RESUMO
The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mucina-4/fisiologia , Neoplasias Pancreáticas/metabolismo , Receptor ErbB-2/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Ligantes , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos SCID , Microscopia Confocal/métodos , Invasividade Neoplásica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Frequent oncogenic alterations occur in the phosphoinositide 3-kinase (PI3K) pathway, urging identification of novel negative controls. We previously reported an original mechanism for restraining PI3K activity, controlled by the somatostatin G protein-coupled receptor (GPCR) sst2 and involving a ligand-regulated interaction between sst2 with the PI3K regulatory p85 subunit. We here identify the scaffolding protein filamin A (FLNA) as a critical player regulating the dynamic of this complex. A preexisting sst2-p85 complex, which was shown to account for a significant basal PI3K activity in the absence of ligand, is disrupted upon sst2 activation. FLNA was here identified as a competitor of p85 for direct binding to two juxtaposed sites on sst2. Switching of GPCR binding preference from p85 toward FLNA is determined by changes in the tyrosine phosphorylation of p85- and FLNA-binding sites on sst2 upon activation. It results in the disruption of the sst2-p85 complex and the subsequent inhibition of PI3K. Knocking down FLNA expression, or abrogating FLNA recruitment to sst2, reversed the inhibition of PI3K and of tumor growth induced by sst2. Importantly, we report that this FLNA inhibitory control on PI3K can be generalized to another GPCR, the mu opioid receptor, thereby providing an unprecedented mechanism underlying GPCR-negative control on PI3K.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Filaminas , Fosforilação , Ligação Proteica , Subunidades Proteicas/genéticaRESUMO
CONTEXT: Among the innovative molecules used to manage neuroendocrine tumors, there is growing interest in combining the somatostatin analogs octreotide or pasireotide (SOM230) and everolimus (RAD001), an inhibitor that targets the protein kinase mammalian target of rapamycin (mTOR). EVIDENCE ACQUISITION: The aims of this review were to describe the signaling pathways targeted independently by somatostatin analogs and everolimus and to summarize the scientific rationale for the potential additive or synergistic antitumor effects of combined therapy. EVIDENCE SYNTHESIS: The somatostatin analogs (octreotide and lanreotide) have potent inhibitory effects on hypersecretion, thereby alleviating the symptoms associated with neuroendocrine tumors. Furthermore, the antitumor potential of octreotide is now well documented. Pasireotide, a somatostatin analog, has the advantage of targeting a wider range of somatostatin receptors (subtypes 1, 2, 3, and 5) than the analogs previously used in clinical practice (which preferentially target subtype 2) and thus has a broader spectrum of activity. Everolimus is a rapamycin analog that inhibits mTOR, but it is more soluble than rapamycin and can be administered orally. mTOR is a protein kinase involved in many signaling pathways, primarily those initiated by tyrosine kinase receptors. Sustained mTOR activity leads to the induction of cell growth, proliferation, and cell survival. Everolimus therefore has obvious potential in cancer therapy. CONCLUSIONS: The combination of somatostatin analogs and everolimus in therapeutic trials offers a promising treatment option for neuroendocrine tumors.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Tumores Neuroendócrinos/tratamento farmacológico , Octreotida/uso terapêutico , Peptídeos Cíclicos/uso terapêutico , Sirolimo/análogos & derivados , Sirolimo/uso terapêutico , Somatostatina/análogos & derivados , Everolimo , Humanos , Tumores Neuroendócrinos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Somatostatina/uso terapêutico , Serina-Treonina Quinases TOR/metabolismoRESUMO
Although contact inhibition is a fundamental process for multicellular organisms, how proliferation is inhibited at high cellular densities remains poorly characterized. Here we show that 4E-BP1, one major repressor of cap-dependent translation, plays a critical role in density-mediated cell cycle arrest. 4E-BP1 promoter is activated and 4E-BP1 protein amount increases as cells reach confluence. Conversely, a much less marked density-dependent inhibition of cell proliferation is observed upon 4E-BP1 silencing. We further show that at high density, progression through the G1 phase of the cell cycle is faster and Cyclin D1 protein is induced in different cell types where 4E-BP1 has been either downregulated (stable shRNA expression or transient siRNA transfection) or removed (knock-out). Thus 4E-BP1 appears as an important mediator of contact inhibition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Citometria de Fluxo , Fase G1/genética , Fase G1/fisiologia , Humanos , Imunoprecipitação , Fosfoproteínas/genética , Regiões Promotoras Genéticas/genéticaRESUMO
The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.
