Virginie Lamour

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-&bgr;) 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-&bgr; gene expression. Because angiogenesis is required for tumor progression, HDAC7 may represent a rational target for therapeutic intervention against cancer.

HDAC4 represses p21 WAF1/Cip1 expression in human cancer cells through a Sp1-dependent, p53-independent mechanism

Cancer cells have complex, unique characteristics that distinguish them from normal cells, such as increased growth rates and evasion of anti-proliferative signals. Global inhibition of class I and II histone deacetylases (HDACs) stops cancer cell proliferation in vitro and has proven effective against cancer in clinical trials, at least in part, through transcriptional reactivation of the p21WAF1/Cip1gene. The HDACs that regulate p21WAF1/Cip1 are not fully identified. Using small interfering RNAs, we found that HDAC4 participates in the repression of p21WAF1/Cip1 through Sp1/Sp3-, but not p53-binding sites. HDAC4 interacts with Sp1, binds and reduces histone H3 acetylation at the Sp1/Sp3 binding site-rich p21WAF1/Cip1 proximal promoter, suggesting a key role for Sp1 in HDAC4-mediated repression of p21WAF1/Cip1. Induction of p21WAF1/Cip1 mediated by silencing of HDAC4 arrested cancer cell growth in vitro and inhibited tumor growth in an in vivo human glioblastoma model. Thus, HDAC4 could be a useful target for new anti-cancer therapies based on selective inhibition of specific HDACs.

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.

Selective osteopontin knockdown exerts anti-tumoral activity in a human glioblastoma model.

Osteopontin (OPN), a member of the SIBLING (Small Integrin‐Binding LIgand N‐linked Glycoprotein) family, is overexpressed in human glioblastoma. Higher levels of OPN expression correlate with increased tumor grade and enhanced migratory capacity of tumor cells. Based on these observations, we explored the possibility that knocking down OPN expression in glioblastoma cells could exert an anti‐tumoral activity using an avian in vivo glioblastoma model that mimics closely human gliobastoma. Human U87‐MG glioma cells transfected with specific anti‐OPN small interfering RNAs (siRNAs) were grafted onto the chicken chorio‐allantoic membrane (CAM). OPN‐deficient U87‐MG cells gave rise to tumors that were significantly smaller than tumors formed from untransfected cells (paired t‐test, p < 0.05). Accordingly, the amount of proliferating cells in OPN‐deficient tumors showed a six‐fold reduction when compared to control tumors. However, OPN inhibition did not affect significantly tumor‐associated angiogenesis. In vitro, OPN‐silenced U87‐MG and U373‐MG cells showed decreased motility and migration. This is the first demonstration that OPN inhibition blocks glioma tumor growth, making this invasion‐related protein an attractive target for glioma therapy.

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 glycoprotein (SIBLING) 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 proangiogenic activities have been demonstrated for 2 SIBLINGs, the role of DMP1 in angiogenesis has not yet been addressed. We demonstrate that this extracellular matrix protein induced the expression of vascular endothelial cadherin (VE-cadherin), a key regulator of intercellular junctions and contact inhibition of growth of endothelial cells that is also known to modulate vascular endothelial growth factor receptor 2 (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 (HUVECs) in a CD44-dependent manner. VEGF-induced proliferation, migration, and tubulogenesis responses were specifically blocked on DMP1 pretreatment of HUVECs. Indeed, after VE-cadherin induction, DMP1 inhibited VEGFR-2 phosphorylation and Src-mediated signaling. However, DMP1 did not interfere with basic fibroblast growth factor-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.

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

Multiple myeloma (MM) is a plasma cell malignancy characterized by the accumulation of tumor cells in the bone marrow (BM) and is associated with immunosuppression, angiogenesis and osteolysis. Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature, immunosuppressive myeloid cells that promote tumor progression through different mechanisms. In this work, we studied the contribution of MDSC subsets to different disease-promoting aspects in MM. We observed an expansion of polymorphonuclear/granulocytic (PMN-)MDSCs in two immunocompetent murine MM models, while this was not observed for monocytic (MO-)MDSCs. Both MDSC subpopulations from MM-bearing mice were immunosuppressive, but PMN-MDSCs displayed a higher suppressive potential. Soluble factors secreted by MM cells increased the viability of MDSCs, whereas the presence of MDSCs did not affect the proliferation of MM cells in vitro or in vivo. Interestingly, we observed a pro-angiogenic effect of PMN-MDSCs in the context of MM using the chick chorioallantoic membrane assay. Consistently, MM-derived PMN-MDSCs showed an up-regulation of angiogenesis-related factors and reduced PMN-MDSC levels were associated with less angiogenesis in vivo. Finally, we identified MO-MDSCs as osteoclast precursors. These results suggest that MDSC subpopulations play diverging roles in MM. We show for the first time that PMN-MDSCs exert a pro-angiogenic role in MM.

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.