[END_REF]. Enforced constitutive expression of miRs-30 in BT-ICs inhibited their self-renewal capacity by reducing Ubc9, and induced apoptosis through silencing ITGβ3. Growing MCF-7 under non-attachment conditions revealed that miRs-30 level was markedly lower than in parental cells that in turn, modulated the expression of apoptosis and proliferation genes [START_REF] Ouzounova | MicroRNA miR-30 family regulates non-attachment growth of breast cancer cells[END_REF].

In this part of work, we asked whether miRs-30 deregulated expression in breast primary tumor might modulate tumor growth and subsequent metastatic dissemination to bone. To answer this question, we used murine breast cancer cell lines that spontaneously metastasize. We have modulated the endogenous miRs-30 level of these cells by lentiviral transduction and by transfection of chemically stabilized oligonucleotides prior to inoculation in mice. We also set up a syngeneic orthotopic mouse model, based on mammary gland injection of breast cancer murine cells. To mimic the in vivo phenotype of CTC, tumor cells have been grown in anchorageindependent conditions, as mammospheres. The impact of 3-dimensional culture on miRs-30 endogenous level of these cells has been analyzed in relation to their target genes. Tumor cells dissociated from mammospheres have been inoculated in caudal artery of mice to study their metastatic potential to bone.

Materials and Methods

Cell lines and cell culture

To study the role of miR-30s in the early steps of metastatic dissemination, we used four murine cell lines: 67NR, 66c14, 4T1 and 4T1.2. Originally, three sublines of breast cancer cells were derived from a spontaneously arising mammary tumor of a BALB/cfC3H mouse: the 67, 66 and 410 (Dexter et al., 1978) (Fig 10). The 67NR were next transfected with the bacterial pSV2 plasmid containing the neomycin resistance gene. They form primary tumors, however, they do not metastasize [START_REF] Aslakson | Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor[END_REF], (Fig 11). The 66 was subjected to mutagenesis with ethyl methanesulfonate. In that way, the 6-thioguanine-resistant mutant, line 66c14 was isolated from a clone of these cells [START_REF] Miller | Quantitative selectivity of contact-mediated intercellular communication in a metastatic mouse mammary tumor line[END_REF]. Later on, 66c14 were transfected with the pGL4 vector to stably express the luciferase gene and were shown to metastasize from the primary tumor to the lung and bone but also to liver [START_REF] Aslakson | Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor[END_REF]. The last subline, the 410, was derived as a primary culture from a single nodule growing in the lung of Balb/c mouse bearing an s.c implant of the tenth in vivo passage of the original, unseparated tumor. The tumor 410 was serially transplanted through 4 in vivo passaged at which time tumor cells were designated 410.4. The 4T1 is a 6-thioguanine resistant variant of 410.4 that was selected without mutagen treatment [START_REF] Aslakson | Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor[END_REF]. From this cell line, a 4T1.2 cell line was established by a single cell cloning [START_REF] Lelekakis | A novel orthotopic model of breast cancer metastasis to bone[END_REF]. 4T1.2 was shown to closely model the sites of metastases seen in human patients including lung, liver, and bone (Fig 11).

Specifically, 4T1.2 cells demonstrated a high incidence of metastasis to spine [START_REF] Bolin | Novel mouse mammary cell lines for in vivo bioluminescence imaging (BLI) of bone metastasis[END_REF]. The 67NR were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1 % penicillin/streptomycin. 66cl4 were cultured in MEM medium supplemented with 10 % fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% sodium pyruvate. 4T1 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 2% penicillin/streptomycin and 0.2 % fungizone. 4T1.2 cells were cultured in MEM medium supplemented with 10 % fetal bovine serum, 1% penicillin/streptomycin and 1% sodium pyruvate. All cells were cultured at 37°C in a humidified atmosphere in 5 % CO2 incubator.

In animals, we inoculated the following cell lines: 4T1 wild-type cell line (4T1) which is from ATCC, UK, 4T1 expressing luciferase2 (4T1luc2) which was engineered in the lab, 4T1.2-luciferase2 cell line (4T1.2luc2) and 66c14-luciferase2 cell line (66c14luc2) which were a generous gift from Celeste Boilin and Cheryl Jorcyk, Boise State University, ID, USA.

Plasmid construction

The pCMV-luciferase2 retroviral vector was constructed by inserting the coding sequence of Firefly luciferase2 (luc2) into the multiple cloning site of pMiRVec. To increase expression and reliability of the firefly luciferase, a synthetic firefly luciferase gene, has been engineered by the constructor (Promega) and inserted in the cloning vector PGL4. The gene was synthetically redesigned by changing the codons to those most frequently used in mammalian cells while simultaneously removing most of the consensus sequences for transcription factor binding sites.

Luc2 coding sequence was amplified by PCR using Accuprime High Fidelilty (Invitrogen) and purified after agarose gel extraction (NucleoSpin, Macherey Nagel). PCR product was inserted into HinDIII/EcoR1-digested pMiRVec vector using In-Fusion HD Cloning Kit (Clontech Laboratories). PCR primers for luc2 were designed with 15 base pairing extensions that are complementary to the ends of the linearized vector. In-fusion cloning reaction was as followed: 2 ml of In-Fusion HD Enzyme Premix (5X), linearized vector (25ng), PCR product (50ng), completed to 10 μl final volume with H 2 O. Reaction was incubated for 15 min. at 50°C and placed on ice, prior to transformation of Stellar Competent cells (Clontech).

The retroviral PmiRVec-miR-30sponge vector was constructed by inserting the miR-30 bulged sponge sequence in the MCS of pMiRVec. Briefly, "miRNA sponges" are miRNA competitive inhibitors that have been developed in Philipp Sharp's laboratory, as an alternative to modified antisens oligonucleotides [START_REF] Ebert | MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells[END_REF]. They function as decoy targets that anneal to the cognate miRNA and derepress miRNA targets in mammalian cells. They are designed as oligonucleotides to miRNAs binding sites with a bulge at the position normally cleaved by Argonaute 2 and are inserted tandemly as heptameric sites into the 3'UTR of a reporter gene.

MiR-30-Sponge have been constructed by Philipp Sharp laboratory by inserting six miR-30 binding sites tandemly arrayed into the 3'UTR of a reporter gene encoding destabilized GFP driven by the CMV promoter. Binding sites for miR-30 seed family were perfectly complementary in the seed region with the bulge at positions 9-12 to prevent RNA interferencetype cleavage and degradation of the sponge, as followed:

The sponges specifically inhibit miRs with a complementary heptameric seed (the sequence indicated in blue, common to the five members of miRs-30), such that a single sponge can be used to block an entire miR seed family. We have digested the DNA with six bulged sequences (in red) from pcDNA5-CMV-d2eGFP vector and cloned this sequence into MCS of pmiRVec to obtain stable expression of this miRs-30 sponge.

Cell transfection and transduction

Transient overexpression or down-regulation of miRs-30 in tumor cell lines was achieved by transfecting the cells with chemically modified double-stranded RNAs that mimic endogenous miRNAs (miR-mimics, Applied BioSystems) or chemically modified single-stranded RNA molecules designed to specifically inhibit endogenous miRNA (miRNA inhibitors, Applied BioSystems). Cell lines were transfected with 10nM of each miR-30s mimics or antagonist sequences or with 50 nM of negative scramble controls (Applied BioSystems). These oligonucleotides were incubated with 10 6 cells in appropriated medium, in the presence of HiPerFect transfection reagent for 30 min at 37°C in a 5% C0 2 incubator, prior to cell plating.

Transfected cells were then grown in culture for 48-72 hours before cell assays.

Tumor cells were transduced using the amphotropic retroviral packaging system (ClonTech Laboratories). Briefly, GP2-293 cell line which has the viral gag and pol genes incorporated in its genome were transfected with the pantropic VSV-G envelope vector and the retroviral pmiR-Vec vector (kindly provided by Dr R. Agami, The Netherlands Cancer Institute, Amsterdam). 4T1 cell line was transduced with pCMV-luciferase2 retroviral vector generating 4T1 cell line stablyexpressing luciferase2 (4T1luc2). 4T1.2 and 66C14 cell lines were transduced with pmiRVec -miR-30-sponge. Virus particles were collected at 72h after transfection, filtered and used to transduce the recipient cells in presence of 8 μg/mL polyprene. Vector transduced cells were selected with blasticidine.

