TGF-β1-induced The Formation Of Cancer-associated Fibroblasts Phenotype In Tumor Microenvironment Of Breast Cancer

Tumor microenvironment has emerged as an important target for cancer therapy. For most solid tumors, particularly carcinomas, their microenvironment consists of the tumor cells themselves, endothelial cells, immune cells and fibroblasts contribute to tumorigenesis by secretion of cytokines and/or direct cell-cell contact. In particular, a subpopulation of fibroblasts, the so-called cancer-associated fibroblasts(CAFs), seems to facilitate tumor initiation, progression, and metastasis. Transforming growth factor-β1(TGF-β1) present in tumor microenvironment acts in a coordinated fashion to either suppress or promote tumor development. However, the molecular mechanisms underlying the effects of TGF-β1 on tumor microenvironment are not well understood. Our clinical data showed a positive association between TGF-β1 expression and cancer-associated fibroblasts(CAFs) in tumor microenvironment of breast cancer patients. Thus we employed starved NIH3T3 fibroblasts in vitro and 4T1 cells mixed with NIH3T3 fibroblasts xenograft model in vivo to simulate nutritional deprivation of tumor microenvironment to explore the effects of TGF-β1.We utilized a vitro model with NIH3T3 mouse embryonic fibroblasts challenged with serum starvation(Star), to investigate TGF-β1(2.5 ng/ml) induced effects on starved NIH3T3 fibroblasts. Experimental results showed that TGF-β1 protected NIH3T3 fibroblasts from Star-induced growth inhibition, mitochondrial damage and cell apoptosis. Interestingly, TGF-β1 induced the formation of CAFs phenotype in starvation(Star)-treated NIH3T3 fibroblasts. Meanwhile, Experimental results showed that TGF-β1 increased the ratio of MDC staining and the expression of autophagy related genes and proteins in Star-stressed cells, which was found to be prevented by autophagy inhibitors such as 3-MA. Our TEM and confocal fluorescence imaging data also clearly indicated that TGF-β1 promoted the mitophagy.Autophagy is one of the main mechanisms of repairing cell damage under the condition of stress. Moreover, autophagy in tumor microenvironment promoted tumor growth and progression in conditions of hypoxia and metabolic stress. Therefore, the current study utilized starvation of NIH3T3 simulates deprivation to investigate the mechanism of TGF-β1 induced protective effects and the formation of CAFs in Star-treated NIH3T3 fibroblasts. Several experiments were performed, including(1) use of autophagy promoter(Rapa) and autophagy inhibitors(3-MA) to explore whether autophagy is involved in TGF-β1 induced protective effects and the formation of CAFs. TGF-β1 treatment could significantly protect NIH3T3 cells from nutrition deprivation and the formation of CAFs phenotype, the effect of TGF-β1 was enhanced by Rapa while abolished by 3-MA treatment.(2) RNA interference was used to investigate autophagy level, cell apoptosis and necrosis and the expression of CAFs phenotypic maker proteins α-SMA and FAP-α after SIRT3 was being silenced by siRNA. Results showed that ATG5 knockdown not only had almost completely inhibited Star- or Star+TGF-β1-induced Beclin 1 expression and LC3β-II/I conversion, but also could block the effect of TGF-β1 on apoptosis protection and the formation of CAFs phenotype. The results revealed the involvement of autophagy in TGF-β1-induced formation of CAFs phenotype in Star-treated NIH3T3 fibroblasts.(3) The use of TGF-βR1/ALK5 inhibitor LY-2157299 to investigate the molecular mechanisms underlying TGF-β1-induced autophagy caused protective effects and the formation of CAFs in Star-treated NIH3T3 fibroblasts. Experimental results showed that LY-2157299 suppressed TGF-β1-induced the upregulation of p-Smad2, p-Smad3, Beclin 1 and LC3β-II/I conversion and the protective effect on mitochondria and formation of CAFs phenotype in Star-treated NIH3T3 fibroblast. These data suggested that TGF-β1 induced protection and formation of CAFs phenotype was through TGF-β/Smad autophagy signaling pathway.In order to confirm that autophagy was involved in TGF-β1 induced cellular protective action and CAFs transformation under nutritional deprivation of tumor microenvironment, we used the mixed xenograft tumor model with the ratio of 1:2(4T1 breast cancer cells: NIH3T3 fibroblast cells) as an in vivo model. Our results showed that treatment of TGF-β1 to starved NIH3T3 cells in the mixed xenograft tumor not only could increase the expression of both autophagy related protein and CAFs markers, but also could promote tumor growth and reduced Star-induced necrosis and apoptosis in mixed xenograft tumor. However, 3-MA treatment reduced the effects of TGF-β1.In conclusion, we found that TGF-β1 promoted the survival of starved NIH3T3 fibroblasts, and activated the formation of CAFs phenotype in the tumor microenvironment, which could promote tumor growth. It is noteworthy that autophagy inhibitor 3-MA, Atg5 siRNA or LY-2157299 blocked TGF-β1-induced protective effects and the formation of CAFs phenotypes, suggesting that TGF-β1-induced these effects through TGF-β/Smad autophagy signaling pathway. The autophagy mechanisms of TGF-β1, thus, make it a unique, potential and more useful to induce formation of CAFs phenotype to promote tumor growth. Therefore, TGF-β1-induced autophagy is a significant determinant in tumor growth and progression for breast cancer therapies.