AC1MMYR2 Impedes Carcinoma Associated Fibroblasts-induced Tumor Metastasis And Chemotherapy Resistance

Objective: Breast cancer is the most common malignancy in women and metastasis is the leading cause of death in breast cancer patients. The metastatic potential of breast cancer cells is closely dependent on the tumour microenvironment. The tumor microenvironment is consisted of cancer cells, immune cells, fibroblasts and other components. Cancer-associated fibroblasts(CAFs) is the most important stromal cells in the tumor microenvironment. CAFs play an important role in promoting tumor metastasis and drug resistance. Remodeling CAFs, namely blocking the interaction between tumor cells and CAFs, which was converted to NFs, is a new strategy for cancer treatment. In addition, our previous studies have shown that miR-21 was overexpessed in CAFs, it was revealed that abnormal miR-21 expression was closely related with the formation and functioning of CAFs. This paper focused on the molecular mechanism of CAFs-induced tumor metastasis and chemotherapy resistance. miR-21 small molecule inhibitor--AC1MMYR2(AMR),with taxol could be explored as a means to improve the effect of chemotherapy and limit tumor metastasis.Methods: 1. We use In situ hybridization and Immunohistochemistry technology to detect the expression of miR-21, FAP-? and ?-SMA in 60 pairs with in situ carcinoma and metastatic carcinoma of human invasive mammary ductal bresst cancer tissues, to study their correlation with the metastasis of breast cancer. 2. Breast cancer cell line MDA-MB-231 and MCF-7, cultured under the following conditions: standard culture medium, CAFs conditioned medium(CAF-CM), AMR treated CAFs for 24 h conditioned medium(AMR-CAF-CM), paclitaxel, AMR-CAF-CM plus paclitaxel; Morphological were observed in photographs by the microscope in bright field. Cell invasion number and migration ability were evaluated by Transwell and Wound Healing Assay, respectively. The expressions of proteins, such as ?-catenin, Vimentin, NF-?B are the detected by western bloting and immunofluorescence. The variation of miR-21 expression in was detected by Real-time PCR. The association between ?-catenin and NF-?B was observed by co-immunoprecipitation. Changes of the half-maximal inhibitory concentration(IC50) of taxol in the cells were detected after drug treatment by MTT. 3. Constructing MDA-MB-231 orthopic tumor model, MDA-MB-231 cells transduced with luciferase lenti-virus and CAFs were injected into mammary fat pads of each nude mouse to investigate the effect of AC1MMYR2 on CAFs-induced tumor metastasis in vivo. The mice were randomly assigned to six groups(n=8 per group): PBS group, AC1MMYR2 group, taxol group and combination-therapy group. Using the animal Living Image Technology to detect the bioluminescence of tumor and lung. Lung metastasis was identified by H&E staining. Immunohistochemistry technology was used to detect the expression levels of FAP-?, ?-SMA, ?-catenin and VHL.Results: Firstly, we studied the effect of cancer-associated fibroblasts(CAFs)-induced metastasis and paclitaxel resistant for breast cancer. The iconic proteins of CAFs are FAP-? and ?-SMA. In this paper we detected 60 groups patient samples with in situ carcinoma and metastatic carcinoma of human invasive mammary ductal bresst cancer tissues and found that high expressions of FAP-?, ?-SMA, and miR-21 were positively correlated with lymph node metastasis. Furthermore, in order to provide CAFs and normal tissue fibroblasts(NFs) for follow-up experiments, we isolated and cultured CAFs and normal tissue fibroblasts(NFs) in primary and found CAFs cultured in primary cells highly expressed FAP-?, ?-SMA and mesenchymal marker protein Vimentin, whereas were negative for the epithelial marker Cytokeratin and E-cadherin. These revealed CAFs cultured in primay retains the characteristics of such cells. In addition, the breast cancer cell line MCF-7 and MDA-MB-231 were co-cultured with CAFs(CAF-CM) or NFs(NF-CM). Compared with NFs group, CAFs can induce significant phenotypic changes in both of these cell lines, from a spindle-like shape with elongated pseudopodia to fibroblast-like morphology. Cell invasion and migration ability was dramatically enhanced compared to the control cells. The expression of Epithelial-Mesenchymal Transition,(EMT) related ?