ABSTRACT
RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.
Subject(s)
Acetylglucosamine , DNA-Binding Proteins , Proliferating Cell Nuclear Antigen , Rad51 Recombinase , Recombinational DNA Repair , Ubiquitin-Protein Ligases , Humans , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , Acetylglucosamine/metabolism , Rad51 Recombinase/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Phosphorylation , DNA Replication , Ubiquitination , DNA Breaks, Double-Stranded , DNA-Directed DNA Polymerase/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , DNA Damage , DNA/metabolism , HEK293 Cells , Ultraviolet Rays , Protein Binding , Glycosylation , Translesion DNA SynthesisABSTRACT
NADPH has long been recognized as a key cofactor for antioxidant defence and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We find that the reduction of cellular NADPH levels, achieved by silencing malic enzyme or glucose-6-phosphate dehydrogenase, impairs global histone acetylation and transcription in both adipocytes and tumour cells. These effects can be reversed by supplementation with exogenous NADPH or by inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator nuclear receptor corepressor 2 (Ncor2; SMRT) or Ncor1, thereby impairing HDAC3 activation. Interestingly, NADPH and the inositol tetraphosphate molecule Ins(1,4,5,6)P4 appear to bind to the same domains on HDAC3, with NADPH having a higher affinity towards HDAC3 than Ins(1,4,5,6)P4. Thus, while Ins(1,4,5,6)P4 promotes formation of the HDAC3-Ncor complex, NADPH inhibits it. Collectively, our findings uncover a previously unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.
Subject(s)
Epigenesis, Genetic/drug effects , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , NADP/pharmacology , Nuclear Receptor Co-Repressor 1/metabolism , Nuclear Receptor Co-Repressor 2/metabolism , Acetylation , Animals , Cell Line, Tumor , Gene Expression Regulation, Enzymologic/drug effects , Histone Deacetylases/biosynthesis , Histone Deacetylases/genetics , Histones/metabolism , Humans , Inositol Phosphates/pharmacology , Malate Dehydrogenase/metabolism , Mice , Mice, Inbred C57BL , Molecular Docking Simulation , NIH 3T3 Cells , Nuclear Receptor Co-Repressor 1/biosynthesis , Nuclear Receptor Co-Repressor 1/genetics , Nuclear Receptor Co-Repressor 2/biosynthesis , Nuclear Receptor Co-Repressor 2/geneticsABSTRACT
The Krüppel-like factor 5 (KLF5) transcription factor is highly expressed in basal type breast cancer and promotes breast cancer cell proliferation, survival, migration, and tumorigenesis. KLF5 protein stability is regulated by ubiquitination. In this study, ubiquitin-specific protease 3 (USP3) was identified as a new KLF5 deubiquitinase by genome-wide siRNA library screening. We demonstrated that USP3 interacts with KLF5 and stabilizes KLF5 via deubiquitination. USP3 knockdown inhibits breast cancer cell proliferation in vitro and tumorigenesis in vivo, which can be partially rescued by ectopic expression of KLF5. Furthermore, we observed a positive correlation between USP3 and KLF5 protein expression levels in human breast cancer samples. These findings suggest that USP3 is a new KLF5 deubiquitinase and that USP3 may represent a potential therapeutic target for breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Kruppel-Like Transcription Factors/metabolism , Ubiquitin-Specific Proteases/metabolism , Ubiquitination , Animals , Cell Line, Tumor , Cell Proliferation , Female , HEK293 Cells , Humans , Mice, Nude , Protein Binding , Protein Stability , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathologyABSTRACT
The transcription factor KLF5 is highly expressed in basal-like breast cancer and promotes breast cancer cell proliferation, survival, migration and tumour growth. Here we show that, in breast cancer cells, KLF5 is stabilized by the deubiquitinase (DUB) BAP1. With a genome-wide siRNA library screen of DUBs, we identify BAP1 as a bona fide KLF5 DUB. BAP1 interacts directly with KLF5 and stabilizes KLF5 via deubiquitination. KLF5 is in the BAP1/HCF-1 complex, and this newly identified complex promotes cell cycle progression partially by inhibiting p27 gene expression. Furthermore, BAP1 knockdown inhibits tumorigenicity and lung metastasis, which can be rescued partially by ectopic expression of KLF5. Collectively, our findings not only identify BAP1 as the DUB for KLF5, but also reveal a critical mechanism that regulates KLF5 expression in breast cancer. Our findings indicate that BAP1 could be a potential therapeutic target for breast and other cancers.
Subject(s)
Breast Neoplasms/metabolism , Cell Proliferation , Kruppel-Like Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin Thiolesterase/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/physiopathology , Cell Cycle , Cell Line, Tumor , Female , Humans , Kruppel-Like Transcription Factors/genetics , Neoplasm Metastasis , Protein Binding , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , UbiquitinationABSTRACT
The Krüppel-like factor 5 (KLF5) has been suggested to promote breast cell proliferation, survival and tumorigenesis. KLF5 protein degradation is increased by several E3 ubiquitin ligases, including WWP1 and SCFFbw7, through the ubiquitin-proteasome pathway. However, the deubiquitinase (DUB) of KLF5 has not been demonstrated. In this study, we identified ATXN3L as a KLF5 DUB by genome-wide siRNA screening. ATXN3L directly binds to KLF5, decreasing its ubiquitination and thus degradation. Functionally, knockdown of ATXN3L inhibits breast cancer cell proliferation partially through KLF5. These findings reveal a previously unrecognized role of ATXN3L in the regulation of KLF5 stability in breast cancer. ATXN3L might be a therapeutic target for breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Endopeptidases/genetics , Endopeptidases/metabolism , Gene Expression Regulation, Neoplastic , Kruppel-Like Transcription Factors/metabolism , Ubiquitination , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Survival , Female , Gene Library , HEK293 Cells , Humans , Plasmids/metabolism , Protein Interaction Mapping , RNA, Small Interfering/metabolism , Ubiquitin-Specific Proteases/metabolismABSTRACT
Growing evidence suggests that YAP/TAZ are mediators of the Hippo pathway and promote breast cancer. However, the roles of YAP/TAZ transcription factor partners TEADs in breast cancer remain unclear. Here we found that TEAD4 was expressed in breast cancer cell lines, especially in triple negative breast cancers (TNBC) cell lines. TEAD4 binds to KLF5. Knockdown of either TEAD4 or KLF5 in HCC1937 and HCC1806 cells induced the expression of CDK inhibitor p27. Depletion of either TEAD4 or KLF5 activated the p27 gene promoter and increased the p27 mRNA levels. Depletion of p27 partially prevents growth inhibition caused by TEAD4 and KLF5 knockdown. TEAD4 overexpression stimulated proliferation in vitro and tumor growth in mice, while stable knockdown of TEAD4 inhibited proliferation in vitro and tumor growth in mice. Thus TEAD4 and KLF5, in collaboration, promoted TNBC cell proliferation and tumor growth in part by inhibiting p27 gene transcription. TEAD4 is a potential target and biomarker for the development of novel therapeutics for breast cancer.
