Calcium Channel Blocker Nifedipine Suppresses Colorectal Cancer Progression And Immune Escape By Preventing NFAT2 Nuclear Translocation

Background and Objective:Colorectal cancer(CRC)is one of the most fatal diseases globally,ranking third in cancer morbidity and second in cancer mortality worldwide.Activation of calcium channels has been demonstrated to be closely related to cancer occurrence and development.However,the role of blockers targeting calcium channels in CRC progression and immune regulation remains unclear,and their clinical applications are still limited.The purpose of this study was to investigate the effect of calcium channel blockers on CRC and tumor immunity,and to provide a new idea for clinical application of calcium channel blockers.Methods:1,Geo Database was used to analyze the relationship between calcium signaling and CRC clinical staging;2,Bioinformatics were used to screen the calcium channels with high expression in CRC,and qRT-PCR was used to detect the expression levels of genes related to different types of calcium channels in CRC cell lines and normal cell lines;3,Functional tests were used to screen Nifedipine(NIFE),which inhibits the progression of CRC,from L type calcium channel blockers.Data analysis and qRT-PCR was used to screen NFAT2 from the target family affected by NIFE;4,Immunofluorescence assay was used to detect the subcellular localization of NFAT2.Overexpression vector,CRISPR/Cas9 technique,Transwell,CCK8,subcutaneous tumor formation in nude mice and subserous in situ tumorigenesis model were used to investigate the effect of NFAT2 on the proliferation and metastasis ability of CRC cells in vitro and in Vivo;5,Immunofluorescence assay was used to detect the effects of NIFE and calcium channel agonist,BAY-K-8644(s)-(-)(BAY),on the subcellular localization of NFAT2;6,Confocal fluorescence assay and Co-immunoprecipitation(CO-IP)was used to investigate the relationship between NFAT2 and CRC oncogene,LASP1.Western blot was used to confirme the mechanism of them;7,Chromatin immunoprecipitation(CHIP)and Luciferase reporter was used to investigate and identify the potential targets of NFAT2;8,Flow cytometry was used to investigate the PD-1 expression of CD8+T cells,and subserous in situ tumorigenesis model were used to detect the anti-tumor effect of NIFE in combination with PD-1 inhibitor.Results:1,The expression of calcium signal related genes in CRC tissues was higher than that in normal tissues and positively correlated with the clinical stages of CRC;2,L-type calcium channels were higher expressed in CRC tissues and cell lines than that in normal tissues and cell lines3,NIFE inhibited the progression of CRC,but BAY did the opposite;4,NFAT2 promoted the malignant phenotype of CRC and could be reversed by NIFE;5,NIFE inhibited calcium influx to inhibit dephosphorylation,activation and nuclear translocation of NFAT2;6,LASP1 promoted NFAT2 nuclear translocation via AKT-GSK3β pathway and could be reversed by NIFE;7,NFAT2 recruited STAT3 to promote downstream signal molecules transcription activation;8,NIFE inhibited the expression of PD-L1 on CRC cells and the expression of PD-1 on CD8+T cellsConclusion:Our findings provide direct evidence that the calcium channel blocker NIFE is a promising clinical therapy to treat patients with advanced CRC by affecting the tumor itself and tumor immunity.Together,NIFE provides promising therapeutic selection and combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in CRC.