And-1 O-Glycosylation Targeting DNA Damage Repair Pathway To Promote Rectal Cancer Radiotherapy Resistance

Background and objective:The incidence and mortality rate of colorectal cancer ranks the top five among all malignant tumors,In China,the incidence rate of colorectal cancer ranks the second only after lung cancer,The mortality ranks fourth,which induced about 200,000 patients died annually.The treatment of colorectal cancer has made rapid progress in the past 10 years,such as the emergence of neoadjuvant chemoradiotherapy,immunotherapy,etc.,also several high-quality clinical studies have provided solid evidence-based medical evidence for the treatment of colorectal cancer.All of them played a major role in improving the prognosis of colorectal cancer patients.Neoadjuvant chemoradiotherapy combined with surgery is the standard treatment for locally advanced rectal cancer currently.Preoperative chemoradiotherapy can reduce the local recurrence rate of patients and even make some patients to achieve p CR(pathological complete remission),which some patients with low rectal cancer can avoid surgical treatment and obtain anus preservation,thereby significantly improving the prognosis and quality of life of patients with rectal cancer.However,the response to radiotherapy varies differently among patients.For patients with locally advanced rectal cancer treated with neoadjuvant chemotherapy + surgery + postoperative adjuvant chemotherapy,the pathological complete remission rate can reach 21%,but after the full course of neoadjuvant therapy(Total Neoadjuvant Therapy),the pathological complete remission rate(p CR)could reach up to 31%,but the results are still unsatisfactory.Therefore,how to improve the response of tumor cells to radiotherapy and chemotherapy to improve the pathological complete remission rate of patients is a major clinical problem that needs to be solved urgently.Radiation therapy causes DNA damage to cancer cells,which prevents tumor cell division and promotes cancer cell apoptosis,then induce tumor shrinkage and even pathologic complete response(p CR).However,after cancer cells exposed to radiation,the DNA damage repair signaling pathway will be activated rapidly to promote the repair of DNA damage,thereby inhibiting cancer cells apoptosis and promoting cancer cells radiotherapy resistance.Therefore,finding key protein molecules involved in DNA damage repair in colorectal cancer cells and study their functions and mechanisms,and then design specific small-molecule drugs for them is an important way to improve the radiosensitivity of cancer patients.Through biochemical methods,we found that the O-linked N-acetylglucosamine(O-GlcNAc)glycosylation of And-1 protein catalyzed by O-GlcNAc glycosyltransferase OGT plays a very important role in DNA damage repair in colorectal cancer.In vitro and in vivo experiments were used to explore its role and molecular mechanism in colorectal cancer radiotherapy resistance,which provided a new idea and theoretical basis for the treatment of colorectal cancer.Methods:1.Through the technology of small interfering RNA to knockdown protein,protein co-immunoprecipitation technology to verify the interaction between proteins,protein O-GlcNAc glycosylation biochemical experiment and DNA homologous recombination repair report experiment to verify And-1 O-GlcNAc glycosylation can affect DNA damage repair.2.Determine the sequence between the O-GlcNAc glycosylation of And-1 and its recruitment to the DNA double-strand damage site by immunofluorescence technique and protein nucleocytoplasmic separation technique.By irradiating cells and detecting the time point of O-GlcNAc glycosylation of FLAG-And-1,and the time point of recruitment of FLAG-And-1 to the DNA double-strand damage site,FLAG-And-1 was identified.The relationship between O-GlcNAc glycosylation and recruitment to DNA damage sites occurs.3.The And-1 O-GlcNAc glycosylation site was identified by Liquid Chromatography-Mass Spectrometry and bioinformatics prediction technology.By overexpressing FLAG-And-1 and Myc-OGT plasmids in293 T cells,and purifying FLAG-And-1,the potential And-1 O-GlcNAc glycosylation site was obtained by liquid chromatography-mass spectrometry.Point,using plasmid site-directed mutagenesis technology,the potential O-GlcNAc glycosylation site amino acid(serine or threonine)of FLAG-And-1 was mutated to alanine,and then FLAG-And was verified by Western Blot.The O-GlcNAc glycosylation level of the mutants,thereby determining the O-GlcNAc glycosylation site of FLAG-And-1.4.Establishment of radiotherapy-resistant colorectal cancer cell lines.We obtained radioresistant colorectal cancer cell lines by long-term,escalating radiation dose treatment of wild-type colorectal cancer cell lines.Western Blot and cell survival experiments were performed on radioresistant cell lines.Results:1.Knockdown of And-1 and N-acetylglucosamine transferase(OGT)by si RNA can significantly reduce the homologous end repair efficiency of cells,and after knockdown of And-1 or OGT in colorectal cancer cells,cells The survival rate after radiation treatment was significantly lower than that of the non-knockdown group.In colorectal cancer cells,And-1and OGT proteins can interact,and after radiation treatment,the interaction between And-1 and OGT is significantly enhanced,and the level of O-GlcNAc glycosylation of And-1 is also significantly enhanced.2.We found that the O-GlcNAc glycosylation level of FLAG-And-1in the cells started to increase after 5 minutes of radiation treatment,while