Finally, cell transduction was also performed with RNAi MISSION TR Lenti microRNA Inhibitor particles. For these experiments, we obtained lentiViral particles from Sigma and performed 4T1.2-luc2 and 66c14-luc2 transduction by incubating 4 x 10 4 cells with 10 TU/cell, in presence of 8 μg/ml of polyprene for 72h. Selection of stable transfectants was further achieved by incubating the cells with puromycin. Briefly, in this technology, expression of the miRNA inhibitors is driven by the hU6 promotor upon genomic integration of the lentiviral transfer vector into the host cell post-transduction [START_REF] Haraguchi | Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells[END_REF]. MiRNA inhibitors are able to competitively bind specific miRNAs and prevent them from regulating their endogenous targets.

Lentiviral vectors have been constructed by integration of decoy RNA molecules in the MCS of pLKO lentiviral vector [START_REF] Haraguchi | Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells[END_REF]. The entire secondary structures of these decoys and the sequence of miRNA-binding site have been optimized to establish a highly potent miRNA inhibitory system (tough decoy design).

Animal studies

Four-week-old female Balb/c mice were purchased from Janier Laboratories. All procedures involving animals, including their housing and care, the method by which they were culled, and experimental protocols were conducted in accordance with a code of practice established by the local ethical committee of the University of Lyon.

Mice were injected orthotopically into the fat pad of the 4th mammary gland with 10 5 cells in 100 L of 50 % PBS-50 % Matrigel for 4T1.2luc2 and 66c14luc2 and with 10 5 cells in 10 L PBS, for 4T1. Tumor size was calculated by external measurement of the width (m1) and length (m2) of tumors using a Vernier caliper. Tumor volume (TV) was calculated using the equation TV = (m1 2 ×m2)/2. On day 14 after tumor cell inoculation, mice were anesthetized and the primary tumors were resected, measured, weighted and cut into two pieces. Half of tumors was fixed into 4 % PFA for immunohistochemistry and the reMayning tissue was placed into liquid nitrogen for RNA extraction.

Injection of 4T1.2luc2 and 66c14luc2 grown under non-attachment conditions and of control cells grown on plates was performed intra-arterially with 10 5 cells in 100 L of PBS.

Bioluminescence imaging of animals was performed weekly, by s.c injection of luciferine and was monitored by the Nightowl imaging system (Berthold, Germany), as previously described [START_REF] Bolin | Novel mouse mammary cell lines for in vivo bioluminescence imaging (BLI) of bone metastasis[END_REF][START_REF] Fradet | Dual function of ERR in breast cancer and bone metastasis formation: implication of VEGF and osteoprotegerin[END_REF][START_REF] Peyruchaud | Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions[END_REF].

At the end of protocols, animals were anesthetized, sacrificed and several organs were dissected.

Hind limbs and spines were placed in medium and lungs in PBS.

Bone marrow micrometastasis isolation

For ex-vivo bone marrow micrometastasis experiments, cells were injected into the mammary gland or into the tail artery of animals. At day of sacrifice, hind limbs, spines and lungs were collected and tibiae and femurs were minced then soaked in an enzyme cocktail containing 300U/mL type-I collagenase and 100 U/ml hyaluronidase (StemCell Technologies) in DMEM medium for 2 hours at 37°C. Lungs were minced and an enzyme solution containing 0.25 mg/mL type-I collagenase (Sigma) was added for 1 hour at 37°C. After incubation, cell suspensions from bone marrow and lungs were seeded in 6-well plates and cultured in complete medium. After 1day culture, cells were placed under 6-thioguanine selection, allowing the selective growth of antibiotic-resistant tumor cells. Colonies of tumor cells were then fixed and stained with crystal violet.

Cell invasion assay

Experiments were conducted in 24-well cell culture plates with 8-m diameter pore-size inserts, coated with 100 l basement membrane Matrigel (0.3 mg/mL), as previously described [START_REF] Fradet | Dual function of ERR in breast cancer and bone metastasis formation: implication of VEGF and osteoprotegerin[END_REF]. 4T1.2luc2 or 66c1luc2 were transfected with either control or miR-30 inhibitors. After 48 h, cells were detached with trypsine/EDTA and a suspension of 5x10 4 cells in 300 L of culture medium containing 0.1 % (w/v) bovine serum albumin was loaded into each insert (upper chamber). The chemoattractant [10 % (v/v) fetal calf serum] was placed in the lower chamber (750 l /well). After a 24-h incubation at 37°C in a 5 % CO2 incubator, inserts were collected, the non-invading cells were removed and the invading cells on the under surface of the inserts were stained with 0.5 % crystal violet for 10 minutes. Cells were then counted under microscope. Total RNA was extracted using Total RNA isolation NucleoSpin RNA II kit, as described in the protocol. 1.2.1.8. b. RNA isolation for microRNA expression analysis

Total RNA, including small RNAs, was extracted using miRNeasy Micro Kit, as described in the protocol.

1.2.1.9 . Real time RT-PCR 1.2.1.9. a. RT-PCR for gene expression analysis cDNA was produced from 1 g total RNA using the iScript cDNA Synthesis Kit. Real-time quantitative PCR reactions were performed using a SYBR Green qPCR kit on a RealPlex (Eppendorf, Durheim). Relative gene expression levels were normalized according to the Ct value of the gene encoding L32 protein and results were expressed as fold differences equal to 2-ΔΔCt.

1.2.1.9. b. RT-PCR for microRNA expression analysis

TaqMan microRNA assays were used to quantify miRs-30 and snU6 levels after RNA isolation.

100 ng of total RNA were reverse-transcribed using the MultiScribe reverse transcriptase and 50 nM stem-loop RT primers specific for miR-30 members and the endogenous control snU6.

Mature miRNAs were amplified using the TaqMan 2X universal PCR Master Mix and miRs-30s level were expressed as fold differences equal to 2 -ΔΔCt .

Results

1.2.2.1.Orthotopic murine model of metastatic dissemination from breast tumor cells: 4T1

We have first set up a mouse model of spontaneous breast cancer metastasis. In this animal model, tumor cells form a primary tumor that is resected, mimicking clinical situation, and tumor cells disseminate to secondary organs to form micro-or macro-metastasis. 4T1-luc2 form primary tumors that are detected by bioluminescence imaging. However, we could not detect tumor cells in bone and lungs by this technique. In regard of the difficulty to detect bioluminescence in lungs, we isolated the DTC from legs and lungs. Micro-metastases were detected in lungs but not in legs. Indeed, when injected in mammary gland in a syngeneic model, some investigators report that 4T1 highly disseminates to lungs, but not to bone. Our results were in accord with these data. We further asked if 4T1 tropism might have been disturbed by luciferase2 labeling or transduction. Therefore, we performed another study using the same mice model, in which 35 mice have been inoculated with 10 5 4T1 cells. Every 7 day, 7 mice were sacrificed, lungs and bone were collected and DTC were isolated from lungs and bone. Despite the fact that lungs were highly metastatic, the presence of micro-metastases in bones was found in only one out of 35 mice, meaning that in our hands, 4T1 metastasizes very weakly, if not at all, to bones.

123 Numbers indicate the day of BLI imaging. At day 14, the primary tumor was resected. The colourful spot on mouse indicates the bioluminescent 4T1-luc2 which forms the primary tumor. After 14 days, tumor was resected and tumor re growth was detected over time.

