RESUMEN
BACKGROUND: Nuclear receptor subfamily 1, group D, member 1 (NR1D1) is a ligand-regulated nuclear receptor and transcriptional factor. Although recent studies have implicated NR1D1 as a regulator of DNA repair and proliferation in breast cancers, its potential as a therapeutic target for breast cancer has not been assessed in terms of clinical outcomes. Thus, this study aims to analyze NR1D1 expression in breast cancer patients and to evaluate its potential prognostic value. METHODS: NR1D1 expression was analyzed by immunohistochemistry using an anti-NR1D1 antibody in 694 breast cancer samples. Survival analyses were performed using the Kaplan-Meier method with the log-rank test to investigate the association of NR1D1 expression with clinical outcome. RESULTS: One hundred thirty-nine of these samples exhibited high NR1D1 expression, mostly in the nucleus of breast cancer cells. NR1D1 expression correlated significantly with histological grade and estrogen receptor status. Overall survival (OS) and disease-free survival (DFS) did not correlate significantly with NR1D1 expression in breast cancer patients regardless of whether they had received chemotherapy. Subgroup analysis performed according to molecular subtype of breast cancer showed a significant influence of high NR1D1 expression on OS (P = 0.002) and DFS (P = 0.007) in patients with triple-negative breast cancer (TNBC) treated with chemotherapy. CONCLUSIONS: High NR1D1 expression level had a favorable impact on OS and DFS in patients with TNBC treated with chemotherapy. NR1D1 should be investigated further as a possible prognostic marker in TNBC patients receiving chemotherapeutic treatment and as a target in the development of chemotherapeutic approaches to treating TNBC.
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Biomarcadores de Tumor , Expresión Génica , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quimioterapia Adyuvante , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Pronóstico , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.
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Reordenamiento Génico , Translocación Genética , Humanos , Animales , Ratones , Mutación , Genómica , Inversión Cromosómica , MamíferosRESUMEN
INTRODUCTION: Trichostatin A (TSA) is a well-characterized histone deacetylase (HDAC) inhibitor. TSA modifies the balance between HDAC and histone acetyltransferase activities that is important in chromatin remodeling and gene expression. Although several previous studies have demonstrated the role of TSA in regulation of estrogen receptor alpha (ERalpha), the precise mechanism by which TSA affects ERalpha activity remains unclear. METHODS: Transient transfection was performed using the Welfect-EX Plus procedure. The mRNA expression was determined using RT-PCR. Protein expression and interaction were determined by western blotting and immunoprecipitation. The transfection of siRNAs was performed using the Oligofectamine reagent procedure. RESULTS: TSA treatment increased acetylation of ERalpha in a dose-dependent manner. The TSA-induced acetylation of ERalpha was accompanied by an increased stability of ERalpha protein. Interestingly, TSA also increased the acetylation and the stability of p300 protein. Overexpression of p300 induced acetylation and stability of ERalpha by blocking ubiquitination. Knockdown of p300 by RNA interference decreased acetylation as well as the protein level of ERalpha, indicating that p300 mediated the TSA-induced stabilization of ERalpha. CONCLUSIONS: We report that TSA enhanced acetylation as well as the stability of the ERalpha protein by modulating stability of p300. These results may provide the molecular basis for pharmacological functions of HDAC inhibitors in the treatment of human breast cancer.
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Proteína p300 Asociada a E1A/metabolismo , Receptor alfa de Estrógeno/metabolismo , Ácidos Hidroxámicos/farmacología , Acetilación/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proteína p300 Asociada a E1A/genética , Receptor alfa de Estrógeno/genética , Células HeLa , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Estabilidad Proteica/efectos de los fármacos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.
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Autofagia , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos , Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismo , Tamoxifeno/farmacología , Adenilato Quinasa/metabolismo , Animales , Autofagia/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/ultraestructura , Resistencia a Antineoplásicos/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Recurrencia Local de Neoplasia/patología , Pronóstico , Regiones Promotoras Genéticas/genética , Transducción de Señal/efectos de los fármacos , Transactivadores , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The regulation of M1/M2 polarization in liver macrophages is closely associated with the progression of nonalcoholic steatohepatitis (NASH); however, the mechanism involved in this process remains unclear. Here, we describe the orphan nuclear receptor retinoic-acid-related orphan receptor α (RORα) as a key regulator of M1/M2 polarization in hepatic residential Kupffer cells (KCs) and infiltrated monocyte-derived macrophages. RORα enhanced M2 polarization in KCs by inducing the kruppel-like factor 4. M2 polarization was defective in KCs and bone-marrow-derived macrophages of the myeloid-specific RORα null mice, and these mice were susceptible to HFD-induced NASH. We found that IL-10 played an important role in connecting the function of M2 KCs to lipid accumulation and apoptosis in hepatocytes. Importantly, M2 polarization was controlled by a RORα activator, JC1-40, which improved symptoms of NASH. Our results suggest that the M2-promoting effects of RORα in liver macrophages may provide better therapeutic strategies against NASH.
