Activation of the heregulin/HER2 pathway in oestrogen receptor (ER)-positive breast-cancer cells leads to suppression of oestrogen-receptor element (ERE)-driven transcription and disruption of oestradiol responsiveness, and thus contributes to progression of tumours to more invasive phenotypes. Here we report the identification of metastatic-associated protein 1 (MTA1), a component of histone deacetylase (HDAC) and nucleosome-remodelling complexes, as a gene product induced by heregulin-beta1 (HRG). Stimulation of cells with HRG is accompanied by suppression of histone acetylation and enhancement of deacetylase activity. MTA1 is also a potent corepressor of ERE transcription, as it blocks the ability of oestradiol to stimulate ER-mediated transcription. The histone-deacetylase inhibitor trichostatin A blocks MTA1-mediated repression of ERE transcription. Furthermore, MTA1 directly interacts with histone deacetylase-1 and -2 and with the activation domain of ER-alpha. Overexpression of MTA1 in breast-cancer cells is accompanied by enhancement of the ability of cells to invade and to grow in an anchorage-independent manner. HRG also promotes interaction of MTA1 with endogenous ER and association of MTA1 or HDAC with ERE-responsive target-gene promoters in vivo. These results identify ER-mediated transcription as a nuclear target of MTA1 and indicate that HDAC complexes associated with the MTA1 corepressor may mediate ER transcriptional repression by HRG.

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.

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.

Epithelial-to-mesenchymal transition (EMT) is a process that plays essential roles in development and wound healing that is characterized by loss of homotypic adhesion and cell polarity and increased invasion and migration. At the molecular level, EMT is characterized by loss of E-cadherin and increased expression of several transcriptional repressors of E-cadherin expression (Zeb-1, Zeb-2, Twist, Snail, and Slug). Early work established that loss of E-cadherin and increased expression of MMP-9 was associated with a poor clinical outcome in patients with urothelial tumors, suggesting that EMT might also be associated with bladder cancer progression and metastasis. More recently, we have used global gene expression profiling to characterize the molecular heterogeneity in human urothelial cancer cell lines (n=20) and primary patient tumors, and unsupervised clustering analyses revealed that the cells naturally segregate into two discrete “epithelial” and “mes-enchymal” subsets, the latter consisting entirely of muscle-invasive tumors. Importantly, sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) or type-3 fibroblast growth factor receptor (FGFR3) was confined to the “epithelial” subset, and sensitivity to EGFR inhibitors could be reestablished by micro-RNA-mediated molecular reversal of EMT. The results suggest that EMT coordinately regulates drug resistance and muscle invasion/metastasis in urothelial cancer and is a dominant feature of overall cancer biology.

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