RESUMEN
BACKGROUND: The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin 1 (mTORC1) signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated glycogen synthase kinase-3ß (GSK-3ß) positively regulated RCC proliferation. The aim of this study was to evaluate the role of GSK-3 in the PI3K/Akt/mTORC1 pathway and regulation of the downstream substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 (S6RP). METHODS: We used human RCC cell lines (ACHN, Caki1, and A498) and, as normal controls, human renal proximal tubular epithelial cell (HRPTEpC) and non-tumorous kidney tissues that were obtained surgically for treatment of RCC patients. Rapamycin-resistant ACHN (ACHN/RR) cells were generated with chronic exposure of ACHN to rapamycin ranging from 1nM finally to 1 µM. Cell viability, cell cycling and direct interaction between GSK-3ß and 4EBP1 were evaluated with MTS assay, flowcytometry and in vitro kinase assay with recombinant GSK-3ß and 4EBP1products, respectively. Protein expression and phosphorylation of molecules associated with the PI3K/Akt/mTORC1 pathway were examined by immunoblotting. Effects of drug combination were determined as the combination index with CompuSyn software. RESULTS: Overexpression and phosphorylation of 4EBP1 and S6RP together with GSK-3 activation were observed in RCC cell lines, but not in human normal kidney cells and tissues. Cell proliferation, p4EBP1 and pS6RP were strongly suppressed by GSK-3 inhibition. Rapamycin and LY294002 sufficiently decreased pS6RP, but only moderately p4EBP1. In vitro kinase assays showed that recombinant GSK-3ß phosphorylated recombinant 4EBP1, and the effect was blocked by GSK-3 inhibitors. Different from rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 and pS6RP in ACHN and ACHN/RR (in 30 min to 1 h). AR- A014418 and rapamycin combination showed additivity at lower concentrations, but antagonism at higher concentrations. CONCLUSIONS: GSK-3ß could directly phosphorylate 4EBP1 and activate the mTORC1 downstream signaling cascades to enhance protein biosynthesis and cell proliferation in RCC cell lines independent of rapamycin sensitivity. The direct GSK-3ß/4EBP1 pathway might be an important subcellular mechanism as an inherent equipment for RCC cells to acquire clinical chemoresistance to mTORC1 inhibitors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma de Células Renales/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neoplasias Renales/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteína S6 Ribosómica/metabolismo , Sirolimus/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma de Células Renales/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Cromonas/farmacología , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/genética , Humanos , Neoplasias Renales/genética , Diana Mecanicista del Complejo 1 de la Rapamicina , Morfolinas/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación , Proteína S6 Ribosómica/genética , Proteínas Quinasas S6 Ribosómicas/genética , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Sorafenib is a multikinase inhibitor approved for the systemic treatment of renal cell carcinoma (RCC). However, sorafenib treatment has a limited effect due to acquired chemoresistance of RCC. Previously, we identified glycogen synthase kinase-3 (GSK-3) as a new therapeutic target in RCC. Here, we observed that sorafenib inhibits proliferation and survival of RCC cells. Significantly, we revealed that sorafenib enhances GSK-3 activity in RCC cells, which could be a potential mechanism of acquired chemoresistance. We found that pharmacological inhibition of GSK-3 potentiates sorafenib antitumor effect in vitro and in vivo. Our results suggest that combining GSK-3 inhibitor and sorafenib might be a potential new therapeutic approach for RCC treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Bencenosulfonatos/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3/administración & dosificación , Neoplasias Renales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma de Células Renales/enzimología , Proliferación Celular/efectos de los fármacos , Humanos , Neoplasias Renales/enzimología , Ratones , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Sorafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , MicroARNs/metabolismo , Factores de Transcripción/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/mortalidad , Núcleo Celular/metabolismo , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/mortalidad , Masculino , MicroARNs/genética , Persona de Mediana Edad , Complejo Represivo Polycomb 2 , Interferencia de ARN , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
PURPOSE: Enhancer of zeste homologue 2 (EZH2), a histone methyltransferase, plays a key role in transcriptional repression through chromatin remodeling. Our objectives were to determine the expression pattern of EZH2 and to assess the anticancer effect of EZH2 depletion in pancreatic cancer cells. EXPERIMENTAL DESIGN: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the expression pattern of EZH2 in normal pancreas and human pancreatic tumors. We used RNA interference, Western blotting, reverse transcription-PCR, and chromatin immunoprecipitation to study the effect of EZH2 depletion on pancreatic cancer cell proliferation and survival. RESULTS: We detected nuclear overexpression of EZH2 in pancreatic cancer cell lines and in 71 of 104 (68%) cases of human pancreatic adenocarcinomas. EZH2 nuclear accumulation was more frequent in poorly differentiated pancreatic adenocarcinomas (31 of 34 cases; P<0.001). We found that genetic depletion of EZH2 results in reexpression of p27(Kip1) and decreased pancreatic cancer cell proliferation. Moreover, we showed that EZH2 depletion sensitized pancreatic cancer cells to doxorubicin and gemcitabine, which leads to a significant induction of apoptosis, suggesting that the combination of EZH2 inhibitors and standard chemotherapy could be a superior potential treatment for pancreatic cancer. CONCLUSIONS: Our results show nuclear accumulation of EZH2 as a hallmark of poorly differentiated pancreatic adenocarcinoma; identify the tumor suppressor p27(Kip1) as a new target gene of EZH2; show that EZH2 nuclear overexpression contributes to pancreatic cancer cell proliferation; and suggest EZH2 as a potential therapeutic target for the treatment of pancreatic cancer.
Asunto(s)
Adenocarcinoma/enzimología , Proteínas de Unión al ADN/fisiología , Resistencia a Antineoplásicos , Neoplasias Pancreáticas/enzimología , Proteína Metiltransferasas/metabolismo , Factores de Transcripción/fisiología , Adenocarcinoma/patología , Línea Celular Tumoral , Núcleo Celular/enzimología , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteína Potenciadora del Homólogo Zeste 2 , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pancreáticas/patología , Complejo Represivo Polycomb 2RESUMEN
PURPOSE: We have shown recently that glycogen synthase kinase-3 (GSK-3) beta regulates nuclear factor-kappaB (NF-kappaB)-mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3beta in pancreatic cancer cells and assess the antitumor effect of GSK-3 inhibition in vivo to improve our understanding of the mechanism by which GSK-3beta affects NF-kappaB activity in pancreatic cancer. EXPERIMENTAL DESIGN: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3beta in human pancreatic tumors. We studied the effect of GSK-3 inhibition on tumor growth, cancer cell proliferation, and survival in established CAPAN2 tumor xenografts using a tumor regrowth delay assay, Western blotting, bromodeoxyuridine incorporation, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. RESULTS: We found nuclear accumulation of GSK-3beta in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3beta nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3beta can accumulate in the nucleus of pancreatic cancer cells and that inhibition of GSK-3 kinase activity represses its nuclear accumulation via proteasomal degradation within the nucleus. Lastly, we have found that inhibition of GSK-3 arrests pancreatic tumor growth in vivo and decreases NF-kappaB-mediated pancreatic cancer cell survival and proliferation in established tumor xenografts. CONCLUSIONS: Our results show the antitumor effect of GSK-3 inhibition in vivo, identify GSK-3beta nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3beta regulates NF-kappaB activity in pancreatic cancer.