The Role And Molecular Mechanism Of The P53-interacting Proteins CCDC106 And ZCCHC10in Cancer

Background:Dysfunction of p53 is an important cause of tumorigenesis.The mutation of p53 gene is the main way of p53 inactivation,and occurs in about half of all cancer cases.In tumors that retain wild-type p53(wtp53),p53 is often inactivated by up regulating the negative regulators or down regulating the positive regulators of p53.In our previous study,we found that CCDC106 can promote the degradation of p53 protein,while ZCCHC10 can inhibit the degradation of p53 protein.This thesis further studies the expressions of CCDC106 and ZCCHC10 in cancer tissues and their effects on the development of cancer.Methods:The levels of mRNA and protein were detected by quantitative RT-PCR and Western blot experiments,respectively.A lentiviral system was used to construct stable cell lines with gene overexpression and interference of the target gene.Co-immunoprecipitation and GST-pull down were used to detect protein interactions.Subcellular fractionation experiment and immunofluorescence staining were used to detect protein subcellular localization.Phosphorylation-specific antibodies were used to detect phosphorylation status of protein.MTT assay was used to determine cell viability,flow cytometry was used to detect cell cycle and apoptosis,plate colony formation assay was used to detect cell proliferation ability,wound healing experiment was used to detect cell migration ability,and TransWell invasion experiment was used to detect cell invasion ability.The effect of CCDC106 or ZCCHC10 on tumor growth in vivo was studied using a mouse model of subcutaneous xenograft.A metastatic mouse model by injection of cells via tail vein was used to study the effect of ZCCHC10 on tumor metastasis.Results:Ⅰ.The role and molecular mechanism of CCDC106 in breast and cervical cancer1.Knockdown of CCDC106 in cervical cancer HeLa cells and breast cancer MCF7 cells containing wild-type p53(wtp53)can enhance the stability of p53 protein,promote apoptosis,and inhibit cell survival,colony formation,migration,and invasion.2.Overexpression of CCDC106 in normal breast epithelial HBL100 and cervical cancer Si Ha cells containing wtp53 promotes the degradation of p53 protein,inhibits apoptosis,and promotes cell survival,colony formation,migration,and invasion.3.CCDC106 has no significant effect on cell survival,colony formation,migration,and invasion of cervical cancer and breast cancer cells(C33A and MDA-MB-231)with p53 mutations.4.The Ser-130 and Ser-147 sites of CCDC106 were previously found to be phosphorylation sites of protein kinase CK2 by in vitro phosphorylation experiments.This paper further confirms that CK2 can indeed phosphorylate CCDC106 at Ser-130 and Ser-147 sites in cells.If Serc-130 of CCDC106 is mutated to Ala,CCDC106 cannot interact with p53 protein;while Ser-147 is mutated to Ala,CCDC106 cannot be located in the nucleus,indicating that phosphorylation of CCDC106 at S130 and S147 is required for the interaction with p53 and localization of CCDC106,respectively.5.Substitution of both Ser-130 and Ser-147 with Ala,or treating cells with CK2 inhibitor CX-4945,can inhibit the phosphorylation and oncogenic role of CCDC106.6.In xenograft mouse models,wild-type CCDC106 can promote the degradation of p53 and tumor growth,while mutant CCDC106 has no effect.7.Knockdown of CCDC106 in cancer cell lines containing wtp53 can increase cell sensitivity to CX-4945.Ⅱ.The role of ZCCHC10 in lung cancer and its molecular mechanism1.By analyzing the expression of ZCCHC10 in 54 pairs of lung cancer tissues and the matched adjacent tissues,it was found that the level of ZCCHC10 protein in lung adenocarcinoma tissues was lower than that of corresponding adjacent tissues.Analysis of the public cancer database found that the expression of ZCCHC10 mRNA in cancer tissues was positively correlated with patient survival.2.Overexpression of ZCCHC10 in lung cancer cells containing wtp53 significantly inhibits cell proliferation,colony formation,migration,invasion and cisplatin resistance,while knockdown of ZCCCHC10 in Normal lung cell line Beas-2b containing wtp53 promotes cell proliferation,colony formation,migration,invasion,and cisplatin resistance.3.ZCCHC10 had no effect on colony formation,proliferation,migration,and invasion of the p53-null cells(H358)or p53-mutated cells(H1437).4.Xenograft tumor formation and metastasis experiments in nude mice showed that overexpression of ZCCHC10 inhibited tumor growth and metastasis.5.Co-immunoprecipitation proved that wild-type ZCCHC10 protein can interact with p53 protein,and mutation of its CCHC domain cannot interact with p53,indicating that ZCCHC10 interacts with p53 through its CCHC domain.6.ZCCHC10 inhibits the interaction between p53 and MDM2 and MDM2-mediated p53 protein ubiquitination,thereby enhancing the stability of p53 protein.7.The p53 inhibitor pifithrin-α attenuates the effects of ZCCHC10 on p53 pathway,cell cycle,apoptosis and epithelial-mesenchymal transition,and the p53 activator Nutlin3 can reverse the effect caused by ZCCHC10 interference,indicating that ZCCHC10 exerts its effect through p53.Conclusion:1.In breast cancer and cervical cancer,CCDC106 plays an oncogenic role by promoting the ubiquitination and degradation of p53 protein.2.Protein kinase CK2 can phosphorylate CCDC106 at Ser-130 and Ser-147.Phosphorylation of CCDC106 at S130 and S147 is required for the interaction with p53 and localization of CCDC106,respectively.3.In lung cancer,ZCCHC10 suppresses lung cancer progression and cisplatin resistance by attenuating MDM2-mediated p53 ubiquitination and degradation.