The Molecule Mechnasim Of Autophagy Stimulated By Macrophages In Breast Cancer

PART I THE EXPRSSION AND CLINICAL SIGNIFICANCE OF MACROPHAGES AND AUTOPHAGY MARKER IN BREAST CANCERObjective: To detect the CD68 and LC3 B expressions among breast tissue chip and analyze the correlation between the expressions and clinicopathological parameters. To investigate the correlation between CD68 and LC3 B.Methods: In this research, there are 160 breast cancer tissue specimens in the breast tissue chip. The expressions of CD68 and LC3 B were investigated by immunofluorescent assay. Chi-square test was used to analyze the correlation between CD68(LC3B) expression and other pathological parameters. The bivariate analysis was used to demonstrate the correlation between the LC3 B and CD68. The prognostic influences was analysed with Kaplan-Meier survival analysis.The Cox regression analysis was carried out to evaluate the hazards of breast cancer patients outcomes.Results: The overexpression of CD68 was correlated to pathology stage. The overexpression of LC3 B was correlated to triple-negative breast cancer. In addition, the data indicated that there was a strong correlation between the density of CD68 and LC3 B expression(r=0.327,p<0.001). The Cox regression analysis and Kaplan-Meier survival analysis demonstrated that CD68 and LC3 B could decrease the 10-year overall survival. LC3 B could not only decrease the 5-year overall survival but also be an independent prognostic factor for breast cancer patient outcomes.Conclusion: There is a linear relationship between CD68 and LC3 B in breast cancer tissue. The results revealed that the increased densities of CD68 distributed in high pathology stage of breast cancer tissues. The overexpression of LC3 B was correlated to triple-negative breast cancer.. LC3 B were demonstrated to be an independent prognostic factor for breast cancer patient outcomes.PART II MACROPHAGES INDUCE AUTOPHAGY AND DECREASE SENSITIVITY OF BREAST CANCER CELLS TO CHEMOTHERAPYObjective: To demonstrate the autophagy in breast cancer cells stimulated by macrophages and observe the chemoresistance induced by macrophages.Methods: In this part, LC3 B was tested by western blot in MCF-7 and MDA-MB-468 cells which were co-cultured with macrophages in different times. Autophagosomes were recorded by confocal microscopy after that MCF-7 was infected with GFP-RFP-LC3 adenovirus and stimulated with macrophages. The Atg7 and Beclin1 gene in breast cancer cells were silenced by si-RNA. The breast cancer cells were treated with autophagy inhibitor, CQ. After that, we investigated the LC3 B in MCF-7 and MDA-MB-468 cells by western blot. The apoptosis of MCF-7 and MD-MB-468 cells was tested by flow cytometry after co-cultured with macrophages, company with paclitaxel(TAX) and /or CQ. In vivo study, the tumor weights of mice were measured to observe the effects of macrophages on chemotherapy in different groups. LC3 B was tested in breast cancer tissues of mice by immunohistochemistry.Results: The western blot results were that macrophages induced autophagy in MCF-7 and MDA-MB-468 cells. Confocal microscopy recorded more autophagosomes in experimental group than in control group. CQ could increased the LC3 B while inhibited autophagy flux in breast cancer cells. LC3 B decreased obviously after silenced the Atg7 and Beclin1 in MCF-7 and MDA-MB-468 cells. The apoptosis rates of breast cancer cells stimulated by macrophages were lower than that cultured alone, respectively(p < 0.001). Inhibition of autophagy in breast cancer increased the apoptosis stimulated by TAX。The vivo study showed that the tumor weight in 4T1+ M? group was larger than alone group. TAX had significant effects on 4T1 group while had no significant effects on 4T1+M? group. The expression of LC3 B was higher in 4T1+ M? group than alone group.Conclusion: Macrophages induced autophagy in breast cancer cells and decreased the sensitivity of breast cancer cells to TAX.PART III THE MOLECULAR MECHANISMS OF AUTOPHAGY INDUCED BY MACROPHAGES IN BREAST CANCER CELLSObjective: To further probe the mechanisms of autophagy induced by macrophages in breast cancer cells.Methods: Cytokines assay was used to screen the functional cytokines and ELLISA was used to verify the results. The LC3 B was tested by western blot after breast cancer cells co-cultured with macrophages which were knocked down the cytokines by si-RNA and the cytokine antagonist was used in co-cultured system. We analyzed the activation of MAPK signal pathway in breast cancer cells by western blot. The ERK、p38 and JNK signal pathways were inhibited by U0126 、 SB203580 and SP600125 respectively. After that, the expression of LC3 B was investgated by western blot.Results: MIP-3α、MCP-3 and ENA-78 were discovered to be cytokines candidates during this progression. After MIP-3α、MCP-3 and ENA-78 si-RNA and cytokines antagonist were used, we found MIP-3α had significant effects on autophagy in breast cancer cells. It was indicated that MAPK signal pathway, especially ERK signal pathway played an essential role in autophagy in breast cancer cells. The expressions of MIP-3α was higher in 4T1+ M? group than alone group.Conclusion: Macrophages induced autophagy through activating ERK signal pathway stimulated by MIP-3α in breast cancer cells.