Orthotopic murine model of metastatic dissemination from breast tumor cells: 4T1.2luc2, 66c14luc2

4T1.2luc2 was injected in 10 μl PBS, according to the Lab's procedure. However, even by increasing the cell amount over 10 5 , we did not get formation of primary tumor, indicating probably a weaker tumorigenic propensity of these cells. Therefore, we have adapted a protocol 125

Despite the fact that injection of 66c14luc2 in 10 μl PBS, according to the Lab's procedure induced primary tumor formation, we used the same procedure than the one used for 4T1.2luc2 study to compare the metastatic behavior between the two cell lines. 66c14luc2 was injected at range from 0.25x10 5 to 1x10 5 cells in 100 μl of 50 % / 50 %, PBS / Matrigel. Finally, injection of 1x10 5 66c14luc2 induced strong dissemination to lungs whereas few mice developed bone metastasis. Moreover, for both cell lines, only the incidence was plotted. It should be noticed that the number of bone micro-metastasis was high in mice inoculated with 4T1.2luc2 but not in lung and inversely for 66c14luc2.

Expression of miRs-30 in breast cancer cell lines

MiRs-30 expression was investigated in the murine cell lines and their levels compared to human breast cancer cell lines, MDA-B02 and its parental MDA-MB-231. MiRs-30 were highly expressed in the three murine cell lines, and this is in agreement with the epithelial phenotype of these cell lines (not shown). Thus, we designed experiments to decrease miRs-30 activity in these cells and to conduct loss-of-function study. Repressing miRs-30 activity was effective to derepress KLF10 and RunX2 expression in 66c14luc2 but was ineffective to do so in 4T1.2luc2. 127 1.2.2.5. Mir-30 silencing in 4T1.2luc2 and 66c14luc2 overexpressing miR-30-decoy 4T1.2luc2 and 66c14luc2 were transduced with lentiviral particles to obtain the stable expression in these cells of miR-30 decoys synthesized from genomic DNA. The expression level of miR-30 target genes, KLF10 and RUNX2 was analysed in 4T1.2luc2-Ctrl and 66c14luc2-Ctrl and in the cells transduced with 4T1.2luc2-decoy-miR-30 and 66c14luc2-decoy-miR-30. The silencing of miRs-30 did not induce any changing in this expression levels. (A) 4T1.2luc2 was transfected with chemically modified double-stranded RNAs that mimic endogenous miRs-30 (miR-30 mimics) or with chemically modified single-stranded RNA designed to specifically inhibit endogenous miRs-30 (miR-30 inhibitors) or with sramble controls (Negative Ctrl). MiRs-30 expression was analyzed by TaqMan RT-qPCR.

(B) Integrin α5 expression was measured by TaqMan-RT-qPCR. (Mean ± SD, *: p<0.05).

Transfection of 4T1.2luc2 with miR-30 mimics increased the miR-30 level over endogenous level whereas miR-30 inhibitors moderately decreased this level (from 31 to only 15 % of inhibition). For data reported on fig 17A, high number of cells was transfected prior to inoculation in mice mammary gland, and we obtained a lower inhibition than in previous transfections. This might be due to the fact that these cells have high dividing rate, thus the amounts of transfectant is diluted very quickly during cell division. Despite this, the expression level of Itgα5, a miR-30 target gene, was increased in 4T1.2luc2 transfected with miR-30 inhibitors. Modulation of invasiveness was analyzed, in vitro on plate coated with Matrigel.

Increasing miRs-30 level inhibited significantly the invasiveness of 4T1.2luc2 whereas trapping miR-30 with miR-30 inhibitors increased cell invasiness. This was not observed in 66c14luc2 To investigate miRs-30 activity in vivo in syngeneic model, Balb/c mice were injected orthotopically, with 4T1.2luc2 transfected with negative control (10 mice), or with 4T1.2luc2 transfected with miR-30 inhibitors (10 mice). No difference was observed in the growth of primary tumor (Figure 19). Mice were sacrified 11 days after tumor resection and micrometastasis were isolated from legs, spine and lungs. We did not observe any occurrence of metastasis in lungs in the two groups of mice. The incidence of metastasis in bone and spine was only of 2 mice having metastasis in the group of mice inoculated with 4T1.2luc2 transfected with miR-30 inhibitors (not shown). The reason of the absence of dissemination may be explained by the short time that we kept the animal after resection, due to the fact that we inoculated cells that were transiently transfected. This study appears only as preliminary experiment. 4T1.2luc2 was transfected with miRs-30 inhibitors (miR-30 inhibitors) and scramble control (Negative Ctrl), prior to inoculation in mammary fad pad. Tumors were resected 11 days after cell inoculation and weighted.

Expression of miRs-30 is down-regulated in 4T1.2luc2 and 66c14luc2 under nonattachment growth conditions

Preclinical evidence suggests that metastasis arises from a small subset of tumor cells, the cancer stem-like cells (CSCs) that have acquired the capacity to self-renew and to differentiate into cell with limited proliferative capacity. CSCs, also named tumor initiating cells (TICs) have migratory capacity and high metastatic potential. Here, we asked if miR-30 level might be modulated in breast TIC (BT-ICs) and impact on their metastatic potential. To enrich 4T1.2luc2 and 66c14luc2 in BT-ICs, we have grown these cell lines without anchorage, in serum-free medium, conditions in which they form mammospheres. Gene expression in 4T1.2luc2 and 66c14luc2 grown under non-attachment conditions (4T1.2 MM, 66c14 MM) was compared to that in 4T1.2luc2 (4T1.2) and 66c14luc2 (66c14) grown in regular culture conditions. 66c14 Att: dissociated mammospheres that have been put back in regular culture conditions (Mean ± SD, *p<0.05). Vimentin and Twifilin are proteins that regulate EMT by promoting F-actin formation, a key component of cellular transition to a more invasive mesenchymal phenotype. Both genes are targets of miRs-30 at the 3'UTR level and their expression is up-regulated in tumor cells that have low levels of miRs-30 (see paper page 59-111 of this manuscript). This result suggests that the miRs-30 depletion in mammospheres promotes Vim and Twf expression and thus would stimulate the invasive mesenchymal phenotype of mammospheres. In primary tumor, BT-ICs acquire mobility to migrate into the surrounding stroma, circulate in blood stream and seed at distant sites to grow. The specific development of bone metastasis requires the recruitment of CTC in bone marrow and their adaptation to survive in the surrounding microenvironment. We hypothesized that miRs-30-depleted mammospheres mimic CTC that have undergone EMT and escaped primary tumors to reach blood flow. To anticipate their propensity to migrate to bone, the expression of genes involved in bone metastasis has been analyzed in these cells.

2009).

Lox expression was increased in miRs-30-depleted-4T1.2luc2, in accord with the fact that this gene is a miRs-30 target. In this model, lysyl oxidase would also stimulate the engraftment of tumor cells in bone marrow. Finally, the expression level of Bcl2-like 11 (Bcl2l11) which is a proapoptotic gene, belonging to Bcl-2 family member [START_REF] Li | MicroRNA-9 regulates neural apoptosis in methylmalonic acidemia via targeting BCL2L11[END_REF], and a predicted target of miR-30s, was also increased in these cells, suggesting a higher level of apoptosis in mammospheres. This was confirmed by preliminary study showing that late apoptosis was increased in mammospheres as compared to cells grown in attachment (not shown). The reason for that is not clear, but it is reasonable to think that the non-attachment culture conditions without serum alter the cells. Despite this, we decided to investigate if miRs-30-induced modulation in expression of these genes would modify metastatic dissemination of 4T1.2luc2 and/or 66c14luc2 in animal model. miRs-30-depleted 4T1.2luc2 and 66c14luc2 in mice. Tumor cells dissociated from mammospheres and tumor cells detached from culture wells were injected intra-arterially in mice. Metastasis dissemination to lung, spine and legs was assessed by occurrence of micro-metastasis in organs. We choose this type of injection that bypasses escape of primary tumor to investigate the steps of bone tropism and homing of CTC. Abbreviations are as in figure 21. (%) represent the percentage of mice that developed micrometastasis in organ.

Metastatic dissemination of

LUNGS

MiRs-30-induced regulation of gene involved in bone metastasis did not impact on metastatic dissemination in vivo (Table 5). We hypothesized that growing cells as mammospheres may induce a loss-of-functions of these cells by stimulation of apoptosis and/or necrosis. In this model, there was a huge amount of tumor cells in lungs that we cannot numerate, making difficult to figure out the impact of gene modulation. Moreover, tumor cells seemed also to have grown outside organs, especially in bone. We concluded, on these preliminary data, that 4T1.2luc2 and 66c14luc12 grown as mammospheres is not adapted to study bone tropism and bone homing in a mice model of bone metastasis.

