Niraparib Enhances Radiotherapy-Induced Ferroptosis And Antitumor Immunity In Colorectal Cancer

Background: In tumor cells,poly(ADP-ribose)polymerase(PARP)-1 and PARP2 initiate and regulate DNA repair pathways to prevent DNA damage and cell death caused by radiotherapy or chemotherapy.Radiotherapy,a common tumor treatment,can induce DNA single-and double-strand damage in tumor cells,which can cause tumor cell death.In recent years,radiotherapy and PARP inhibitors have often been used in combination in clinical trials to improve the therapeutic effect on certain cancers.However,the exact mechanism is not clear.Therefore,an in-depth study of the mechanism of the combination of radiotherapy and PARPi will help us to better understand its efficacy,optimize its clinical application,and develop more effective cancer treatment strategies.Niraparib,a widely used PARPi,can exert antitumor effects by targeting DNA repair pathways.In this study,we investigated the potential mechanisms of niraparib enhancing radiotherapy-induced ferroptosis and anti-tumor immune in colorectal cancer.Objective: To investigate the effects of combined treatment with radiotherapy and niraparib on the development of colorectal cancer using colorectal cancer as the disease model;and to elucidate the underlying mechanisms of niraparib enhancing radiotherapy-induced antitumor effects.Methods: The effects of radiotherapy and niraparib on CRC cell proliferation were examined by cell clone formation assay;the cGAS stable knockdown MC38 cell line was constructed using shRNA and a mouse colorectal cancer subcutaneous tumor model was constructed to examine the inhibition of tumors in mice by radiotherapy and niraparib;the downstream target genes regulated by cGAS were analyzed by transcriptome sequencing technology and bioinformatics;qRT-PCR,Western Blot,ELISA,immunohistochemistry and immunofluorescence to detect cGAS-STING-IFNβ and cGAS-ATF3-SLC7A11 signaling axis genes and protein expression;transmission electron microscopy to observe and record the mitochondrial morphology as well as submicroscopic structure of ferroptosis cells;and flow cytometry to detect the infiltration of immune cells in mouse CRC tumors.In addition,to further investigate the clinical correlation between antitumor immunity and ferroptosis and the efficacy of radiotherapy in patients,we collected pre-radiotherapy biopsies and matched surgical resection samples from rectal cancer patients who underwent neoadjuvant radiotherapy and radical rectal cancer surgery,and detected cGAS-mediated antitumor immunity and ferroptosis-related protein expression by immunohistochemistry and immunofluorescence.Tracking the patients’ postoperative disease-free survival(DFS),patients’ survival curves were plotted using Kaplan-Meier survival analysis.Results: Niraparib enhanced radiotherapy-induced proliferation inhibition of CRC cells and CRC tumors in mice.The inhibitory effects induced by niraparib and radiotherapy were attenuated after cGAS knockdown;RNA-seq assay revealed that differential genes of ferroptosis and type Ⅰ interferon β pathways were significantly enriched and ATF3 expression was significantly downregulated after cGAS knockdown;niraparib promoted the radiotherapy-induced expression of CRC ferroptosis markers PTGS2,increased expression of MDA and decreased expression of SLC7A11-GPX4 in CRC induced by radiotherapy,and the corresponding change effect was attenuated after knockdown of cGAS;niraparib promoted enhanced expression of type Ⅰ interferon β induced by radiotherapy,and the corresponding enhancement effect was attenuated after knockdown of cGAS;Transmission electron microscopy analysis of cellular submicrostructure and ultrastructure revealed that mitochondria of tumor cells were crinkled by radiotherapy,and the radiotherapy-induced structural alteration of mitochondria was reversed after knockdown of cGAS.Flow cytometry assay revealed that niraparib promoted enhanced infiltration of CRC antitumor immune cells in mice induced by radiotherapy,and the enhancement effect was attenuated after knockdown of cGAS.Clinical correlation study found that radiotherapy increased cGAS,IFNβ,CD8 a,ATF3,and PTGS2 expression in tumor tissues of rectal cancer patients,and the prognosis(DFS)of patients with high cGAS,IFNβ,CD8 a,ATF3,and PTGS2 expression in tumor tissues after surgery was better than that of patients with low expression.Conclusion: Radiotherapy can modulate the cGAS-ATF3-SLC7A11 signaling axis to promote CRC tumor ferroptosis,as well as activate CRC antitumor immunity by enhancing the cGAS-STING-IFNβ signaling pathway,and niraparib can further enhance the above effects of radiotherapy.23 figures,20 tables,156 references...