Prostate cancer (PCa)-related deaths are caused by the emergence of castrate-resistant prostate cancer (CRPC) and subsequent metastasis, suggesting the need for better mechanistic understanding of tumor aggressiveness in order to advance the development of novel therapies. Emerging evidence suggests that acquisition of the epithelial-to-mesenchymal transition (EMT), a process that resembles the genesis of cancer stem-like cells, contributes to tumor aggressiveness and is mediated by deregulated expression of microRNAs (miRNAs), such as miR-200 and let-7 family. Loss of miR-200 expression results in the over-expression of Lin28B, which is prevalent in human PCa. Lin28B is also known to block the processing of another miRNA (pre-let-7 and pri-let-7), resulting in decreased mature let-7, thereby leads to increased Suz12 and EZH2 expression, which are important components of the polycomb repressive complex 2 (PRC2). Thus, over- expression of Lin28B and loss of miR-200 and let-7 appear to be responsible for PCa aggressiveness. We found over-expression of Lin28B in PDGF-D-over-expressing PCa cells with the EMT phenotype (PC3 PDGF- D cells) concomitant with decreased expression of miR-200 and let-7 family and increased expression of Suz12 and EZH2, which is consistent with findings obtained from human PCa tissue specimens. Moreover, we found differential expression of these markers between African-American and Caucasian-American patients. We also found that the re-expression of miR-200b, miR-200c, and let-7 either by a genetic approach or by treating cells with our newly developed agent (3,4-difluorobenzo-curcumin or CDF) down-regulated the expression of Lin28B and EZH2. Based on our preliminary results, we hypothesize that over-expression of Lin28B leads to the acquisition of invasive and metastatic characteristics in PCa cells (EMT-phenotype cells) via down-regulation of miR-200b and miR-200c, resulting in increased expression of Suz12, ZEB1, and ZEB2. We also hypothesize that over-expression of Lin28B represses the maturation of let-7 family, leading to increased expression of EZH2, and these processes can be attenuated by treatment of cells with CDF. We will test our hypotheses by three specific aims.
Aim -1: Establish the mechanism(s) by which Lin28B regulates miRNAs and causes tumor cell aggressiveness (i.e., increased cell proliferation, migration, invasion, clonogenic growth, and self-renewal capacity).
Aim 2 : Determine whether CDF-mediated inhibition of tumor cell growth is due to deregulation of Lin28B, miR-200, let-7, Suz12, and EZH2 expression both in vitro and in an animal model in vivo.
Aim -3: Assess the relevance of Lin28B, EZH2, miR-200, and let-7 expression for characterizing tumor aggressiveness in human PCa tissue specimens in African-American compared to Caucasian-American patients. Our pre-clinical mechanistic and PCa-tissue-based research will provide insights into the roles of miRNAs and their targets in characterizing PCa aggressiveness, and will also provide a novel approach (i.e., use of CDF) toward designing targeted therapy for PCa patients.
The overall objective of this application is to test the hypotheses: (i) that over-expression of Lin28B leads to the acquisition of the invasive and metastatic characteristics of prostate cancer (PCa) cells (EMT-phenotype cells) via down-regulation of miR-200b and miR-200c, resulting in increased expression of Suz12, ZEB1, and ZEB2; and (ii) that over-expression of Lin28B represses the maturation of let-7 family, leading to increased expression of EZH2, and (iii) that these processes can be attenuated by our novel synthetic analogue of curcumin [(3, 4- difluoro-benzo-curcumin); in short difluorinated-curcumin or CDF, a synthetically derived compound with greater bioavailability than curcumin]. Moreover, the aim of the study is to investigate the relevance of deregulated expression of Lin28B, EZH2, miR-200, and let-7 in archival human PCa tissue specimens to assess their roles in tumor aggressiveness in African American versus Caucasian-American patients.
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