-catenin, ZEB1 and ZEB2 were significantly elevated in CAF-CM cultured breast cancer cells.These above results suggest that CAFs can promote breast cancer cells EMT,thus speeding up the metastasis. Finally, the half-maximal inhibitory taxol concentrations(IC50) for native MCF-7 cells and MDA-MB-231 were 0.1 and 0.4 ?M, respectively. Treatment with CAF-CM increased IC50 values by 8–10 folds. These results indicated CAFs improved breast cancer cells the resistance to paclitaxel. The above results indicate that CAFs in the breast cancer patients samples were positively correlated with lymph node metastasis, and CAFs induced metastasis of breast cancer cells and chemotherapy resistance to paclitaxel at the cellular level. Secondly, we focused on the effect of miR-21 on CAFs induced breast cancer metastasis. RT-PCR was performed and the result showed that the levels of miR-21 were increased by 1.8 and 2.4-fold in MCF-7 and MDA-MB-231 cells after CAF-CM treatment, respectively, proving CAFs can induce cancer cells highly expressing of miR-21. In addition, breast cancer cells, cultured with NFs-CM transfected by miR-21 mimics-enhanced migration and invasion abilities and are induced the transition from epithelial to mesenchymal. EMT related ?-catenin and vimentin expression levels were increased. An inhibitor of miR-21--AMR could knockdown the expression of miR-21 in CAFs and effectively reversed CAFs induced tumor invasion and proliferation, reduction of ?-catenin and Vimentin expression, which was similar to the results of tumor cells co-cultured with normal fibroblasts. These results suggest that the high expression levels of miR 21 in CAFs are key factors promoting EMT and metastasis of breast cancer cells. Thirdly, we studied the effects of miR-21 inhibitor--AMR on CAFs induced breast cancer cell metastasis and resistance to paclitaxel and its molecular mechanism. AMR-CAF-CM treatment can reduce the CAFs induced metastasis and invasion of MCF-7 and MDA-MB-231. In addition, paclitaxel can inhibit metastasis and invasion of breast cancer cells, but in the CAF-CM treated breast cancer cells, paclitaxel can not play a role in promoting metastasis and invasion of cancer cells. Again, AMR –CAF-CM treated breast cancer cells, while adding paclitaxel, resulted in significant reduction of ?-catenin, ZEB1, ZEB2 expression and increase E-cadherin expression levels, reducing ?-catenin nuclear translocation in CAF-CM cultured cancer cells, namely reducing the phenomenon of EMT. These results suggest that, AMR can reprogram CAFs to inhibit CAFs-induced breast cancer metastasis and enhance the sensitization to paclitaxel. Further, we studied the mechanism of CAFs promoted breast cancer cells the transition from endothelial cells to mesenchymal cells and found that: AMR-CAF-CM treated MCF-7 and MDA-MB-231 can inhibit the expression of miR-21 and miR-21 promoted the expression of the target gene VHL, thus reducing the expression of NF-?B and the activity of NF-?B, reducing co-precipitation of NF-?B and ?-catenin, and finally to inhibit metastasis of tumor cells and enhance the chemotherapy effect of paclitaxel. These results demonstrated AMR can inhibit CAFs-induced breast cancer metastasis and enhance the sensitization to paclitaxel by NF-?B / miR-21 / VHL. Fourthly, in vivo study, bioluminescence imaging showed that taxol suppressed tumor growth to some extent, compared with the PBS group, whereas the chemotherapy was attenuated in the CAF-treated group. A significant reduction in tumor growth was detected in the AMR plus taxol treated mice.Furthermore, CAFs promoted lung metastasis, with extensive tumor foci found compared to the control cells. In contrast, lung metastasis of the combination treated mice was dramatically lower than that of control cells, which was further demonstrated by H&E staining. In addition, the VHL expression levels were increased, whereas NF-?B and ?-catenin were significantly decreased, most importantly, levels of FAP-? and ?-SMA were significantly decreased in AMR plus taxol treated mice.Conclusion This study proved interactions between breast cancer epithelial cells and CAFs are critical for tumor invasion and metastasis. Simultaneously, we reported miR-21 is a key factor in maintaining the effect of CAFs on breast cancer cells in stromal cells. AC1MMYR2, a small molecule inhibitor of miR-21, can hinder promoting cancer cell invasion and metastasis of cancer cells induced by CAFs and enhanced resistance to paclitaxel. The molecular mechanism is that AC1MMYR2 reprogrammed CAFs to decrease miR-21 expression of breast cancer cells by impeding miR-21 expression of CAFs and regulated NF-?B signaling pathway by directly targeting VHL, thereby blocking the co-precipitation of NF-?B and ?-catenin and nuclear translacation, reprogramming CAFs and preventing the occurrence of EMT in cancer cells. Our research laid the theoretical and experimental basis for clinical tumor therapy of AC1MMYR2.