Discussion

In the course of this study we have first confirmed that 4T1 is not a model of metastatic dissemination to bone after cell inoculation in fat pad since animals developed strong macrometastasis in lung after tumor resection but not in bone. Moreover, mice would suffer rapidly from lung metastasis before developing bone metastasis. We found that 4T1.2luc2 forms reproducible breast primary tumors when inoculated in mammary gland mixed to Matrigel and disseminates more in spine and leg than in lung. 66c14luc2 disseminate more in lungs than in bone. In regard of high levels of miRs-30 expression in these murine cells, we used a loss-offunction approach to study miRs-30 activity. The miRs-sponge technology that has been specifically design to target entire miR family seemed very attractive for our study, in regard of the five members of the family. From the sponge sequences in a cloning vector designed by Philippe Sharp laboratory, we obtained a stable cell line from the entire 4T1.2luc2 and 66c14luc2 population. This technical approach allows avoiding clonal selection of these very heterogeneous cell populations. We did not obtain the derepression of miRs-30 target genes in 4T1.2luc2. The reason for that is not clear, since the technology is efficient to derepress expression of several genes (RUNX2, ITGβ3, CDH11 and CX43) in human MDA-MB-231 and MCF-7. It is possible that the transduction was not efficient in 4T1.2luc2 or that these genes were not efficiently regulated by miRs-30 in these cells. We used the miR-30s mimics and miR-30s inhibitors to modulate gene expression in these cells. Here we choose to target the 5 miRs-30 by mixing equimolar concentrations of each. Both, mimics and inhibitors modulate 4T1.2luc2 invasiveness efficiently indicating that exogenous miRs-30 can still repress gene targets, despite high levels of endogenous miRs-30. In this, miRs-30 in these murine cell lines might be not directly linked to their spontaneous dissemination. This would agree with results reporting that overexpression of miR-30a suppresses the migration and invasive phenotype of breast cancer cell line, MDA-MB-231. When we transfected MDA-MB-231 with miR-30 mimics, cell invasiveness was decreased but transfection with miR-30 inhibitors did not increase cell invasiveness (See paper page 59-111). Another complexity of miRs-30 activity is that there are five members that share similar seed regions but that may have difference in activity. They are also expressed at very high levels in cells that would explain why miR-30 inhibitors were inefficient to modulate tumor growth in vivo. Further, we found that growing these cells without anchorage, to mimic CTC in vivo, 140 induced a depletion of endogenous level of miRs-30. In preliminary results, this miRs-30 depletion up-regulated the expression of several genes involved in bone metastasis. Although these experiments need to be repeated, we hypothesize that the down-regulation of miRs-30 is not occurring only in primary tumor, through the occurrence of EMT and/or Dicer down-regulation.

This process may be modulated during dissemination steps. DICER expression will be analyzed in mammospheres to find out if the tumorogenicity was altered. It also turn out that such grown cells are not suitable to be inoculated to mouse, probably due to an enhanced oncogenic-induced failsafe program in cells.

Overall, the orthotopic mouse model set up with 4T1.2luc2 is suitable to investigate metastatic dissemination to bone. However, we did not succeed to efficiently downregulate miRs-30 in these cells. Transient transfection of short oligonucleotides is not suitable, in regard of the rapid high dilution of these molecules in highly dividing cells. In 4T1.2luc2, the efficiency of sponge technology that allows continuous and stable expression of miRs-30 inhibitors, counteracting all members of the family need to be improved. some discrepancy in the literature. It is of special importance in regard of the very high expression of each miR-30. This means that for a loss-of-function study, a high amount of "trapping activity" is needed to derepress the expression of targeted genes. In these experiments, we are going to use the sponge technology that seems promising, using MCF-7 cells.

Surprisingly, preliminary study conducted with MDA-MB-231 and MCF-7 showed that increasing miRs-30 levels by transfecting these cells with equimolar concentrations of each miR-30 modulate gene expression, in a way that resembles that obtained with MDA-B02. Neverthe less, this approach will allow to compare the activity of each miR-30, as they may modulate differently the expression of specific genes.

In the course of our study, we have also shown that genes associated to various solid cancer metastases were down-regulated in MDA-B02-miR-30s by direct miRs-30 targeting (NEDD4, 5NTE) or indirect regulation (TNC). These genes were also up-regulated in bone metastasis at the transcriptomic level and appear as bone metastasis targets. We also found that the oncogenes Nid1 and Vimentin that are in-silico predicted direct targets of miR-30s are up-regulated in bone metastasis at transcriptomic levels and could promote bone metastasis. We are going to investigate the role of some of these targets in bone metastasis.

Finally, another interesting approach to characterize the role of miRs-30 in bone metastasis would be to treat mice with miRs-30 mimics, using our mice model of human bone metastasis. Les métastases osseuses sont des complications fréquentes des tumeurs solides, notamment du cancer du sein. Sur le plan clinique, elles sont associées à une morbidité sévère, induisant une hypercalcémie, des compressions médullaires, des douleurs osseuses, une fragilité osseuse et des fractures. Ces complications entrainent une réduction importante de la qualité de vie mais aussi de l'espérance de vie des patientes. Avec 50 000 à 70 000 personnes concernées par an en France, les métastases osseuses constituent un problème majeur de santé publique pour lequel il faut envisager des traitements curatifs, car il n'existe à l'heure actuelle que des traitements palliatifs. Les cellules tumorales de carcinomes mammaires qui métastasent au site osseux expriment des gènes qui favorisent le tropisme osseux de ces cellules ainsi que leur ancrage et développement dans la moelle osseuse. Au niveau de l'os, les cellules métastatiques altèrent la fonction des cellules osseuses formatrices, les ostéoblastes, mais aussi résorbantes, les ostéblastes, de la matrice osseuse. En perturbant ainsi l'équilibre entre formation et résorption osseuse, les cellules métastatiques induisent la destruction de l'os. Les mécanismes moléculaires sous-jacents à ces processus sont contrôlés par l'expression génique des cellules tumorales qui interagissent avec le microenvironnement et les cellules osseuses. Dans ce contexte, les microARNs (miRs) en tant que régulateur endogène de l'expression génique, interfèrent avec les différentes étapes de la formation des métastases osseuses, incluant l'échappement des cellules tumorales de la tumeur primaire, la dissémination et l'invasion du site osseux, ainsi que l'apparition de lésions ostéolytiques.

Conference Presentations

Notre travail expérimental a débuté par l'analyse des profils transcriptomiques des miRs dans des cellules de tumeurs mammaires à fort tropisme osseux. Nous montrons que l'expression d'une famille de miRs (miRs-30), composée de miR-30a, miR-30b, miR-30c, miR-30d et miR-30e, est inhibée dans ces cellules. Sur le plan clinique, la faible expression des miRs-30 dans la tumeur de patientes atteintes de cancer du sein est associée à un mauvais diagnostic de rechute métastatique, à la taille élevée de la tumeur primaire, à l'envahissement ganglionnaire, au stade avancé de la maladie et au statut hormono-résistant. Nous avons alors étudié l'activité des miRs-30 dans le développement des métastases osseuses en utilisant un modèle expérimental de métastases osseuses humaines dans lequel des cellules tumorales sont inoculées dans la circulation sanguine des souris. L'expression forcée des miR-30s dans ces cellules tumorales qui métastasent fortement et spécifiquement à l'os, inhibe la formation des métastases osseuses. Nous montrons que les miRs-30 inhibent l'invasion et stimulent l'ostéoblastogenèse, in vitro et réduisent la charge tumorale et l'ostéoclastogenèse, in vivo. En accord avec ces résultats, l'expression de gènes qui stimulent les métastases osseuses est inhibée par les miRs-30. Parmi ces gènes, l'expression du CTGF (connective tissue growth factor) est augmentée dans les métastases osseuses humaines.

