Background-We studied Dicer and Drosha, components of the RNA-interference machinery, in ovarian cancer.

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.

Constitutive activation of MEK-ERK signaling is often found in melanomas. Here, we identify a mechanism that links ERK with JNK signaling in human melanoma. Constitutively active ERK increases c-Jun transcription and stability, which are mediated by CREB and GSK3, respectively. Subsequently, c-Jun increases transcription of target genes, including RACK1, an adaptor protein that enables PKC to phosphorylate and enhance JNK activity, enforcing a feed-forward mechanism of the JNK-Jun pathway. Activated c-Jun is also responsible for elevated cyclin D1 expression, which is frequently overexpressed in human melanoma. Our data reveal that, in human melanoma, the rewired ERK signaling pathway upregulates JNK and activates the c-Jun oncogene and its downstream targets, including RACK1 and cyclin D1.

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