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1.
bioRxiv ; 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38168208

RESUMO

R loops are RNA-DNA hybrid containing structures involved in diverse cellular processes, including DNA double-strand break (DSB) repair. R loop homeostasis involving the formation and resolution of R loops is critical for DSB repair, and its dysregulation leads to genome instability. Here we show that the HELZ helicase promotes R loop resolution to facilitate DSB repair by homologous recombination (HR). HELZ depletion causes hypersensitivity to DSB-inducing agents, and HELZ localizes and binds to DSBs. HELZ depletion further leads to genomic instability in a R loop dependent manner and the accumulation of R loops globally and at DSBs. HELZ binds to R loops in response to DSBs and promotes their resolution, thereby facilitating HR to promote genome integrity. Our findings thus define a role for HELZ in promoting the resolution of R loops critical for DSB repair by HR.

2.
Oncogene ; 39(25): 4798-4813, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32457468

RESUMO

Small cell lung cancer (SCLC) is a highly aggressive malignancy with poor outcomes associated with resistance to cisplatin-based chemotherapy. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2), which silences transcription through trimethylation of histone H3 lysine 27 (H3K27me3) and has emerged as an important therapeutic target with inhibitors targeting its methyltransferase activity under clinical investigation. Here, we show that EZH2 has a non-catalytic and PRC2-independent role in stabilizing DDB2 to promote nucleotide excision repair (NER) and govern cisplatin resistance in SCLC. Using a synthetic lethality screen, we identified important regulators of cisplatin resistance in SCLC cells, including EZH2. EZH2 depletion causes cellular cisplatin and UV hypersensitivity in an epistatic manner with DDB1-DDB2. EZH2 complexes with DDB1-DDB2 and promotes DDB2 stability by impairing its ubiquitination independent of methyltransferase activity or PRC2, thereby facilitating DDB2 localization to cyclobutane pyrimidine dimer crosslinks to govern their repair. Furthermore, targeting EZH2 for depletion with DZNep strongly sensitizes SCLC cells and tumors to cisplatin. Our findings reveal a non-catalytic and PRC2-independent function for EZH2 in promoting NER through DDB2 stabilization, suggesting a rationale for targeting EZH2 beyond its catalytic activity for overcoming cisplatin resistance in SCLC.


Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , DNA/genética , DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Complexo Repressor Polycomb 2/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo
3.
Clin Cancer Res ; 24(2): 420-432, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29208669

RESUMO

Purpose: Vimentin is an epithelial-to-mesenchymal transition (EMT) biomarker and intermediate filament protein that functions during cell migration to maintain structure and motility. Despite the abundance of clinical data linking vimentin to poor patient outcome, it is unclear if vimentin is required for metastasis or is a correlative biomarker. We developed a novel genetically engineered mouse model (GEMM) to probe vimentin in lung adenocarcinoma metastasis.Experimental Design: We used the LSL-KrasG12D/Lkb1fl/fl/Vim-/- model (KLV-/-), which incorporates a whole-body knockout of vimentin and is derived from the Cre-dependent LSL-KrasG12D/Lkb1fl/fl model (KLV+/+). We compared the metastatic phenotypes of the GEMMs and analyzed primary tumors from the KLV models and lung adenocarcinoma patients to assess vimentin expression and function.Results: Characterization of KLV+/+ and KLV-/- mice shows that although vimentin is not required for primary lung tumor growth, vimentin is required for metastasis, and vimentin loss generates lower grade primary tumors. Interestingly, in the KLV+/+ mice, vimentin was not expressed in tumor cells but in cancer-associated fibroblasts (CAFs) surrounding collective invasion packs (CIPs) of epithelial tumor cells, with significantly less CIPs in KLV-/- mice. CIPs correlate with tumor grade and are vimentin-negative and E-cadherin-positive, indicating a lack of cancer cell EMT. A similar heterotypic staining pattern was observed in human lung adenocarcinoma samples. In vitro studies show that vimentin is required for CAF motility to lead tumor cell invasion, supporting a vimentin-dependent model of collective invasion.Conclusions: These data show that vimentin is required for lung adenocarcinoma metastasis by maintaining heterotypic tumor cell-CAF interactions during collective invasion. Clin Cancer Res; 24(2); 420-32. ©2017 AACR.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Fibroblastos Associados a Câncer/metabolismo , Transição Epitelial-Mesenquimal/genética , Vimentina/genética , Quinases Proteína-Quinases Ativadas por AMP , Adenocarcinoma de Pulmão/metabolismo , Animais , Biomarcadores Tumorais , Fibroblastos Associados a Câncer/patologia , Comunicação Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos Knockout , Metástase Neoplásica , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Vimentina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Cell Rep ; 20(8): 1921-1935, 2017 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-28834754

RESUMO

DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.


Assuntos
Reparo do DNA por Junção de Extremidades , Recombinação Homóloga , Proteína 1 com Domínio SAM e Domínio HD/genética , Quebras de DNA de Cadeia Dupla , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteína 1 com Domínio SAM e Domínio HD/deficiência , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Transfecção
5.
J Biol Chem ; 292(24): 9919-9931, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28461331

RESUMO

Sirtuin 2 (SIRT2) is a sirtuin family deacetylase, which maintains genome integrity and prevents tumorigenesis. Although Sirt2 deficiency in mice leads to tumorigenesis, the functional significance of somatic SIRT2 mutations in human tumors is unclear. Using structural insight combined with bioinformatics and functional analyses, we show that naturally occurring cancer-associated SIRT2 mutations at evolutionarily conserved sites disrupt its deacetylation of DNA-damage response proteins by impairing SIRT2 catalytic activity or protein levels but not its localization or binding with substrate. We observed that these SIRT2 mutant proteins fail to restore the replication stress sensitivity, impairment in recovery from replication stress, and impairment in ATR-interacting protein (ATRIP) focus accumulation of SIRT2 deficiency. Moreover, the SIRT2 mutant proteins failed to rescue the spontaneous induction of DNA damage and micronuclei of SIRT2 deficiency in cancer cells. Our findings support a model for SIRT2's tumor-suppressive function in which somatic mutations in SIRT2 contribute to genomic instability by impairing its deacetylase activity or diminishing its protein levels in the DNA-damage response. In conclusion, our work provides a mechanistic basis for understanding the biological and clinical significance of SIRT2 mutations in genome maintenance and tumor suppression.


Assuntos
Instabilidade Genômica , Modelos Moleculares , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Sirtuína 2/metabolismo , Acetilação , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Linhagem Celular , Biologia Computacional , Sequência Conservada , Reparo do DNA , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Humanos , Micronúcleos com Defeito Cromossômico , Mutação de Sentido Incorreto , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Conformação Proteica , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/química , Sirtuína 2/genética
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