The Effect Of NS1-BP On ESCC Radiotherapy Response And The Underlying Mechanisms

Objective Neoadjuvant or definitive chemoradiotherapy(CRT)is a standard treatment for advanced-stage oesophageal squamous cell carcinoma(ESCC),and radiotherapy is critical to the therapeutic strategy.NS1-BP(a member of the BTB-Kelch protein family)has been shown to inhibited Hela cell proliferation by suppressing c-Myc.However,its significance in human cancers remains unclear.Our study was to explore the expression level and clinical significance of NS1-BP in ESCC,and the effect of NS1-BP on ESCC radiosensitivity.Methods We used immunohistochemistry to detect the expression dynamics of NS1-BP in a learning cohort(n=98)and a validation cohort(n=46)of patients with ESCC treated with CRT.The present study aimed to investigate NS1-BP expression in ESCC and its clinical prognostic significance.Furthermore,ESCC cell line were stably transferred with retroviruses coexpressing the c DNA of NS1-BP,and then overexpression of NS1-BP cell line was constructed.The Control group is this ESCC cell line with a lentivirus carrying the corresponding control scrambled c DNA sequence.Blank group is the normal ESCC cell line.By Western blotting assay,MTT proliferation assay and Annexin-V-FITC ? PI flow cytometry apoptosis assay,we detected the effect of NS1-BP overexpression on cell growth and apoptosis in ESCC.Subsequently,we used in vitro and in vivo experiments to study the underlying mechanism of NS1-BP's effect on ESCC tumourigenesis and radiotherapy response.Results1.NS1-BP was frequently down-regulated in ESCC tissues and cells NS1-BP expression was also examined using immunohistochemistry(IHC)in 144 ESCC tumor tissues and 30 normal esophageal mucosa tissues.And NS1-BP was strongly down-regulated in these ESCC samples.Western blotting found that all five ESCC cell lines expressed lower levels of NS1-BP than control cells. 2.Down-regulation of NS1-BP predicted CRT resistance According to our IHC evaluation criteria,cases were classified into two different groups: low expression cases and cases with high expression.The analysis of clinicopathological characteristics show low NS1-BP expression was positively associated with CRT resistance in both the learning cohort(P = 0.007)and the validation cohort(P= 0.003).And down-regulation of NS1-BP predicted a shorter disease-specific survival.3.NS1-BP suppressed cell growth and promoted apoptosis in ESCC The NS1-BP construct was cloned and stably transfected into TE-1 and Kyse-30 cells,which were demonstrated to have moderate NS1-BP expression levels among our available ESCC cell lines.The MTT assay showed that ectopic overexpression of NS1-BP inhibited cell growth and flow cytometry analysis indicated that NS1-BP significantly promoted apoptosis in TE-1 and Kyse-30 cells compared with control cells under normal conditions.And loss-of-function studies revealed a consistent result.4.NS1-BP levels modulated ESCC radiosensitivity in vitro and in vitro The colony formation assay showed that the survival capacity of NS1-BP-overexpressing ESCC cells was lower than that of control cells after IR treatment.Compared with negative controls,the enhanced expression of NS1-BP significantly increased the apoptosis rate of cells treated with IR.We next evaluated the effect of NS1-BP on the repair of IR-induced DNA damage in ESCC cells.Increased numbers of ?H2AX foci were present in NS1-BP-overexpressing TE-1 and Kyse-30 cells after IR treatment.And loss-of-function studies revealed a consistent result.In vivo experiment,NS1-BP enhanced the radiosensitivity of ESCC cells.5.NS1-BP inhibited MYC signaling pathways by transcriptionally suppressing c-Myc Our study consistently showed NS1-BP have a suppressive effect on c-Myc expression.Subsequent western blot analysis confirmed that key proteins required for the cell cycle checkpoint were also altered following a c-Myc decrease.In particular,enhanced expression of NS1-BP significantly inhibited radiation-induced ATM/Chk1 phosphorylation,but had no effect on Chk2 phosphorylation.To further evaluate the mechanism by which NS1-BP suppresses c-Myc expression,we used luciferase reporter assay and Ch IP assay to test that NS1-BP transcriptionally suppressed the expression of c-Myc.Conclusions Our findings highlight the etiology and importance of NS1-BP in ESCC and radiotherapy.Targeting the NS1-BP/c-Myc pathway may provide a novel therapeutic strategy for ESCC.