RESUMO
PIP4K2A is an insufficiently studied type II lipid kinase that catalyzes the conversion of phosphatidylinositol-5-phosphate (PI5P) into phosphatidylinositol 4,5-bisphosphate (PI4,5P2). The involvement of PIP4K2A/B in cancer has been suggested, particularly in the context of p53 mutant/null tumors. PIP4K2A/B depletion has been shown to induce tumor growth inhibition, possibly due to hyperactivation of AKT and reactive oxygen species-mediated apoptosis. Herein, we report the identification of the novel potent and highly selective inhibitors BAY-091 and BAY-297 of the kinase PIP4K2A by high-throughput screening and subsequent structure-based optimization. Cellular target engagement of BAY-091 and BAY-297 was demonstrated using cellular thermal shift assay technology. However, inhibition of PIP4K2A with BAY-091 or BAY-297 did not translate into the hypothesized mode of action and antiproliferative activity in p53-deficient tumor cells. Therefore, BAY-091 and BAY-297 serve as valuable chemical probes to study PIP4K2A signaling and its involvement in pathophysiological conditions such as cancer.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Naftiridinas/química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Genomic variants in both ADTRP and TFPI genes are associated with risk of coronary artery disease (CAD). ADTRP regulates TFPI expression and endothelial cell functions involved in the initiation of atherosclerotic CAD. ADTRP also specifies primitive myelopoiesis and definitive hematopoiesis by upregulating TFPI expression. However, the underlying molecular mechanism is unknown. Here we show that transcription factor POU1F1 is the key by which ADTRP regulates TFPI expression. Luciferase reporter assays, chromatin-immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) in combination with analysis of large and small deletions of the TFPI promoter/regulatory region were used to identify the molecular mechanism by which ADTRP regulates TFPI expression. Genetic association was assessed using case-control association analysis and phenome-wide association analysis (PhenGWA). ADTRP regulates TFPI expression at the transcription level in a dose-dependent manner. The ADTRP-response element was localized to a 50 bp region between -806 bp and -756 bp upstream of TFPI transcription start site, which contains a binding site for POU1F1. Deletion of POU1F1-binding site or knockdown of POU1F1 expression abolished ADTRP-mediated transcription of TFPI. ChIP and EMSA demonstrated that POU1F1 binds to the ADTRP response element. Genetic analysis identified significant association between POU1F1 variants and risk of CAD. PhenGWA identified other phenotypic traits associated with the ADTRP-POU1F1-TFPI axis such as lymphocyte count (ADTRP), waist circumference (TFPI), and standing height (POU1F1). These data identify POU1F1 as a transcription factor that regulates TFPI transcription in response to ADTRP, and link POU1F1 variants to risk of CAD for the first time.
Assuntos
Doença da Artéria Coronariana/metabolismo , Lipoproteínas/biossíntese , Proteínas de Membrana/metabolismo , Fator de Transcrição Pit-1/metabolismo , Aterosclerose/genética , Estudos de Casos e Controles , Linhagem Celular , Imunoprecipitação da Cromatina/métodos , Doença da Artéria Coronariana/genética , Bases de Dados Genéticas , Células Endoteliais/metabolismo , Genes Homeobox , Células HeLa , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Regiões Promotoras Genéticas , Elementos de Resposta , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
The mutation of K-RAS represents one of the most frequent genetic alterations in cancer. Targeting of downstream effectors of RAS, including of MEK and ERK, has limited clinical success in cancer patients with K-RAS mutations. The reduced sensitivity of K-RAS-mutated cells to certain MEK inhibitors (MEKi) is associated with the feedback phosphorylation of MEK by C-RAF and with the reactivation of mitogen-activated protein kinase (MAPK) signaling. Here, we report that the RAF dimer inhibitors lifirafenib (BGB-283) and compound C show a strong synergistic effect with MEKi, including mirdametinib (PD-0325901) and selumetinib, in suppressing the proliferation of K-RAS-mutated non-small-cell lung cancer and colorectal cancer (CRC) cell lines. This synergistic effect was not observed with the B-RAFV600E selective inhibitor vemurafenib. Our mechanistic analysis revealed that RAF dimer inhibition suppresses RAF-dependent MEK reactivation and leads to the sustained inhibition of MAPK signaling in K-RAS-mutated cells. This synergistic effect was also observed in several K-RAS mutant mouse xenograft models. A pharmacodynamic analysis supported a role for the synergistic phospho-ERK blockade in enhancing the antitumor activity observed in the K-RAS mutant models. These findings support a vertical inhibition strategy in which RAF dimer and MEKi are combined to target K-RAS-mutated cancers, and have led to a Phase 1b/2 combination therapy study of lifirafenib and mirdametinib in solid tumor patients with K-RAS mutations and other MAPK pathway aberrations.
Assuntos
Antineoplásicos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Regulação Alostérica/efeitos dos fármacos , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Multimerização Proteica/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Vemurafenib/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of ADTRP was regulated by androgen, but the molecular mechanism is unknown. In this study, we identified the molecular mechanism by which androgen regulates ADTRP expression and tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression. Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulated ADTRP transcription in a dose- and time-dependent manner, and the effect was abolished by three different androgen inhibitors, including pyrvinium pamoate, bicalutamide, and cyproterone acetate. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE, TGTTCT) located at +324bp from the ADTRP transcription start site. The ARE is required for concentration-dependent transcriptional activation of ADTRP. HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was rescued by ADTRP siRNA and exacerbated by overexpression of ADTRP and its downstream genes PIK3R3 and MIA3. These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of ADTRP expression.
