RESUMEN
Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat-fed, and high-fat-fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina/fisiología , Insulina/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Animales , Animales Modificados Genéticamente , Diabetes Mellitus Tipo 2/etiología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Fosforilación , Proteína Quinasa C-epsilon/genética , Proteómica/métodos , ARN Interferente Pequeño/metabolismo , Ratas , Receptor de Insulina/metabolismo , Proteína S6 Ribosómica/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.
Asunto(s)
Adenoma/tratamiento farmacológico , Neoplasias de las Glándulas Suprarrenales/tratamiento farmacológico , Aldosterona/biosíntesis , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Macrólidos/farmacología , Mutación Missense , Proteínas de Neoplasias/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Neoplasias de las Glándulas Suprarrenales/patología , Aldosterona/genética , Sustitución de Aminoácidos , Línea Celular Tumoral , Citocromo P-450 CYP11B2/biosíntesis , Citocromo P-450 CYP11B2/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genéticaRESUMEN
Resistance to cancer chemotherapies leads to deadly consequences, yet current research focuses only on the roles of somatically acquired mutations in this resistance. The mutational status of the germline is also likely to play a role in the way cells respond to chemotherapy. The carrier status for the POLB rs3136797 germline mutation encoding P242R DNA polymerase beta (Pol ß) is associated with poor prognosis for lung cancer, specifically in response to treatment with cisplatin. Here, it is revealed that the P242R mutation is sufficient to promote resistance to cisplatin in human cells and in mouse xenografts. Mechanistically, P242R Pol ß acts as a translesion polymerase and prefers to insert the correct nucleotide opposite cisplatin intrastrand cross-links, leading to the activation of the nucleotide excision repair (NER) pathway, removal of crosslinks, and resistance to cisplatin. In contrast, wild-type (WT) Pol ß preferentially inserts the incorrect nucleotide initiating mismatch repair and cell death. Importantly, in a mouse xenograft model, tumors derived from lung cancer cells expressing WT Pol ß displayed a slower rate of growth when treated with cisplatin, whereas tumors expressing P242R Pol ß had no response to cisplatin. Pol ß is critical for mediating crosstalk in response to cisplatin. The current data strongly suggest that the status of Pol ß influences cellular responses to crosslinking agents and that Pol ß is a promising biomarker to predict responses to specific chemotherapies. Finally, these results highlight that the genetic status of the germline is a critical factor in the response to cancer treatment.Implications: Pol ß has prognostic biomarker potential in the treatment of cancer with cisplatin and perhaps other intrastrand crosslinking agents. Mol Cancer Res; 15(3); 269-80. ©2017 AACR.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , ADN Polimerasa beta/genética , Mutación de Línea Germinal , Neoplasias/tratamiento farmacológico , Células A549 , Animales , Reparación de la Incompatibilidad de ADN , ADN Polimerasa beta/metabolismo , Resistencia a Antineoplásicos , Células HCT116 , Humanos , Células MCF-7 , Ratones , Neoplasias/enzimología , Neoplasias/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lysine demethylase 5A (KDM5A/RBP2/JARID1A) is a histone lysine demethylase that is overexpressed in several human cancers including lung, gastric, breast and liver cancers. It plays key roles in important cancer processes including tumorigenesis, metastasis, and drug tolerance, making it a potential cancer therapeutic target. Chemical tools to analyze KDM5A demethylase activity are extremely limited as available inhibitors are not specific for KDM5A. Here, we characterized KDM5A using a homogeneous luminescence-based assay and conducted a screen of about 9,000 small molecules for inhibitors. From this screen, we identified several 3-thio-1,2,4-triazole compounds that inhibited KDM5A with low µM in vitro IC50 values. Importantly, these compounds showed great specificity and did not inhibit its close homologue KDM5B (PLU1/JARID1B) or the related H3K27 demethylases KDM6A (UTX) and KDM6B (JMJD3). One compound, named YUKA1, was able to increase H3K4me3 levels in human cells and selectively inhibit the proliferation of cancer cells whose growth depends on KDM5A. As KDM5A was shown to mediate drug tolerance, we investigated the ability of YUKA1 to prevent drug tolerance in EGFR-mutant lung cancer cells treated with gefitinib and HER2+ breast cancer cells treated with trastuzumab. Remarkably, this compound hindered the emergence of drug-tolerant cells, highlighting the critical role of KDM5A demethylase activity in drug resistance. The small molecules presented here are excellent tool compounds for further study of KDM5A's demethylase activity and its contributions to cancer.
