Abstract LB-351: Comprehensive miRNA profiling of taxane-resistant human breast cancer variants using the SmartChip Real-Time PCR System
Authors: Duran George E. , Wang Cindy Y. , Francisco E. Brian , Wong Kee , Robinson Nicole , Sikic Branimir I. 
Published: July 2012

Abstract
Background: We derived taxane-resistant variants from four human breast cancer cell lines (MCF-7, BT-549, MDA-MB-231 and T-47D) by continuous exposure to docetaxel. Rt-PCR assays indicated that 4/4 of these variants were positive for the multidrug resistance gene MDR1 (ABCB1). In order to study non-MDR1 mechanisms of taxane resistance, we derived another set of taxane variants by co-selecting with docetaxel in the presence of the P-glycoprotein inhibitor, PSC 833 (2 μM). Rt-PCR assays indicate that all of these taxane variants are negative for MDR1, and BODIPY-labeled fluorescent paclitaxel and [3H]-docetaxel accumulation assays indicate that the resistance observed is unlikely to be due to transporter-mediated mechanisms. No mutations were found in the taxane cellular target, class I β-tubulin (TUBB), but we found elevated class III β-tubulin (TUBB3) in the majority of the non-MDR1 taxane variants by Q-RT-PCR and immunoblotting. Furthermore, we have identified reduced BRCA1 and elevated CDKN1A (p21) in these variants among other alterations by microarray profiling, and are pursuing the functional significance of key alterations using RNAi and gene transfection.
Objective of the Study: MiRNAs are post-transcriptional regulators of cell proliferation, tissue differentiation, embryonic development and apoptosis. The objective of this study is to profile the expression of miRNAs in these breast cancer variants relative to parental controls using the recently developed SmartChip Human MicroRNA Panel by WaferGen Biosystems, Inc. The miRNA expression profiles that identify taxane resistance in these cell models may be useful to predict chemoresistance in human breast cancer patients. Methodology: 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.
Preliminary Results: MiRNA profiling is complete and a preliminary analysis of these data revealed reduced miR-200 family expression, consistent with published findings that miR-200c directly targets and suppresses TUBB3. Furthermore, Significance Analysis of Microarrays (SAM) software identified reduced miR-635 and miR-296–3P in both MDR1(+) and MDR1(−) taxane variants relative to parental controls.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-351. doi:10.1158/1538-7445.AM2011-LB-351