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.

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.

Downregulation of GRK5 hampers the migration of breast cancer cells.

Sunitinib is a multispecific kinase inhibitor and one of its targets is the kinase GRK5, which is regulating a multitude of G protein-coupled receptors (GPCRs). In this study we demonstrate that a decreased GRK5 expression induced by knock-down experiments or sunitinib treatment hampers the migration of cancer cell lines. A proteomic analysis revealed many pathways related to cell migration which were down regulated upon the GRK5 knock-down. Furthermore, we found in MDA-MB-231 breast cancer cells that the inhibition of migration is mediated by the GPCR gastrin releasing peptide receptor (GRPR) leading to a reduced expression of migration regulating downstream targets like CDC42 and ROCK1. An in silico Kaplan Meier analysis revealed that GRK5 and GRPR overexpression reduces the distant metastasis free survival in triple-negative breast cancer (TNBC) patients. Thus, we suggest a novel anti-migratory effect of impaired GRK5 expression which induces a negative feedback loop on GRPR signalling.

MiRNA-27a sensitizes breast cancer cells to treatment with Selective Estrogen Receptor Modulators.

BACKGROUND: MicroRNA-27a (miR-27a) is a small non-coding RNA, shown to play a role in multiple cancers, including the regulation of ERα expression in breast cancer. Most ERα positive tumors are treated with Selective Estrogen Receptor Modulators (SERMs) and thus the role of miR-27a expression in response to SERM treatment is of interest. METHODS: Tamoxifen resistant cells were generated by molecular evolution with six cycles of tamoxifen treatment. MCF7 and T47D luminal A breast cancer cell lines were either treated with miR-27a mimics, or ER-signaling was modulated ectopically. The changes were analyzed with RT-qPCR, western blotting and transcriptional activity ERE-reporter assays. Moreover, the response to SERM treatments (tamoxifen, endoxifen and toremifen) was investigated by cell viability and apoptosis measurements. An in silico analysis of survival data from the METABRIC study was performed in order to assess the prognostic value of miR-27a for response to SERM treatment. RESULTS: Tamoxifen-resistant cells showed decreased expression of ERα and miR-27a. The overexpression of miR-27a increased the levels of ERα, while modulation of ERα decreased miR-27a expression. High miR-27a expression increased the sensitivity of MCF7 and T47D cells to SERM treatments and re-sensitized the cells to tamoxifen. Patient survival of luminal A breast cancer patients that underwent endocrine therapies was better in groups with high miR-27a expression. CONCLUSION: MiR-27a sensitizes luminal A breast cancer cells to SERM treatments based on a positive feedback loop with ERα. An increased overall-survival of ER-positive breast cancer patients that underwent endocrine treatments and displayed high miR-27a levels was found.

A proteomic analysis of chemoresistance development via sequential treatment with doxorubicin reveals novel players in MCF­7 breast cancer cells.

Breast cancer exhibits the highest incidence of all cancer types and is the 2nd leading cause of cancer mortality in women. Up to 82% of breast cancer patients receive a chemotherapy­containing treatment regimen. However, numerous breast tumors recur within 10 years following an initial response and are frequently resistant to previous therapeutic agents. Thus, to analyze the crucial factors, and whether the development of resistance in tumor cells follows certain patterns, is of great importance. In the present study, the clinical treatment schedule of the frequently used chemotherapeutic drug doxorubicin was applied in an in vitro model, the Molecular Evolution Assay (MEA), leading to resistance formation. By investigating the alterations in protein expression in MCF­7 breast cancer cells with three biological replicates, it was observed that the development of resistance to doxorubicin is a multi­directed process. The number and composition of the differentially expressed proteins varied, in addition to the pathways involved in chemoresistance, leading to only a small number of proteins and pathways being commonly regulated in all the MEAs. The proteins 60S ribosomal export protein NMD3 and 4F2 cell­surface antigen heavy chain (SLC3A2) were identified to be the most promising differentially expressed targets; the gene ontology term 'apoptotic signaling pathway' was reduced and 'cell redox homeostasis' was upregulated. Based on the present findings in vitro, it may be hypothesized that the development of resistance in patients is an even more complex process, emphasizing the need for further investigations of resistance development in the clinic to eventually improve patient outcomes.

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.

Salinomycin co-treatment enhances tamoxifen cytotoxicity in luminal A breast tumor cells by facilitating lysosomal degradation of receptor tyrosine kinases.

Luminal A breast cancer is the most common breast cancer subtype which is usually treated with selective estrogen receptor modulators (SERMS) like tamoxifen. Nevertheless, one third of estrogen receptor positive breast cancer patients initially do not respond to endocrine therapy and about 40% of luminal A breast tumors recur in five years. In this study, we investigated an alternative treatment approach by combining tamoxifen and salinomycin in luminal A breast cancer cell lines. We have found that salinomycin induces an additional cytotoxic effect by inhibiting the ligand independent activation of ERα. Thereby salinomycin increases the intracellular calcium level. This leads to a premature fusion of endosomes with lysosomes and thus to the degradation of Egfr family members. Since this process is essential for luminal A breast cancer cells to circumvent tamoxifen treatment, the combination of both drugs induces cytotoxicity in tamoxifen sensitive as well as resistant luminal A breast cancer cell lines.