Stéphane Gobeil

A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene

Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.

MicroRNA-196a promotes an oncogenic effect in head and neck cancer cells by suppressing annexin A1 and enhancing radioresistance

Radiotherapy is a major treatment modality for head and neck squamous cell carcinoma (HNSCC). Up to 50% of patients with locally advanced disease relapse after radical treatment and there is therefore a need to develop predictive bomarkers for clinical use that allow the selection of patients who are likely to respond. MicroRNA (miRNA) expression profiling of a panel of HNSCC tumours with and without recurrent disease after surgery and radiotherapy detected miR‐196a as one of the highest upregulated miRNAs in the poor prognostic group. To further study the role of miR‐196a, its expression was determined in eight head and neck cancer cell lines. Overexpression of miR‐196a in HNSCC cells, with low endogenous miR‐196a expression, significantly increased cell proliferation, migration and invasion, and induced epithelial to mesenchymal transition. Conversely, miR‐196a knockdown in cells with high endogenous expression levels significantly reduced oncogenic behaviour. Importantly, overexpression of miR‐196a increased radioresistance of cells as measured by gamma H2AX staining and MTT survival assay. Annexin A1 (ANXA1), a known target of miR‐196a, was found to be directly modulated by miR‐196a as measured by luciferase assay and confirmed by Western blot analysis. ANXA1 knockdown in HNSCC exhibited similar phenotypic effects to miR‐196a overexpression, suggesting the oncogenic effect of miR‐196a may at least be partly regulated through suppression of ANXA1. In conclusion, this study identifies miR‐196a as a potential important biomarker of prognosis and response of HNSCC to radiotherapy. Furthermore, our data suggest that miR‐196a and/or its target gene ANXA1 could represent important therapeutic targets in HNSCC.

The RAD51 paralogs ensure cellular protection against mitotic defects and aneuploidy.

Summary The interplay between homologous DNA recombination and mitotic progression is poorly understood. The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) are key enzymes for DNA double-strand break repair. In our search for specific functions of the various RAD51 paralogs, we found that inhibition of XRCC3 elicits checkpoint defects, while inhibition of RAD51B or RAD51C induces G2/M cell cycle arrest in HeLa cells. Using live-cell microscopy we show that in XRCC3-knockdown cells the spindle assembly checkpoint persists and there is a higher frequency of chromosome misalignments, anaphase bridges, and aneuploidy. We observed centrosome defects in the absence of XRCC3. While RAD51B and RAD51C act early in homologous recombination, XRCC3 functions jointly with GEN1 later in the pathway at the stage of Holliday junction resolution. Our data demonstrate that Holliday junction resolution has critical functions for preventing aberrant mitosis and aneuploidy in mitotic cells.

BCAT1 expression associates with ovarian cancer progression: possible implications in altered disease metabolism

Previously, we have identified the branched chain amino-acid transaminase 1 (BCAT1) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that BCAT1 is strongly overexpressed in both LMP and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Knockdown of the BCAT1 expression in epithelial ovarian cancer (EOC) cells led to sharp decrease of cell proliferation, migration and invasion and inhibited cell cycle progression. BCAT1 silencing was associated with the suppression of numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, and the induction of some tumor suppressor genes (TSGs). Moreover, BCAT1 suppression resulted in downregulation of numerous genes implicated in lipid production and protein synthesis, suggesting its important role in controlling EOC metabolism. Further metabolomic analyses were indicative for significant depletion of most amino acids and different phospho- and sphingolipids following BCAT1 knockdown. Finally, BCAT1 suppression led to significantly prolonged survival time in xenograft model of advanced peritoneal EOC. Taken together, our findings provide new insights about the functional role of BCAT1 in ovarian carcinogenesis and identify this transaminase as a novel EOC biomarker and putative EOC therapeutic target.

Inhibition of RUNX2 Transcriptional Activity Blocks the Proliferation, Migration and Invasion of Epithelial Ovarian Carcinoma Cells

Previously, we have identified the RUNX2 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from serous epithelial ovarian cancer (EOC) patients, when compared to primary cultures derived from matched primary (prior to CT) tumors. However, we found no differences in the RUNX2 methylation in primary EOC tumors and EOC omental metastases, suggesting that DNA methylation-based epigenetic mechanisms have no impact on RUNX2 expression in advanced (metastatic) stage of the disease. Moreover, RUNX2 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. Knockdown of the RUNX2 expression in EOC cells led to a sharp decrease of cell proliferation and significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as various genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX2 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the RUNX2 gene in serous EOC progression and suggest that RUNX2 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX2 and other members of the RUNX gene family in ovarian tumorigenesis.