Ces résultats sont présentés dans la première partie du travail expérimental d'un papier intitulé : « The Tumor Suppressor miRs-30-5p Family Regulates Human Breast Cancer Cell Colonization in Bone », qui est en préparation de soumission à publication.

Dans la seconde partie du travail, nous avons étudié l'activité des miRs-30 dans la formation de la tumeur primaire et dans la dissémination métastatique des cellules tumorales au site osseux. Des expériences de xénogreffes tumorales orthotopiques ont été menées, en inoculant des cellules tumorales qui métastasent spontanément dans la glande mammaire de la souris. Dans ce modèle, les miRs-30 n'altèrent pas la croissance tumorale et l'apparition des micrométastases dans l'os. Cependant les miRs-30 inhibent l'invasion et le caractère de cellules souches tumorales de ces cellules métastatiques, in vitro.

Globalement, cette étude montre que les miRs-30 inhibent les étapes du tropisme, de l'ancrage de l'invasion et de la formation de macro-métastases dans l'os, mais pas de la croissance tumorale et/ou de l'échappement des cellules cancéreuses de la tumeur primaire.

Ce travail nous permet d'envisager plusieurs perspectives. Nous allons modifier l'expression des miRs-30 dans des cellules de cancer du sein hormono-sensibles et étudier l'impact de cette modification sur le phénotype cellulaire, la croissance tumorale et la dissémination métastatique vers les sites secondaires. L'intérêt thérapeutique des miRs-30 sera testé en injectant les miRs-30 sous forme d'oligonucléotides stabilisés chimiquement, chez la souris, en présence ou non d'agents pharmacologiques utilisés dans le traitement des ostéolyses malignes, les bisphosphonates. Finalement, ce travail a mis en évidence des gènes fortement exprimés dans les cellules tumorales à tropisme osseux et dont l'expression est inhibée par les miRs-30. L'expression de certains de ces gènes est élevée dans les métastases osseuses humaines. Nous étudierons le rôle de ces gènes au niveau des métastases osseuses.

La dernière partie de la thèse est consacrée à l'activité de l'oncomir miR-10b, un miR dont l'expression est augmentée par le facteur de transcription Twist1, ce qui facilite la formation des métastases osseuses. Le papier intitulé « TWIST1 expression in breast cancer cells facilitates bone metastasis formation » est inséré en dernière partie du manuscrit.

Après avoir replacé ce travail dans son contexte bibliographique en présentant l'état des connaissances actuelles sur le cancer du sein, le tissu osseux, les métastases osseuses et les microARNs, nous présentons les résultats obtenus au cours des travaux de thèse, ainsi que les perspectives qui en decoulent.

  1.2.1.6. Cell invasion assay ................................................................................................... 1.2.1.7. Cell culture under-non attachment condition ............................................................. 1.2.1.8. RNA isolation ........................................................................................................... 1.2.1.8. a. RNA isolation for gene expression analysis ............................................ 1.2.1.8.b. RNA isolation for microRNA expression analysis ................................... 1.2.1.9 . Real time RT-PCR ................................................................................................... 1.2.1.9.a RT-PCR for gene expression analysis ....................................................... 1.2.1.9.b RT-PCR for microRNA expression analysis .............................................. 1.2.1.10. Reagents ................................................................................................................. 1.2.2. Results .................................................................................................................... 1.2.2.1.Orthotopic murine model of metastatic dissemination from breast tumor cells: 4T1 ... 1.2.2.2. Orthotopic murine model of metastatic dissemination from breast tumor cells: 4T1.2luc2, 66c14luc2. ........................................................................................................... 1.2.2.3. Expression of miRs-30 in breast cancer cell lines. .................................................... 1.2.2.4. MiRs-30 silencing in 4T1.2luc2 and 66c14luc2 by overexpressing miR-30-decoy ..... 1.2.2.5. Effect of miR-30 antagonism on 4T1.2luc2 invasiveness and tumor growth in murine xenograph model................................................................................................................... 1.2.2.6. Expression of miRs-30 is down-regulated in 4T1.2luc2 and 66c14luc2 under nonattachment growth conditions. ............................................................................................... 1.2.2.7. Metastatic dissemination of miRs-30-depleted 4T1.2luc2 and 66c14luc2 in mice ...... 1.2.3. Discussion .............................................................................................................. PART 3 ......................................................................................................................................... GENERAL DISCUSSION AND PERSPECTIVES .................................................................... PART 4 ......................................................................................................................................... ADDITIONAL WORK ................................................................................................................ 1. TWIST1 Expression in Breast Cancer Cells Facilitates Bone Metastasis Formation. .......... PART 5 ......................................................................................................................................... REFERENCES ............................................................................................................................. PART 6 ......................................................................................................................................... CONFERENCE PRESENTATIONS AND AWARDS ............................................................... 1. Conference Presentations ..................................................................................................... 1.1. BONE-NET Meetings: .................................................................................................. 1.2. International Conferences: ............................................................................................ 2. Awards ................................................................................................................................

Figure 1 .

 1 Figure1. Incidence of new cancer cases and death in women in more developed and less developed regions of the world in 2012. Figure2. Histological section of a mouse tibia, stained with the Masson's trichrome. Figure3. Osteoblast differentiation. Figure4. Possible fate of mature osteoblast. Figure5. Osteoclast-osteoblast interactions in the basic multicellular unit (BMU). Figure6. Classification of bone metastases. Figure7. The vicious cycle. Figure8. MicroRNA biogenesis. Figure9. MicroRNA genomic locations. Figure10. Derivation of murine breast cancer cell lines. Figure11. Metastatic dissemination of breast cancer cell lines after inoculation of these cells in mice mammary gland. Figure12. Follow-up of bioluminescence imaging from a mouse inoculated with 4T1luc2 in mammary gland. Figure13. Spontaneous metastatic dissemination of 4T1.2luc2 and 66c14luc2 to bone and lung after cell inoculation into the mice mammary fad pad. Figure14. Expression of miRs-30 in human and murine breast cancer cell lines. Figure15. Expression of miRs-30 target genes in 4T1.2luc2 and 66cluc2 expressing miRssponge-30. Figure16. Expression of miRs-30 target genes in 4T1.2luc2 and 66cluc2 stably transduced with lentiviral microRNA inhibitors. Figure17. Expression of miRs-30 in 4T1.2luc2 after transient transfection of miR-30 mimics and miR-30 inhibitors. Figure18. MiRs-30 decreased 4T1.2luc2 invasion whereas trapping miRs-30 increased 4T1.2luc2 invasion. Figure19. MiRs-30 did not affect 4T1.2luc2 growth in mice mammary fad pad. Figure20. Expression of miRs-30 in mammospheres obtained by growing 67NR, 66c14luc2 and 4T1.2luc2 without anchorage and without serum. Figure21. Expression of cancer stem cell markers in 4T1.2luc2 and 66c14luc2. Figure22. Expression of Vimentin and Twifilin in 4T1.2luc2 and 66c14luc2. Figure23. Expression of bone metastasis-related genes in 4T1.2luc2 (A) and 66c14luc2 (B).

Figure 1 .

 1 Figure 1. Incidence of new cancer cases and death in women in more developed and less developed regions of the world in 2012 (Ferlay et al., 2015).

Figure 2 .

 2 Figure 2. Histological section of a mouse tibia, stained with the Masson's trichrome. Black arrows indicate a row of osteoblasts on a bone trabecula (blue staining), white arrow indicate bone-lining cells, bar = 10 μm (Capulli et al., 2014).

Figure 3 .

 3 Figure 3. Osteoblast differentiation. The expression of BMPs and Wnt pathway promotes MSCs commitment towards an osteo/chondroprogenitor. Next, RUNX2 and DLX5 direct MSCs into osteoprogenitor differentiation. A pre-osteoblast arising from an osteoprogenitor expresses high amount of ALP and Type I Collagen. A mature osteoblast additionally expresses bone matrix proteins, such as OCN, OPN and BSP II (Capulli et al., 2014).

Figure 4 .

 4 Figure 4. Possible fate of mature osteoblast (Capulli et al., 2014).

Figure 5 .

