VAV2 Is Required For DNA Repair And Implicated In Cancer Radiotherapy Resistance

Background&Aims:Radiotherapy remains the mainstay for treatment of various types of human cancer;however,the clinical efficacy is often limited by radioresistance,in which the underlying mechanism is largely unknown.Esophageal squamous carcinoma(ESCC)is one of highly prevalent malignant tumors in China.Here,using ESCC as a model,we established ESCC patient-derived xenografts(PDXs),primary cells and radioresistant cell lines.Integrated 149 amplified and overexpressed genes in 94 ESCC compared with normal tissues and differentially expressed genes in radioresistant ESCC,we demonstrate that guanine nucleotide exchange factor 2(VAV2),which is overexpressed in most human cancers,plays an important role in primary and secondary radioresistance.This study was to identify differential genes whose aberrant expression may cause radioresistance through PDXs and investigate the functional roles leading to radioresistance in order to provide the efficient therapeutic targets for ESCC.Methods:The radiosensitivity of VAV2 was verified in ESCC cells with VAV2 overexpression or knock out using the cell proliferation,colony formation and extreme limiting dilution assays(ELDA).Western blot was used to confirm the role of VAV2 in DNA damage response by collecting the protein at the same time points of different IR doses or different time points of same IR dose in ESCC cell lines.We then established the radioresistant ESCC cell lines and detected the expression of VAV2 from mRNA and protein levels to confirm its role in secondary resistance.RNA sequencing was performed of KYSE150 cells with VAV2 overexpression or knock down to analyze the differentially expressed genes and pathways using gene set enrichment analysis(GSEA).Western blot,immunofluorescence and comet assays were used to examine the levels of target gene expression and DNA double-strand breaks.The proteins interacting with VAV2 were analyzed by immunoprecipitation and target proteins potentially regulated by VAV2 were analyzed by proteomics.In vitro and in vivo studies were conducted using the inhibitor to analyze the sensitivity.The survival of patients accepted radiotherapy from our ESCC and TCGA 20 tumors were calculated hazard ratios by Cox models.Results:We identified VAV2 plays a radioresistant oncogene in ESCC and found that cells overexpressing VAV2 were less sensitive to IR compared with cells without VAV2 overexpression and silencing VAV2 expression in primary radioresistant ESCC cells re-conferred sensitivity to IR.VAV2 levels increased gradually with irradiation time and dose.The mRNA and protein expression levels of VAV2 increased in radioresistant cells compared with the paternal cell line.GSEA reveals G2/M and DNA repair is upregulated in cells with VAV2 overexpression and downregulated in cells with VAV2 knock down.The interaction of VAV2 with the Ku70/Ku80 complex was confirmed by mass spectrometry and immunoprecipitation assays.Immunofluorescent staining of ESCC cells and multiple immunofluorescent staining of ESCC tumor specimens showed the stronger co-localization of VAV2 with Ku70/Ku80.Furthermore,VAV2 overexpression excessively activates signal transducer and activator of transcription 1(STAT1)signaling and Fludarabine,a STAT1 inhibitor and anticancer drug,significantly increases the sensitivity of VAV2 overexpressing ESCC to irradiation indicated by DNA damage and tumor growth.We also show that VAV2 level in cancer may predict radiotherapy vulnerability.Conclusion:VAV2 functions as an oncogene that plays an important role in primary and secondary radioresistance.VAV2 is required for the Ku70/Ku80 complex formation and participates in non-homologous end joining repair(NHEJ)of DNA damages,which substantially upregulates ST AT1.VAV2 is significantly overexpressed in most cancers and is associated with poor prognosis after radiotherapy.Targeting VAV2-STAT1 pathway significantly increases radiosensitivity and can be used as an effective treatment to improve radiosensitivity of ESCC.