LOXL2 Mediates PARP Inhibitor Resistance In Ovarian Cancer By Promoting Enhancer Activity

Objective: Epigenetic regulation plays an important role in determining cell fate and affecting the prognosis of patients.This paper intends to screen and study the mechanism of epigenetic regulators that play a key role in ovarian cancer and the value of targeted therapy.Methods : Gene expression analysis and prognostic gene meta-analysis、Kaplan-Meier survival curve analysis were performed through public databases.The m RNA expression level of the genes were detected by RNA-seq and RT-qPCR,and the protein expression level of the genes were detected by Western blot and immunohistochemistry.H3K4me3 and H3K27 ac histone modification level were detected by ChIP-seq.H3K27 ac ChIP-seq data were used to identify super enhancers by Rank Ordering of Super-Enhancers(ROSE)method,and visualized by Integrative Genomics Viewer(IGV)software.CCK8、colony formation and flow cytometry were used to detect tumor cell proliferation and apoptosis.The degree of DNA damage was detected by γH2AX immunofluorescence and alkaline comet assay.The subcutaneous tumor of ovarian cancer and ovarian orthotopic transplantation model were used to observe tumor volume by in vivo bioluminescence imaging.Results: Through the combined analysis of the expression level of epigenetic regulationrelated genes and the prognosis of patients in the public database,it was found that LOXL2 was significantly increased in ovarian cancer and significantly associated with poor prognosis.Immunohistochemical results of normal fallopian tubes and tumor tissues of high-grade serous ovarian cancer patients showed that LOXL2 expression was significantly increased in tumor tissues,and the expression level of LOXL2 in patients with recurrence within 5 years was significantly higher than that in patients without recurrence.In HR proficiency ovarian cancer cell lines,silencing LOXL2 and blocking LOXL2 with small molecule inhibitor BAPN significantly enhanced PARP inhibitor-induced DNA damage,promoted apoptosis,and inhibited cell survival after PARP inhibitor treatment.Animal experiments also showed that targeting LOXL2 significantly enhanced the therapeutic effect of PARP inhibitors on ovarian tumors.On the mechanism,LOXL2 promotes the transcriptional activity of enhancer / super enhancer by up-regulating key genes of super enhancer complexes such as BRD4,thereby up-regulating multiple DNA damage repairrelated genes such as MDC1、BAZ1B、KAT5、USP7 and CDK5.The regulation of LOXL2 on MDC1 and other genes and the enhancement of PARP inhibitor resistance in vitro and in vivo depends on the super enhancer complex.Conclusion: LOXL2 up-regulates key genes of super enhancer complexes such as BRD4,and enhances PARP inhibitor resistance by promoting the epigenetic-expression regulation of enhancers/super enhancers of multiple DNA damage repair-related genes.The results of this study suggest that targeting LOXL2 to enhance the sensitivity of PARP inhibitors may not only be used for the combined treatment of drug-resistant ovarian cancer,but also may create targets for the treatment of HR proficiency tumors using PARP inhibitors.Targeting LOXL2 has similar effects as targeting super enhancer complexes,but LOXL2 is highly expressed in tumor cells and may become a more accurate therapeutic target.Part Ⅰ: LOXL2 promotes PARP inhibitor resistance in ovarian cancerObjective: To explore the epigenetic regulatory factors that play a key role in ovarian cancer and clarify their main biological functions.Methods: Gene expression analysis and prognostic gene meta-analysis、Kaplan-Meier survival curve analysis were performed through public databases.The relationship between the expression level with the pathogenesis and prognosis of patients was verified by immunohistochemistry of normal fallopian tubes and tumor tissues from patients with highgrade serous ovarian cancer.Tumor cell proliferation and apoptosis were detected by CCK8、 colony formation and flow cytometry.The m RNA expression level of the gene was detected by RNA-seq and RT-qPCR,and the protein expression level of the gene was detected by Western blot.The