RESUMO
Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked ß-N-acetylglucosamine transferase (OGT), which adds an O-linked ß-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid-induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.
Assuntos
Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Piranos/farmacologia , Receptores de Glucocorticoides/metabolismo , Tiazóis/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Dexametasona/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Concentração Inibidora 50 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , N-Acetilglucosaminiltransferases/metabolismo , Prednisolona/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células U937RESUMO
Homologous recombination (HR) is a cellular mechanism for accurate repair of double-strand DNA breaks, often deregulated in cancer. Development of novel cancer therapeutics targeting HR pathways would benefit from a quantitative and rapid means of measuring HR. Here, we describe a zinc finger nuclease (ZFN) assay that can quantify HR. Knockdown of BRCA1 or inactivation of BRCA2 decreased HR activity in cells, whereas gene restoration induced activity 8-fold. HR activity was also reflective of BRCA1/2 status in cells with known endogenous mutations.
Assuntos
Endonucleases/metabolismo , Ensaios Enzimáticos , Recombinação Homóloga , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Dedos de ZincoRESUMO
Glioblastoma (GBM) is the most common malignant brain tumor that is characterized by high proliferative rate and invasiveness. Since dysregulation of Notch signaling is implicated in the pathogenesis of many human cancers, here we investigated the role of Notch signaling in GBM. We found that there is aberrant activation of Notch signaling in GBM cell lines and human GBM-derived neurospheres. Inhibition of Notch signaling via the expression of a dominant negative form of the Notch coactivator, mastermind-like 1 (DN-MAML1), or the treatment of a γ-secretase inhibitor, (GSI) MRK-003, resulted in a significant reduction in GBM cell growth in vitro and in vivo. Knockdown of individual Notch receptors revealed that Notch1 and Notch2 receptors differentially contributed to GBM cell growth, with Notch2 having a predominant role. Furthermore, blockade of Notch signaling inhibited the proliferation of human GBM-derived neurospheres in vitro and in vivo. Our overall data indicate that Notch signaling contributes significantly to optimal GBM growth, strongly supporting that the Notch pathway is a promising therapeutic target for GBM.
RESUMO
BACKGROUND: Cancer stem cells (CSCs) are believed to be responsible for breast cancer formation and recurrence; therefore, therapeutic strategies targeting CSCs must be developed. One approach may be targeting signaling pathways, like Notch, that are involved in stem cell self-renewal and survival. MATERIALS AND METHODS: Breast cancer stem-like cells derived from cell lines and patient samples were examined for Notch expression and activation. The effect of Notch inhibition on sphere formation, proliferation, and colony formation was determined. RESULTS: Breast cancer stem-like cells consistently expressed elevated Notch activation compared with bulk tumor cells. Blockade of Notch signaling using pharmacologic and genomic approaches prevented sphere formation, proliferation, and/or colony formation in soft agar. Interestingly, a gamma-secretase inhibitor, MRK003, induced apoptosis in these cells. CONCLUSION: Our findings support a crucial role for Notch signaling in maintenance of breast cancer stem-like cells, and suggest Notch inhibition may have clinical benefits in targeting CSCs.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores Notch/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Óxidos S-Cíclicos/farmacologia , Feminino , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptor ErbB-2/biossíntese , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Esferoides Celulares , Tiadiazóis/farmacologiaRESUMO
ERBB2/neu and Notch signaling are known to be deregulated in many human cancers. However, pathway cross-talk and dependencies are not well understood. In this study, we use an ERBB2-transgenic mouse model of breast cancer (neuT) to show that Notch signaling plays a critical role in tumor maintenance. Inhibition of the Notch pathway with a gamma-secretase inhibitor (GSI) decreased both the Notch and the mammalian target of rapamycin/AKT pathways. Antitumor activity resulting from GSI treatment was associated with decreased cell proliferation as measured by Ki67 and decreased expression of glucose transporter Glut1. Positron emission tomography (PET) imaging showed that the functional consequences of decreased Glut1 translated to reduced glucose uptake and correlated with antitumor effects as measured by micro-computed tomography imaging. The decrease of Glut1 in neuT tumors was also observed in several human breast cancer cell lines following GSI treatment. We provide evidence that approximately 27% of ERBB2-positive human breast cancer specimens display high expression of HES1, phospho-S6RP, and GLUT1. Together, these results suggest that pathways downstream of Notch signaling are, at least in part, responsible for promoting tumor growth in neuT and also active in both neuT and a subset of human breast cancers. These findings suggest that GSI may provide therapeutic benefit to a subset of ERBB2-positive breast cancers and that [(18)F]FDG-PET imaging may be useful in monitoring clinical response.