Mechanism Investigation Of HGF/c-Met Signal Pathway Modulates Tamoxifen Resistance In Breast Cancer Cells

【Background】Breast cancer is a common female malignancy which accounts for a quarter of tumors in female. Breast cancer has become a leading killer in female, because of its continuous increasing incidence and high mortality. As we know, breast cancer is a kind of hormone-dependent tumor, in which estrogen is the main factor to drive the growth of cancer. About 70% patients of breast cancer show estrogen receptor(ER) positive, which plays a key role in treatment of breast cancer. Tamoxifen(TAM), as a selective estrogen receptor modulator, has become the primary endocrine therapy for breast cancer. It can inhibit the growth of breast cancer through competitive combination with estrogen receptors. But now, primary and secondary Tamoxifen resistances have become an important challenge in endocrine treatment of breast cancer.HGF/c-Met signaling pathway plays an important role in tumor development, invasion and metastasis. The combination of HGF and its c-Met protein ligand is characterized by a series of signal transduction of enzymatic reactions. So, how HGF/c-Met involved in breast cancer Tamoxifen resistance need to study. The in-depth researches on the mecha-nism of HGF/c-Met signaling pathway participating in breast cancer cell Tamoxifen resistance will lead to development of new ideas for prevention and therapy for breast cancer. 【Objective】The research on the mechanism of HGF/c-Met signaling pathway will reveal the biological functions and mechanisms of the regulatory network in breast cancer cell Tamoxifen resistance system, which may provide a novel drug target site for monitoring and reversing the Tamoxifen resistance more effectively. 【Methods】1. To establish the model of MCF-7, the human breast cancer cell line, drug-resistance ability. Transmission electron microscopic(TEM) observe cell morphology. MTT assay, Colony formation assay, apoptosis test and cell scratch assay detect the breast cancer resistant cells biological function.2. Cytokines microarray text the difference of Cytokines in breast cancer cells and Tamoxifen-resistant breast cancer cells. ELISA assays analyze the expression of HGF in serum samples of both initial diagnosed and relapsed metastatic patients of ER-positive breast cancer. RT-PCR assays text on and WB assays show the c-Met and its downstream signaling pathways activated degree.3. After intervening HGF and overexpressing c-Met expressions, MTT, EDU, and cell apoptosis assays are used to analyze cells biological function.4. Through bioinformatics analysis, we predict the possible micro RNA molecule that may involved in breast cancer Tamoxifen-resistance by regulating the HGF expression. Dual luciferase reporter gene experiments are used to verify the combination of miR-141, miR-200 a and HGF. 【Results】1. Transmission electron microscopic examination have found that screened drug resistant breast cancer cells were significantly different with the parent cells in microstructure, such as the increase in mitotic figures and endoplasmic reticulum, etc. in the former. The screened drug-resistant breast cancer cells could obtain the ability to promote cells proliferation, anti-apoptosis actions, and increase cells migration.2. Cytokines microarray have revealed that the Tamoxifen-resistant breast cancer cells can secrete HGF independently and activate c-Met and theirs downstream signaling pathways in both autocrine and paracrine ways. ELISA assays have demonstrated HGF level in relapsed metastatic patients elevated significantly. RT-PCR also confirmed that HGF expression was higher in the Tamoxifen-resistant cells than the parent cells. WB assays have showed that the c-Met and its downstream signaling pathways of drug-resistant breast cancer cells were activated. MTT assays have showed that the Tamoxifen-resistance of breast cancer cells was enhanced after the administration of pure HGF and conditional medium when compared to the untreated group. WB tests have indicated that the c-met and its downstream molecules were activated after the pure HGF stimulation.3. After using c-Met small molecule inhibitors to block its downstream proteins pathway, the activity of Tamoxifen-resistant breast cancer cells were significantly reduced. Cell apoptosis was highly increased when HGF neutralizing antibody was used to treat Tamoxifen sensitivity-cells. Biological functions of cells, such as proliferation and anti-apoptosis, were increased when c-Met gene was overexpressed.4. MiR-141 and miR-200 a can be matched with 3’UTR region of HGF to initiate the negative feedback control. By further experiments, we also have verified the HOTAIR can inhibit the expressions of miR-141 and miR-200 a. CHIP experiments have showed that H3K27 and the EZH2 may exert transcriptional repression functions through the combination with the promoter region of miR-141 and miR-200 a. 【Conclusion】HGF/c-Met-HOTAIR-miR-141/200 a feedback signaling pathways participate in breast cancer cells Tamoxifen resistance, which may provide a novel drug target site for monitoring and reversing the Tamoxifen resistance effectively.