 5 Figure 5. Osteoclasts-osteoblast interactions in the basic multicellular unit (BMU).

Figure 7 .

 7 Figure 7. The vicious cycle. The figure illustrates the many factors that stimulate on one hand osteoclast, osteoblast and vascular cells activity, and on the other hand tumor cell growth and survival in the bone microenvironment.

  tumors that give rise to metastasis. It has been shown that inhibition of ROCK activity reduces cell migration and metastasis to bone in vivo. A downstream target of ROCK is c-myc which facilitates the dissemination of MDA-MB-231 breast cancer cells to bone. Coupled to increased c-myc, miR-17-92 cluster expression is increased in metastatic MDA-MB-231 breast cancer cells in vitro and in vivo. The intratumoral administration of anti-miR-17 molecules into mice bearing MDA-MB-231 tumor xenografts greatly repressed bone metastasis without significant effect on primary tumor growth (Liu et al., 2009). Therefore, inhibition of ROCK/c-myc by anti-miR-17 strategy might prevent breast cancer bone metastasis. Another miR with reported oncogene role is miR-218. It appears as an osteo-miR by controlling bone formation through the upregulation of RUNX2 and the downregulation of Wnt signaling inhibitors (sclerostin, dickkopf-2, and secreted frizzled-related protein2) in osteoblasts. Additionaly, miR-218 stimulates the expression of Wntrelated proteins (bone sialoprotein and osteopontin) in metastatic breast cancer cells. Moreover, miR-218 may facilitate MDA-MB-231 breast cancer bone metastasis by upregulating CXCR4, a chemokine receptor supporting breast cancer cell migration to bone and mediating tumor growth in bone

  miR-30 family is made up of five members in human, encoded by six genes: a, b, c-1, c-2, d and e which share a conserved seed sequence. They are encoded in the genome in three clusters: miR-30 e and c-1 cluster together on chromosome 1 which is found within a nuclear transcription factor Y gamma (NFYC) gene; miR-30d and b on chromosome 8 and miR-30a and c-2 cluster on chromosome 6. In mouse, there are six members of the family, encoded by seven genes: a, b, c-1, c-2, d, e and f. MiR-30e, f and c-1 cluster together, like in human, in the exon of Nfyc gene, but on chromosome 4. Mir-30d and b cluster on chromosome 15 and miR-30a and c-2 cluster on chromosome 1.

Figure 10 .

 10 Figure 10. Derivation of murine breast cancer cell lines.

Figure 11 .

 11 Figure 11. Metastatic dissemination of breast cancer cell lines after inoculation of these cells in mice mammary gland.

Figure 12 .

 12 Figure 12. Follow-up of bioluminescence imaging from a mouse inoculated with 4T1luc2 in mammary gland.

(

  from Christophe Ginestier, Centre de Recherche en Cancérologie de Marcheille, Laboratoire d'Oncologie Moléculaire, UMR891 Inserm/Institut Paoli-Calmettes, Marcheille, France), in which 4T1.2luc2 was injected in 100 μl of 50 % / 50 % mixture of PBS and Matrigel . In these conditions, 4T1.2luc2 forms primary tumor and disseminate to secondary organs (Figures 13).

Figure 13 .

 13 Figure 13. Spontaneous metastatic dissemination of 4T1.2luc2 and 66c14luc2 to bone and lung after cell inoculation into the mice mammary fad pad. The diagram represents the percentage of mice having developed bone and lung metastases. (n=12 animals).

Figure 14 .

 14 Figure 14 . Expression of miRs-30 in human and murine breast cancer cell lines. MiRs-30 level in breast cancer cell lines was analyzed by TaqMan RT-qPCR and expressed as relative level compared to MDA-MB-231.

Figure 15 .

 15 Figure 15. Expression of miRs-30 target genes in 4T1.2luc2 and 66cluc2 expressing miRssponge-30.Both cell lines were transduced by pMiRVec-sponge-30 and pMiRvec-Ctrl to stably express miR-sponge-30 (4T1.2luc2-sponge-30, 66cluc2-spone-30) or miRs-scramble (4T1.2luc2-Ctrl, 66cluc2-Ctrl). Gene expression was measured by TaqMan-RT-qPCR. (*: p<0.05).

Figure 16 .BFigure 17 .

 1617 Figure 16. Expression of miRs-30 target genes in 4T1.2luc2 and 66cluc2 stably transduced with lentiviral miR inhibitors. Both cell lines were transduced by lentiviral miR scramble control (Ctrl) and by lentiviral miR inhibitors (-decoy-miR-30) Ctrl or miR-30 inhibitors (4T1.2luc2-Ctrl, 4T1.2-decoy-miR-30, 66c14.luc2-Ctrl, 66c14luc2-decoy-miR-30). Gene expression was measured by TaqMan-RT-qPCR. (*: p<0.05).Repressing miR-30 activity was ineffective to de-repress KLF10 and RunX2 expression in 66c14luc2 and in 4T1.2luc2.

(

  Fig 18).

Figure 18 .

 18 Figure 18. MiRs-30 decreased 4T1.2luc2 invasion whereas trapping miRs-30 increased 4T1.2luc2 invasion. 4T1.2luc2 was cultured in 24-well culture plates with 8-m diameter pore-size inserts, coated with basement membrane Matrigel. (Mean of n=3, p<0.05). No significant effect was observed with 66c14luc2.

Figure 19 .

 19 Figure 19. MiRs-30 did not affect 4T1.2luc2 growth in mice mammary fad pad.

Figure 21 .

 21 Figure 21. Expression of cancer stem cell markers in 4T1.2luc2 and 66c14luc2.

Figure 22 .

 22 Figure 22. Expression of Vimentin and Twifilin in 4T1.2luc2 and 66c14luc2.

Table 5 .

 5 Incidence of metastasis in mice inoculated with 4T1.2luc2 and 66c14luc2 grown under nonattachment conditions and with 4T1.2luc2 and 66c14luc2 cultured in regular conditions.

  At 10 years from the diagnosis, 70 % of patients with luminal A tumors were alive compared with 62.6 % of nonbasal TN patients, 54.4

% of luminal B, 52.6 % of basal-like, 48.1 % of HER2-enriched and 46.1 % of luminal/HER2

Table

Table 1 . Frequency of site specific metastases according to molecular breast cancer subtype.

 1 ).

	Subtype	bone	lung Metastatic organ [%] liver brain distal nodal
	Luminal A	66	23.8	28.6	7.6	15.9
	Luminal B	71.4	30.4	32	10.8	23.3
	HER2 positive,
	ER/PR positive	65	36.8	44.4	15.4	22.2
	HER2 positive,
	ER/PR negative	59.6	47.1	45.6	28.7	25
	Basal-like	39	42.8	21.4	25.2	39.6
	TN nonbasal	43.1	35.8	32.1	22	35.8

Table 2 . Arterial blood supply in target organs of cancer metastasis (Weiss et al., 1980)

 2

	Lung

-5p miR-30b-5p miR-30c-5p miR-30d-5p miR-30e-5p miR-30e-3p let-7c Post-menopausal status

