Integrative genomics and proteomics analysis reveals engagement of cooperating oncogenic pathways driven by the Polycomb Group (PcG) proteins BMI1 and Ezh2: Evidence for activation of the PcG chromatin silencing network in human prostate carcinoma metastasis precursor cells

Abstract

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The Ezh2 and BMI1 proteins are members of Polycomb group protein (PcG) chromatin silencing complexes conferring genome scale transcriptional repression via covalent modification of histones. Here we demonstrate that an apparent functional co-activation of the BMI1- and Ezh2-associated pathways is one of the defining genotypic and phenotypic features of circulating human prostate carcinoma metastasis precursor (MP) cells isolated from blood of nude mice bearing orthotopic metastatic human prostate carcinoma (PC) xenografts. A quantitative immunofluorescence co-localization analysis revealed that MP cells are markedly enriched for dual-positive PC cells over-expressing both BMI1 and Ezh2 proteins compared to the parental PC cells comprising a bulk of primary tumors. Nuclear accumulation of histones H3metK27 and UbiH2A confirmed activated states of the PRC1 and PRC2 chromatin repressive complexes in the MP cells. Increased BMI1 and Ezh2 expression was corroborated by microarray and Q-RT-PCR gene expression analyses indicating significantly higher mRNA expression levels of both BMI1 and Ezh2 in MP cells. Importantly, DNA copy number analysis revealed a high-level gene amplification of both BMI1 and Ezh2 genes in MP cells, highly metastatic human PC variants established from lymph node metastases, as well as bone marrow-resident human PC cells. Applying mouse/human comparative translational genomics approach, we identified the 11-gene BMI1-pathway signature, which consistently display a stem cell-like expression profile in distant metastatic lesions as revealed by the analysis of 9 metastatic and 23 primary prostate tumors from patients. Gene silencing experiments using BMI1 siRNA confirmed that expression of 8 of 11 signature genes is BMI1-dependent in MP cells. Ezh2 siRNA-mediated silencing modified expression of 2 of 3 remaining signature genes. MP cells manifest increased resistance to anoikis, suggesting selection for enhanced survival in detached conditions during metastatic dissemination in vivo. Using functional genomics approaches based on siRNA-mediated gene silencing, we demonstrated that increased expression of the BMI1 and Ezh2 contributes to the anoikis resistance in vitro and is essential for metastasis in vivo of MP cells. Tissue microarray (TMA) analysis of clinical samples demonstrated a concomitant over-expression of the BMI1 and Ezh2 proteins in a majority (∼ 85%) of prostate carcinomas. Our data indicate co-engagement in MP cells of seemingly functionally complementary oncogenic pathways associated with activation of the BMI1 and Ezh2 genes, suggesting an important role of chromatin silencing network in defining genomic pathology of metastatic human prostate carcinoma cells.