RESUMEN
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.
Asunto(s)
Neoplasias Encefálicas , Glioma , Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/farmacología , Resistencia a Antineoplásicos/genética , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/metabolismo , Histona Desacetilasas/farmacología , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/farmacología , Temozolomida/farmacología , Temozolomida/uso terapéutico , Ubiquitina-Proteína Ligasas/farmacologíaRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas Fluorescentes Verdes/genética , Histona Desacetilasa 1/genética , Inhibidores de Histona Desacetilasas/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/antagonistas & inhibidores , Proteínas Fluorescentes Verdes/metabolismo , Histona Desacetilasa 1/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Melanocitos/citología , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Puromicina/farmacología , Proteínas Recombinantes de Fusión , Ácido Valproico/farmacologíaRESUMEN
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.
Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Histona Desacetilasas/fisiología , Animales , Roturas del ADN de Doble Cadena , Daño del ADN/efectos de los fármacos , Reparación del ADN por Unión de Extremidades , Reparación del ADN/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Recombinación Homóloga , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal , Sirtuinas/genética , Sirtuinas/metabolismoRESUMEN
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.
Asunto(s)
Resistencia a Antineoplásicos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Melanoma/patología , Recombinasa Rad51/metabolismo , Neoplasias Cutáneas/patología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/antagonistas & inhibidores , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Técnica del Anticuerpo Fluorescente , Histona Desacetilasas/química , Histona Desacetilasas/genética , Recombinación Homóloga/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Mensajero/genética , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Melanoma Cutáneo MalignoRESUMEN
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.