MiR-200c Suppresses TGF-β Signaling And Counteracts Trastuzumab Resistance And Metastasis By Targeting ZNF217 And ZEB1 In Breast Cancer

【Background】Breast cancer is one of the most common malignancies for women and has gained great attentions from the public and worldwide researchers. Human epidermal growth factor receptor 2(HER2), a key therapeutic target, is over-expressed in 20~25% of human breast cancers, and is associated with poor prognosis, metastasis, and reduced disease-free survival. Trastuzumab(trade name: herceptin), a humanized monoclonal antibody against HER2, has been successfully used for therapy of early-stage and metastatic breast cancer. However, trastuzumab resistance accompanied with enhanced metastatic potential in breast cancer is the most intractable issue in clinic. Despite accumulating studies unraveling the mechanisms underlying trastuzumab resistance and metastasis of breast cancer recently, it remains elusive whether these malignant phenotypes are connected and how they are regulated by canonical oncogenic pathways.TGF-β signaling plays key and dual roles in carcinogenesis and progression in cancer. On one hand, it suppresses proliferation and promotes apoptosis of cancer cells in early stages of carcinogenesis; on the other hand, it induces and sustains epithelial-mesenchymal transition(EMT) which promotes invasion and metastasis in late stages of cancer progression. Additionally, TGF-β signaling, an emerging important player in resistance to radiotherapy and medication, draws more and more attention and is suspected to be a key therapeutic target in cancers. However, the roles of TGF-β signaling in trastuzumab resistance accompanied with enhanced metastatic potential of breast cancer are unknown, and the mechanisms underlying the signaling regulation remain elucidated. Mi RNAs, a cohort of small noncoding RNAs, play important roles in all the stages of cancers by post-transcriptionally silencing target genes. So deciphering the roles and mechanisms of mi RNAs in TGF-β signaling regulation, trastuzumab resistance and metastasis will provide novel insights and strategies into reversing the multifaced malignant phenotypes in breast cancer. 【Objectives】We aim to elucidate the roles and mechanisms of mi RNAs in TGF-β signaling regulation, trastuzumab resistance and metastasis simultaneously in breast cancer, thus providing rationale for combined treatment of breast cancer patients. 【Methods】Wide type(WT) breast cancer cells were cultured in medium contained trastuzumab continuously to establish trastuzumab resistant(TR) model in vitro. In WT and TR breast cancer, MTT assay was used to analyze the trastuzumab sensitivity of cells; soft-agar colony-formation assay was used to analyze the anchorage-independent growth; transwell assay was used to analyze the invasion; morphological observation was used to observe the morphological changes; quantitative realtime PCR(q PCR) was used to analyze the m RNA levels of EMT associated transcriptional factors; western blot(WB) was used to analyze the protein levels of EMT markers.In WT and TR breast cancer cells, WB was used to analyze the activation of TGF-β signaling; q PCR and ELISA assays were used to detect the m RNA and secreted protein levels of TGF-β, respectively. After silencing TGF-β receptor Ⅱ(TGFBR2) in TR cells, MTT assay was used to analyze the trastuzumab sensitivity, transwell assay was used to analyze the invasion, and q PCR was used to detect the m RNA levels of EMT associated transcriptional factors.Mi RNA array was used to screen differentially expressed mi RNAs between WT and TR cells. After introduction of mi R-200 c, MTT assay and apoptosis assay by flow cytometry were used to analyze the trastuzumab sensitivity, transwell and wound-healing assay were used to analyze the invasion and migration respectively, cell morphology was observed, WB was used to analyze the protein levels of EMT markers, WB was used to analyze the activation of TGF-β signaling, and q PCR and ELISA assays were used to detect the m RNA and secreted protein levels of TGF-β respectively. Tumorigenesis and lung metastasis assay in nude mice were used to analyze the trastuzumab resistance and metastasis of breast cancer cells in vivo.Soft wares were used to predict targets of mi R-200c; dual luciferase reporter assay, q PCR, and WB were used to confirm the targets of mi R-200 c. After expression manipulation of targets of mi R-200 c, MTT assay and apoptosis assay by flow cytometry were used to analyze the trastuzumab sensitivity; transwell and wound-healing assay were used to analyze the invasion and migration respectively; cell morphology was vasualized by microscopy; WB was used to analyze the protein levels of EMT markers. QPCR, WB, and ELISA were used to evaluate the existence of nested loops of mi R-200c/ZEB1 and mi R-200c/ZNF217/TGF-β/ZEB1. 【Results】We established trastuzumab resistance model of breast cancer in vitro by culturing WT cell in medium contained 5ng/m L trastuzumab for 6 months. Compared to WT cells, MTT assay indicates TR cells resistant to trastuzumab; soft-agar colony-formation assay and transwell assay indicate enhanced anchorage-independent growth and invasion in TR cells; morphological observation, q PCR, and WB indicate significant EMT induction and maintenance in TR cells.Compared to WT cells, WB indicates elevated activation of TGF-β signaling in TR cells; q PCR and ELISA assays indicate elevated m RNA and secreted protein levels of TGF-β isoforms in TR cells respectively. MTT, transwell assay and q PCR indicate that silencing of TGFBR2 sensitizes TR cells to trastuzumab, inhibits invasion, and downregulates m RNA levels of EMT-associated transcriptional factors in TR cells.Mi RNA array indicates that mi R-200 c is the most significantly downregulated mi RNA in TR cells compared to in WT cells. MTT assay and apoptosis assay by flow cytometry indicate mi R-200 c sensitize TR cells to trastuzumab; transwell and wound-healing assay indicate mi R-200 c inhibits invasion and migration in TR cells; Morphological observation and WB indicate mi R-200 c reverses EMT in TR cells; WB indicates mi R-200 c suppresses TGF-β signaling; q PCR and ELISA assays indicate mi R-200 c down regulates the m RNA and secreted protein level of TGF-β2 and TGF-β3. Tumorigenesis and lung metastasis assays in nude mice indicate that mi R-200 c counteracts trastuzumab resistance and metastasis of TR breast cancer cells in vivo.Softwares prediction suggests that ZNF217 and ZEB1 are 2 targets of mi R-200c; dual luciferase reporter assay, q PCR, and WB confirm mi R-200 c does directly target these 2 targets. Silencing of ZNF217 and ZEB1 reverses trastuzumab resistance and EMT, and inhibit invasion and migration in TR cells. QPCR, WB and ELISA assays indicate that EMT and trastuzumab sensitivity are regulated by nested loops of mi-R200c/ZEB1 and mi R-200c/ZNF217/TGF-β/ZEB1 in breast cancer. 【Conclusions】In this study, we found that acquisition of trastuzumab resistance is accompanied by more malignant phenotypes including EMT, enhanced anchorage-independent growth and invasion of breast cancer cells. TGF-β signaling was first demonstrated to play key roles in induction and maintenance of those aforementioned malignant phenotypes in breast cancer. Additionally, we found mi R-200 c regulates TGF-β signaling and counteracts trastuzumab resistance and metastasis by targeting ZNF217 and ZEB1 in breast cancer. Finally, we found that mi R-200 c, ZNF217, TGF-β and ZEB1 constitute nested regulatory loops of mi R-200c/ZEB1 and mi R-200c/ZNF217/TGF-β/ZEB1 in breast cancer. These results provide new insights into molecular mechanisms underlying malignant phenotypes and potential targets for treatment of in breast cancer.