PRDM15 Promotes DNA Damage Non-homologous End Joining Repair To Regulate Radiotherapy Resistance In Rectal Cancer

1.BackgroundColorectal cancer is the third most common cause of cancer worldwide and the second leading cause of cancer-related deaths.At present,the multidisciplinary treatment model of neoadjuvant radiotherapy followed by surgery for locally advanced rectal cancer has become the new treatment standard.This approach improves tumor outcomes,including local control and long-term survival.Nevertheless,it is possible that some subsets of cancer cells which are radiologically resistant may survive such treatment.In addition,during radiation therapy,the cancer cells gradually develop radiation resistant subsets.Successful colonization of cancer cells in local or distant organs can lead to local recurrence and distant metastasis,which can result in poor prognosis.Therefore,it is very important to solve radiation resistance in patients with rectal cancer to improve their prognosis.The main mechanism of tumor radiation therapy is to induce DNA damage in cancer cells,and the DNA damage response(DDR)of cancer cells determines the radiosensitivity of cancer cells.DDR is a complete DNA repair system possessed by cells.DNA double-strand break(DSB)is the most serious type of DNA damage in cancer cells after radiotherapy,which often leads to apoptosis,autophagy and cell cycle arrest.However,cancer cells have a stronger DNA repair ability than normal cells.So they may survive radiation therapy.There are two main repair pathways for DSB,namely non-homologous end joining(NHEJ)and homologous recombination(HR).Currently,several drugs have been applied to block these two repair pathways,but the clinical application of DDR is still in its infancy.Thus,the identification of novel molecules that may be involved in DDR could provide new targets for radiosensitization.PRDM family is a zinc finger sequence-specific chromatin factor that plays an important role in embryonic development and cell differentiation determination.In all members of the PRDM family,PRDM15 maintained the naive state of mouse embryonic stem cells.PRDM15 regulates signal transduction in WNT and MAPK-ERK pathways by directly promoting Rspo1 and Spry1 expression.In B-cell lymphoma,PRDM15 regulates the PI-3K /AKT/m TOR pathway and the transcription program of glycolysis activity.PRDM15 knockout can cause metabolic crisis and selective death of lymphoma cells.In addition,PRDM15 regulates the transcription of key effectors in the NOTCH and WNT/PCP pathways to preserve early midline structures during embryonic development.Although PRDM15 has a good characteristic role in stem cell biology,its role in cancer has not been clearly reported.In this study,we found that PRDM15 can promote NHEJ through interacting with DNA-PK to regulate radiotherapy resistance of rectal cancer.We found that knockdown of PRDM15 in tumor cell and patient tissue-derived xenograft models(CDX and PDX)increased the radiosensitivity of the models.In addition,high expression of PRDM15 was associated with poor prognosis in patients with locally advanced rectal cancer who received neoadjuvant chemoradiotherapy.This study provides a new idea for the radiotherapy of rectal cancer.2.Methods2.1 PRDM15 regulates the DNA damage response of colorectal cancer cellsColorectal cancer cell lines from different sources,such as HCT116,HT29 and Wi Dr,were selected to extract cell proteins at 0 h(blank control without intervention),0.5 h,4 h,8 h and 12 h after receiving ionizing radiation.Western Blot was used to detect the changes of PRDM15 in these cells in response to ionizing radiation.In order to further refine the factors that may stimulate PRDM15 to participate in DDR,we also used clinically chemotherapy drugs,etoposide(ETO),camptothecin(CPT)and Olaparib to induce DNA damage,and the detection technology was the same as ionizing radiation group.RNA was extracted at 0 h(blank control),4 h,8 h,12 h and 24 h after receiving radiation.The transcriptional changes of PRDM15 in HCT116 and HT29 cells in response to ionizing radiation were detected by RT-PCR.In terms of subcellular localization of PRDM15,immunofluorescence was performed to confirm whether it was located in the nucleus under a confocal fluorescence microscope.After preliminary exploration,we found that PRDM15 might be involved in DDR.Then we constructed HCT116 cells with knockdown and overexpression of PRDM15.PRDM15 NC,KD and OE cells were used for cell proliferation,clonogenesis rate,apoptosis and cell cycle detection to confirm that PRDM15 promotes the radiation resistance in colorectal cancer cells.2.2 PRDM15 promotes DNA damage NHEJ repair to regulate radiotherapy sensitivity in colorectal cancer cellsFoci of γ-H2 AX and 53BP1 in PRDM15 NC and PRDM15 KD HCT116 was detected by immunofluorescence at 0 h,0.5 h,12 h and 24 h after irradiation to evaluate the effect of PRDM15 knockdown on DNA damage repair in colorectal cancer cells.In order to further explore the specific effects of PRDM15 on the DDR pathway of colorectal cancer cells,we induced DNA damage in colorectal cancer cells of PRDM15 NC,PRDM15 KD and PRDM15 OE respectively.At 0 h(blank