| Background and objectiveBreast cancer is the most commonly diagnosed type of cancer, and the second leading canuse of cancer-related deaths smong women. As a result of improved treatement, the survival rates for breast cancer have improved markedly over the decades. However, there are still more than1.3million women worldwide are diagnosed with breast cancer and more than500,000women die from breast cancer. The appearance of drug resistance and metastasis is almost always a harbinger of eventual coancer mortality. Clinically as well as biologically, metastasis is intricately linked with resistance to chemotherapy, drug-resistant tumor cells are more susceptible to metastasis and metastatic tumor cells are more resistant to chemotherapy. However, an integrated understanding of acquired drug resistance and metastasis is lacking. This combined clinical and biological problem prompted us to explore the mechanisms responsible for the association between resistance and metastasis. According to recent reports, epithelial-mesenchymal transition (EMT) maybe link resistance and metastasis. The EMT affects critical steps of morphogenesis by interconverting epithelial cell types into cells with mesenchymal attributes. EMT programs activated in carcinoma cells enable them to acquire cellular traits associated with high-grade malignancy, including the ability to complete various steps of the metastatic cascade. In addition, certain epithelial cells that pass through an EMT acquire the self-renewing trait associated with nomal tissue and cancer stem cells (CSCs), especially in breast or breast cancer. Clinically, CSCs have been associated with higher rates of recurrence and metastasis. Importantly, CSCs have been found to be relatively resistant to radiation and chemotherapy compared to their nontumorigenic progeny. Recent evidence showed chemotherapy could induce EMT and a series of drug-resistant cancer cells exhibited EMT phenotype companied with CSC feature. So the vicious circle among chemotherapy-EMT-CSCs promotes the cancer progression. Despite of diverse factors have been reported to induce EMT in various cell types, the signal mechanisms that induce and then maintain the resistnce/mesenchymal/CSC state remains unclear. Furthermore, recent evidence strongely points to tumor microenvironment components as potential participants in the generation of chemoresistance and promotion of metastasis. Luckly,5-Fu resistant breast cancer cells (MCF-7/5-Fu) derived from MCF-7, showed typical EMT phenotype with higher resistanc, invasion and BCSC (breast cancer stem cell) traits. And a series of differential genes between MCF-7and MCF-7/5-Fu cells, such as TGFβ2have been screened. Here, in regard to the paracrine and autocrine signal nets in the extracellular microenvironment based on the role of TGF(32and the regulation of itself expression in breast cancer cell lines, we aimed to investigate the effect of resistant breast cancer cells on primary cancer cells and to explore the mechanisms responsible for the induction and maintance of the resistnce/mesenchymal/CSC state and to provide new biomarkers and evidence for resistance and metastasis reversion.Methods(1) Using miRNA microarray combined with bioinformatic analysis identified miR-141, miR-200a and miR-145maybe target TGFβ2. cDNA sequence containing two human pre-miR-141, pre-miR-200a and pre-miR-145units were inserted into pLEX-MCS lentivirus expression system. MCF-7/5-Fu, MDA-MB-231and Hs578T cells were infected with miRNA expression lentivirus, and TGFβ2expression, cell phenotype, drug sensitivity, EMT markers, invasive potential and BCSC traits were determined. Luciferase activity assays were also performed to confirm miR-141, miR-200a and miR-145directly target TGFβ2.(2) qRT-PCR and ELISA were used to determine the expression stutas of TGFβ2in selected breast cancer cell lines. TGFβ2was used to treat MCF-7, T47D, BT474, SKBR3and MDA-MB-453cells with endogenously low expression of TGFβ2and TGFβR inhibitors SD208and SB525334were used to treat MCF-7/5-Fu, MDA-MB-231and Hs578T cells with endogenously high expression of TGFβ2. Then, the phenotype change, drug sensitivity, EMT markers, invasive potential and BCSC traits were determined.(3) miR-141, miR-200a and miR-145expression was examined in MCF-7cells treated with cytokine TGFβ2and in MDA-MB-231and Hs578T cells treated with TGFβR inhibitors SD208and SB525334. Snail1expression in MCF-7, MDA-MB-231and Hs578T cells were knockdowned using sh-RNA expression vector pRNAT-U6.1/sh-snaill, then miR-141/200a and miR-145expression was examined after treated as above. Luciferase activity assays were performed to indentify potential promoters for miR-141, miR-200a and miR-145, respectively. CHIP assay was performed to confirm whether snail1physically binds to the promoters. Then, miR-141, miR-200a and miR-145expression was examined in MDA-MB-231and Hs578T cells treated with DKK1or restorted expression of GSK3β with pLV-Ubc-GSK3β expression vector.