Chapter 12 Identification and analysis of caspase substrates: Proteolytic Cleavage of poly(ADP-rib ose)polymerase and DNA fragmentation factor 45

Publisher Summary This chapter details specific techniques that identify caspase substrates. Historically, random screening for in vivo and in vitro cleavage of proteins known or suspected to be implicated in apoptosis was routinely used. Sequencing of proteins processed during apoptosis was used to identify putative caspase cleavage sites. The chapter discusses the recently targeted assays for caspase substrates employed, such as the yeast two- and three-hybrid system to screen for new caspase substrates. The chapter focuses on protocols that are used to characterize the cleavage of poly (ADP-ribose)polymerase (PARP-1) during apoptosis. PARP- 1 is a nuclear enzyme that catalyzes the transfer of ADP-ribose polymers onto itself and other nuclear proteins in response to DNA strand breaks. PARP- 1 is a protein of 113 kDa and cleaved to produce fragments of 89 and 24 kDa in a wide variety of cells undergoing apoptosis and as a result, this cleavage pattern has become a hallmark of apoptosis. The chapter also describes the techniques used to determine the cleavage of DNA fragmentation factor 45 (DFF45/ICAD).

The mannose receptor LY75 (DEC205/CD205) modulates cellular phenotype and metastatic potential of ovarian cancer cells

The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. Previously, we identified the mannose receptor LY75 gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. LY75 represents endocytic receptor expressed on dendritic cells and so far, has been primarily studied for its role in antigen processing and presentation. Here we demonstrate that LY75 is overexpressed in advanced EOC and that LY75 suppression induces mesenchymal-to-epithelial transition (MET) in EOC cell lines with mesenchymal morphology (SKOV3 and TOV112), accompanied by reduction of their migratory and invasive capacity in vitro and enhanced tumor cell colonization and metastatic growth in vivo. LY75 knockdown in SKOV3 cells also resulted in predominant upregulation of functional pathways implicated in cell proliferation and metabolism, while pathways associated with cell signaling and adhesion, complement activation and immune response were mostly suppressed. Moreover, LY75 suppression had an opposite effect on EOC cell lines with epithelial phenotype (A2780s and OV2008), by directing epithelial-to-mesenchymal transition (EMT) associated with reduced capacity for in vivo EOC cell colonization, as similar/identical signaling pathways were reversely regulated, when compared to mesenchymal LY75 knockdown EOC cells. To our knowledge, this is the first report of a gene displaying such pleiotropic effects in sustaining the cellular phenotype of EOC cells and points to novel functions of this receptor in modulating EOC dissemination. Our data also support previous findings regarding the superior capacity of epithelial cancer cells in metastatic colonization of distant sites, compared to cancer cells with mesenchymal-like morphology.

Amplification of Adipogenic Commitment by VSTM2A

SUMMARY Despite progress in our comprehension of the mechanisms regulating adipose tissue development, the nature of the factors that functionally characterize adipose precursors is still elusive. Defining the early steps regulating adipocyte development is needed for the generation of tools to control adipose tissue size and function. Here, we report the discovery of V-set and transmembrane domain containing 2A (VSTM2A) as a protein expressed and secreted by committed preadipocytes. VSTM2A expression is elevated in the early phases of adipogenesis in vitro and adipose tissue development in vivo. We show that VSTM2A-producing cells associate with the vasculature and express the common surface markers of adipocyte progenitors. Overexpression of VSTM2A induces adipogenesis, whereas its depletion impairs this process. VSTM2A controls preadipocyte determination at least in part by modulating BMP signaling and PPARγ2 activation. We propose a model in which VSTM2A is produced to preserve and amplify the adipogenic capability of adipose precursors.

Suppression of the grainyhead transcription factor 2 gene (GRHL2) inhibits the proliferation, migration, invasion and mediates cell cycle arrest of ovarian cancer cells

ABSTRACT Previously, we have identified the Grainyhead transcription factor 2 gene (GRHL2) as notably hypomethylated in high-grade (HG) serous epithelial ovarian tumors, compared with normal ovarian tissues. GRHL2 is known for its functions in normal tissue development and wound healing. In the context of cancer, the role of GRHL2 is still ambiguous as both tumorigenic and tumor suppressive functions have been reported for this gene, although a role of GRHL2 in maintaining the epithelial status of cancer cells has been suggested. In this study, we report that GRHL2 is strongly overexpressed in both low malignant potential (LMP) and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Suppression of the GRHL2 expression led to a sharp decrease in cell proliferation, migration and invasion and induced G1 cell cycle arrest in epithelial ovarian cancer (EOC) cells displaying either epithelial (A2780s) or mesenchymal (SKOV3) phenotypes. However, no phenotypic alterations were observed in these EOC cell lines following GRHL2 silencing. Gene expression profiling and consecutive canonical pathway and network analyses confirmed these data, as in both these EOC cell lines, GRHL2 ablation was associated with the downregulation of various genes and pathways implicated in cell growth and proliferation, cell cycle control and cellular metabolism. Taken together, our data are indicative for a strong oncogenic potential of the GRHL2 gene in EOC progression and support recent findings on the role of GRHL2 as one of the major phenotypic stability factors (PSFs) that stabilize the highly aggressive/metastatic hybrid epithelial/mesenchymal (E/M) phenotype of cancer cells.

The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L

Epithelial cells that lose attachment to the extracellular matrix undergo a specialized form of apoptosis called anoikis. Here, using large-scale RNA interference (RNAi) screening, we find that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase, plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. We find that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis we show that knockdown of Kdm3a enhances metastatic potential. Finally, we find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, our results reveal a novel transcriptional regulatory program that mediates anoikis. DOI: http://dx.doi.org/10.7554/eLife.16844.001