The Role Of SGK1 In Modulation Radiosensitivity In Locally Advanced Rectal Cancer

Background:Colorectal cancer(CRC)is a common malignant tumor and there is an upward trend in the incidence,whether in western countries or China.Neoadjuvant chemoradiotherapy(chemoradiotherapy+surgery+postoperative+adjuvant+chemother-py)has become the standard treatment for locally advanced rectal cancer(LARC).After neoadjuvant chemoradiotherapy,about 20%-25%show pathological complete response(pCR),30%of them can not reach pCR because of radioresistences.SGK1 plays a critical role in regulating apoptosis,DNA damage,signal transduction,and ion transmembrane transport.However,There are few report on the relationship between SGK1 and radiotherapy and the underlying mechanism.Therefore,we studied the correlation and mechanism between SGK1 and radiosensitivity,in order to explore its value and clinical significance as a radiosensitizing target.Methods:1.The biopsy specimens were obtained from the local advanced rectal patients admitted to the Sun Yat-sen University Cancer Center.And divided into pCR group(radiosensitive group)and non-pCR group(radioresistance group).Expression of SGK1 in human colorectal cancer biopsy tissue samples and cell lines were detected by transcriptome sequencing,immunoblotting,RT-PCR and IHC.2.The mean lethal dose(DO)and 20%survival fratration(SF2)were calculated according to the platelet clone formation experimental fit survival curve(click multi-target model)to analyze the radiosensitivity of different cells.3.Transcriptome sequencing and mutantions were used to explore the molecular mechanism of SGK1 affecting radiosensitivity4.Statistical analysis:Differences between groups were compared using non-paired T test or one-way ANOVA,and a significant difference was considered when P<0.05.Results:1.Expression of SGK1 in colorectal cancer and its correlation with radiosensitivityThe transcriptome sequencing of rectal tumor tissue of newly diagnosed biopsy showed that the expression level of SGK1 in non-pCR group(n=6)was significantly higher than that in pCR group(n=6)(p<0.05).There was a significant correlation between the level of SGK1 and the radiosensitivity in each four intestinal cancer cell lines HT29,SW837,DLD1 and LOVO.Regardless of western or RT-PCR,SGK1 levels in HT29 cells were the highest in six colon cancer cell lines,and SF2 was likewise.The expression of SGK1 was higher in the resistance group by IHC.2.Overexpression of SGK1 increases cell resistance to radiationWhen overexpress SGK1-T1 or SGK1-T3 transcripts were inbuced successfully in LOVO cells,the proliferation of LOVO cells were promoted.At the same time,the DO of LOVO-SGK1-T1 and LOVO-SGK1-T3 were increased,indicating that the cells were ray-dependent.The sensitization ratios(SER)of LOVO-SGK1-T1 and LOVO-SGK1-T3 were 0.718 and 0.594,indicating that the sensitivity was reduced.3.In vitro SGK1 inhibitor can increase the sensitivity of cells to radiationThe SER of the in vitro inhibitor GSK650394 against LOVO cells reached 3.59.Similarly,HT29 with higher expression of SGK1 has higher sensitivity to radiation after SGK1 inhibitor GSK650394 and ray combination,and the SER was 1.45.All results showed that the radioresistant of cells was reduced after the combination treatment with SGK1 inhibitor GSK650394 and radiation.4.In vivo SGK1 inhibitors increase cell sensitivity to radiationThe tumor volume of the combination group was statistically different from that of the control group(p=0.009).The tumor volume of the SGK inhibitor treated group and the ray combination treated group were also smaller than that of the ray treated alone group,They all demonstrated that the use of in vivo SGK1 inhibitors combined with radiation can increase the therapeutic effects of radiation.5.The molecular mechanism of SGK1 affecting radiosensitivityBased on functional enrichment and analysis,the following three pathways were found to be the mainly participated in affecting radiosensitivity:transcription factor ATF3,sputum interferon pathway,and P53 signaling pathway.Further RT-PCR verificating results showed that the core genes involved in these pathways changed their expression levels in SGK1 inhibitors treated and ray-linked cells.Conclusion:This study showed that SGK1 was an important metabolic molecule affecting the sensitivity of colorectal radiotherapy.The possible mechanism may be achieved by transcription factor ATF3,sputum interferon pathway and P53 signaling pathway.This study also showed that inhibition of SGK1 could improve the therapeutic effect of intestinal cancer radiotherapy.In conclusion,this study reveals the role of SGK1 in influencing radiotherapy response and provides potential sensitization measures for patients with radiotherapy resistance.