FLAG-And-1 was 10 minutes after radiation treatment.It starts to recruit to DNA damage sites within minutes,and affects proteins such as Ct IP and RPA32 to participate in DNA damage repair.3.Among the different truncations of And-1,O-GlcNAc glycosylation can only occur in the Sep B domain(home from the 336 th amino acid to the 984 th amino acid).Using Liquid Chromatography-Mass Spectrometry,we identified 9 potential O-GlcNAc glycosylation sites in the Sep B domain,and verified them with biochemical experiments.Among them,we found that the 575 th Serine and the 893 th serine are O-GlcNAc glycosylation sites.After mutating them,it was found that FLAG-And-1 could not be O-GlcNAc glycosylation anymore.4.In And-1 knockdown colorectal cancer cell lines,wild-type FLAG-And-1 can be rapidly recruited to DNA damage sites after radiation treatment,while FLAG-And-1 that cannot O-GlcNAc glycosylation The mutant(2SA)was unable to recruit to DNA damage sites after DNA damage.Using wild-type FLAG-And-1 for backfilling experiments,it was found that it can re-recruit DNA repair-related proteins(such as Ct IP,RPA32)to the DNA damage site,while the mutant FLAG-And-1(2SA)lost this function.5.The Sep B domain and HMG domain of And-1 can interact,and the interaction between these two domains is significantly attenuated after radiation treatment,while treatment with N-acetylglucosaminyltransferase inhibitor(OSMI-1)Afterwards,the interaction between these two domains was not affected by radiation treatment.And the interaction between the truncation of the FLAG-And-1Sep B domain and the HMG domain after using the O-GlcNAc glycosylation site mutation after radiation treatment had no significant change compared with the treatment group.6.In the colon cancer cell line SW620 with sh RNA knockdown of endogenous And-1,the use of radiotherapy in cells was found to promote the phosphorylation of Chk1 protein and RPA32 protein in cells transfected with wild-type FLAG-And-1,while transfected with inactive mutant(2SA)could not promote the phosphorylation of Chk1 protein and RPA32 protein.And found that in the colon cancer cell line SW620,which knocked down endogenous And-1 with sh RNA,in cells transfected with wild-type FLAG-And-1,the interaction of Ct IP and NBS1 protein was higher than that in transfected with inactive mutant.Body(2SA)was significantly increased.7.In radioresistant colorectal cancer cell lines,the expression of N-acetylglucosamine transferase(OGT)was significantly higher than wild-type cell lines,while the expression of And-1 was significantly higher in wild-type and There was no significant change in radioresistant cell lines;however,the level of O-GlcNAc glycosylation in And-1 was significantly higher in radioresistant cell lines than in wild-type colorectal cancer cell lines.In colorectal cancer radioresistant cells transfected with wild-type FLAG-And-1 or a functionally inactive mutant FLAG-And-1(2SA),after radiation treatment,transfected wild-type FLAG-And-1group The expression rate of γH2AX decreased significantly faster than that of the inactive mutant group.Comet electrophoresis experiments also found that the comet electrophoresis tail of the wild-type FLAG-And-1group was significantly shorter than that of the functional inactive mutant FLAG-And-1 at 24 hours.1(2SA)group.Moreover,the cell survival rate of the wild-type FLAG-And-1 group after receiving radiation treatment was significantly higher than that of the inactive mutant FLAG-And-1(2SA)group,and apoptosis-related proteins(such as cleaved-PARP)were significantly increased.and cleaved-Caspase3)were significantly decreased.After knocking down OGT with small interfering RNA in cells expressing wild-type FLAG-And-1,it was found that the cell survival rate after radiation treatment was higher than that of the inactive mutant FLAG.-And-1(2SA)group cells had no significant difference.And we treated radioresistant cell lines with And-1 inhibitors to regain radiosensitivity.Conclusions:1.Knockdown of And-1 and OGT can significantly reduce the homologous recombination repair function of cells to DNA double-strand damage,which can promote the sensitivity of colorectal cancer cells to radiation treatment.And-1 and OGT can interact,and their interaction is significantly enhanced after radiation treatment,and OGT mediates the O-GlcNAc glycosylation of And-1,and the O-GlcNAc glycosylation of And-1 is significantly enhanced after radiation treatment.2.And-1 can be rapidly glycosylated by O-GlcNAc after radiation treatment,and the O-GlcNAc glycosylation of And-1 is earlier than its recruitment to DNA damage sites.3.The O-GlcNAc glycosylation of And-1 occurs in its Sep B domain,and the specific O-GlcNAc glycosylation sites are No.575 serine and No.893 serine.4.And-1 O-GlcNAc glycosylation can promote its recruitment to DNA damage sites,and influence proteins such as Ct IP and RPA32 to participate in repairing DNA double-strand damage.5.And-1 O-GlcNAc glycosylation affects the interaction between its own domains,thereby promoting its recruitment to DNA damage sites.6.And-1 O-GlcNAc glycosylation affects the phosphorylation of Chk1 and promotes cellular repair of DNA double-strand damage.7.OGT is highly expressed in radioresistant colorectal cancer cell lines,and the O-GlcNAc glycosylation of And-1 is also increased.O-GlcNAc glycosylation of And-1 can significantly improve the radioresistance of cells.Both And-1 inhibitors BZA or CH3 could significantly reduce the radioresistance of colorectal cancer radioresistance cell lines.