Abstract 2062: A quantitative proteomic investigation of microRNA-145 in breast cancer
Authors: Nicholas W. Bateman, Brian L. Hood, Mai Sun, Thomas P. Conrads
Journal: Cancer Research (2010)

Abstract MicroRNAs (miRNAs) represent an endogenous, post-transcriptional gene expression regulatory mechanism that is mediated by small, non-coding double-stranded RNAs (dsRNA) which target discrete messenger RNAs (mRNAs) resulting in their degradation or translational repression. Current computational methodologies predict that an average of 200 unique genes may be targeted by a single miRNA with the vast majority remaining, as yet, experimentally unvalidated. Furthermore, as miRNAs regulate their gene targets post-transcriptionally, the application of global, quantitative proteomic analysis to miRNA target discovery will yield a more comprehensive assessment of miRNA activity than gene expression analysis alone. Several miRNAs have been shown to be robustly downregulated in breast cancer, including microRNA-145 (miR-145). Recent investigations of miR-145 in breast cancer have revealed tumor suppressor activities for this miRNA and that loss of miR-145 expression may be associated with early stages of breast cancer pathogenesis. To elucidate targets of miR-145 and its role in breast cancer, we restored expression in the human breast cancer cell lines MDA-MB-231 and SKBR3 and conducted both phenotypic and global, quantitative protein expression analysis utilizing mass spectrometry-based proteomics. Phenotypic analyses indicated that MDA-MB-231 and SKBR3 cells stably expressing miR-145 exhibit a growth advantage relative to control cells. In support of this evidence, GO enrichment analysis of significantly differentially expressed proteins in miR-145-expressing MDA-MB-231 (MDA-145) cells revealed modulation of factors associated with cell cycle regulation, such as increased expression of the G2/M-phase regulatory factors anaphase promoting complex subunit 1 (ANAPC1) and cell division cycle 20 homolog (CDC20) expression and decreased expression of DnaJ (Hsp40) homolog (DNAJA3). Investigation of miR-145-specific effects via sequence analysis of 3’ untranslated regions derived from genes corresponding to proteins which were decreased in abundance in MDA-145 cells revealed 33 target candidates that contained a miR-145 “seed” motif. Of these 33 candidates, 21 have been previously predicted as miR-145 targets (miRNAMap). Target candidates are associated with cell cycle regulation and mitochondrial function and include integrin linked kinase (ILK), the cytokine growth factor chromosome 19 open reading frame 10 (C19ORF10) and microsomal glutathione S-transferase 1 (MGST1). Validation of target candidates and exploration of their role in the miR-145-mediated growth advantage phenotype are currently underway. In conclusion, these results indicate that restoration of stable miR-145 expression in breast cancer cells confers a growth advantage which is underscored by modulation of proteins associated with cell cycle regulation and mitochondrial activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2062.