Abstract 1095: Comprehensive miRNA profiling of taxane-resistant human breast and ovarian cancer cell lines using the SmartChip real-time PCR system

Abstract

MiRNAs are post-transcriptional regulators of cell proliferation, tissue differentiation, embryonic development and apoptosis. We profiled the expression of miRNAs in a panel of taxane-resistant human breast and ovarian cancer cell lines using the recently developed WaferGen SmartChip System. Four breast cancer cell lines (MCF-7, BT-549, MDA-MB-231 and T-47D) and four ovarian cancer cell lines (1A9/A2780, ES-2, MES-OV and OVCAR-3) were selected for resistance to either docetaxel or paclitaxel alone or co-selected with taxane in the presence of the P-glycoprotein inhibitor, PSC-833 (valspodar, 2 µM). All of the variants established by exposure to taxanes alone are MDR1/ABCB1(+), whereas the resistance observed in the co-selected variants is not transporter-mediated. We have previously reported elevated class III β-tubulin (TUBB3) content, reduced BRCA1 and elevated CDKN1A (p21) in the majority of the non-MDR1 taxane variants relative to parental controls. For miRNA profiling, total RNA was analyzed using the SmartChip Human MicroRNA Panel version 2.0 containing 1,150 unique real-time PCR reactions in quadruplicate for a total of ∼4,600 reactions per sample. Following ligation to a miRNA cloning linker (Integrated DNA Technology, Coraville, IA), the equivalent of 500 ng of starting RNA per sample was added to a one-step cDNA and real-time PCR cocktail (Applied Biosystems, Foster City, CA). The total volume per reaction was 100 nL containing an equivalent of 96 pg of RNA. Forty cycles of real-time PCR were performed on the SmartChip Cycler and a quality screen was performed to remove any outlier data. MiRNA profiling is complete and an analysis of these data revealed a clear separation between the two sets of non-MDR1 variants based on tumor type (breast vs. ovarian), and selection conditions (non-MDR1 vs. parental). We observed reduced miR-200 family expression in the majority of these taxane-resistant variants, consistent with published findings that miR-200c directly targets and suppresses TUBB3. Furthermore, Significance Analysis of Microarrays (SAM) software identified reduced miR-635, miR-296-3P, and let-7B in both MDR1(+) and MDR1(-) variants of breast origin, and elevated let-7 family (let-7B, let-7F and let-7I) and reduced miR-1225-5P and miR-4286 in the ovarian cell lines relative to parental controls. The functional significance of these observations will be studied by introducing miRNA inhibitors or mimics specific to key alterations with the goal of sensitizing cells to taxanes. 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 1095. doi:1538-7445.AM2012-1095