FAM162A Promotes Treatment Resistance And Self-renewal Of GSC Via The Fanconi Anemia Pathway In Hypoxia

Objective: High rates of treatment resistance and recurrence are important causes of poor prognosis in patients with glioblastoma multiforme(GBM).Although existing studies have demonstrated that chromosomal instability(CIN),highly activated DNA damage response(DDR)and DNA repair pathways,and the synergistic effects of hypoxia and glioma stem cells(GSC)are key factors mediating GBM chemoresistance.However,the specific molecular mechanisms by which these factors interact to mediate GBM chemoresistance remain unclear.In this study,I screened FAM162 A as an important molecule involved in the promotion of GSC treatment resistance by alkylating agents in hypoxic environment based on understanding and evaluating the relationship between the activation status of CIN or DDR and its treatment resistance in GBM.I further investigated the specific molecular mechanism of FAM162 A to promote GSC self-renewal and GBM treatment resistance,and tried to evaluate the effect and mechanism of curcumin alone and in combination with cisplatin in anti-GSC therapeutic application.Methods: The incidence of atypical mitotic events,quantitative CIN scores and somatic mutation status in glioma patients in TCGA and CGGA were first analyzed using bioinformatics methods to reflect CIN levels,and immunofluorescence staining of clinical tumor tissues was used to determine the activation status of DDR in GBM.We used gene set variation analysis(GSVA)and survival analysis in combination with in vitro western blot(WB),quantitative real-time reverse transcription polymerase chain(RT-q PCR),chromatin immunoprecipitation(Ch IP),and other multiple laboratory tests to detect hypoxia-induced changes in FAM162 A expression and DDR status after silencing of FAM162 A in GSC.Based on these results,FAM162 A was tried to be screened as an important molecule involved in alkylating agent-induced treatment resistance of GSC in hypoxic environment,and to be regulated by hypoxia.Further,RT-q PCR,WB,immunofluorescence staining and immunohistochemistry were used to verify the specificity of FAM162 A expression in GSC/GBM and correlation with DNA damage repair response in the level of GSC and GBM tumor tissue.Then,a series of cell phenotypic assays and intracranial tumorigenesis assays in nude mice with GSC were used to detect the effects of silencing FAM162 A on the self-renewal ability such as cell proliferation,sphere formation,colony formation and tumorigenic ability in vivo.Combined with the above experimental methods and co-inmunoprecipitation(Co-IP),the specific molecular mechanism of FAM162 A in promoting GSC self-renewal and GBM treatment resistance was explored.Finally,based on a large number of existing studies on the application of curcumin in the treatment of solid tumors in vivo and in vitro,I designed and conducted a series of in vivo and in vitro experiments on curcumin alone and in combination with cisplatin for the treatment of GSC in an attempt to understand and evaluate the effects and mechanisms of curcumin alone and in combination with cisplatin for anti-GSC treatment.Results: Analyses based on the incidence of atypical mitotic events,quantitative CIN scores and somatic mutation status indicate that GBM tumor tissues had significantly higher CIN levels than other grades of glioma,and that this level of CIN increased significantly with the increasing grade of glioma.The abnormally increased DNA damage and activated DDR in GBM tumor tissues are closely related to the higher level of CIN,with the highly activated HR-mediated DNA repair pathway possibly being the reason for the high level of CIN in GBM tumor tissues.FAM162 A is significantly associated with DNA damage repair,hypoxia and cell cycle processes in GBM,and is significantly highly expressed in GBM,acting as an important poor prognostic factor in GBM patients.Under hypoxic conditions,HIF1α induces significant high expression of FAM162 A in GSC.Silencing FAM162 A expression caused a significant increase in DNA damage and DNA replication blockage in GSC.Silencing FAM162 A expression under hypoxic conditions significantly inhibited stemness maintenance and tumorigenicity of GSC in vitro and in vivo.In vitro and in vivo experiments showed that FAM162 A promotes HR-mediated DNA repair in response to DNA damage caused by hypoxia and alkylating agents by inducing FANCD2 monoubiquitination in GSCs in hypoxic environments.FAM162 A directly interacts with FANCG,regulates its expression by affecting the ubiquitin-proteasome pathway of FANCG protein degradation,and then regulates DNA damage repair mediated by the FA pathway induced by hypoxia and alkylating agents by affecting the level of FANCD2 mono-ubiquitination.In vitro and in vivo experiments with curcumin alone and in combination with cisplatin for the treatment of GSC confirmed that curcumin alone and in combination with cisplatin had good anti-GSC treatment activity,which could inhibit GSC self-renewal and GSC tumorigenicity in vivo and in vitro by targeting FANCD2,a molecule downstream of FAM162 A,rather than FAM162 A itself,and enhance the anti-GSC therapeutic effect of cisplatin.Conclusions: CIN is a fundamental characteristic of GBM,and the aberrantly activated DNA damage repair response in GBM is closely related to tumor chemoresistance.FAM162 A is regulated by HIF1α in hypoxic environment and is involved in maintaining the self-renewal ability and chemoresistance of GSC.This is achieved by directly binding and interacting with FANCG to activate FANCD2 mono-ubiquitination-mediated FA pathway which enhances the repairing of DNA damage caused by alkylating agents and hypoxia in GSC.Curcumin alone and in combination with cisplatin targeting FANCD2 have exerted good anti-GSC therapeutic effects.