RESUMO
Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.
Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/patologia , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/patologia , Epigênese Genética , Cromatina/genéticaRESUMO
The Wnt signaling pathway is a critical mediator of the development and maintenance of several tissues. The adrenal cortex is highly dependent upon Wnt/ß-catenin signaling for proper zonation and endocrine function. Adrenocortical cells emerge in the peripheral capsule and subcapsular cortex of the gland as progenitor cells that centripetally differentiate into steroid hormone-producing cells of three functionally distinct concentric zones that respond robustly to various endocrine stimuli. Wnt/ß-catenin signaling mediates adrenocortical progenitor cell fate and tissue renewal to maintain the gland throughout life. Aberrant Wnt/ß-catenin signaling contributes to various adrenal disorders of steroid production and growth that range from hypofunction and hypoplasia to hyperfunction, hyperplasia, benign adrenocortical adenomas, and malignant adrenocortical carcinomas. Great strides have been made in defining the molecular underpinnings of adrenocortical homeostasis and disease, including the interplay between the capsule and cortex, critical components involved in maintaining the adrenocortical Wnt/ß-catenin signaling gradient, and new targets in adrenal cancer. This review seeks to examine these and other recent advancements in understanding adrenocortical Wnt/ß-catenin signaling and how this knowledge can inform therapeutic options for adrenal disease.
Assuntos
Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Doenças das Glândulas Suprarrenais/metabolismo , Doenças das Glândulas Suprarrenais/patologia , beta Catenina/metabolismo , Animais , Humanos , Ligantes , Regeneração , Via de Sinalização WntRESUMO
ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/patologia , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Cães , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Esterol O-Aciltransferase/metabolismoRESUMO
Expression of the Growth Hormone (GH)-stimulated gene Socs2 (Suppressor of Cytokine Signaling 2) is mediated by the transcription activator STAT5 (Signal Transducer and Activator of Transcription 5) and the transcription repressor BCL6 (B-Cell Lymphoma 6). ChIP-Sequencing identified Cish (Cytokine-Inducible SH2-containing protein) and Bcl6 as having similar patterns of reciprocal occupancy by BCL6 and STAT5 in response to GH, though GH stimulates Cish and inhibits Bcl6 expression. The co-activator p300 occupied Socs2, Cish and Bcl6 promoters, and enhanced STAT5-mediated activation of Socs2 and Cish. In contrast, on Bcl6, p300 functioned as a repressor and inhibited in conjunction with STAT5 or BCL6. The co-repressor HDAC3 (Histone deacetylase 3) inhibited the Socs2, Cish and Bcl6 promoters in the presence of STAT5. Thus transcriptional outcomes on GH-regulated genes occupied by BCL6 and STAT5 are determined in a promoter-specific fashion by co-regulatory proteins which mediate the distinction between activating and repressive transcription factors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína p300 Associada a E1A/metabolismo , Hormônio do Crescimento/metabolismo , Histona Desacetilases/metabolismo , Elementos de Resposta/fisiologia , Fator de Transcrição STAT5/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteína p300 Associada a E1A/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hormônio do Crescimento/farmacologia , Histona Desacetilases/genética , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6 , Fator de Transcrição STAT5/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The transcriptional repressor B-cell Lymphoma 6 (Bcl6) was recently identified in a profile of genes regulated in adipocytes, suggesting a relationship between Bcl6 and metabolic regulation. As a representative target gene repressed by Bcl6, Suppressor of Cytokine Signaling (Socs) 2 expression was elevated in Bcl6 deficient (KO) mice, including metabolic tissues liver, adipose tissue and muscle, as well as in spleen and thymus. Bcl6 occupied the Socs2 promoter in wild-type, but not Bcl6 KO mice, suggesting direct regulation of Socs2 by Bcl6 in vivo. Mice deficient in Bcl6 were found to exhibit multiple features of dysregulated lipid metabolism. Adipose tissue mass was dramatically reduced or absent in Bcl6 KO mice. Further, hepatic and serum triglycerides were low. Bcl6 deficiency was accompanied by decreased hepatic expression of Stearoyl-CoA desaturase 1 (Scd1) and Fatty acid synthase (Fasn) genes which encode lipogenic enzymes. Expression of the gene for the transcription factor Carbohydrate-Responsive Element Binding Protein (Chrebp), which regulates expression of lipogenic genes, was also reduced in liver of Bcl6 KO mice. Bcl6 deficiency disrupted fasting-induced increases in hepatic triglyceride deposition, but not decreases in lipogenic gene expression. Taken together, these findings suggest that in addition to its well-recognized roles in immune regulation, Bcl6 plays a role in regulatory events of lipid metabolism, and that in the absence of Bcl6, lipid metabolism in liver and adipose tissue is dysregulated.
