RESUMO
Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin-induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1-/- MEF cells, and not in wild-type MEFs. Similarly, resveratrol alone or in combination with rapamycin induced cell death in human bladder cancer cell lines. These data indicate that administration of resveratrol together with rapamycin may be a promising therapeutic option for treatment of bladder cancer. J. Cell. Physiol. 232: 436-446, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sirolimo/uso terapêutico , Estilbenos/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Estilbenos/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Lymphangioleiomyomatosis (LAM) is a rare neoplastic metastatic disease affecting women of childbearing age. LAM is caused by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) as a consequence of tuberous sclerosis complex (TSC) 1/2 inactivation. Clinically, LAM results in cystic lung destruction. mTORC1 inhibition using rapamycin analogs (rapalogs) is partially effective in reducing disease progression and improving lung function. However, cessation of treatment results in continued progression of the disease. In the present study, we investigated the effectiveness of the combination of rapamycin treatment with resveratrol, an autophagy inhibitor, in the TSC2-null xenograft tumor model. We determined that this combination inhibits phosphatidylinositol-4,5-bisphosphate 3-kinase PI3K/Akt/mTORC1 signaling and activates apoptosis. Therefore, the combination of rapamycin and resveratrol may be an effective clinical strategy for treatment of LAM and other diseases with mTORC1 hyperactivation.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Linfangioleiomiomatose/tratamento farmacológico , Sirolimo/farmacologia , Estilbenos/farmacologia , Proteínas Supressoras de Tumor/genética , Neoplasias Uterinas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Humanos , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/imunologia , Linfangioleiomiomatose/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos SCID , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Ratos , Resveratrol , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Resultado do Tratamento , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/imunologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/imunologia , Neoplasias Uterinas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of CD4 T cells. The class II phosphatidylinositol 3 kinase C2ß (PI3KC2ß) is activated by the T-cell receptor (TCR) and is critical for KCa3.1 channel activation. Tripartite motif containing protein 27 (TRIM27) is a member of a large family of proteins that function as Really Interesting New Gene (RING) E3 ubiquitin ligases. We now show that TRIM27 functions as an E3 ligase and mediates lysine 48 polyubiquitination of PI3KC2ß, leading to a decrease in PI3K enzyme activity. By inhibiting PI3KC2ß, TRIM27 also functions to negatively regulate CD4 T cells by inhibiting KCa3.1 channel activity and TCR-stimulated Ca(2+) influx and cytokine production in Jurkat, primary human CD4 T cells, and Th0, Th1, and Th2 CD4 T cells generated from TRIM27(-/-) mice. These findings provide a unique mechanism for regulating class II PI3Ks, and identify TRIM27 as a previously undescribed negative regulator of CD4 T cells.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ubiquitinação , Animais , Cálcio/metabolismo , Citocinas/biossíntese , Proteínas de Ligação a DNA/deficiência , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Ativação do Canal Iônico , Células Jurkat , Camundongos , Mucoproteínas/metabolismo , Proteínas Nucleares/deficiência , Fosfatidilinositol 3-Quinases/metabolismo , Poliubiquitina/metabolismo , Ligação Proteica , Proteólise , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Células Th1/imunologia , Células Th2/imunologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína LigasesRESUMO
Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.
Assuntos
Resistência à Insulina , Mitocôndrias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/fisiologia , Linhagem Celular Tumoral , Classe III de Fosfatidilinositol 3-Quinases , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Técnicas de Introdução de Genes , Glucose/análise , Glucose/metabolismo , Teste de Tolerância a Glucose , Glicólise/fisiologia , Hepatócitos , Heterozigoto , Humanos , Insulina/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Cultura Primária de CélulasRESUMO
Homologous recombination (HR) is a conserved process that maintains genome stability and cell survival by repairing DNA double-strand breaks (DSBs). The RAD51-related family of proteins is involved in repair of DSBs; consequently, deregulation of RAD51 causes chromosomal rearrangements and stimulates tumorigenesis. RAD51C has been identified as a potential tumor suppressor and a breast and ovarian cancer susceptibility gene. Recent studies have also implicated estrogen as a DNA-damaging agent that causes DSBs. We found that in ERα-positive breast cancer cells, estrogen transcriptionally regulates RAD51C expression in ERα-dependent mechanism. Moreover, estrogen induces RAD51C assembly into nuclear foci at DSBs, which is a precursor to RAD51 complex recruitment to the nucleus. Additionally, disruption of ERα signaling by either anti-estrogens or siRNA prevented estrogen induced upregulation of RAD51C. We have also found an association of a worse clinical outcome between RAD51C expression and ERα status of tumors. These findings provide insight into the mechanism of genomic instability in ERα-positive breast cancer and suggest that individuals with mutations in RAD51C that are exposed to estrogen would be more susceptible to accumulation of DNA damage, leading to cancer progression.
Assuntos
Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Estrogênios/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Bases de Dados Genéticas , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Transporte Proteico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Resultado do TratamentoRESUMO
Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110ß, and p110δ), p110ß has unique properties. Unlike the other family members, p110ß directly binds to Gßγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110ß binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for defining their role in p110ß signaling. This chapter describes the approach that was used to identify the Rab5 binding site in p110ß, and discusses methods for the analysis of p110ß-Rab5 interactions.
Assuntos
Domínio Catalítico , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas rab5 de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina Difosfato/química , Células HEK293 , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/isolamento & purificação , Proteínas Imobilizadas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/isolamento & purificaçãoRESUMO
In contrast to the class I phosphoinositide 3-kinases (PI3Ks), the organismal roles of the kinase activity of the class II PI3Ks are less clear. Here, we report that class II PI3K-C2ß kinase-dead mice are viable and healthy but display an unanticipated enhanced insulin sensitivity and glucose tolerance, as well as protection against high-fat-diet-induced liver steatosis. Despite having a broad tissue distribution, systemic PI3K-C2ß inhibition selectively enhances insulin signaling only in metabolic tissues. In a primary hepatocyte model, basal PI3P lipid levels are reduced by 60% upon PI3K-C2ß inhibition. This results in an expansion of the very early APPL1-positive endosomal compartment and altered insulin receptor trafficking, correlating with an amplification of insulin-induced, class I PI3K-dependent Akt signaling, without impacting MAPK activity. These data reveal PI3K-C2ß as a critical regulator of endosomal trafficking, specifically in insulin signaling, and identify PI3K-C2ß as a potential drug target for insulin sensitization.
Assuntos
Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Insulina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagia , Glicemia/análise , Células Cultivadas , Classe II de Fosfatidilinositol 3-Quinases/genética , Dieta Hiperlipídica , Endossomos/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Técnicas de Introdução de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Insulina/sangue , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Synergistic activation by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110ß from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110ß is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110ß-dependent functions, we identified the site in p110ß that binds to the Gßγ subunit of G proteins. Mutation of this site eliminated Gßγ-dependent activation of PI3Kß (a dimer of p110ß and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide-mediated activation. Disrupting the p110ß-Gßγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kß in fibroblasts and reduced the proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kß could provide a therapeutic approach for tumors that depend on p110ß for growth and metastasis.