subcutaneous tumor of ovarian cancer and ovarian orthotopic transplantation model were used to observe tumor volume by in vivo bioluminescence imaging.Results: Through the combined analysis of the expression levels of epigenetic regulationrelated genes and the prognosis of patients in the public database,it was found that LOXL2 was significantly increased in ovarian cancer and was significantly associated with poor prognosis.The immunohistochemical results of tumor tissues collected from patients with high-grade serous ovarian cancer showed that the expression level of LOXL2 in patients with recurrence within 5 years was significantly higher than that in patients without recurrence.The genes up-regulated by LOXL2 were significantly enriched in cell responses to DNA damage stimulation,suggesting that LOXL2 may affect the sensitivity of PARP inhibitors.Indeed,LOXL2 expression itself did not significantly affect tumor cell proliferation,but LOXL2 promoted cell survival,colony formation,and reduced apoptosis after PARP inhibitor treatment.The results of subcutaneous tumor model of ovarian cancer and ovarian orthotopic transplantation model showed that silencing LOXL2 and blocking LOXL2 function by BAPN significantly enhanced the therapeutic effect of PARP inhibitors in ovarian cancer,and BAPN had no significant toxicity to important organs.Conclusion: LOXL2 is significantly associated with poor prognosis in patients with ovarian cancer.Reducing the expression of LOXL2 can significantly increase the sensitivity of PARP inhibitors in vivo and in vitro.Part Ⅱ: The mechanism of LOXL2 promoting PARP inhibitor resistance in ovarian cancerObjective: To explore the mechanism of LOXL2 promoting ovarian cancer PARP inhibitor resistance at the cellular level and molecular level.Methods: RNA-seq and RT-qPCR were used to detect the m RNA expression level of genes,and Western blot was used to detect the protein expression level of genes.The histone modification level of H3K4me3 and H3K27 ac were detected by ChIP-seq.Super enhancers were identified by Rank Ordering of Super-Enhancers(ROSE)method using H3K27ac ChIP-seq data,and visualized by Integrative Genomics Viewer(IGV)software.DNA damage was detected by γH2AX immunofluorescence and alkaline comet assay.CCK8、colony formation,and flow cytometry were used to detect tumor cell proliferation and apoptosis.The subcutaneous tumor model of ovarian cancer was used to observe tumor growth by regular survey tumor volume.Results: Alkaline comet assay and γH2AX immunofluorescence showed that LOXL2 resisted PARP inhibitor-induced DNA damage.LOXL2 up-regulated multiple DNA damage repair-related genes such as MDC1、BAZ1B、KAT5、USP7 and CDK5,and up-regulated multiple super enhancer complex genes such as BRD4.Through the combined analysis of ChIP-seq results of H3K4me3 and H3K27 ac sites and RNA-seq results,it was found that LOXL2 increased the expression level of downstream enhancer and super enhancer-related genes,including DNA damage repair genes such as MDC1,without significantly changing the H3K27 ac modification level.Through silencing or blocking the key protein BRD4 in the super enhancer complex by si RNA or small molecule inhibitors,it was proved that LOXL2 promotes DNA damage repair and related gene expression,and the promotion of LOXL2 on PARP inhibitor resistance both depends on the super enhancer complex.Conclusion: LOXL2 up-regulates multiple DNA damage repair-related genes by promoting the transcriptional activity of enhancer/super enhancer,thereby resisting PARP inhibitorinduced cell damage.The epigenetic-expression regulation of LOXL2 on these DNA damage repair genes depends on its promotion of the expression of key genes of the super enhancer complex,such as BRD4.The results of this study suggested that targeting LOXL2 to enhance the sensitivity of PARP inhibitors may not only be used for the combined treatment of drug-resistant ovarian cancer,but also may create a target for PARP inhibitor treatment for HR proficiency tumors.Targeting LOXL2 has similar effects as targeting super enhancer complexes,but LOXL2 is highly expressed in tumor cells and may become a more accurate therapeutic target.