	PCDH20	protocadherin 20	-0,24	0.56	1
	67 ± 1.17 (n=9) unc-5 homolog C (C. elegans) 0.01 protocadherin 19 Gene name SMAD family member 2 Tumorigenic proliferation UNC5C PCDH19 Variable SMAD2 LIN28B lin-28 homolog B Migration / invasion Post-Menopausal Status Yes RARG retinoic acid receptor, gamma SMAD2 SMAD family member 2 NEDD4 neural precursor cell expressed, developmentally 20.88 ± 5.30 (n=5) N HR 69 1.00 No 40 1.05 down-regulated 4 NT5E Tumor size < 20 mm 36 1.00 5'-nucleotidase, ecto (CD73 KLF9 Kruppel-like factor 9 TWF1 twinfilin, actin-binding protein > 20 mm 69 2.35	0.01 0.49-2.23 14.74 ± 5.63 (n=5) Score -0,21 0.93 PCT -0,04 0.67 CI P-value -0,25 0,91 -0.46 0.98 -0.30 0.97 -0.25 0.91 -1.00 > 0.99 0.909 -0.47 0.93 -0.20 0.67 -0.42 0.98 0.89-6.21 0.086	60.25±2.19 (n=5) μ 7-8 1 mer mer 0 0 1 0 0 2 2 1 1	Exp Valid 0.05 (33)
	MDA-B02-pmiR30b-c B02-pmiR30e-c-d-b-a CALB2 calbindin 2 6.00 ± 0.96 (n=7) 5.74 ± 0.76 (n=6) B-cellCll lymphoma Morphology Other ERG v-ets erythroblastosis virus E26 oncogene homolo 0.01 27.55 ± 2.65 (n=5) 0.02 26.64 ± 2.70 (n=4) 20 1.00 Apoptosis / senescence / cell survival AVEN apoptosis, caspase activation inhibitor SRGAP3 SLIT-ROBO Rho GTPase activating protein 3 Ductal 89 1.32 PLAGL2 pleiomorphic adenoma gene-like 2 Grade GI 11 No events 0.01 0.02 0.46-3.81 21.75 ± 2.51 (n=5) 21.75 ± 6.13 (n=4) -0.20 0.85 -0.10 0.71 > -0.03 0.93 -0.34 0.15 -0.18 0.96 0.605 -0.10 > 0.99 MTDH metadherin -0.24 > 0.99 Matrix TIMP3 TIMP metallopeptidase inhibitor 3 -0.22 0.93 GII 42 1.00	48.0 ± 1.50 (n=4) 41.67 ± 16.52 (n=4) 1 0 0 0 1 2 2 1	0.01 0.02 (49) (39) (57) (40)
	Variable NFIB LOX GLI2 NID1 pN Immune surveillance N nuclear factor I/B lysyl oxidase GLI family zinc finger 2 GIII μ nidogen 1 Neg μ B3GNT5 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 5 GALNT7 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-28 2.18 μ 49 1.00 μ ADAM12 ADAM metallopeptidase doMayn 12 1-3 pos 36 1.54 ADAM22 ADAM metallopeptidase doMayn 22 ADAM11 ADAM metallopeptidase doMayn 11 >4 pos 29 6.89 2.52-18.81 -0.18 -0.22 0.94-5.05 μ > -0.05 -0.05 -1.07 -0.84 > -0.03 0.49-4.86 -0,17 > -0.01 ADAM19 ADAM metallopeptidase doMayn 19 -0,32 pN Neg 49 1.00 acetylgalactosaminyltransferase UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase ADAM9 ADAM metallopeptidase doMayn 9 -0,18 Pos 65 3.39 1.29-8.92 α PIK3CD phosphoinositide-3-kinase, catalytic, delta polypeptide -0,23 Bone Osteogenesis ER Pos 85 1.00 Neg 24 2.09 0.95-4.64 miR-30aYes 0.511 0.952 1.000 1.000 1.019 No 1.043 (0.895) 1.805 (0.875) 1.009 (0.692) 0.954 (0.669) 1.008 (0.597) 0.972 (0.855) 1.319 (0.245) 0.99 3 0.94 1 0.070 μ μ 0.95 1 0.96 1 > 0.99 0 > 0.99 0.64 0 0.73 1 0.84 <0.001 0 0.93 1 0 (58) μ 0.91 1 0.014 0,92 0 0.068 1.060 0.899 Cancer stemness ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) -0,25 0,7 0 RUNX2 runt-related transcription factor 2 -0.52 0.84 1 34,35,41 μ RUNX1 runt-related transcription factor 1 -0.12 0.96 PR Pos 74 1.00 1 SATB1 SATB homeobox 1 -0.06 0.76 Neg 35 1.47 0.69-3.15 0.319 1 Tumor Size < 20 mm 1.239 1.027 1.17 1.309 1.499 1.217 1.432 IL1A interleukin 1, alpha -0,24 0,62 0 SATB2 SATB homeobox 2 -0.05 0.44 1 ER and PR Pos 69 1.00
	>= 20 mm Grade ITGA6 EMT WNT7B ER and/or PR integrin, alpha 6 0.865 (0.002) wingless-type MMTV integration site family, member 0.905 (0.150) 0.880 (0.001) 0.926 (0.020) 7B Neg 40 1.93 0.92-4.05 0.938 (0.001) 0.925 (0.002) 0.762 (0.007) -0,2 0,85 0 -0.01 0.76 1 0.083
	GI GII GIII LN status EED Osteoclast SNAI1 CALCR SNAI2 OSTM1	1.210 1.050 0.697 (0.071) embryonic ectoderm development 1.042 0.986 0.895 (0.071) μ calcitonin receptor nuclear factor of activated T-cells 1.238 0.973 0.970 1.155 0.758 (0.028) 0.811 (0.295) snail homolog snail homolog 2 osteopetrosis associated transmembrane protein 1 > -0.03 1.689 1.115 0.748 (0.006) 0.848 (0.076) 0.748 (0.071) 1.306 1.553 1.053 1.167 -0.61 0.76 0 -0.25 0.76 0 -0.26 0.81 1 _ 0.51 1 0,84 0	(38)
	pN neg pN pos ER/PR status ER/PR pos ER/PR neg CELSR3 CELSR3 Osteoblast GJA1 Adhesion BMP7	1.132 0.812 (0.028) 1.082 0.752 (0.02) cadherin, EGF LAG cadherin, EGF LAG seven-pass G-type receptor 3 0.952 1.072 1.062 0.977 (0.942) 0.799 (0.114) 0.889 (0.252) 1.012 1.050 1.000 0.872 (0.09) 0.804 (0.050) 0.975 (0.343) gap junction protein, alpha 1 (flamingo homolog, Drosophila) bone morphogenetic protein 7 > -0.02 1.292 0.948 (0.012) 0.899 (0.014) 0.747 (0.048) 1.168 1.350 1.219 1.082 0.771 (0.006) 0.901 (0.382) 0.641 (0.128) -1.50 > 0.99 0 1.195 -0,55 > 0.99 0 -0.06 0.84 1 0,4 0
	S100PBP	S100P binding protein	-0.53	0.74		1
	ITGA4	integrin, alpha 4	Ct) using RQ Manager software. -0.25 0.70	0
	PCDH10	protocadherin 10	-0.23	0.86		1
	ITGA6	integrin, alpha 6	-0.20	0.85		0
	ITGB3	integrin, beta 3	-0.19	0.93		0	(59)
	ITGA5	integrin, alpha 5	-0.14	0.77		0
							90 91 92 93 94 95 96 97 100 101 102 103

  and 500 μM L-glutamine for 5 days. At this time, cell aggregates were mechanically and enzymatically disrupted, filtered through 70 or 40 μm filter, counted and cultured again in 10 cm ultralow attachment plates. The same was repeated twice and at each stage, some cells were cultured under attachment condition. RNA was extracted from both conditions to analyze miR-30s and gene expression levels.

	1.2.1.7. Cell culture under-non attachment condition
	66c14luc2 and 4T1.2luc2 cells were cultured on ultralow attachment 10 cm plates (Nunclon
	Sphera) at 1×10 4 cells in 10 mL Mammocult Basal Medium, supplemented with 10 %
	Mammocult Proliferation Supplement, 0.0002 % heparin, 1 μM hydrocortisone, 0.5 %
	penicillin/streptomycin 1.2.1.8. RNA isolation
	1.2.1.8. a. RNA isolation for gene expression analysis

Table 5 . Incidence of metastasis in mice inoculated with 4T1.2luc2 and 66c14luc2 grown under non-attachment conditions and with 4T1.2luc2 and 66c14luc2 cultured in regular conditions.

