The Role And Mechanism Of SENP5 In Promoting Homologous Recombination Repair To Regulate Radiotherapy Resistance In Rectal Cancer

Backgroud:Colorectal cancer is the third most common cancer and the second most leading cause of cancer-related mortality worldwide.Currently,neoadjuvant radiotherapy combining surgery has become a routine therapeutic strategy for patients with rectal cancer(stage (Ⅱ/Ⅲ).Preoperative radiotherapy can effectively reduce the tumor volume and increase the anal preservation rate,thus effectively improving the prognosis of patients.However,there are still some patients with rectal cancer who are irresponsive or even resistant to radiotherapy in clinical practice,posing a major challenge to the efficacy of radiotherapy.Therefore,elucidating the molecular mechanisms of tumor cell radiosensitivity has become a major challenge in the field of radiation oncology,and in-depth studies of these mechanisms are essential to improve the effectiveness of radiotherapy.Previously,our group used CRISPR Cas9 genomic library to screen the radiotherapy sensitization targets,combining bioinformatics analysis and high-content screening,SUMO specific peptidase 5(SENP5)was selected as the molecule with significant effect on radiosensitivity of colorectal cancer cells.SENP5,a member of SENP-specific protease family,participates in a variety of biological processes such as cell cycle,autophagy and apoptosis by mediating de-SUMOylation of proteins.Dysregulation of SENP5 expression may lead to cellular dysfunction and induce cancer.Several studies based on clinical tumor samples have reported that SENP5 is highly expressed in tumors such as hepatocellular carcinoma,nasopharyngeal carcinoma and ovarian cancer and is associated with poor prognosis.However,studies on how SENP5 regulates tumor radiosensitivity are limited,and it is unclear whether and how de-SUMOylation mediated by which are involved in the regulation of tumor radiosensitivity.Therefore,this study is dedicated to investigate the role of SENP5 in colorectal cancer radio-resistance and its potential mechanisms.Content and Methods:1.Effect of SENP5 knockdown on radiosensitivity of colorectal cancer cellsThe expression levels of SENP5 protein and mRNA in normal intestinal epithelium and colorectal cancer cells were detected by western blot and real-time fluorescence quantitative polymerase chain reaction(q PCR)assays.The expression and subcellular localization of SENP5 in colorectal cancer cells after irradiation were detected by western blot and immunofluorescence assays.We constructed SENP5 knockdown colorectal cancer cell lines in HCT116 and HT29 cells to explore the role of SENP5 in tumor radio-resistance.CCK8 assay,colony formation assay,apoptosis assay and cell cycle assay were used to compare the radiosensitivity of negative control(NC)and SENP5 knockdown cells.2.Role of SENP5 in DNA damage repairTranscriptome sequencing analysis of NC and knockdown SENP5 HCT116 cells revealed that the expression of DNA replication and damage repair-related genes were downregulated.DNA damage in SENP5 knockdown cells were examined using comet assay and immunofluorescence γ-H2AX staining.Homologous recombination(HR)and nonhomologous end-joining(NHEJ)reporter systems were used to determine the repair efficiency of HR and NHEJ in SENP5 knockdown cells.Expression changes of key proteins in the DNA damage repair pathway were further detected by immunofluorescence and western blot.The role of SENP5 in regulating tumor tissue radiosensitivity were also explored in vivo using a colorectal cancer cell derived xenograft(CDX).Finally,in SENP5 knockdown cells that overexpressing wild-type and de-SUMOylation loss-of-function SENP5,rescue experiments were performed,and the role of SENP5-mediated deSUMOylation in cellular DNA damage repair were evaluated by CCK8,clone formation assay,comet assay,and western blot.3.Identification and functional study of target molecules regulated by SENP5 for DNA damage repairH2A.Z were initially identified as the target protein regulated by SENP5 via SUMO proteomics combining SENP5 immunoprecipitation mass spectrometry analysis.Wild-type SENP5 was overexpressed in H2A.Z knockdown cells and colony formation assays were performed to detect tumor radiosensitivity.Then,the interaction of SENP5 and H2A.Z was detected by immunoprecipitation using H2A.Z antibodies in HCT116 and HT29 cells.The interaction of these proteins was verified by exogenous immunoprecipitation assay in 293 T cells.In addition,H2A.Z immunoprecipitation assay was used to detect the changes of SUMOylation in NC and SENP5 knockdown cells after irradiation.Subsequently,Chromatin Immunoprecipitation sequencing(ChIP-seq)was used to explore the effect of SENP5 knockdown on the regulation of H2A.Z-mediated gene transcription.The effects of SENP5 knockdown on the HR repair pathway were explored by combining H2A.Z ChIPseq data with transcriptome sequencing.Finally,q PCR,western blot and immunofluorescence experiments were used to investigate the DNA damage response of HR repair key proteins in SENP5 knockdown cells after irradiation.4.The role and correlation of SENP5 in patients’ resistance to rectal cancer radiotherapyWe explored the correlation between SENP5 and radiosensitivity of rectal cancer by performing SENP5 immunohistochemical detection on tumor tissue microarrays of rectal cancer patients and combined it with tumor