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Diferenciación Celular , Macrófagos del Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Animales , Células Cultivadas , Dieta Alta en Grasa/efectos adversos , Interleucina-10/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Macrófagos del Hígado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genéticaRESUMEN
Mitochondrial dysfunction may play a key role in the progression of steatosis to nonalcoholic steatohepatitis (NASH); however, the molecular mechanism that controls the structure and function of mitochondria in NASH is not clearly understood. Here, we demonstrated that RORα is a regulator of expression of Bnip3 and PGC-1α, and thereby enhances mitochondrial quality. First, we observed that liver-specific RORα knockout mice (RORα-LKO) were more susceptible to high-fat diet-induced NASH compared with control, probably due to mitochondrial dysfunction. Concordantly, mitochondrial fission in response to nutrient stimuli was abolished with downregulation of Bnip3 and phospho-Drp1 in the hepatocytes of RORα-LKO. RORα enhanced oxygen consumption rate and expression of genes associated with mitochondrial quality control. Finally, we observed the positive correlation of the expression levels of Bnip3 and PGC-1α with those of RORα in patients with steatohepatitis. Together, we demonstrated that RORα mediates mitochondrial quality under nutrient-overloaded conditions and propose RORα as a potential therapeutic target in treatment of NASH.
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Hígado/metabolismo , Mitocondrias/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Animales , Hepatocitos/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genéticaRESUMEN
DNA repair capacity is critical for survival of cancer cells upon therapeutic DNA damage and thus is an important determinant of susceptibility to chemotherapy in cancer patients. In this study, we identified a novel function of nuclear receptor NR1D1 in DNA repair, which enhanced chemosensitivity in breast cancer cells. NR1D1 inhibited both nonhomologous end joining and homologous recombination double-strand breaks repair, and delayed the clearance of γH2AX DNA repair foci that formed after treatment of doxorubicin. PARylation of NR1D1 by PARP1 drove its recruitment to damaged DNA lesions. Deletion of the ligand binding domain of NR1D1 that interacted with PARP1, or treatment of 6-(5H)-phenanthridinone, an inhibitor of PARP1, suppressed the recruitment of NR1D1 to DNA damaged sites, indicating PARylation as a critical step for the NR1D1 recruitment. NR1D1 inhibited recruitment of the components of DNA damage response complex such as SIRT6, pNBS1, and BRCA1 to DNA lesions. Downregulation of NR1D1 in MCF7 cells resulted in resistance to doxorubicin, both in vitro and in vivo Analysis of four public patient data sets indicated that NR1D1 expression correlates positively with clinical outcome in breast cancer patients who received chemotherapy. Our findings suggest that NR1D1 and its ligands provide therapeutic options that could enhance the outcomes of chemotherapy in breast cancer patients. Cancer Res; 77(9); 2453-63. ©2017 AACR.
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Neoplasias de la Mama/tratamiento farmacológico , Reparación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Doxorrubicina/administración & dosificación , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Proteínas Nucleares/genética , Sirtuinas/genéticaRESUMEN
AIMS: To elucidate the role of nuclear receptor subfamily 1, group D, member 1 (Nr1d1) in hepatic lipid metabolism and pathogenesis of nonalcoholic fatty liver diseases, Nr1d1 gene mutant mice, in which the DNA-binding domain (exons 3 and 4) was deleted (Nr1d1 Δex3/4), were challenged with a high-fat diet (HFD), and the gene expression patterns that responded to this alteration were profiled. MAIN METHODS: The Nr1d1 Δex3/4 mice were fed an HFD for 12weeks. Liver tissues were examined by histology, and lipid droplets were detected by Oil-Red O staining. Serum biochemical analyses were performed to assess markers of liver injury. Microarray analysis was used to profile hepatic gene expression patterns. Functional annotation, upstream prediction, and gene coexpression prediction analyses were performed. KEY FINDINGS: The Nr1d1 Δex3/4 mice showed enhanced hepatic steatosis after being challenged with an HFD, but not with a low-fat diet, indicating an interaction between diet and genotype for this phenotypic change. Gene expression profiling revealed that this interaction might involve neutrophil recruitment and the cyclic adenosine monophosphate metabolic pathway. A study of transcription factor binding site enrichment suggested that CCAAT/enhancer-binding protein alpha and hepatocyte nuclear factor 4 alpha were associated with this phenotypic change. SIGNIFICANCE: Loss of DNA binding of Nr1d1 was associated with a deterioration in hepatic steatosis. The interaction between the Nr1d1 Δex3/4 genotype with an HFD might mediate these phenotypic changes, probably through a nonclassical transcriptional function of Nr1d1.
