Abstract
Abstract We established that elevated COX-2 expression by human and murine breast cancer cells promotes tumor progression and metastasis via multiple mechanisms primarily due to activation of the PGE-2 receptor EP4. COX-2 and HER-2, often co-expressed in human breast cancer, are both major determinants of cancer progression. Many of the HER-2 actions were shown to be COX-2 dependent, but the roles of COX-2 in the absence or presence of HER-2 in breast cancer initiation and sustenance remain unclear. To define these roles, we examined the consequences of introducing COX-2 gene into COX-2-ve, HER-2-ve, ER+ve, non-metastatic MCF-7 and COX-2-ve, ER-ve, HER-2-over-expressing SK-BR-3 human breast cancer cell lines. After stable integration of COX-2 cDNA, these two cell lines were named MCF-7-COX-2 and SKBR3-COX-2. When compared with their empty vector-transfected counterparts, they showed (1) epithelial-mesenchymal transition (down regulation of E Cadherin and up regulation Vimentin by qRT-PCR), (2) higher proliferative activity (BrdU uptake), (3) higher migratory and invasive abilities across microporous membranes (8μM pore size) in Boyden chambers, (4) upregulation of EP4 and angiogenic and lymphangiogenic factors VEGF-A, C and D (qRT PCR and western blot), (5) markedly increased stem like cell populations in vitro (spheroid forming ability of single cells in ultra-low attachment plates for successive generations) and an increase in ALDH activity (flow cytometry). (6) Above changes could be abrogated with specific COX-2 inhibitor NS-398 (Pfizer) or an EP4 antagonist ONO-AE3-208 (ONO Pharmaceuticals, Japan) at non toxic concentrations, indicating that all these functions are dependent on COX-2 and EP4 activity. (7) Intravenous injection of MCF-7-COX2 cells into immuno-deficient mice revealed a dramatic increase in their lung colony forming capacity at 4-6 weeks, as compared to empty vector-transfected cells. (8) Using differential gene and micro-RNA (miRNA) arrays we identified two miRNAs upregulated and their target fourteen tumor-suppressor like genes down-regulated by introducing COX-2 into MCF-7 cells. (9) We observed a positive co-relation between the miRNA and COX-2 expression in multiple COX-2 disparate cell lines, in which expression of miRNAs was inhibited by treatment with COX-2 inhibitors or EP4 antagonists. Current studies are testing the functions of the miRNAs by knockdown and knock-in studies, and utilizing human breast cancer tissues to explore the clinical relevance of these findings. Our model system allowing identification and characterization of COX-2 induced stem like cells in breast cancer should help defining COX-2 and EP4 mediated pathways controlling their biology and utilization of the novel miRNA markers for prognostic and therapeutic applications in the clinic. (Supported by the CBCF, Ontario Chapter and the OICR funds from the Government of Ontario to PKL). 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 3324. doi:1538-7445.AM2012-3324
Published Version
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