control without intervention),0.5 h,4 h,8 h and 12 h after intervention,cell proteins were extracted,and Western Blot was used to detect the effects of knockdown or overexpression of PRDM15 on specific molecules in HR and NHEJ signaling pathways.In addition,we used inhibitors of upstream key kinases of the DDR pathway to further explore the way PRDM15 participates in DDR.We selected DNA-PK inhibitor NU7441,ATM inhibitor KU55933 and ATR inhibitor VE821 to pretreat HCT116 cells for 2 h before 8 Gy radiation dose.Cell proteins were extracted at 0 h(control group with inhibitor intervention but without radiation intervention),0.5 h and 8 h after treatment.Western Blot was used to detect the effect of inhibitor of upstream kinase of DDR pathway on PRDM15 expression level after DNA damage.Finally,we conducted co-IP using protein lysates of HCT116 cells to verify the hypotheses proposed from the above research.2.3 Effect of PRDM15 on radiosensitivity in rectal cancer animal model and clinical analysisThe CDX and PDX radiotherapy model for rectal cancer in nude mice were constructed,and the sensitization effect of PRDM15 knockdown on radiotherapy for rectal cancer was determined in vivo by tumor growth curve,gross anatomy,pathological section and immunohistochemistry.In addition,we analyzed the expression difference of PRDM15 in tumor tissues and para-cancer tissues of rectal cancer patients by TCGA database,and collected postoperative samples from clinical rectal cancer patients in our department,which were made into a tissue microarray for immunohistochemical detection of PRDM15,and explored the correlation between the expression level of PRDM15 and tumor regression grade(TRG).Finally,according to the expression level of PRDM15,patients from the tissue microarray were divided into high expression and low expression.Kaplan-meier curves of these patients were analyzed with the clinical follow-up data to further clarify the radiotherapy resistance of PRDM15 from the clinical level.3.Results3.1 PRDM15 regulates the DNA damage repair in colorectal cancer cellsAfter 8 Gy radiation,the expression of PRDM15 was significantly increased in different colorectal cancer cells.At the transcriptional level,PRDM15 m RNA was increased in irradiated colorectal cancer cells.After immunofluorescence co-location detection,we found that PRDM15 was localized to the nucleus,formed foci after DNA damage and co-located with γ-H2 AX.After constructing HCT116 cells with knockdown and overexpression of PRDM15,it was confirmed that knockdown PRDM15 could reduce the proliferation of colorectal cancer cells after radiation by CCK-8 assay,clone formation assay,cell apoptosis and cell cycle assay.Knockdown of PRDM15 can reduce the cell survival rate and increase the apoptosis rate after radiation.Knockdown of PRDM15 resulted in G2/M phase arrest failure in irradiated colorectal cancer cells.3.2 PRDM15 interacts with DNA-PK to regulate NHEJBy detecting the foci numbers of γ-H2 AX and 53BP1 in HCT116 cells after radiation,we found that knocking down PRDM15 significantly inhibited the DNA damage repair of colorectal cancer cells after radiation.The number of γ-H2 Ax and53BP1 foci in PRDM15 KD cells was more than that in PRDM15 NC cells.Western Blot further confirmed that knocking down PRDM15 inhibited the NHEJ pathway.To further verify this hypothesis,we treated cells with an upstream kinase inhibitor of DDR.We found that PRDM15 expression was decreased after radiation only after treatment with NU7441,a DNA-PK inhibitor.These results indicate that PRDM15 is highly likely to mediate NHEJ repair through interacting with DNA-PK.Finally,we confirmed the interaction between PRDM15 and DNA-PK by co-IP.3.3 Effect of PRDM15 on radiosensitivity of rectal cancer animal models and clinical analysisAfter the successful construction of CDX and PDX models of rectal cancer in nude mice,we conducted radiation intervention on them.It was found that knocking down PRDM15 had significant radiotherapy sensitization effect on CDX and PDX models of rectal cancer in nude mice.In addition,by immunohistochemical staining,we confirmed that knocking down PRDM15 significantly inhibited the proliferation and promoted the apoptosis of rectal cancer tissues after radiotherapy.On the other hand,through immunohistochemical detection of clinical tissue microarray,we found that the expression of PRDM15 was highly correlated with TRG.Based on the above results,we divided the tissue microarray derived patients into two groups,which were high and low expression of PRDM15.The following results confirmed that high expression of PRDM15 was related to radiotherapy resistance and poor prognosis clinically.4.ConclusionIn this study,PRDM15 as a new target of radiotherapy resistance to colorectal cancer was found to regulate the DDR in colorectal cancer cells.The mechanism of PRDM15 is to mediate NHEJ repair through interaction with DNA-PK,thus regulating radioresistance in HCT116.In vivo,knocking down of PRDM15 improves the radiosensitivity of PDX models.In terms of clinical effect,high expression of PRDM15 is associated with inferior TRG and prognosis after radiotherapy in rectal cancer patients.