Abstract 139: The role of miRNAs in cyclooxygenase-2-mediated breast cancer progression

Abstract

Abstract We had shown that over-expression of cyclo-oxygenase (COX)-2 in human, as well as murine breast cancer cells, promotes tumor progression and metastasis by multiple mechanisms: host immune cell inactivation and a stimulation of cancer cell migration, invasion, tumor-associated angiogenesis and lymphangiogenesis, which support blood-borne and lymph-borne metastasis. Most of these events resulted from activation of the prostanoid receptor EP4 by endogenous PGE2. Recently, by stable transfection of COX-2 cDNA into a non-metastatic, COX-2 negative human breast cancer cell line MCF-7, we showed that COX-2 induces all the phenotypic properties of stem-like or “tumor initiating cells” (TIC) in MCF-7-COX-2 cells, as defined by in vitro studies and validated in vivo. Through combined gene expression and microRNA (miRNA) micro array analysis, we identified two miRNAs (miR-526b and miR-655) that are up-regulated in MCF-7-COX-2 cells, associated with a down-regulation of 14 target genes linked with tumor-suppressor functions. We hypothesize that these miRNAs are important for COX-2 mediated TIC associated functions in human breast cancer. As a first step, we validated their expression in several COX-2 disparate human breast cancer cell lines: MCF-7 (COX-2 negative), MCF-7-COX-2, SKBR-3 (HER-2 over-expressing but COX-2 negative) and SKBR-3-COX-2 (COX-2 over-expressing following stable transfection of COX-2). The expression levels of miR-655 were strongly correlated with COX-2 mRNA (quantified RT-PCR) expression in these cell lines. Furthermore, the migratory and invasive capacities of the cell lines went hand in hand with miR-655 expression. Expression of miR-655 was markedly inhibited by treating MCF-COX-2 cells with a COX-2 inhibitor NS398 or an EP4 antagonist ONO-AE3-208, indicating that the expression depends on both COX-2 and EP4 activity. Finally, we discovered that cells derived from tumorspheres (in vitro correlate of TIC growth) produced by various human breast cancer cell lines (Hs578T, T47D, MDA-MB-231, SKBR-3, SKBR-3-COX-2, and MCF-7-COX-2) grown on low attachment plates, exhibited a dramatic increase in COX-2 expression in comparison to the cells grown as monolayer. We also found that the tumorspheres over-express miR-655. These findings, taken together, fortify the notion that COX-2, EP4 and COX-2 induced miR-655 expression, play important roles in promoting and maintaining the TIC phenotype in breast cancer cells. In support, COX-2 inhibitors and EP4 antagonists were highly effective in abrogating tumor growth, tumor- associated angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in our mouse breast cancer model. Currently we are testing whether EP4 and miR-655 serve as useful prognostic markers and therapeutic targets in human breast cancer. (Supported by the CBCF, Ontario chapter and the OICR funds to PKL. LD is a TBCRU scholar.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 139. doi:1538-7445.AM2012-139