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Glucose/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Oncogênica v-akt/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor ErbB-2/metabolismo , Receptores Notch/metabolismo , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Óxidos S-Cíclicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Fluordesoxiglucose F18/farmacocinética , Transportador de Glucose Tipo 1/biossíntese , Humanos , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Oncogênica v-akt/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Tiadiazóis/farmacologiaRESUMO
To identify dysregulated pathways in distinct phases of NOTCH1-mediated T-cell leukemogenesis, as well as small-molecule inhibitors that could synergize with or substitute for gamma-secretase inhibitors (GSIs) in T-cell acute lymphoblastic leukemia (T-ALL) therapy, we compared gene expression profiles in a Notch1-induced mouse model of T-ALL with those in human T-ALL. The overall patterns of NOTCH1-mediated gene expression in human and mouse T-ALLs were remarkably similar, as defined early in transformation in the mouse by the regulation of MYC and its target genes and activation of nuclear factor-kappaB and PI3K/AKT pathways. Later events in murine Notch1-mediated leukemogenesis included down-regulation of genes encoding tumor suppressors and negative cell cycle regulators. Gene set enrichment analysis and connectivity map algorithm predicted that small-molecule inhibitors, including heat-shock protein 90, histone deacetylase, PI3K/AKT, and proteasome inhibitors, could reverse the gene expression changes induced by NOTCH1. When tested in vitro, histone deacetylase, PI3K and proteasome inhibitors synergized with GSI in suppressing T-ALL cell growth in GSI-sensitive cells. Interestingly, alvespimycin, a potent inhibitor of the heat-shock protein 90 molecular chaperone, markedly inhibited the growth of both GSI-sensitive and -resistant T-ALL cells, suggesting that its loss disrupts signal transduction pathways crucial for the growth and survival of T-ALL cells.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Genes myc , Humanos , Técnicas In Vitro , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/etiologia , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/genética , Transdução de Sinais , Especificidade da EspécieRESUMO
NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19(INK4d)) and CDKN1B (p27(Kip1)), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Óxidos S-Cíclicos/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Inibidores de Proteases/farmacologia , Receptor Notch1/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Tiadiazóis/farmacologia , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p19/biossíntese , Inibidor de Quinase Dependente de Ciclina p27 , Fase G1/efeitos dos fármacos , Fase G1/genética , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , TransfecçãoRESUMO
Malignant mesothelioma (MM) is a cancer of the lining of the lungs, heart, and intestine and is known to respond poorly to chemotherapy. Here we show that malignant mesothelial cells have an elevated Notch signaling pathway compared with normal human mesothelial cells. We studied the role of Notch in MM under normoxic and hypoxic conditions, the latter condition best recapitulating the MM microenvironment. Genetic and chemical modulation of the Notch pathway indicated that MM cells are dependent on Notch signaling. More specifically, this signaling was Notch-1 dependent as the result of its negative transcriptional regulation on phosphatase and tensin homologue (PTEN), which led to activation of the prosurvival phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. Our study also provides evidence that whereas Notch-1 is elevated in the malignant setting, Notch-2 is diminished. This differential expression of the two Notch isoforms benefits cancer cell survival because reexpression of Notch-2 was toxic to MM cells. The mechanism of Notch-2 toxicity to MM cells countered that of Notch-1, as it was the result of positive transcriptional regulation of PTEN and inhibition of the PI3K/Akt/mTOR signaling pathway. These results provide new insight into the role of Notch in MM and suggest that Notch pathway inhibitors may be useful in the treatment of this deadly disease.
