Epithelial-mesenchymal Transition Of Colon Cancer Cells Induced By FAK Pathway In Cancer-associated Fibroblasts

Objective To investigate the role and mechanism of cancer-associated fibroblasts in colon cancer Epithelial-Mesenchymal Transition(EMT)under three-dimensional culture conditions,and to study the role of Tetrathiomolybdate(TM)in the development of colon cancer.Methods(1)The cancer-associated fibroblasts(CAFs)and normal fibroblasts(NFs)from colon cancer were cultured and identified by immunofluorescence and Western blot.(2)The expression of LOXL2(lysyl oxidase-like protein 2)in CAFs and NFs cells was detected by ELISA.(3)Matrigel was used as a scaffold material to establish a 3D co-culture model of colon cancer LOVO cells with CAFs and NFs.(4)Flame atomic absorption spectrometry was used to determine the trace elements in the supernatant of each group.(5)Western blot was used to detect the expression of EMT-related proteins such as E-cadherin and N-cadherin,the expression of signal pathway-related proteins such as FAK(focal adhesion kinase)and P-FAK.(6)And the effect of chelator of copper TM on CAFs on the expression of related proteins in LOVO cells.Results(1)The expression of α-smooth muscle actin(α-SMA)in CAFs was significantly higher than that of NFs,suggesting that the CAFs frozen in the previous experiments still retain their biological characteristics and can be used as follow-up experimental studies.(2)The level of LOXL2 secreted by CAFs is higher than that NFs(P<0.05);(3)Compared with trace elements in LOVO cell group,CAFs+LOVO co-culture group,copper content increased significantly,zinc content decreased significantly,and the difference was statistically significant;calcium,magnesium and iron had no significant difference;(4)In the CAFs+LOVO three-dimensional cell co-culture group,the expression level of E-cadherin was significantly decreased(P<0.05),and N-cadherin was significantly increased(P<0.05),indicating that CAFs promoted epithelial-mesenchymal transition in colon cancer LOVO cells;FAK was activated during this process and phosphorylation occurs.(5)The results also showed that TM can reduce copper in the tumor microenvironment and inhibit the occurrence of EMT by inhibiting the activation of lysyl oxidase FAK,thereby significantly reducing the invasion and metastasis of tumor cells.Conclusion Therefore,CAFs stimulates epithelial-mesenchymal transition(EMT)of human colon cancer LoVo cells by secreting LOXL2 to activate FAK signaling pathway,thereby promoting tumor metastasis.TM can inhibit the occurrence of EMT in the CAFs-induced colon cancer LoVo cell line,thereby reducing the invasion and metastasis of colon cancer cells.