(4) The phenotype and molecules changes were observed in MCF-7, T47D, BT474, SKBR3and MDA-MB-453epithelial type breast cancer cells cultured with conditioned media from MCF-7/5-Fu cells. MTS assay was used to drug sensitivity; qRT-PCR, wesetern blot and immunofluorescence assays were used to examine the expression of EMT markers; Transwell assay was used to determine the invasive potential; Mammosphere Cullture and FACS Analysis were used to investigate the BSCS traits.(5) Differential cytokines in culture media from MCF-7and MCF-7/5-Fu cells were compared with L-series507-antibody array, and qRT-PCR and ELISA assays were used to confirm the results form array and determine the expression pattern of differential cytokines in selected breast cancer cell lines. TNFa, NOV, FGF2and IL-6was used singly or combined (termed:cytokines cocktail) to stimulate MCF-7, T47D, BT474, SKBR3and MDA-MB-453epithelial type breast cancer cells, and cell phenotype, drug sensitivity, EMT markers, invasive potential and BCSC traits were determined. Furthermore, luciferase activity assays and western blot were used to ovserve the activation of signal pathways after cytokines cocitail treatment, and the effect of signal pathways inhibitors in EMT reversion also observed.Results(1) Both mRNA and protein levels of TGFβ2were much higher in MCF-7/5-Fu, MDA-MB-231and Hs578T cells, accompanied with miR-141, miR-200a and miR-145downregulation. pLEX-MCS lentivirus expression system with two pre-miRNA units significantly enhanced miR-141, miR-200a and miR-145expression accompanied with TGFβ2downregulated. These results combined with luciferase activity assay strongly suggested miR-141, miR-200a and miR-145could directly target TGFβ2expression. Low-dose TGFβ2could upregulate miR-141, miR-200a and miR-145expression snail1dependently. However, TGFp signal inhibition, snail1knockdown, GSK3β restoration and DKK1treatment significantly upregulated miR-141, miR-200a and miR-145expression. In addition, snail1could physically bind to the promoters of miR-141, miR-200a and miR-145to repress them transcription. However, GSK3β could reverse the repressive effect of snail1on miR-141, miR-200a and miR-145expression.(2) TGFβ signal inhibition with inhibitors or miR-141, miR-200a and miR-145overexpression only partially reversed the EMT of MCF-7/5-Fu cells, but significantly reversed cellular phenotype and drug resistance of MDA-MB-231and Hs578T cells, and inhibited invastion and BCSCs traits. As the same, long-term TGFβ2treatment only induced partially EMT phenotype in MCF-7and MDA-MB-453cells, but not in T47D, BT474and SKBR3cells.(3) MCF-7, T47D, BT474, SKBR3and MDA-MB-453cells exhibited typical EMT phenotype after cultured with conditioned media from MCF-7/5-Fu cells and this change was supported by expression analysis of EMT markers. We termed these changed cell lines XX/media, such as MCF-7/media. XX/media cell lines exhibited more resistanc to5-Fu, more proliferation potential, more invasive potential, and the BCSCs content (CD44+CD24-subpopulation) was much enriched as measured by mammosphere assay and FACS analysis.(4) Using antibodies array combined qRT-PCR and ELISA confirmation, TGFβ2, TNFα, NOV, CXCL10, FGF2and IL-6was foud to be upregulated in MCF-7/5-Fu cells, these cytokines except TGFβ2, were also upregulated in XX/media cell lines. We found only cytokines formed with TNFa, NOV, FGF2and IL-6could induce MCF-7, T47D, BT474, SKBR3and MDA-MB-453cell lines to be typical EMT (termed:XX/cocktail) and changes of EMT markers. XX/cocktail cells showed more drug resistance, invasive potential, and BCSCs traits. In addition, NF-κB, Wnt/β-catenin, Hedgehog, MAPK/ERK and p38MAPK pathways were more active in XX/cocktail cells, and treatment with combined inhibitors with NF-kB inhibitor Bay11-7082, Wnt/β-catenin, ERK inhibitor PD98059and p38inhibitor SB203580significangly reversed the EMT phenotype of XX/cocktail cells.Conclusion(1) TGFβ2/miR-141, miR-200a and miR-145/snaill regulatory loop plays key role in maintaining the high expression of TGFβ2in mesenchymal cell type breast cancer cell lines.(2)GSK3β or DKK1can reverse the repressive role of snail1on miR-141, miR-200a and miR-145expression. (3) The role of TGFβ in breast cancer is cell type dependent. TGFβ signal inhibition can significantly reverse the biological characteristics of breast cancer cells with primary mesenchymal state, but only very partially reverse EMT phenotype in acquired resistant breast cancer cells.(4) Conditioned culture media from MCF-7/5-Fu cells can induce EMT in epithelial type breast cancer cells, and this is the firs report about the effect of acquired resistant breast cancer cells with resistnce/mesenchymal/BCSCs traits on primary breast cancer cells.(5) TNFα, NOV, FGF2and IL-6maybe play key role in maintaining chemotherapy induced EMT phenotype and multiple signal pathways such as NF-κB, Wnt/β-catenin, Hedgehog, MAPK/ERK and p38MAPK pathways responsible for the effects. |