Assuntos
Mesotelioma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Processos de Crescimento Celular/fisiologia , Hipóxia Celular , Sobrevivência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA de Neoplasias/biossíntese , Humanos , Mesotelioma/genética , Mesotelioma/patologia , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/biossíntese , Receptor Notch2/biossíntese , Transdução de Sinais , Células Tumorais CultivadasRESUMO
PURPOSE: For many tumor cells, de novo lipogenesis is a requirement for growth and survival. A considerable body of work suggests that inhibition of this pathway may be a powerful approach to antineoplastic therapy. It has recently been shown that inhibition of various steps in the lipogenic pathway individually can induce apoptosis or loss of viability in tumor cells. However, it is not clear whether quantitative differences exist in the ability of lipogenic enzymes to control tumor cell survival. We present a systematic approach that allows for a direct comparison of the control of lipogenic pathway enzymes over tumor cell growth and apoptosis using different cancer cells. EXPERIMENTAL DESIGN: RNA interference-mediated, graded down-regulation of fatty acid synthase (FAS) pathway enzymes was employed in combination with measurements of lipogenesis, apoptosis, and cell growth. RESULTS: In applying RNA interference titrations to two lipogenic enzymes, acetyl-CoA carboxylase 1 (ACC1) and FAS, we show that ACC1 and FAS both significantly control cell growth and apoptosis in HCT-116 cells. These results also extend to PC-3 and A2780 cancer cells. CONCLUSIONS: Control of tumor cell survival by different steps in de novo lipogenesis can be quantified. Because ACC1 and FAS both significantly control tumor cell growth and apoptosis, we propose that pharmacologic inhibitors of either enzyme might be useful agents in targeting cancer cells that critically rely on fatty acid synthesis. The experimental approach described here may be extended to other targets or disease-relevant pathways to identify steps suitable for therapeutic intervention.
Assuntos
Acetiltransferases/metabolismo , Neoplasias do Colo/enzimologia , Ácido Graxo Sintases/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Células HCT116 , Humanos , Lipogênese , Transdução de Sinais , TransfecçãoRESUMO
NOTCH signaling is an evolutionarily conserved signaling pathway that regulates cell fate during development and postnatal life. It has been increasingly linked to carcinogenesis, although its role in cancer seems to be highly context and tissue specific. Although NOTCH signaling is required for lung development, little is known about its role in lung cancer. In this study, we show that NOTCH signaling, as measured by the gamma-secretase cleavage product N(IC)-1, is active in both normal human and lung tumor samples; however, downstream NOTCH readouts (i.e., HES-1 and HES-5) are elevated in lung tumors. Levels of NOTCH signaling components in primary human lung cells reflect observations in tissue samples, yet lung tumor cell lines showed little NOTCH signaling. Because oxygen concentrations are important in normal lung physiology and lung tumors are hypoxic, the effect of low oxygen on these lung tumor cell lines was evaluated. We found that hypoxia dramatically elevates NOTCH signaling (especially NOTCH-1) in lung tumor cell lines and concomitantly sensitizes them to inhibition via small-molecule gamma-secretase inhibitors or NOTCH-1 RNA interference. gamma-Secretase inhibitor-induced apoptosis of lung tumor cells grown under hypoxic conditions could be rescued by reintroduction of active NOTCH-1. Our data strengthen the role of NOTCH in lung cancer and as a therapeutic target for the treatment of lung and other hypoxic tumor types.
Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/genética , Oxigênio/farmacologia , Receptor Notch1/fisiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Hipóxia Celular/fisiologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Oxigênio/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
In this report, inhibitors of the gamma-secretase enzyme have been exploited to characterize the antiproliferative relationship between target inhibition and cellular responses in Notch-dependent human T cell acute lymphoblastic leukemia (T-ALL) cell lines. Inhibition of gamma-secretase led to decreased Notch signaling, measured by endogenous NOTCH intracellular domain (NICD) formation, and was associated with decreased cell viability. Flow cytometry revealed that decreased cell viability resulted from a G(0)/G(1) cell cycle block, which correlated strongly to the induction of apoptosis. These effects associated with inhibitor treatment were rescued by exogenous expression of NICD and were not mirrored when a markedly less active enantiomer was used, demonstrating the gamma-secretase dependency and specificity of these responses. Together, these data strengthen the rationale for using gamma-secretase inhibitors therapeutically and suggest that programmed cell death may contribute to reduction of tumor burden in the clinic.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/patologia , Receptores Notch/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Óxidos S-Cíclicos/farmacologia , Citometria de Fluxo , Humanos , Leucemia-Linfoma de Células T do Adulto/enzimologia , Leucemia-Linfoma de Células T do Adulto/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiadiazóis/farmacologiaRESUMO
A series of 3-aryl-4-hydroxyquinolin-2(1H)-ones with fatty acid synthase inhibitory activity was prepared. Starting from a derivative with an IC(50) = 1.4 microM, SAR studies led to compounds with more than 70-fold increase in potency (IC(50) < 20 nM).