Asunto(s)
Neoplasias de la Mama/genética , Resistencia a Antineoplásicos , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proliferación Celular , Tolerancia a Medicamentos , Epigénesis Genética , Células HeLa , Histonas/química , Humanos , Concentración 50 Inhibidora , Luminiscencia , Células MCF-7 , Metástasis de la Neoplasia , Péptidos/química , Proteína 2 de Unión a Retinoblastoma/metabolismo , Resultado del TratamientoRESUMEN
JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC50 of about 3 µm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/química , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Animales , Antineoplásicos/farmacología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Química Farmacéutica/métodos , Diseño de Fármacos , Epigénesis Genética , Histona Demetilasas/metabolismo , Histonas/química , Humanos , Insectos , Neoplasias/tratamiento farmacológico , Péptidos/química , Unión Proteica , Proteínas Recombinantes/química , Tiazoles/químicaRESUMEN
The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new pharmacophores. Hookworms are blood-feeding, intestinal nematode parasites that infect up to 600 million people worldwide. Vaccination with recombinant Ancylostoma ceylanicum macrophage migration inhibitory factor (rAceMIF) provided partial protection from disease, thus establishing a "proof-of-concept" for targeting AceMIF to prevent or treat infection. A high-throughput screen (HTS) against rAceMIF identified six AceMIF-specific inhibitors. A nonsteroidal anti-inflammatory drug (NSAID), sodium meclofenamate, could be tested in an animal model to assess the therapeutic efficacy in treating hookworm disease. Furosemide, an FDA-approved diuretic, exhibited submicromolar inhibition of rAceMIF tautomerase activity. Structure-activity relationships of a pharmacophore based on furosemide included one analog that binds similarly to the active site, yet does not inhibit the Na-K-Cl symporter (NKCC1) responsible for diuretic activity.
Asunto(s)
Ancylostomatoidea/metabolismo , Reposicionamiento de Medicamentos , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Ancylostomatoidea/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Diuréticos/química , Diuréticos/farmacología , Diuréticos/uso terapéutico , Furosemida/química , Furosemida/farmacología , Furosemida/uso terapéutico , Ensayos Analíticos de Alto Rendimiento , Infecciones por Uncinaria/tratamiento farmacológico , Humanos , Cinética , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Ácido Meclofenámico/química , Ácido Meclofenámico/farmacología , Ácido Meclofenámico/uso terapéutico , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
The acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content. In this study, comparative genomics has allowed us to infer the roles of genomic rearrangement and changes in both distribution and copy number of the insertion element, IS1096, in the evolution of Mycobacterium smegmatis mc2155 from its progenitor, M. smegmatis ATCC 607. Comparative analysis revealed that the ATCC 607 genome contains only 11 IS1096 elements against the 24 reported in mc2155. As mc2155 evolved, there was a considerable expansion in the copy number of IS1096 (+13) as well as duplication of a 56-kb fragment flanked on both sides by IS1096; concurrently, a single IS1096 element and its flank were deleted. This study demonstrates that insertion sequence (IS) expansion and IS-induced rearrangements such as duplication, deletion and shuffling are major forces driving genomic diversity and evolution.
Asunto(s)
Elementos Transponibles de ADN/genética , Reordenamiento Génico/genética , Mycobacterium smegmatis/genética , ADN Bacteriano/genética , Evolución Molecular , Duplicación de Gen , Variación Genética , Humanos , Mutagénesis Insercional/métodos , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
We describe a transcriptional analysis platform consisting of a universal micro-array system (UMAS) combined with an enzymatic manipulation step that is capable of generating expression profiles from any organism without requiring a priori species-specific knowledge of transcript sequences. The transcriptome is converted to cDNA and processed with restriction endonucleases to generate low-complexity pools (approximately 80-120) of equal length DNA fragments. The resulting material is amplified and detected with the UMAS system, comprising all possible 4,096 (4(6)) DNA hexamers. Ligation to the arrays yields thousands of 14-mer sequence tags. The compendium of signals from all pools in the array-of-universal arrays comprises a full-transcriptome expression profile. The technology was validated by analysis of the galactose response of Saccharomyces cerevisiae, and the resulting profiles showed excellent agreement with the literature and real-time PCR assays. The technology was also used to demonstrate expression profiling from a hybrid organism in a proof-of-concept experiment where a T-cell receptor gene was expressed in yeast.