Translocation Of Cx43 In Cancer Cells Promoted By Cancer Associated Fibroblasts In Colorectal Cancer

Background: Colorectal cancer is one of the most common malignant tumors in the world.Metastasis is still the leading cause of death in patients with colorectal cancer.Studies have shown that tumor metastasis is closely related to tumor microenvironment.Cancer associated fibroblasts(CAFs)are the major components of tumor stroma,which can secrete a variety of cytokines to interact with tumor cells.The function of Cx43 in tumors is still unclear.Some studies have revealed that Cx43 could inhibit tumor progression while other researches have shown that Cx43 acted as an oncogene.Our previous researches have found that when colorectal cancer cells were co-cultured with CAFs,the expression of Cx43 would be up-regulated and Cx43 would gather towards the nuclei in tumor cells,thus enhancing their migration and invasion.The clinical samples showed that patients with Cx43 nuclear expression had poorer prognosis than those without Cx43 nuclear expression.However,Cx43 localized at the cytoplasm of SW480 and SW620 cells when overexpressed and inhibited their migration and invasion.Purposes: To elucidate the relationship between the subcellular localization of Cx43 and its dual functions,to clarify how CAFs up-regulate the expression of Cx43,to find the possible molecular mechanism of Cx43 translocation from cytoplasm to nuclei,and to dig out the potential of Cx43 to be a new indicator of clinical diagnosis and treatment,prognostic assessment and efficacy monitoring.Methods: Survival analysis and forest plots were used to evaluate the relationship between Cx43 expression in CRC patients and their prognosis in TCGA and GEO databases.CRC cell lines overexpressing Cx43 stably were constructed by lentivirus.Nuclear and Cytoplasmic Protein Extraction Kit and confocal microscopy were applied to detect Cx43 localization.Transwell and CCK8 assays were used to compare the invasion,migration and proliferation properties of tumor cells.The effect of Cx43 on subcutaneous tumor formation in vivo was detected in nude mice.Transcriptome sequencing and phosphorylation site mutation were applied to explore the molecular mechanism of Cx43 nucleation.Results: In this study,we found that colorectal cancer patients with high Cx43 expression had poor overall and disease-free survival in the TCGA and GEO databases.Different localizations of Cx43 were found in colorectal cancer cell lines.Cx43 was expressed at cytoplasm in SW480 and SW620 cells,while it was expressed at nuclei as well as cytoplasm in RKO cells.Cx43 inhibited the invasion,migration and proliferation of SW480 and SW620 cells,but it promoted the invasion,migration and proliferation of RKO cells,performing dual functions.TGF-β secreted by CAFs could mediate the up-regulation and nuclear aggregation of Cx43.PKC kinase could phosphorylate Cx43 S368 site,thus increasing its nuclear expression and promoting the invasion and migration of tumor cells.Conclusions: 1.High expression of Cx43 is associated with poor prognosis of patients with colorectal cancer in TCGA and GEO databases.2.Cx43 with different subcellular localization has different functions.3.TGF-β secreted by CAFs can promote the up-regulation and nuclear aggregation of Cx43 in colorectal cancer cells.4.The phosphorylation of Cx43 S368 site could increase Cx43 nuclear expression,thus increasing the migration and invasion properties of tumor cells.