Acquired resistance to classical chemotherapeutics is a major obstacle in cancer treatment. Doxorubicin is frequently used in breast cancer therapy either as single-agent or in combination with other drugs like docetaxel and cyclophosphamide. All these chemotherapies have in common that they are administered sequentially and often result in chemoresistance. Here, we mimicked this pulse therapy of breast cancer patients in an in vitro cell culture model, where the epithelial breast cancer cell line BT474 was sequentially treated with doxorubicin for several treatment cycles. In consequence, we obtained chemoresistant cells displaying a mesenchymal-like phenotype with decreased levels of miR-200c. To investigate the involvement of miR-200c in resistance formation, we inhibited and overexpressed miR-200c in different cell lines. Thereby, the cells were rendered more resistant or susceptible to doxorubicin treatment. Moreover, the receptor tyrosine kinase TrkB and the transcriptional repressor Bmi1 were identified as miR-200c targets mediating the drug resistance. Hence, we provide a mechanism of acquired resistance to doxorubicin that is caused by the loss of miR-200c. Along with this, our study demonstrates the complex network of microRNA mediated chemoresistance highlighting the challenges in cancer therapy and the importance of novel microRNA-modulating anticancer agents.

The GTPase K-ras is involved in a variety of cellular processes such as differentiation, proliferation and survival. However, activating mutations, which frequently occur in many types of cancer, turn KRAS into one of the most prominent oncogenes. Likewise, miR-200c is a key player in tumorigenesis functioning as a molecular switch between an epithelial, non-migratory, chemosensitive and a mesenchymal, migratory, chemoresistant state. While it has been reported that KRAS is modulated by several tumor suppressor miRNAs, this is the first report on the regulation of KRAS by miR-200c, both playing a pivotal role in oncogenesis. We show that KRAS is a predicted target of miR-200c and that the protein expression of KRAS inversely correlates with the miR-200c expression in a panel of human breast cancer cell lines. KRAS was experimentally validated as a target of miR-200c by Western blot analyses and luciferase reporter assays. Furthermore, the inhibitory effect of miR-200c-dependent KRAS silencing on proliferation and cell cycle was demonstrated in different breast and lung cancer cell lines. Thereby, the particular role of KRAS was dissected from the role of all the other miR-200c targets by specific knockdown experiments using siRNA against KRAS. Cell lines harboring an activating KRAS mutation were similarly affected by miR-200c as well as by the siRNA against KRAS. However, in a cell line with wild-type KRAS only miR-200c was able to change proliferation and cell cycle. Our findings suggest that miR-200c is a potent inhibitor of tumor progression and therapy resistance, by regulating a multitude of oncogenic pathways including the RAS pathway. Thus, miR-200c may cause stronger anti-tumor effects than a specific siRNA against KRAS, emphasizing the potential role of miR-200c as tumor suppressive miRNA

A major obstacle in the successful treatment of cancer is the occurrence of chemoresistance. Cancer cells surviving chemotherapy and giving rise to a recurrence of the tumor are termed cancer stem cells and can be identified by elevated levels of certain stem cell markers. Eradication of this cell population is a priority objective in cancer therapy. Here, we report elevated levels of stem cell markers in MCF‐7 mammospheres. Likewise, an upregulation of HER2 and its differential expression within individual cells of mammospheres was observed. Sorting for HER2^high and HER2^low cells revealed an upregulation of stem cell markers NANOG, OCT4 and SOX2 in the HER2^low cell fraction. Accordingly, HER2^low cells also showed reduced proliferation, ductal‐like outgrowths and an increased number of colonies in matrigel. Xenografts from subcutaneously injected HER2^low sorted cells exihibited earlier onset but slower growth of tumors and an increase in stem cell markers compared to tumors developed from the HER2^high fraction. Treatment of mammospheres with salinomycin reduced the expression of SOX2 indicating a selective targeting of cancer stem cells. Trastuzumab however, did not reduce the expression of SOX2 in mammospheres. Furthermore, a combinatorial treatment of mammospheres with trastuzumab and salinomycin was superior to single treatment with each drug. Thus, targeting HER2 expressing tumors with anti‐HER2 therapies will not necessarily eliminate cancer stem cells and may lead to a more aggressive cancer cell phenotype. Our study demonstrates efficient killing of both HER2 positive cells and cancer stem cells, hence opening a possibility for a new combinatorial treatment strategy.

Tumor spreading is the major threat for cancer patients. The recently published anti-cancer drug salinomycin raised hope for an improved treatment by targeting therapy-refractory cancer stem cells. However, an unambiguous role of salinomycin against cancer cell migration and metastasis formation remains elusive.

During the progression from ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC), cells must overcome the physically restraining basement membrane (BM), which compartmentalizes the epithelium from the stroma. Since the extracellular matrix (ECM) of the epithelial and stromal compartments are biochemically and physically distinct from one another, the progression demands a certain degree of cellular plasticity for a primary tumor to become invasive. The epithelial-to-mesenchymal transition (EMT) depicts such a cell program, equipping cancer cells with features allowing for dissemination from the epithelial entity and stromal invasion at the single-cell level. Here, the reciprocal interference between an altering tumor microenvironment and the EMT phenotype was investigated in vitro. BM-typical collagen IV and stroma-typical collagen I coatings were applied as provisional 2D matrices. Pro-inflammatory growth factors were introduced to improve tissue mimicry. Whereas the growth on coated surfaces only slightly affected the EMT phenotype, the combinatorial action of collagen with growth factor TGF-β1 induced prominent phenotypic changes. However, EMT induction was independent of collagen type, and cellular accessibility for EMT-like changes was strongly cell-line dependent. Summarizing the entire body of data, an EMT-phenotyping model was used to determine cellular EMT status and estimate EMT-like changes. The miR200c-mediated reversion of mesenchymal MDA-MB-231 cells is reflected by our EMT-phenotype model, thus emphasizing its potential to predict the therapeutic efficacy of EMT-targeting drugs in the future.