TGFβ2/Snail-mediated Emt Confers5-FU Resistance In Human Breast Cancer

Background and objectiveBreast cancer is the most commonly diagnosed type of cancer, the second leading cause of cancer-related deaths among women in the world. Chemotherapy plays an important role in the management of breast cancer, but the efficacy of this treatment is limited by drug resistance.5-Fluorouracil (5-FU) is a first-line chemotherapeutic agent effective in the treatment of breast cancer. However, in spite of the comparatively high sensitivity of breast cancer to5-FU, the prognosis for advanced or recurrent cases remains poor since most deaths are the result of metastasis that is refractory to conventional chemotherapy. The mechanisms of5-FU resistance in breast cancer are not clear by now.Epithelial-mesenchymal transition is a process by which epithelial cells lose many of their mature characteristics (ie, tight junctions) and acquire properties commonly associated with mesenchymal cells, with increased motility. Emerging evidence associates chemoresistance with acquisition of EMT and cancer stem-like cell phenotype. There is some evidence that the chemoresistant cells displayed an EMT phenotype, and induction of EMT with TGF-β may lead to the decreased efficacy of the sensitivity of tumor cells to chemotherapy. However, it is not clear whether this phenomenon is involved in acquired resistance to5-FU.In this study, in orde to elucidate the relatioship between the chemoresistace of5-FU and EMT, we used a previously established in vitro cell model of5-fluorouracil-resistant MCF7cells (MCF7/5-FU), according to the previous Gene Chip expression data, and determine whether the cellular morphology, molecular changes, migration and invasion consistent with EMT, as well as cancers stem cell features. Methods(1) The morphological changes of MCF7/5-FU cells were observed. The EMT markers were detected by qRT-PCR and westem blot. Immunofluorescence staining for the expression and cellular localization of E-cadherin and Vimentin. The migratory and invasive capacity were assessed by attachment and detachment assays and migration and invasion assays respectively. The CD44+CD24-/low cells were separated by FACS. The capacity of self-renewal were assessed by mammosphere formation.the percentage of SP analyzed by FACS.(2) Silence the expression of Snail by RNAi. The cellular morphology, molecular changes, migration and invasion were compared between the Snail scilence and silence control cells. The downstream targets of Snail, MMPs were detected by qRT-PCR and western blot. The enzymatic activity of MMP-9was analyzed by gelatin zymography. The response to5-FU was detemined by MTS and Annexin V/PI apoptosis assay. P53and the related apoptosis pathway were detected by westem blot. Following, transfection with wilde type p53in MCF7/5-FU cells, and to determine wether the EMT penotype and chemoresistance were reversived(3) The exression and secreted TGF|32was detected by qRT-PCR, westem blot andELSIA. Treat the MCF7/5-FU cells with neutralizing antibody and determine whether the properties of the mesenchymal,stem cell and chemoresistance were reversed。The activation of Smad,NF-κB and Erk pathways were detected by western blot. The Smad2inhibitor Staurosporine, the ERK inhibitor PD9805,the NF-κB inhibitor, Bay-117082were used to treated with MCF7/5-FU cells and determine whether the properties of the mesenchymal,stem cell and chemoresistance were reversed。Western blot analysis the TGF(32/Smad signaling pathway in MCF7/5-FU with Snail knockdown. MTS for response to5-FU. The response of different breast cells to5-FU was detemined by MTS.The morphological changes of MCF7/5-FU cells were observed. The EMT markers and TGF(32/Smad signaling pathway were detected by westem blot.(4) Treatment the MCF7, MCF7/5-FU and MDA-MB-231cells with different does of metformin, the cell viability was assessed with MTS. The EMT and related breast CSC phenotype were assessed. The efficacy of5-FU and metformin alone and in combination on the viability of these cells was evaluate by MTS.Results(1) we observed that MCF-7/5-FU cells exhibit an altered phenol-type whereby cells disperse, develop pseudopodia, and assume a spindle shape-properties associated with the EMT phenotype. This observation was further supported by expression analysis of classic EMT markers and detection of cellular motility. Furthermore, it have an increased amount of cancer stem cells in MCF7/5-FU cell population.(2) knockdown of Snail in MCF7/5-FU cells resulted in changes from fibroblast-like shapes to cobblestone morphology, a significant increased in the expression of epithelial markers and decreased the levels of mesenchymal markers, decline in cell migration and invasion, characteristic of mesenchymal-to-epithelial transition, the reverse of EMT.The cancer stem cell penotype was also decreased. Snail up-regulated the expression and enzymatic activity of MMP9to enhance the invasion abilty of the MCF7/5-FU cells. Snail attenuated the chemosensity to5-FU by suppressing the P53. While transfection of MCF7/5-FU cells with wilde type p53led to the reversal of EMT and upregulation of Snail gene expression. (3) Expression and secretion of TGFβ2were increased in MCF7/5-FU cells. Treatment with TGF(32neutralizing antibody reversed the EMT, depleted stem-like cancer cells, and enhance the chemosensity to5-FU. In MCF7/5-FU cells, the Smad2was activated. Conversely, inhibition of smad2resulted in reversal of the EMT phenotype. Overexpression of Snail in MCF7cells feedback increased the TGFβ2/Smad signaling pathway. Furthermore, TGFβ2/Smad/Snail-mediated EMT was also associated with the primary resistance to5-FU.(4) Treatment with metformin lead to the reversal of EMT and chemoresistance to5-FU, and reduction of the putative CD44+CD24-breast cancer stem cell population, through inactivation of the TGF(32/Smad/Snail signaling pathway.Conclusion(1) This is the first description of chronic5-FU resistance cells with mesenchymal features as well as cancers stem cell characteristics.(2) Snail plays a crucial role in maintaining mesenchymal, cancer stem-like and chemoresistant properties in MCF7/5-FU cells.(3) P53repress Snail, and overexpression P53in MCF7/5-FU cells lead to the reversal of EMT and chemoresistance to5-FU.(4) We show for the first time that TGF(32/Snail signailing loop-mediated EMT was related to the primary and acquired chemoresitance to5-FU.(5) Metformin reversed the EMT in5-FU-resistant breast cancer cells by weakening the activation of TGFβ2/Snail signailing loop.