TGF-β Combined With An FGFR Inhibitor Induces The Differentiation Of Hepatoma Cells Into Fibroblast-like Cells

Objective: Tumors are also known as "Wounds that never heal," because they mimic the chronic inflammation that occurs when a wound reacts.In the early stages of normal wound healing,upregulation of TGF-β and bFGF expression promotes tissue regeneration.However,late in wound healing,activation of the TGF-β pathway and downregulation of the bFGF pathway lead to fibers scarring.However,the inactivation of the TGF-β pathway in tumor cells and the continued activation of the bFGF signaling pathway are among the important factors leading to tumor proliferation,so tumors never heal like normal wounds.Tumor formation involves the continuous activation of bFGF pathway,while tissue damage repair is a self-limiting process.At the end of wound healing,sufficient TGF-β and decreasing bFGF in the tissue lead to the cessation of cell proliferation,matrix deposition,and fibrous scar.However,TGF-β1 inhibits tumor growth in early cancer,but in late cancer TGF-β1 promotes tumor cell proliferation and metastasis by inducing EMT and interacting with other growth factors.In this study,we used TGF-β1 in combination with AZD4547 to inhibit the tumor bFGF pathway and activate the tumor TGF-β pathway at the same time,to explore whether this regulatory mode inhibit tumor growth and promote the formation of fibrous scar.Methods 1.CCK8 cytotoxic activity test and table blue exclusion test were used to detect the effects of TGF-β1 and AZD4547 used alone or in combination on the proliferation of SMMC-7721 and HepG2 cell lines of liver cancer.2.Transwell and wound healing assay were used to detect the effect of TGF-β1 and AZD4547 on the migration of SMMC-7721 and HepG2 cell.Immunofluorescence assay was used to further detect the effect of TGF-β1 and AZD4547 on tumor cell migration related proteins,such as E-cadherin and N-cadherin.3.Flow cytometry(FCM)was used to detect the phenotypic changes of the TGF-β1 and AZD4547 treated cells and the markers commonly used in fibroblasts and mesenchymal cells,such as CD90,CD105,CD140 B,CD13 and HLA-ABC.4.Subcutaneous xenograft of nude mouse liver cancer cell line(HepG2)was used to detect the inhibitory effect of single use and combined use of TGF-β1 and AZD4547 on the in vivo xenograft tumor of liver cancer cell line,and PCNA immunohistochemistry was used to detect the effect of drugs on the proliferation of xenograft tumor.5.Immunohistochemistry and collagen staining were used to detect the degree of fibrosis and the expression of fibroblast markers in the transplanted tumor sections after TGF-β1 and AZD4547 treatment.6.Gene sequencing results confirmed that there were significant differences in gene expression between TGF-β1 and AZD4547 induced differentiation cells and HCC cells.After drug addition,the expression of hepatocellular carcinoma specific genes in tumor cells decreased,the expression of fibroblast related genes in tumor cells increased,and the change of gene expression after drug addition was more similar to fibroblast.7.By summarizing the results of gene sequencing,bioinformatics analysis was carried out,and then the relevant pathway proteins induced by liver cancer cell transdifferentiation were experimentally verified.The protein expression in TGF-β pathway was verified by Western blot to search for the pathway proteins that may be closely related to the induced differentiation and tumor inhibition.Results: 1.In vitro experiments with TGF-β1 and AZD4547 significantly inhibit the proliferation and migration of liver cancer cells.The inhibitory effect of TGF-β combined with AZD4547 on hepatoma cells was stronger than of AZD4547 alone.2.The combination of TGF-β and AZD4547 inhibited the xenograft tumor of hepatocarcinoma cells in vivo(inhibition rate was 55%),which was better than AZD4547 alone(35% inhibition rate).3.In vivo experiments,TGF-β and AZD4547 combined drugs can promote tumor cell fibrosis while inhibiting tumor growth.4.Cellular experiments have shown that the combination of TGF-β and AZD4547 can induce tumor cell morphology to become spindle-shaped and promote the expression of vimentin,type I collagen and fibronectin in tumor cells.5.The combination of TGF-β and AZD4547 can significantly change the phenotype of tumor cells and promote the expression of stromal cell markers CD90,CD105,CD140 B,CD13 and HLA-ABC.6.The results of gene sequencing confirmed that the expression of TGF-β1 and AZD4547 induced differentiation was significantly different from that of the original hepatoma cells.The expression of hepatocarcinoma-specific genes in tumor cells was decreased after TGF-β1 and AZD4547 administration.The expression of fibroblast-related genes in tumor cells was increased after induction,and the expression of induced genes was closer to fibroblasts.7.The study found that TGF-β1 combined with AZD4547 can maintain the level of p-Smad3 in tumor cells while decreasing the level of p-Erk,indicating that Smad pathway plays an important role in the differentiation process.8.The same growth inhibition and differentiation induction effects was achieved with other FGFR inhibitors.The morphological changes of tumor cells and the increased expression of Vim and COL I were still observed using FGFR inhibitor(BGJ398)and MEK inhibitor(PD98059)instead of AZD4547 in combination with TGF-β1.Conclusion Combined use of TGF-β1 and AZD4547 can induce hepatocellular carcinoma cells to differentiate into normal fibroblasts,and the morphology,molecular phenotype,functional protein and gene expression of the differentiated cells are similar to that of normal fibroblasts.In vivo experiments showed that TGF-b1 combined with AZD4547 not only inhibited tumor growth,but also promoted tumor parenchymal fibrosis.Increased phosphorylation of Smad3 protein and decreased phosphorylation of Erk protein are key processes for TGF-β1 combined with AZD4547 to induce transdifferentiation of hepatoma cells.The induction of differentiation may not be achieved by TGF-β1 or AZD4547 alone.Our results suggest that FGFR inhibitors reverse the tumor-promoting effect of TGF-β1 in hepatoma cells into tumor inhibition and induce tumor cells to express fibroblast-related genes.In vivo and in vitro experiments showed that TGF-β1 combined with AZD4547 had stronger inhibitory effect on tumor growth than AZD4547 alone.This study provides a new strategy for liver cancer transformation research and multi-target cancer target therapy research.