 5

			%	LEGS	%	SPINE	%
	4T1.2 MM	3/5	60	4/5	80	4/5	80
	66c14 MM	5/5	100	5/5	100	5/5	100
	4T1.2	4/5	80	4/5	80	4/5	80
	66c14	5/5	100	3/5	60	4/5	80

1.1. BONE-NET Meetings: 19-21 January 2012, Liege, Belgium 18-22 June 2012, Nantes, France 4-9 February 2013, Kuopio, Finland 18-21 June 2013, Leiden, Netherlands 3-6 February 2014, Sheffield, UK 18-20 June 2014, Hamburg, Germany 7 October 2014, Crete, Greece 18 November 2014, Barcelona, Spain 1.2. International Conferences: ECTS PhD training Course, 27-30 June 2014 at St Catherine's College, Oxford, UK, ORAL PRESENTATION The 2nd International Symposium on Advances in Circulating Tumor Cells (ACTC) From Basic Research to Clinical Practice, (ACTC 2014) 8-11 October 2014, Crete, Greece, POSTER 2. Awards La journée des DOctorants de la SFR Santé Lyon-Est, 5 September 2013, Lyon, France, POSTER, AWARD for the best poster Implication des MicroRNAs-30 dans la Dissémination Métastatique des Cellules Tumorales de Cancer du Sein au Site Osseux.

Courtabeuf, France

Acknowledgements

I would like to express my special appreciation and thanks to Dr Philippe Clézardin for recruiting me as a PhD student for the BONE NET project. These three years were a real professional and personal adventure. I thank Dr Martine Croset for the scientific support. I would also like to thank Dr Martine Duterque and Dr Catherine Alix-Panabières for serving as the Rapporteurs of my PhD thesis. I thank Prof. Pierre Boulanger for chairing the Jury. Especially, thank all of you for willing to come back from your holidays to be the Jury Members for my PhD thesis. I am thankful to Caroline, Olivier, Edith, Chantal and Ismahène for your ideas and advice during the lab meetings.

I thank Laura for her personal support during all these days spent together in the lab and outside the lab. I want to acknowledge Casina and Debashish for amazing time spent together during the BONE NET project. These moments will always be in my memory. You are great companions for work and for fun! I thank to Lamia and Lise for your guidance over the lab from the beginning until the end and for your precious advice on the great spots to visit in Lyon and in France. I thank to Bénédicte, Anais, Anaid, Francesco, Raphael, Sophie, Délphine, Johnny, Mathilde, François, Geoffrey, Sacha, Elodie, Tristan and Manon for being so awesome and helpful to me. Special thanks to Sandra for sharing the conversations during long days in piece blanche and for being always there for me when needed for the animal protocols. I also thank all other team members for their support.

I would also like to express my gratitude to all of you for your patience with my French. Thanks

Abbreviations

resulting precursor hairpin, the pre-miR, is exported from the nucleus by Exportin-5-Ran-GTP.

In the cytoplasm, the RNase III enzyme, Dicer in complex with the double-stranded RNAbinding protein TRBP cleaves the pre-miR hairpin to its mature length [START_REF] Bernstein | Role for a bidentate ribonuclease in the initiation step of RNA interference[END_REF].

The functional strand of the mature miR is loaded together with Argonaute 2 (AGO2) proteins into the RNA-induced silencing complex (RISC), where it guides RISC to silence target messenger RNAs (mRNAs) through either mRNA cleavage, translational repression or deadenylation, whereas the passenger strand is often degraded [START_REF] Winter | Many roads to maturity: microRNA biogenesis pathways and their regulation[END_REF].

Figure 8. MicroRNA biogenesis.

The primary miR transcript (pri-miR) is produced by RNA polymerase II or III and is cleaved by the microprocessor complex Drosha-DGCR8 in the nucleus. The resulting precursor hairpin, the pre-miR, is exported from the nucleus by Exportin-5-Ran-GTP. In the cytoplasm, the RNase Dicer in complex with the double-stranded RNA-binding protein TRBP cleaves the pre-miR hairpin to its mature form. The functional strand of the mature miR is loaded together with Argonaute (Ago2) proteins into the RNA-induced silencing complex (RISC), where it guides RISC to silence target mRNAs through mRNA cleavage, translational repression or deadenylation. The passenger strand is often degraded (adapted from [START_REF] Winter | Many roads to maturity: microRNA biogenesis pathways and their regulation[END_REF]). MiR-30 level in cells grown under non-attachment conditions (MMO) was compared to the corresponding cell line cultured on plastic and in medium supplemented with FBS (Att). p is the number of passages for MMO, (Mean ± SD of 3 measurements).

The three cell lines harbored reduced levels of the five miRs-30 when grown as mammospheres and this was partly reversed when these mammospheres were dissociated, plated and cultured in presence of FBS. Moreover, the CSCs phenotype was markedly up-regulated in tumor cells

GENERAL DISCUSSION AND PERSPECTIVES

According to data reported in literature, miRs-30 appear as tumor suppressors, in regard of their EMT repressor properties. These miRs inhibit tumorogenesis, tumor cell invasion and survival.

We have shown that they inhibit breast cancer bone metastasis, mostly by repressing the expression of osteomimetic genes. To show that, we have increased miRs-30 in MDA-B02, a cell line that specifically invades bone, in animal model. These cells have been previously selected by selection pressure in animal and exhibit a mesenchymal phenotype. They disseminate to bone after injection in blood circulation and mimic breast cancer circulating tumor cells that have escaped primary tumor. In accord to its mesenchymal phenotype, this cell line is only weakly tumorogenic and do not escape primary tumor to disseminate in mice. Therefore, our observation is still restricted to a specific cell line with osteomimicry competence and need to be extended to others cell lines. We need to find out if miRs-30 would decrease bone metastasis in rather less osteomimetic cell lines before concluding that this miR family is negatively associated to a specific bone metastasis signature. This is also sustained by the fact that some members of miRs-30 prevents tumor growth and/or cancer cells escape and lung metastasis in animal models.

In the clinic, we show that miR-30a-5p, miR-30d-5p and miR30e-5p in primary tumors from patients with breast cancer is negatively associated with poor distant metastasis-free survival.

From this observation that also partially confirms previously published data, we conclude that a miRs-30 signature exists in vivo in breast tumors. Whether this confers the CTC to have an enhanced ability to invade bone rather than lungs or others metastatic sites reMayns to be investigated. It seems also, from our results, obtained with 4T1.2 model, that the expression of pro-metastatic genes is increased in breast cancer CTC. Therefore, searching for a gene signature of bone metastasis in CTC from breast cancer patients would be of interest. It would also inform on whether osteomimicry is relevant for all types of breast cancer subtypes or restricted to hormone-responsive breast cancer. We and others have also shown that miRs-30 in primary tumors from patients with breast cancer are negatively associated with hormone-insensitive status. For further study, we will work with the hormone-responsive and epithelial cell line, MCF-7 that expresses relatively high levels of miRs-30 and does not present osteomimicry competency. The aim will be to find out if modulation of miRs-30 levels in these cells would modulate osteomimetic genes and bone metastasis, in an hormone-responsive model.

Another point that needs to be addressed is to find out whether the five different members of the miR family differ in their anti-tumoral and anti-metastatic activity. This has been suggested by PART 4 ADDITIONAL WORK

TWIST1 Expression in Breast Cancer Cells Facilitates Bone Metastasis Formation.

Twist1 is a basic helix-loop-helix transcription factor initially identified as a major regulator of cell movement and tissue reorganisation during early embryogenesis that is aberrantly reactivated in human cancers. Twist1 promotes tumour cell invasion through activation of miR-10b, facilitating the intravasation of breast cancer cells in the systemic circulation and their dissemination to the lungs. Twist1 has been reported as a link between EMT and stemness in breast carcinomas. We have shown, by microArray transcriptomic analysis, that Twist1 expression level is significantly higher in bone metastasis from breast cancer patients than in others type of metastasis.

Twist1 expression in bone metastasis from breast cancer patients (from Croset M, Pantano F, Driouch K and Clezardin P; unpublished results)

(from the Gene Expression Omnibus, under IDs GSE11078 (MSK) and GSE14020 (Institut Curie).

We have studied the activity of Twist1 in bone metastasis formation in a mice model of human bone metastasis.

We have shown that Twist1 enhances the engraftment of tumor cells to bone marrow without affecting osteomimicry and EMT/MET-dependent functions but through a miR-10b dependent mechanism.

PART 5

REFERENCES PART 6 CONFERENCE PRESENTATIONS AND AWARDS