Assuntos
Proteínas de Ligação a DNA/deficiência , Metabolismo dos Lipídeos , Proteínas Repressoras/deficiência , Transcrição Gênica , Tecido Adiposo/patologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas de Ligação a DNA/metabolismo , Jejum/sangue , Ácidos Graxos/metabolismo , Feminino , Regulação da Expressão Gênica , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Fígado/metabolismo , Masculino , Metaboloma/genética , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Tamanho do Órgão , Oxirredução , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/metabolismo , Soro/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triglicerídeos/metabolismoRESUMO
Regulation of c-Fos transcription by GH is mediated by CCAAT/enhancer binding protein ß (C/EBPß). This study examines the role of C/EBPß in mediating GH activation of other early response genes, including Cyr61, Btg2, Socs3, Zfp36, and Socs1. C/EBPß depletion using short hairpin RNA impaired responsiveness of these genes to GH, as seen for c-Fos. Rescue with wild-type C/EBPß led to GH-dependent recruitment of the coactivator p300 to the c-Fos promoter. In contrast, rescue with C/EBPß mutated at the ERK phosphorylation site at T188 failed to induce GH-dependent recruitment of p300, indicating that ERK-mediated phosphorylation of C/EBPß at T188 is required for GH-induced recruitment of p300 to c-Fos. GH also induced the occupancy of phosphorylated C/EBPß and p300 on Cyr61, Btg2, and Socs3 at predicted C/EBP-cAMP response element-binding protein motifs in their promoters. Consistent with a role for ERKs in GH-induced expression of these genes, treatment with U0126 to block ERK phosphorylation inhibited their GH-induced expression. In contrast, GH-dependent expression of Zfp36 and Socs1 was not inhibited by U0126. Thus, induction of multiple early response genes by GH in 3T3-F442A cells is mediated by C/EBPß. A subset of these genes is regulated similarly to c-Fos, through a mechanism involving GH-stimulated ERK 1/2 activation, phosphorylation of C/EBPß, and recruitment of p300. Overall, these studies suggest that C/EBPß, like the signal transducer and activator of transcription proteins, regulates multiple genes in response to GH.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Hormônio do Crescimento/metabolismo , Animais , Butadienos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Cricetinae , Cricetulus , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes fos/genética , Immunoblotting , Camundongos , Mutação , Nitrilas/farmacologia , Fosforilação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Elementos de Resposta , Transdução de Sinais/genética , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Resistance to chemotherapy is a major problem facing breast cancer patients. Cisplatin, a highly effective DNA-damaging drug, has shown only little success in breast cancer treatment. We are reporting that low nanomolar doses of bisphenol A (BPA) or estradiol antagonize cisplatin cytotoxicity in breast cancer cells, with their effects not mediated via classical estrogen receptors. Although both compounds increase the expression of Bcl-2, a Bcl-2 inhibitor completely blocked the protective effects of BPA while only partially affecting those of estradiol. Blockade of BPA and E2 actions should sensitize ER-negative breast tumors to anti-cancer drugs and allow for the inclusion of cisplatin in treatment regimens.
