Inducible microRNA-200c decreases motility of breast cancer cells and reduces filamin A

Cancer progression and metastases are frequently related to changes of cell motility. Amongst others, the microRNA-200c (miR-200c) was shown to maintain the epithelial state of cells and to hamper migration. Here, we describe two miR-200c inducible breast cancer cell lines, derived from miR-200c knock-out MCF7 cells as well as from the miR-200c-negative MDA-MB-231 cells and report on the emerging phenotypic effects after miR-200s induction. The induction of miR-200c expression seems to effect a rapid reduction of cell motility, as determined by 1D microlane migration assays. Sustained expression of miR200c leads to a changed morphology and reveals a novel mechanism by which miR- 200c interferes with cytoskeletal components. We find that filamin A expression is attenuated by miRNA-200c induced downregulation of the transcription factors c-Jun and MRTF/SRF. This potentially novel pathway that is independent of the prominent ZEB axis could lead to a broader understanding of the role that miR200c plays in cancer metastasis

A proteomic analysis of an in vitro knock-out of miR-200c

Loss of miR-200c is correlated to advanced cancer-subtypes due to increased EMT and decreased treatment efficacy by chemotherapeutics. As miRNAs regulate a multitude of targets, the analysis of differentially expressed proteins upon a genomic knock-out (KO) is of interest. In this study, we generated a TALENs KO of miR-200c in MCF7 breast cancer cells, excluded its compensation by family-members and evaluated the impact on the proteome by analyzing three individual KO-clones. We identified 26 key proteins and a variety of enrichments in metabolic and cytoskeletal pathways. In six of these targets (AGR2, FLNA/B, ALDH7A1, SCIN, GSTM3) the differential expression was additionally detected at mRNA level. Together, these alterations in protein abundance accounted for the observed biological phenotypes, i.e. increased migration and chemoresistance and altered metabolism, found in the miR-200c-KO clones. These findings provide novel insights into miR-200c and pave the way for further studies

Combating Drug Resistance by Exploiting miRNA-200c-Controlled Phase II Detoxification

Acquired drug resistance constitutes a serious obstacle to the successful therapy of cancer. In the process of therapy resistance, microRNAs can play important roles. In order to combat resistance formation and to improve the efficacy of chemotherapeutics, the mechanisms of the multifaceted hsa-miR-200c on drug resistance were elucidated. Upon knockout of hsa-miR-200c in breast carcinoma cells, a proteomic approach identified altered expression of glutathione S-transferases (GSTs) when cells were treated with the chemotherapeutic drug doxorubicin. In different hsa-miR-200c expression systems, such as knockout, inducible sponge and inducible overexpression, the differential expression of all members of the GST family was evaluated. Expression of hsa-miR-200c in cancer cells led to the repression of a multitude of these GSTs and as consequence, enhanced drug-induced tumor cell death which was evaluated for two chemotherapeutic drugs. Additionally, the influence of hsa-miR-200c on the glutathione pathway, which is part of the phase II detoxification mechanism, was investigated. Finally, the long-term effects of hsa-miR-200c on drug efficacy were studied in vitro and in vivo. Upon doxycycline induction of hsa-miR-200c, MDA-MB 231 xenograft mouse models revealed a strongly reduced tumor growth and an enhanced treatment response to doxorubicin. A combined treatment of these tumors with hsa-miR-200c and doxorubicin resulted in complete regression of the tumor in 60% of the animals. These results identify hsa-miR-200c as an important player regulating the cellular phase II detoxification, thus sensitizing cancer cells not expressing this microRNA to chemotherapeutics and reversing drug resistance through suppression of GSTs