miR-200 Expression Regulates Epithelial-to-Mesenchymal Transition in Bladder Cancer Cells and Reverses Resistance to Epidermal Growth Factor Receptor Therapy

Abstract

Purpose The epithelial to mesenchyme transition (EMT) is a cell development-regulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNAs) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFR therapy. Experimental Design miRNA array screening and real-time reverse transcription-polymerase chain reaction were used to identify and validate the differential expression of miRNAs involved in EMT in 9 bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and c were expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation as well as intracellular distribution of EGFR and ERRFI-1 were validated through western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutanty 3′UTR/ERRFI-1/Luciferse reporters. Results We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFR inhibitors-induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB-1, ZEB-2, ERRFI-1, and cell migration, and increased sensitivity to EGFR blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3′UTR/ERRFI-1/Luc and miR-200 co-transfections demonstrated that the direct down-regulation of ERRFI-1 was miR-200-dependent since mutations in the two putative miR-200-binding sites have rescued the inhibitory effect. Conclusions Members of the miR-200 family appear to control the EMT process and sensitivity to EGFR therapy, in bladder cancer cells and that expression of miR-200 is sufficient to restore EGFR dependency, at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.

miR-24-3p regulates bladder cancer cell proliferation, migration, invasion and autophagy by targeting DEDD

“…A handful of studies has found that various miRNAs are involved in the development of bladder cancer (31). For example, miR-126 and miR-182 are related to urinary bladder cancer (12); miR125b inhibits the development of bladder cancer through E2F3 (32); miR-143 serves as a tumor inhibitor of bladder cancer (13); miR-200 regulates EMT in bladder cancer cells and reverses the resistance to epidermal growth factor (EGF) receptor (33).…”

miR-200bc/429 cluster targets PLCγ1 and differentially regulates proliferation and EGF-driven invasion than miR-200a/141 in breast cancer

“…A strong increase in the phosphorylation of Cdc25C at the Ser-216 site, which prevents Cdc25C dual-specificity phosphatase from dephosphorylating and activating cyclin B1/Cdc2 (Peng et al, 1997) There is increasing evidence that EMT may contribute to the resistance to different therapies in cancer (Thomson et al, 2005;Yauch et al, 2005;Arumugam et al, 2009). The miR-200 family, which regulates EMT, has also been reported in the sensitization of cancer cells to different drugs, for example, to the EGFR-targeting molecule gefitinib in bladder carcinoma (Adam et al, 2009) or to paclitaxel in breast, ovarian and endometrial cancers (Cochrane et al, 2010). A current focus in drug discovery is to develop agents that target the cell cycle checkpoints, aiming at increasing or decreasing the amount of arrested cells at these points (reviewed in DiPaola, 2002).…”

Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity

“…Their silencing confers resistance to EGFR in mesenchymal bladder cancer cells that can be reversed by the expression of miR-200 163 .…”