Contribution à l’Etude du Rôle des Protéines SIBLINGs au Cours de la Progression Tumorale

La famille des protéines SIBLINGs comprend la sialoprotéine osseuse (BSP), l’ostéopontine (OPN), la sialophosphoprotéine de dentine (DSPP), la protéine de matrice de dentine 1 (DMP1), la phosphoglycoprotéine de matrice extracellulaire (MEPE) et l’énameline (ENAM). Comme leur nom l’indique, ces protéines ont d’abord été identifiées au niveau de la matrice minéralisée de l’os et de la dent. Durant la dernière décennie, notre Laboratoire et d’autres équipes ont démontré que cette famille de protéines est également exprimée par un certain nombre de tissus tumoraux. Nous avons entrepris ce doctorat dans la continuité des projets de recherche menés au Laboratoire s’intéressant à l’étude des SIBLINGs au cours de la progression tumorale et métastatique. La première partie de notre projet a consisté à investiguer les mécanismes de régulation de l’expression du gène de la BSP humaine au niveau de cellules ostéoblastiques. Le facteur de transcription Runx2 est un facteur clé de la régulation des gènes osseux. Dès lors, nous avons émis l’hypothèse selon laquelle l’induction de l’expression du gène de la BSP observée au cours de la différenciation ostéoblastique pourrait être sous la dépendance de ce facteur. Nous avons ensuite étudié la régulation du gène de la BSP au niveau de cellules cancéreuses mammaires. En effet, nous avons voulu déterminer si l’expression de la BSP était sous la dépendance de mécanismes de régulation transcriptionnelle différents de ceux observés au niveau de cellules d’origine osseuse. L’objectif final étant de bloquer spécifiquement l’expression de la BSP au niveau des tumeurs. Notre stratégie a d’abord consisté à identifier les principaux facteurs transcriptionnels impliqués dans cette régulation puis à en étudier l’impact sur l’activité du promoteur de la BSP au niveau des deux types cellulaires considérés.Dans la deuxième partie de ce projet, nous nous sommes consacrés à l’étude d’un autre membre de la famille des SIBLINGs, l’OPN, afin d’en identifier le rôle au niveau des gliomes humains. Précédemment, il a été démontré que l’OPN est surexprimée dans les gliomes et ce, en corrélation avec le grade de la tumeur. Cependant, il n’y a que peu d’études décrivant le rôle de l’OPN dans les gliomes. Dès lors, nous avons voulu vérifier l’importance de l’OPN au cours du développement des gliomes en utilisant la technique d’interférence à l’ARN

Creb-1 and Ap-1 Transcription Factors Jund and Fra-2 Regulate Bone Sialoprotein Gene Expression in Human Breast Cancer Cells

Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a -56/-84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells

Granulocytic myeloid-derived suppressor cells promote angiogenesis in the context of multiple myeloma

peer reviewe

Histone deacetylase 7 silencing alters endothelial cell migration, a key step in angiogenesis

Global inhibition of class I and II histone deacetylases (HDACs) impairs angiogenesis. Herein, we have undertaken the identification of the specific HDAC(s) with activity that is necessary for the development of blood vessels. Using small interfering RNAs, we observed that HDAC7 silencing in endothelial cells altered their morphology, their migration, and their capacity to form capillary tube-like structures in vitro but did not affect cell adhesion, proliferation, or apoptosis. Among several factors known to be involved in angiogenesis, platelet-derived growth factor-B (PDGF-B) and its receptor (PDGFR-beta) were the most upregulated genes following HDAC7 silencing. We demonstrated that their increased expression induced by HDAC7 silencing was partially responsible for the inhibition of endothelial cell migration. In addition, we have also shown that treatment of endothelial cells with phorbol 12-myristate 13-acetate resulted in the exportation of HDAC7 out of the nucleus through a protein kinase C/protein kinase D activation pathway and induced, similarly to HDAC7 silencing, an increase in PDGF-B expression, as well as a partial inhibition of endothelial cell migration. Collectively, these data identified HDAC7 as a key modulator of endothelial cell migration and hence angiogenesis, at least in part, by regulating PDGF-B/PDGFR-beta gene expression. Because angiogenesis is required for tumor progression, HDAC7 may represent a rational target for therapeutic intervention against cancer

Overexpression of CD9 in human breast cancer cells promotes the development of bone metastases.

BACKGROUND: Bone is a preferred target for circulating metastatic breast cancer cells. We found that the CD9 protein was up-regulated in the B02 osteotropic cell line, derived from the aggressive parental MDA-MB-231 breast cancer cell line. Here, we investigated the putative relationship between CD9 expression and the osteotropic phenotype. MATERIALS AND METHODS: Overexpression of CD9 was analyzed by immunoblotting in different cell lines. Immunohistochemistry was used to assess CD9 expression in primary tumors and metastatic lesions. In vivo experiments were conducted in mice using a monoclonal antibody against CD9. RESULTS: CD9 overexpression was confirmed in osteotropic cells. CD9 was significantly overexpressed in bone metastases versus primary tumors and visceral metastatic lesions. Finally, in vivo experiments showed that an antibody against CD9 delays homing of B02 cells in bone marrow, slowing down bone destruction. CONCLUSION: Our study reveals a potential implication of CD9 in the formation of bony metastases from breast cancer cells

Targeting osteopontin suppresses glioblastoma stem-like cell character and tumorigenicity in vivo

Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence

Dentin Matrix Protein 1 induces membrane expression of VE-cadherin on endothelial cells and inhibits VEGF-induced angiogenesis by blocking VEGFR-2 phosphorylation.

Dentin matrix protein 1 (DMP1) is a member of the Small Integrin-Binding LIgand N-linked Glycoproteins (SIBLINGs) family, a group of proteins initially described as mineralized extracellular matrices components. More recently, SIBLINGs have been implicated in several key steps of cancer progression, including angiogenesis. Although pro-angiogenic activities have been demonstrated for two SIBLINGs, the role of DMP1 in angiogenesis has not been addressed yet. We demonstrated that this extracellular matrix protein induced the expression of VE-cadherin, a key regulator of intercellular junctions and contact inhibition of growth of endothelial cells that is also known to modulate VEGFR-2 activity, the major high affinity receptor for VEGF. DMP1 induced VE-cadherin and p27(Kip1) expression followed by cell cycle arrest in human umbilical vein endothelial cells (HUVEC) in a CD44-dependent manner. VEGF-induced proliferation, migration and tubulogenesis responses were specifically blocked upon DMP1 pre-treatment of HUVEC. Indeed, subsequently to VE-cadherin induction, DMP1 inhibited VEGFR-2 phosphorylation and Src-mediated signaling. However, DMP1 did not interfere with bFGF-induced angiogenesis. In vivo, DMP1 significantly reduced laser-induced choroidal neovascularization lesions and tumor-associated angiogenesis. These data enable us to put DMP1 on the angiogenic chessboard for the first time and to identify this protein as a new specific inhibitor of VEGF-induced angiogenesis

Runx2-and histone deacetylase 3-mediated repression is relieved in differentiating human osteoblast cells to allow high bone sialoprotein expression

Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralization-competent phenotype by osteoblast cells