TRPM4 Inhibited Colorectal Cancer Metastasis Through Ca²⁺/Calpain-mediated Suppression Of FAK-PI3K/Akt/mTOR Signaling Pathway

Background:Colorectal cancer(CRC)is a common digestive malignancy clinically with high morbidity and mortality rates.It is frequently diagnosed in the advanced stages with metastasis,which made it of great significance to find potential target for CRC metastatic therapy.The expression of membrane protein Transient Receptor Potential Cation Channel Subfamily M Member 4(TRPM4)is remarkably downregulated or silenced in CRC due to the epigenetic methylation in its promoter region,and the calcium level is markedly higher in CRC cell lines after TRPM4 transfection.Both DNA methylation and calcium signaling pathway play vital roles in tumorigenesis,tumor invasion and migration.However,there is no evidence for whether TRPM4 could be identified as a novel methylated Tumor Suppressor Gene(TSG)and affects CRC development and metastasis through regulating calcium signal.Objective:We aim to investigate the biological function and molecular mechanism of methylated TRPM4,particularly to elucidate whether TRPM4 could affect CRC metastasis by calcium mediated downstream signaling pathway,which provide scientific evidence for CRC diagnosis and therapy based on TRPM4.Methods:The methylation of TRPM4 was evaluated by methylation specific PCR.TRPM4 expression was determined by semi-quantitative RT-PCR and immunohistochemistry.After stably transfection of TRPM4 expression plasmid into CRC cell lines,the efforts of TRPM4 on tumor cell proliferation,migration,cytoskeletal reorganization and calcium homeostasis were tested in vitro and in vivo.The signaling pathway regulated by TRPM4 was assessed by Western Blot.Results:The expression of TRPM4 was reduced or silenced in most CRC tissues and cell lines with high methylation on promoter region.Low expression of TRPM4 was strongly correlated with high clinical stage(p<0.001),more lymph node metastases(p=0.008),more distant metastases(p=0.025)and invasion(p<0.001).The overall survival rate was considerably higher in patients with TRPM4 overexpression versus those with lower expression.After TRPM4 transfection,tumor cell proliferation and migration were significantly inhibited in vitro and in vivo,and TRPM4 restructured CRC cytoskeleton.The results of flow cytometry and Western Blot showed that TRPM4 activated Ca3+-mediated calpain pathway through enhancing calcium influx,inducing Focal Adhesion Kinase(FAK)proteolysis and suppression of Phosphatidylinositol 3-kinase/Protein Kinase B/Mammalian Target of Rapamycin(PI3K/Akt/mTOR)signaling cascade,resulting in CRC metastasis.Simultaneously,TRPM4 changed expression of multiple migration-related proteins.The inhibition of protease calpain effectively relieve the retard of FAK/Akt signals and reverse the metastatic suppression of TRPM4.Conclusions:As a novel methylated TSG,TRPM4 could markedly inhibit CRC growth,invasion and migration and triggered cytoskeletal remodeling.TRPM4 activated calcium-calpain signaling to impede FAK-PI3K/AKT/mTOR pathway,leading to metastatic suppression.Consequently,TRPM4 is likely to be a promising biomarker and potential target for CRC diagnosis and metastatic therapy.