Assuntos
Antineoplásicos/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Cisplatino/antagonistas & inibidores , Estradiol/farmacologia , Estrogênios não Esteroides/farmacologia , Fenóis/farmacologia , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Compostos Benzidrílicos , Western Blotting , Linhagem Celular Tumoral , Separação Celular , Cisplatino/toxicidade , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estrogênios/farmacologia , Feminino , Citometria de Fluxo , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin (PRL) acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with PRL caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. PRL prevented cisplatin-induced G(2)/M cell cycle arrest and apoptosis. In the presence of PRL, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by gamma-H2AX staining. PRL dramatically increased the activity of glutathione-S-transferase (GST), which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and mitogen-activated protein kinase inhibitors. PRL upregulated the expression of the GSTmu, but not the pi, isozyme. A GST inhibitor abrogated antagonism of cisplatin cytotoxicity by PRL. In conclusion, PRL confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both PRL and its receptor. Suppression of PRL production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Glutationa Transferase/metabolismo , Prolactina/farmacologia , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Doxorrubicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Glutationa/metabolismo , Histonas/metabolismo , Humanos , Paclitaxel/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Vimblastina/farmacologiaRESUMO
IL-6 is an important cytokine that regulates both immune and metabolic functions. Within adipose tissue, preadipocytes produce significant amounts of IL-6, but little is known about the factors or mechanisms that regulate IL-6 production in these cells. Using LS14, a newly developed human adipocyte cell line, our objective was to determine the mechanisms by which insulin stimulates IL-6 production and release in preadipocytes. Insulin increased IL-6 gene expression and secretion in a time- and dose-dependent manner. Insulin decreased cyclic AMP (cAMP) but increased cyclic GMP (cGMP) levels, and IL-6 expression/release was stimulated by a cGMP analog. The stimulatory effect of insulin and cGMP was abrogated by a specific inhibitor of protein kinase G (cyclic GMP-dependent protein kinase). Both insulin and cGMP rapidly induced phosphorylation of cAMP response element binding protein. Insulin also activated the MAPK signaling pathway, and its blockade prevented the insulin-stimulated increases in IL-6 cell content and release, but not IL-6 gene expression. Although inhibition of the proteosome increased IL-6 cell content and release, proteosome activity was unaffected by insulin. These data suggest that the stimulatory effects of insulin on IL-6 release involve several interrelated components: transcription, intracellular releasable pool, and secretion, which are differentially regulated and, thus, determine the size of the releasable pool of IL-6. Insulin-induced IL-6 gene expression is mediated by cGMP/cyclic GMP-dependent protein kinase/cAMP response element binding protein, whereas MAPK is involved in the insulin-stimulated IL-6 synthesis/release.
Assuntos
Adipócitos/efeitos dos fármacos , Linhagem Celular , Insulina/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Adipócitos/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Humanos , Isoquinolinas/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
Prolactin (PRL) is a 23-kDa protein hormone that binds to a single-span membrane receptor, a member of the cytokine receptor superfamily, and exerts its action via several interacting signaling pathways. PRL is a multifunctional hormone that affects multiple reproductive and metabolic functions and is also involved in tumorigenicity. In addition to being a classical pituitary hormone, PRL in humans is produced by many tissues throughout the body where it acts as a cytokine. The objective of this review is to compare and contrast multiple aspects of PRL, from structure to regulation, and from physiology to pathology in rats, mice, and humans. At each juncture, questions are raised whether, or to what extent, data from rodents are relevant to PRL homeostasis in humans. Most current knowledge on PRL has been obtained from studies with rats and, more recently, from the use of transgenic mice. Although this information is indispensable for understanding PRL in human health and disease, there is sufficient disparity in the control of the production, distribution, and physiological functions of PRL among these species to warrant careful and judicial extrapolation to humans.