Mechanism Of Porcupine-dependent Ku70 Palmitoylation In DNA Damage Response

Objectives: Genomic instability is one of the hallmarks of cancer.The DNA Damage Response(DDR)is the major mechanism in place to guard against genomic instability and it is also a contributing factor of cellular sensitivity to ionizing radiation(IR)and many chemotherapeutic drugs.Elucidating mechanisms involved in DDR pathways is of great importance both for understanding tumorigenesis and for intervention of cancer therapies.Protein palmitoylation is an important post-translational modification required for protein membrane association,subcellular trafficking,and protein–protein interactions.Abnormal palmitoylation has been documented in various cancers,however,whether and how palmitoylation is involved in the DDR and genomic stability is not known.My thesis study focuses on elucidating the nuclear function of Porcupine(PORCN),a membrane-bound O-acyltransferase,and commonly known to reside on the endoplasmic reticulum membrane,in the DDR and radiosensitivity.Methods: For characterization of the nuclear fraction of PORCN in the DDR,we first established stable cell lines with lentiviral PORCN or control sh RNA.Then we reintroduced the sh RNA resistant,wild-type or ?NLS form of PORCN into the PORCN sh RNA HT1080 cell line.We analyzed activation of the DDR by several methods,including the clonogenic survival assay to assess radiosensitivity,immunofluorescence microscopy for formation of nuclear ?-H2 AX foci,and reporter assays for Non-homologous end joining(NHEJ)and Homologous Recombination(HR)repair.We used mass spectrometry to identify PORCN interacting proteins after co-immunoprecipitation.To analyze protein palmitoylation,we used the acyl-biotinyl exchange(ABE)assay to substitute the reversible acyl-group with a detectable and irreversible biotin adduct on cysteine residues.To study the functional significance of palmitoylation,we generated mutations of the KU70 palmitoylation sites with cysteine(Cys)mutated to serine(Ser).We examined the palmitoylation capacity of these mutants in HT1080 cells.We also used the co-IP experiment to determine the effect of Ku70 palmitoylation on the formation of the DNA-PKcs/Ku70/Ku80 complex.To study in vivo activity of palmitoylation induced by IR,we established a xenograft model using cell lines with KU70 palmitoylation sites mutated,and assessed tumor growth delay after the xenografts were irradiated.Results: Initially we found that PORCN-deleted cells had a suboptimal DDR(prolonged ?-H2 AX foci,mitotic slippage,and defective NHEJ)and they were hypersensitive to IR.We found that there was a fraction of PORCN presents in the nuclear.This nuclear fraction requires the nuclear localization sequence(NLS)(the KKRKAR sequence spanning residues 230-235.Confocal microscopy showed that,in the absence of the NLS domain,there was accumulation of ?-H2 AX foci during mitosis.The DNA damage repair assays showed that PORCN-?NLS cells had a much lower NHEJ activity.However,the HR was normal in PORCN-?NLS cells.Clonogenic assays showed that,as compared to PORCN-wt cells,PORCN-?NLS cells were hypersensitive to IR.Immunofluorescence microscopy showed that,in PORCN-?NLS cells the ?-H2 AX foci remained elevated 16 hours after IR,when ?-H2 AX foci in wt cells had already returned to the normal level,indicating a delayed DNA repair process in PORCN-?NLS cells.In vivo results showed that PORCN-?NLS tumors were significantly sensitive to IR,as compared to that of PORCN-WT.A mass spectrometry analysis identified the nuclear PORCN complex with many DNA repair proteins icnlduing the DNA-PKcs/Ku70/Ku80 complex.Further experiments showed that PORCN led to Ku70 palmitoylation and this process is dependent on the NLS domain of PORCN.Individual mutation of one of the five Cys to Ser in Ku70 showed reduced palmitoylation.However,when all the Cys were mutated,KU70 palmitoylation was abrogated.We found that,compared to Ku70-WT cells,Ku70-MU cells showed a significant radiosensitive phenotype.Immunofluorescence results showed that,as compared to Ku70-WT cells,Ku70-MU cells had a delayed DNA repair process.DNA damage repair detection system showed that Ku70-MU cells showed a much lower NHEJ activity in response to IR.Confocal microscopy showed that in Ku70-MU cells,there was accumulation of ?-H2 AX foci during mitosis after IR.Further,we found that in KU70-MU cells,Ku80 interacted less efficiently with non-palmitoylable Ku70.Meanwhile,we failed to detect DNA-PKcs in the Ku70-MU immune-precipitates.A human tumor xenograft model showed that the tumor carrying Ku70-MU has significantly higher radiosensitivity,compared to that of Ku70-WT.Conclusion: There is a fraction of PORCN presents in the nuclear.The nuclear PORCN is required for Ku70 palmitoylation and this process is essential for activation of an optimal DDR.Further,Ku70 palmitoylation is required for formation of the DNA-PKcs/Ku70/Ku80 complex and NHEJ.Collectively,our data highlight mechanisms of a critical function of Porcupine-dependent Ku70 Palmitoylation in DDR.