Assuntos
Androgênios/farmacologia , Aterosclerose/metabolismo , Doença da Artéria Coronariana/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/biossíntese , Elementos de Resposta , Transcrição Gênica/efeitos dos fármacos , Aterosclerose/genética , Aterosclerose/patologia , Técnicas de Cocultura , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Células Endoteliais/metabolismo , Estudo de Associação Genômica Ampla , Células HL-60 , Células HeLa , Humanos , Proteínas de Membrana/genética , Monócitos/metabolismo , Monócitos/patologia , Migração Transendotelial e Transepitelial/efeitos dos fármacosRESUMO
Oncogenic BRAF, which drives cell transformation and proliferation, has been detected in approximately 50% of human malignant melanomas and 5% to 15% of colorectal cancers. Despite the remarkable clinical activities achieved by vemurafenib and dabrafenib in treating BRAF(V600E) metastatic melanoma, their clinical efficacy in BRAF(V600E) colorectal cancer is far less impressive. Prior studies suggested that feedback activation of EGFR and MAPK signaling upon BRAF inhibition might contribute to the relative unresponsiveness of colorectal cancer to the first-generation BRAF inhibitors. Here, we report characterization of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently under clinical investigation. In vitro, BGB-283 potently inhibits BRAF(V600E)-activated ERK phosphorylation and cell proliferation. It demonstrates selective cytotoxicity and preferentially inhibits proliferation of cancer cells harboring BRAF(V600E) and EGFR mutation/amplification. In BRAF(V600E) colorectal cancer cell lines, BGB-283 effectively inhibits the reactivation of EGFR and EGFR-mediated cell proliferation. In vivo, BGB-283 treatment leads to dose-dependent tumor growth inhibition accompanied by partial and complete tumor regressions in both cell line-derived and primary human colorectal tumor xenografts bearing BRAF(V600E) mutation. These findings support BGB-283 as a potent antitumor drug candidate with clinical potential for treating colorectal cancer harboring BRAF(V600E) mutation.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Naftiridinas/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Quinases raf/antagonistas & inibidores , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Modelos Moleculares , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/metabolismoRESUMO
Reactive oxygen species (ROS) promote tumor cell proliferation and survival by directly modulating growth-regulatory molecules and key transcription factors. The signal transducer and activator of transcription 3 (STAT3) is constitutively active in a variety of tumor cell types, where the effect of ROS on the Janus kinase/STAT pathway has been examined. We report here that STAT3 is directly sensitive to intracellular oxidants. Oxidation of conserved cysteines by peroxide decreased STAT3 binding to consensus serum-inducible elements (SIE) in vitro and in vivo and diminished interleukin (IL)-6-mediated reporter expression. Inhibitory effects produced by cysteine oxidation in STAT3 were negated in redox-insensitive STAT3 mutants. In contrast, ROS had no effect on IL-6-induced STAT3 recruitment to the c-myc P2 promoter. Expression of a redox-insensitive STAT3 in breast carcinoma cells accelerated their proliferation while reducing resistance to oxidative stress. Our results implicate STAT3 in coupling intracellular redox homeostasis to cell proliferation and survival.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição STAT3/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Neoplasias da Mama/patologia , Divisão Celular , Sequência Conservada , Cisteína/genética , Cisteína/metabolismo , Feminino , Genes Reporter , Humanos , Dados de Sequência Molecular , Estresse Oxidativo , Fator de Transcrição STAT1/genética , Especificidade da Espécie , Quimeras de Transplante , VertebradosRESUMO
The Signal Transducer and Activator of Transcription 3 (STAT3) is necessary for ES cell renewal, plays critical roles during vertebrate development, and has oncogenic potential. STAT3 also mediates cytokine responses notably in the induction of acute phase response genes in the liver. Thus STAT3 is a pleiotropic regulator during cell proliferation and a cell-specific mediator of pro-inflammatory responses. How STAT3 fulfils both roles is unclear. To address this question we attempted to characterise pre-initiation complexes (PICs) on STAT3-responsive promoters containing the c-myc P2 promoter element (P2E) or c-fos Serum-Inducible Element (SIE). Although both promoters mediated cytokine responses in HepG2 cells, poor binding of STAT1 and STAT3 in vitro precluded isolation of active promoter complexes on the P2E. The inability of STAT3 to bind the P2E in vitro correlated with failure of the P2E to mediate cytokine-responsive gene expression in several other cell types. Thus the c-myc P2E behaves as a dual-purpose STAT3 element with anomalous characteristics in HepG2 cells.
Assuntos
Proteínas de Ligação a DNA/genética , Hepatócitos/fisiologia , Regiões Promotoras Genéticas/genética , Fator de Transcrição STAT3/genética , Fatores de Transcrição/genética , Sítios de Ligação , Linhagem Celular , Humanos , Ligação ProteicaRESUMO
The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor.