RESUMO
Overexpression of histone deacetylases (HDACs) in cancer commonly causes resistance to genotoxic-based therapies. Here, we report on the novel mechanism whereby overexpressed class I HDACs increase the resistance of glioblastoma cells to the SN1 methylating agent temozolomide (TMZ). The chemotherapeutic TMZ triggers the activation of the DNA damage response (DDR) in resistant glioma cells, leading to DNA lesion bypass and cellular survival. Mass spectrometry analysis revealed that the catalytic activity of class I HDACs stimulates the expression of the E3 ubiquitin ligase RAD18. Furthermore, the data showed that RAD18 is part of the O6-methylguanine-induced DDR as TMZ induces the formation of RAD18 foci at sites of DNA damage. Downregulation of RAD18 by HDAC inhibition prevented glioma cells from activating the DDR upon TMZ exposure. Lastly, RAD18 or O6-methylguanine-DNA methyltransferase (MGMT) overexpression abolished the sensitization effect of HDAC inhibition on TMZ-exposed glioma cells. Our study describes a mechanism whereby class I HDAC overexpression in glioma cells causes resistance to TMZ treatment. HDACs accomplish this by promoting the bypass of O6-methylguanine DNA lesions via enhancing RAD18 expression. It also provides a treatment option with HDAC inhibition to undermine this mechanism.
Assuntos
Neoplasias Encefálicas , Glioma , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Glioma/tratamento farmacológico , Glioma/genética , Glioma/metabolismo , Histona Desacetilases/farmacologia , Humanos , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/farmacologia , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Ubiquitina-Proteína Ligases/farmacologiaRESUMO
Here we describe the method used in our laboratory for determining the activity of homologous recombination repair of DNA double-strand breaks in cell lines. This plasmid-based method, first published by Pierce et al. 1999 from Maria Jasin's laboratory, is used along with flow cytometry for demonstrating the positive regulation of class I histone deacetylases on the repair of DNA double-strand breaks by homologous recombination.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Fluorescência Verde/genética , Histona Desacetilase 1/genética , Inibidores de Histona Desacetilases/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/antagonistas & inibidores , Proteínas de Fluorescência Verde/metabolismo , Histona Desacetilase 1/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Puromicina/farmacologia , Proteínas Recombinantes de Fusão , Ácido Valproico/farmacologiaRESUMO
Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD+ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD+, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Histona Desacetilases/fisiologia , Animais , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades , Reparo do DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Recombinação Homóloga , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais , Sirtuínas/genética , Sirtuínas/metabolismoRESUMO
DNA-damaging anticancer drugs remain a part of metastatic melanoma therapy. Epigenetic reprogramming caused by increased histone deacetylase (HDAC) activity arising during tumor formation may contribute to resistance of melanomas to the alkylating drugs temozolomide, dacarbazine, and fotemustine. Here, we report on the impact of class I HDACs on the response of malignant melanoma cells treated with alkylating agents. The data show that malignant melanomas in situ contain a high level of HDAC1/2 and malignant melanoma cells overexpress HDAC1/2/3 compared with noncancer cells. Furthermore, pharmacologic inhibition of class I HDACs sensitizes malignant melanoma cells to apoptosis following exposure to alkylating agents, while not affecting primary melanocytes. Inhibition of HDAC1/2/3 caused sensitization of melanoma cells to temozolomide in vitro and in melanoma xenografts in vivo HDAC1/2/3 inhibition resulted in suppression of DNA double-strand break (DSB) repair by homologous recombination because of downregulation of RAD51 and FANCD2. This sensitized cells to the cytotoxic DNA lesion O(6)-methylguanine and caused a synthetic lethal interaction with the PARP-1 inhibitor olaparib. Furthermore, knockdown experiments identified HDAC2 as being responsible for the regulation of RAD51. The influence of class I HDACs on DSB repair by homologous recombination and the possible clinical implication on malignant melanoma therapy with temozolomide and other alkylating drugs suggests a combination approach where class I HDAC inhibitors such as valproic acid or MS-275 (entinostat) appear to counteract HDAC- and RAD51/FANCD2-mediated melanoma cell resistance. Cancer Res; 76(10); 3067-77. ©2016 AACR.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Melanoma/patologia , Rad51 Recombinase/metabolismo , Neoplasias Cutâneas/patologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/antagonistas & inibidores , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Imunofluorescência , Histona Desacetilases/química , Histona Desacetilases/genética , Recombinação Homóloga/efeitos dos fármacos , Humanos , Técnicas Imunoenzimáticas , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Mensageiro/genética , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Melanoma Maligno CutâneoRESUMO
In the treatment of metastatic melanoma, a highly therapy-refractory cancer, alkylating agents are used and, for the subgroup of BRAFV600E cancers, the B-Raf inhibitor vemurafenib. Although vemurafenib is initially beneficial, development of drug resistance occurs leading to tumor relapse, which necessitates the requirement for combined or sequential therapy with other drugs, including genotoxic alkylating agents. This leads to the question whether vemurafenib and alkylating agents act synergistically and whether chronic vemurafenib treatment alters the melanoma cell response to alkylating agents. Here we show that a) BRAFV600E melanoma cells are killed by vemurafenib, driving apoptosis, b) BRAFV600E melanoma cells are neither more resistant nor sensitive to temozolomide/fotemustine than non-mutant cells, c) combined treatment with vemurafenib plus temozolomide or fotemustine has an additive effect on cell kill, d) acquired vemurafenib resistance of BRAFV600E melanoma cells does not affect MGMT, MSH2, MSH6, PMS2 and MLH1, nor does it affect the resistance to temozolomide and fotemustine, e) metastatic melanoma biopsies obtained from patients prior to and after vemurafenib treatment did not show a change in the MGMT promoter methylation status and MGMT expression level. The data suggest that consecutive treatment with vemurafenib and alkylating drugs is a reasonable strategy for metastatic melanoma treatment.