Nuclear Accumulation Of UBC9 Contributes To SUMOylation Of Lamin A/C And Nucleophagy In Response To DNA Damage

BackgroundMacroautophagy(hereafter referred to as autophagy)is an evolutionarily conserved intracellular mechanism for lysosomal degradation of damaged cellular components.Selective forms of autophagy,including reticulophagy,mitophagy,ribophagy and nucleophagy,can be activated when cells undergo extra stress,such as genotoxicity and nutritional starvation.A selective autophagy may target various nuclear components(laminas)even parts of nuclei through a series of processes.This specific degradation of nuclear components and nuclei by the autophagy pathway is called nucleophagy.Nucleophagy can maintenance genomic stability by removing damaged nuclei and nuclear materials,but excessively nucleophagy can also result in cell death.Recent studies indicated that nucleophagy has an important significance in physiological and pathological processes during the treatment of cancer.Most studies have focused on autophagic turnover of cytoplasmic materials,and little is known about the role of autophagy in the degradation of nuclear components.Here,we find a new direction in the molecular processes and regulatory mechanisms in nucleophagy and provide insight into tumor suppression by natural products.MethodsHuman MDA-MB-231 and MCF-7 breast cancer cell lines were used as model systems in vitro.Nuclear DNA leakage was determined by western blot and immunofluorescence analyses.Induction of nucleophagy was determined by confocal microscopy.The interaction and colocalization of LC3 and lamin A/C was determined by immunoprecipitation and immunofluorescence.The role of the SUMO E2 ligase,UBC9,on the regulation of SUMOylation of lamin A/C and nucleophagy was determined by siRNA silencing of UBC9,and analyzed by immunoprecipitation and immunofluorescence.Results1.Induction of nuclear DNA leakage activates nucleophagy.Treatment of MDA-MB-231 and MCF-7 cells with DOX resulted in increases in the levels of phosphor-H2AX(Ser139)and nuclear DNA leakage.Western blot showed that treatment with DOX increasde the levels of LC3B-II and decreased the levels of SQSTM1 in either whole-cell or nuclear extracts.Confocal images showed that the leaked nuclear DNA as the cargo was packed into EGFP-LC3 puncta and fusion with lysosome.DOX-mediated nucleophagy also occurs in other cancer cell lines,such as human hepatocellular carcinoma cell line,prostate cancer cell line and lung adenocarcinoma cell line.2.LC3-lamin A/C interaction is required for nucleophagy.Treating cells with DOX decreased the levels of lamin A/C in nucleus both in MDA-MB-231 and MCF-7 cells.Immunofluorescence analysis showed that DOX treatment led to leaked nuclear DNA colocalized with lamin A/C,suggesting that cleavage of lamin A/C contribute to nuclear DNA leakage.Immunoprecipitation and immunofluorescence assays revealed that the interaction of LC3 with lamin A/C was increased in leaked nuclear of cells treatment with DOX.The colocalization of leaked nuclear DNA,lamin A/C,mRFP-LC3 puncta and mGFP-LAMP1 revealed that LC3-lamin A/C interaction was required for nucleophagy.3.SUMOylation of lamin A/C is required for LC3-Lamin A/C interaction.The level of co-precipitation of SUMO1 and lamin A/C was increased in nucleus of cells treated with DOX.Immunofluorescence analysis showed that leaked lamin A/C colocalized with EGFP-LC3,SUMO1 and leaked nuclear DNA,and indicated that SUMOylation of lamin A/C contributed to LC3-Lamin A/C interaction.4.Nuclear accumulation of UBC9 contributes to SUMOylation of lamin A/C.The proteomic approach indicated that UBC9 was upregulated after MDA-MB-231 cells treated with DOX.Western blot and immunofluorescence assays revealed that UBC9 was accumulation in nucleus after DNA damage,and this accumulation promoted SUMOylation of laminA/C through interaction with lamin A/C.5.Knockdown of UBC9 attenuates SUMOylation of lamin A/C and nucleophagy mediated by DNA damage.Knockdown of UBC9 inhibited SUMOylation of lamin A/C and attenuated the interaction of LC3 with lamin A/C,which finally inhibited nucleophagy.6.Natural products induce nucleophagy.Treatment of MDA-MB-231 and A549 with benzyl isothiocyanate(BITC)and triptolide(TPL)resulted in DNA damage and nuclear DNA leakage.Confocal images showed that the leaked nuclear DNA as the cargo was packed into EGFP-LC3 puncta and fusion with lysosome.Western blot showed that treatment with BITC and TPL increased the levels of LC3B-II and decreased the levels of lamin A/C in nuclear extracts.ConclusionsOur findings suggest that nuclear DNA leakage activates nucleophagy through UBC9-mediated SUMOylation of lamin A/C,leading to degradation of nuclear components including lamin A/C and leaked nuclear DNA.Natural products such as BITC and TPL can also induce nuclear DNA leakage and nucleophagy.