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Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Animales , Dieta Alta en Grasa/efectos adversos , Exones , Femenino , Eliminación de Gen , Metabolismo de los Lípidos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , TranscriptomaRESUMEN
Metastasis-associated protein 1 (MTA1) is a component of the nucleosome remodeling and histone deacetylase (HDAC) complex, which plays an important role in progression of breast cancer. Although MTA1 is known as a repressor of the transactivation function of estrogen receptor α (ERα), its involvement in the epigenetic control of transcription of the ERα gene ESR1 has not been studied. Here, we show that silencing of MTA1 reduced the level of expression of ERα in ERα-positive cells but increased it in ERα-negative cells. In both MCF7 and MDA-MB-231, MTA1 was recruited to the region +146 to +461 bp downstream of the transcription start site of ESR1 (ERpro315). Proteomics analysis of the MTA1 complex that was pulled down by an oligonucleotide encoding ERpro315 revealed that the transcription factor AP-2γ (TFAP2C) and the IFN-γ-inducible protein 16 (IFI16) were components of the complex. Interestingly, in MCF7, TFAP2C activated the reporter encoding ERpro315 and the level of ERα mRNA. By contrast, in MDA-MB-231, IFI16 repressed the promoter activity and silencing of MTA1 increased expression of ERα. Importantly, class II HDACs are involved in the MTA1-mediated differential regulation of ERα. Finally, an MDA-MB-231-derived cell line that stably expressed shIFI16 or shMTA1 was more susceptible to tamoxifen-induced growth inhibition in in vitro and in vivo experiments. Taken together, our findings suggest that the MTA1-TFAP2C or the MTA1-IFI16 complex may contribute to the epigenetic regulation of ESR1 expression in breast cancer and may determine the chemosensitivity of tumors to tamoxifen therapy in patients with breast cancer.
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Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Histona Desacetilasas/genética , Proteínas Represoras/genética , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Tamoxifeno/farmacología , Transactivadores , Factor de Transcripción AP-2/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
Although the roles of Nur77, an orphan member of the nuclear hormone receptor superfamily, in the control of cellular proliferation, apoptosis, inflammation, and glucose metabolism, are well recognized, the molecular mechanism regulating the activity and expression of Nur77 is not fully understood. Acetylation of transcription factors has emerged recently as a major post-translational modification that regulates protein stability and transcriptional activity. Here, we examined whether Nur77 is acetylated, and we characterized potential associated factors. First, Nur77 was found to be an acetylated protein when examined by immunoprecipitation and western blotting using acetyl protein-specific antibodies. Second, expression of p300, which possesses histone acetyltransferase activity, enhanced the acetylation and protein stability of Nur77. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, also increased Nur77 acetylation. Among the several types of HDACs, HDAC1 was found as the major enzyme affecting protein level of Nur77. HDAC1 decreased the acetylation level, protein level, and transcriptional activity of Nur77. Interestingly, overexpression of Nur77 induced expression of both p300 and HDAC1. Finally, the expression of Nur77 increased along with that of p300, but decreased with induction of HDAC1 after treatment with epithelial growth factor, nerve growth factor, or 6-mercaptopurine, suggesting that the self-control of the acetylation status contributes to the transient induction of Nur77 protein. Taken together, these results demonstrate that acetylation of Nur77 is modulated by p300 and HDAC1, and suggest that acetylation is an important post-translational modification for the rapid turnover of Nur77 protein.
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Proteína p300 Asociada a E1A/fisiología , Histona Desacetilasa 1/fisiología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Acetilación , Animales , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Estabilidad ProteicaRESUMEN
The interplay between hypoxia-inducible factor-1alpha (HIF-1alpha) and histone deacetylase (HDACs) have been well studied; however, the mechanism of cross-talk is unclear. Here, we investigated the roles of HDAC4 and HDAC5 in the regulation of HIF-1alpha function and its associated mechanisms. HDAC4 and HDAC5 enhanced transactivation by HIF-1alpha without stabilizing HIF-1alpha. HDAC4 and HDAC5 physically associated with HIF-1alpha through the inhibitory domain (ID) that is the binding site for factor inhibiting HIF-1 (FIH-1). In the presence of these HDACs, binding of HIF-1alpha to FIH-1 decreased, whereas binding to p300 increased. These results indicate that HDAC4 and HDAC5 increase the transactivation function of HIF-1alpha by promoting dissociation of HIF-1alpha from FIH-1 and association with p300.