| The stem cell self-renewal pathway is often hijacked and over-activated in cancer cells. The polycomb group protein BMI-1 is a known regulator of stemness and its over-expression is associated with a number of malignancies. In this study, we have examined the role of BMI-1 in the pathogenesis of Ewing's sarcoma family of tumors (ESFT). We hypothesize that deregulation of BMI-1-driven signaling pathway is central to the initiation and maintenance of this family group of aggressive pediatric tumor. To assess our hypothesis, we have performed gain and loss of function studies and provided evidence to support the role of BMI-1 in promoting anchorage-independent growth of ESFT in vitro and tumorigenicity in vivo independent of the genetic status of the cells. Contrary to early indications in stem cells and other caner cell types, BMI-1 promotes growth of ESFT cells independent of CDKN2A repression. To identify novel BMI-1 downstream targets involved in BMI-1 mediated tumorigenicity, we have conducted gene expression profiling on ESFT cells following BMI-1 knockdown. Significantly, we discover hundreds of putative BMI-1 targets including genes involved in cell adhesion, development and differentiation.;To further dissect the role of BMI-1 in influencing ESFT phenotype, we investigated the potential contribution of BMI-1 to other hallmarks of cancer, in particular, loss of cell contact inhibition, a fundamental transforming property of cancer cells. Using cells that stably express an shRNA against BMI-1, we have discovered that loss of BMI-1 expression restores contact inhibition to ESFT cells. Significantly, although proliferation of cells in log phase growth is unaffected by loss of BMI-1, at high cell density BMI-1 knockdown cells undergo cell cycle arrest and death. In contrast, control vector-transduced cells continue to enter cell cycle and proliferate, avoiding contact inhibition. Although many signaling pathways are involved in mediating the cell contact inhibition response, inactivation of the Yes-Associated Protein (YAP), a key downstream target of the Hippo pathway, has been implicated in both Drosophila and mammalian cells. Intriguingly, our data show that loss of BMI-1 expression results in loss of YAP protein at high cell density coincident with induction of contact inhibition. Further, silencing of YAP markedly decreases the tumorigenic potential of ESFT cells, implicating its role as a cooperative oncogene in ESFT. Together, our finding suggests that YAP is a novel downstream target of BMI-1 and that BMI-1-mediated stabilization of YAP renders cancer cells impervious to cell contact inhibition. Cancer cells escape cell contact inhibition to achieve unrestrained proliferation and enhanced invasive and metastatic properties. Our findings suggest that BMI-1 may be a key upstream mediator of this critical cancer-associated phenotype.;Finally, the cell of origin of ESFT remains elusive. We hypothesize that high levels of BMI-1 expression is an inherent feature of its cell of origin and its activity is required in cooperation with EWS-FLI1, a characteristic molecular feature of the majority of ESFT cells, for ESFT initiation. To this end, we have investigated the role of BMI-1 in EWS-FLI1-driven malignant transformation in primary human fibroblasts. Studies by Lessnick et al. have shown that EWS-FLI1 is toxic to primary human fibroblasts in a p53-dependent manner. Intriguingly, our study shows that co-expression of BMI-1 and EWS-FLI1 in primary human fibroblasts renders cells tolerant for EWS-FLI1 expression. Importantly, we find that EWS-FLI1 induces a senescent phenotype in MRC5 cells in the absence of BMI-1 over-expression, as evidenced by positive staining for senescence-associated-beta-galactosidase activity. Together, these data suggests that BMI-1 abrogates EWS-FLI1-induced oncogene-induced senescence (OIS). Studies are currently ongoing to dissect pathways involved in BMI-1-mediated protection against EWS-FLI1-induced OIS.;In an attempt to identify other cooperative oncogenes of EWS-FLI1 in addition to BMI-1 in primary human cells, we have found that hTERT-immortalized primary human fibroblasts tolerated EWS-FLI1 expression just as well as BMI-1-over-expressing primary human cells. This implicates a role of hTERT in EWS-FLI1-mediated malignant transformation. It will be interesting to determine if co-expression of BMI-1, hTERT, and EWS-FLI1 together will be sufficient to reprogram primary human fibroblasts into malignant ESFT-like cells. (Abstract shortened by UMI.)... |
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