regression grading score.Patients were divided into high and low expression groups according to the expression level of SENP5,and survival curves were plotted based on clinical follow-up data.To further investigate the role of down-regulation of SENP5 on radiosensitivity of rectal cancer,we constructed patient derived xenografts(PDX)and monitored the body weight curve,tumor growth curve,tumor weight and immunohistochemical staining in nude mice to gain insight into the role of SENP5 in regulating rectal cancer.Results:1.Knockdown of SENP5 increased the radiosensitivity of colorectal cancer cells.Compared to normal intestinal epithelial cells,both protein and mRNA expression levels of SENP5 were significantly elevated in colorectal cancer cells.Therefore,we selected HCT116 and HT29 with high SENP5 expression for subsequent experimental studies.After irradiation,SENP5 expression were increased in the cells and localized to the nucleus.Compared with NC cells,the cell proliferation ability of colorectal cancer cells with SENP5 down-regulation was inhibited and clone formation rate was significantly reduced after irradiation.Flow cytometry analysis showed that SENP5-knockdowned cells had reduced G2/M cycle arrest and increased apoptosis rate after irradiation.2.SENP5 promotes HR repair to enhance tumor radiation resistance through its deSUMOylation domainThe comet electrophoresis assay suggested that knockdown of SENP5 aggravated DNA damage in irradiated cells,and γ-H2AX immunofluorescence staining showed that the DNA damage repair ability of SENP5 knockdown cells was attenuated.Subsequently,the HR/NHEJ reporter system suggested that SENP5 down-regulation had no significant effect on NHEJ repair,but could significantly inhibit HR repair.Further immunofluorescence experiments revealed that RAD51 foci,a key protein for HR repair,was significantly reduced in SENP5 knockdown cells,while 53BP1 foci,a key protein for NHEJ repair,was not significantly changed.Western blot experiments also revealed that phosphorylation of ATR,CHK1 and CHK2,key proteins for HR repair,was reduced in SENP5 knockdown cells.Similarly,in the CDX model,tumors with low SENP5 expression showed retarded growth and were more sensitive to radiation.Immunohistochemical results also showed that HR repair was significantly inhibited in SENP5 knockdown colorectal cancer transplant tumors after irradiation.Finally,rescue experiments performed in SENP5 down-regulated cells showed that overexpression of wild-type SENP5 can lift the growth inhibition induced by irradiation and promote HR repair.However,these effects were not observed in cells overexpressed with SENP5 that lost the function of de-SUMOylation.3.SENP5 promotes the transcriptional regulation of key proteins in HR repair by mediating H2A.Z de-SUMOylationH2A.Z was screened out by SUMOylation proteomics combining with SENP5 protein mass spectrometry as a SENP5 target protein and its SUMOylation sites were identified at lysine 121,122 and 126.Rescue experiments performed in H2A.Z down-regulated cells showed that H2A.Z is the main effector protein of SENP5 in promoting radioresistance.Subsequently,endogenous and exogenous co-immunoprecipitation assays demonstrated SENP5 binding to H2A.Z after irradiation.However,the SUMOylation site mutanted H2A.Z(K121R/K122R/K126R)did not interact with SENP5.Immunoprecipitation assays showed that SENP5 bound to H2A.Z and regulated its de-SUMOylation after irradiation,and H2A.Z ChIP assays revealed increased binding of H2A.Z to the promoters of HR repair genes TOPBP1,BRCA2 and BARD1 in SENP5 knockdown cells compared to NC cells.In irradiated SENP5 knockdown cells,the mRNA and protein expression levels of TOPBP1,BRCA2 and BARD1 were decreased,and the number of foci in the nucleus was reduced.4.SENP5 can be used as a clinical radiotherapy marker and therapeutic target.Tissue microarray results of clinical rectal cancer patients showed that patients with high expression of SENP5 in tumor tissues has higher radiotherapy resistance and poorer prognosis.In PDX model,knockdown of SENP5 significantly inhibited the growth rate of transplanted tumors.Immunohistochemical staining showed that knockdown of SENP5 could exacerbated tumor cell DNA damage and inhibited tumor proliferation.Conclusions:In summary,this study shows that SENP5 is a key molecule regarding radio-resistance in colorectal cancer cells.Knockdown of SENP5 was can inhibit HR repair and aggravate DNA damage in irradiated tumor cells in in vivo and in vitro experiments.Rescue experiments illustrated that SENP5-mediated de-SUMOylation plays an important role in radio-resistance.In addition,we found that SENP5 can promote tumor radio-resistance by regulating the de-SUMOylation of H2A.Z.In SENP5 knockdown cells,increased binding of H2A.Z to the promoters of TOPBP1,BRCA2 and BARD1,key genes for HR repair,resulted in lower mRNA and protein expression levels of these molecules and reduced recruitment at DNA damage sites,thereby inhibited HR repair.Clinical tissue microarray data showed that SENP5 expression was negatively correlated with prognosis.Through the PDX model of rectal cancer,it was found that knockdown of SENP5 can increase tumor radiosensitivity.Our work reveals the molecular mechanism of SENP5 in the DNA damage response and provide a new theoretical basis for the development of potential biomarkers and therapeutic targets for colorectal cancer radiotherapy.