Study On The Mechanism Of TGF-β1 Induces CAFs Formation And Promotes Breast Cancer Invasion And Metastasis

Background:Breast cancer is a malignant tumor that threatens the life and health of women in the worldwide,invasion and metastasis is one of the maker of malignant tumor,more than 90%of the cancer patients died for cancer metastasis.Breast cancer is a highly complex microenvironment system that composed of tumor cells and mesenchymal cells,tumor cells can produce high levels of reactive oxygen species（ROS）,which induces stromal fibroblasts to undergo oxidative stress and affect the stability of tumor cell genomes,mesenchymal cells can secreting some growth factors to regulate the tumorigenesis and development,which plays an important role in the process and metastasis of tumor.In the tumor microenvironment,fibroblasts are activated to cancer-associated fibroblasts（CAFs）,CAFs are the most abundant cellular components in the tumor stroma,they can promote tumor proliferation,invasion and metastasis,but the mechanism of it still unknow.Epithelial-mesenchymal transition（EMT）of tumor cells is considered to be a critical step in tumor metastasis.Transforming growth factor-β1（TGF-β1）signaling pathway is a classic pathway for EMT.TGF-β1 is also a potential autophagy activator,it can promote the generation of fibrosis.Previous research found that TGF-β1 can induce autophagy in serum-starved fibroblasts,and it plays a crucial role in the formation of CAFs.However,what is the mechanism of CAFs formation?How CAFs promote tumor invasion and metastasis?Which deserves our further discussion.Objectives:1.To explore the mechanism of cancer-associated fibroblasts（CAFs）formation in the tumor microenvironment.2.To detect the effect of cancer-associated fibroblasts（CAFs）on breast cancer invasion and metastasis,and investigate the mechanism of CAFs in breast cancer invasion and metastasis.Methods:1.Detecting cell survival rate by MTS method,detecting MDA content,SOD activity by microplate reader,and detecting ROS content by flow cytometry.2.Investigating mRNA and protein expression ofα-SMA,FAP-α,Cav-1,Beclin1,LC3β,E-Cadherin,N-Cadherin by RT-qPCR and Western blotting.3.Observing the autophagic structure of the cells by transmission electron microscopy and detecting FAP-αand LC3βCo-localization by confocal laser.4.The invasion and migration abilities of 4T1 cells were investigated by wound healing and migration and invasion assay.5.Interfering the expression of FAP-αby siRNA method.6.Establishing a mixed xenograft tumor model of NIH3T3 cells and 4T1 cells,Results:1.After pretreatment with 100μM H₂O₂ on NIH3T3 cells for 24 hours,the cell survival rate was between 50%and 70%that suggested the cell was sensitive with H2O2,MDA content and ROS level were significantly augmented by H2O2 while SOD activity was weaken by it（P<0.05）,which resulting in oxidative stress on NIH3T3 cells.2.The survival rate and SOD activity of NIH3T3 cells were increased significantly by 2.5 ng/mL TGF-β1 while the content of MDA and ROS were reduced by it,Which alleviated the oxidative damage of the cells.In addition,the mRNA and protein expressions ofα-SMA and FAP-αwere up-regulated significantly by TGF-β1 while Caveolin was down-regulated by it on NIH3T3 cells that treated by H2O2,Which suggested TGF-β1 promoted the NIH3T3 cells that pretreated by H₂O₂ convert into CAFs.3.TGF-β1 significantly up-regulated the mRNA and protein expression of Beclin1and LC3β,enhanced LC3II accumulation as the falling of LC3I.RaPa enhanced the effects of TGF-β1 while 3-MA inhibited it.The deformation of mitochondria were packaged by Autophagosome in NIH3T3 cells treated by H2O2,TGF-β1 increased autophagosomes and alleviates mitochondrial damage caused by oxidative stress.In addition,TGF-β1 enhanced the expressions of FAP-αand LC3βon NIH3T3 cells and increased colocalization of FAP-αand LC3β.However,the autophagy inhibitor 3-MA inhibited it.All the results suggested TGF-β1 may promote the conversion of NIH3T3cells that pretreated by H2O2 to CAFs by autophagy.4.After 4T1 cell cocultured with NIH3T3 condition medium for 24 hours,TGF-β1 can not only significantly increased the healing percentage of cancer cell but also the number of invasion and metastasis cells.5.In the NIH3T3 cells and 4T1 cell coculture system,the protein expression of E-Cadherin was down-regulated by TGF-β1 while N-Cadherin up-regulated by it,RaPa enhanced the effects of TGF-β1 while 3-MA inhibited it.All the results suggested that TGF-β1 may induce CAFs formation by stimulating fibroblast autophagy.that promote the tumor cells invasion and metastasis After interfering FAP-αexpression,the number of invasion and metastasis cells decreased significantly,the process of EMT was reversed by it,Which suggested TGF-β1 may promote EMT by inducing CAFs formation that promote cancer cells invasion and metastasis.6.Tumor volume,tumor weight increased significantly and the necrosis area of tumor was decreased after injected with mixed cells of NIH3T3 and 4T1.and gene expression of CAFs markers（α-SMA and FAP-α）and autophagy markers（Beclin1 and LC3β）were increased.The mRNA and protein expression of the E-Cadherin were decreased while N-Cadherin was increased significantly.All the results suggested fibroblasts can conversion to CAFs and promote EMT process of tumor in mixed transplantation tumor of animal.In addition,lung metastases were increased compared with 4T1 group,Which further illustrated that the conversion of fibroblasts to CAFs can promote the tumor cells invasion and metastasis by EMT process.Conclusions:1.Autophagy was involved in TGF-β1-induced protection and CAFs phenotype formation in NIH3T3 cells with H2O2-treated.2.TGF-β1 can promote CAFs to initiate epithelial-mesenchymal transition（EMT）by increasing FAP-αexpression that promoting tumor invasion and metastasis.3.4T1:NIH3T3=1:2 mixed vaccination can promote tumor growth and lung metastasis.