Assuntos
Fator de Iniciação Eucariótico 4G/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADPH Desidrogenase/metabolismo , Fragmentos de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Fator de Iniciação Eucariótico 4G/genética , Humanos , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NADPH Desidrogenase/genética , Estresse Oxidativo , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Ligação Proteica , Biossíntese de Proteínas , Estabilidade ProteicaRESUMO
The somatostatin receptor subtype 2 (sst2) behaves as a tumor suppressor when expressed and stimulated by its ligand somatostatin in pancreatic cancer. We reveal a mechanism underlying oncosuppressive action of sst2, whereby this inhibitory receptor upregulates the expression of the secreted angioinhibitory factor thrombospondin-1 (TSP-1), as demonstrated in exocrine BxPC-3 and endocrine BON pancreatic cancer cells. The sst2-dependent upregulation of TSP-1 occurs through the inhibition of the PI3K pathway. It depends on transcriptional and translational events, involving a previously undescribed IRES in the 5'-UTR of TSP-1 mRNA. Chick chorioallantoic membrane was used as an in vivo model to demonstrate that TSP-1 is a critical effector of the inhibitory role of sst2 on the neoangiogenesis and oncogenesis induced by pancreatic cancer cells. TSP-1 reduced in vitro tubulogenesis of endothelial cells when grown in conditioned medium from pancreatic cancer cells expressing sst2, as compared to those expressing the control vector. TSP-1 inhibited tumor cell-induced neoangiogenesis by directly sequestering the proangiogenic factor VEGF, and inactivating the angiogenesis initiated by VEGFR2 phosphorylation in endothelial cells. Using human pancreatic tissue-microarrays, the expression of both sst2 and TSP-1 was shown to be correlated during the pancreatic neoplastic program. Both proteins are nearly undetectable in normal exocrine pancreas and in most invasive cancer lesions, but their expression is strikingly upregulated in most preinvasive cancer-adjacent lesions. The upregulation of both sst2 and TSP-1 tumor suppressors may function as an early negative feedback to restrain pancreatic carcinogenesis.
Assuntos
Neoplasias Pancreáticas/fisiopatologia , Receptores de Somatostatina/fisiologia , Trombospondina 1/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/prevenção & controle , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptores de Somatostatina/genética , Trombospondina 1/genética , Transplante Heterólogo , Proteínas Supressoras de Tumor/genética , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Assembly of the multi-subunit eukaryotic translation initiation factor-4F (eIF4F) is critical for protein synthesis and cell growth and proliferation. eIF4F formation is regulated by the translation-inhibitory protein 4E-BP1. While proliferation factors and intracellular pathways that impinge upon 4E-BP1 phosphorylation have been extensively studied, how they control 4E-BP1 expression remains unknown. Here, we show that Smad4, a transcription factor normally required for TGFbeta-mediated inhibition of normal cell proliferation, enhances 4E-BP1 gene-promoter activity through binding to a conserved element. 4E-BP1 expression is specifically modulated by treatment with TGFbeta and by manipulations of the natural Smad4 regulators (co-Smads) in cells isolated from Smad4(+/+) human tumours, whereas no response is observed in cells isolated from Smad4(-/-) human tumours or in cells where Smad4 has been knocked down by specific siRNAs. In addition, cells where 4E-BP1 has been knocked down (inducible shRNAs in human pancreatic cancer cells or siRNAs in non-malignant human keratinocytes) or has been knocked out (mouse embryonic fibroblasts isolated from 4E-BP1(-/-) mice) proliferate faster and are resistant to the antiproliferative effect of TGFbeta. Thus, 4E-BP1 gene appears critical for TGFbeta/Smad4-mediated inhibition of cell proliferation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proliferação de Células/efeitos dos fármacos , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Proteína Smad4/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Sequência de Bases , Proteínas de Ciclo Celular , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Fosfoproteínas/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Elementos de Resposta , Proteína Smad4/genética , Proteína Smad4/metabolismo , Transfecção , Fator de Crescimento Transformador beta/fisiologiaRESUMO
BACKGROUND: Somatostatin receptor scintigraphy (SRS) has been reported for receptor (SSTR) screening in advanced hepatocarcinoma (aHC) prior to somatostatin analogue treatment. AIMS: To evaluate SSTR screening with SRS in aHC patients. RESULTS: Seventy aHC patients (63 men) aged 65 +/- 11 y were included, with alcohol, viral or other causes cirrhosis in 35 (50%), 23 (33%), 12 (17%) cases respectively. CLIP score was 2.7 +/- 1.7, with more than three nodules in 37 (53%) cases. Largest nodule measured 7.6 +/- 4.5 cm. Median alpha-fetoprotein was 574 UI/mL. SRS was positive in 25/70 (35.7%) livers and 7/17 (41.2%) metastatic sites. Positive SRS patients differed from others for tumor size (9.2 +/- 4 vs. 6.7 +/- 4.6 cm, p = 0.03), prothrombin time (PT) (75.2 +/- 15.2 vs. 61.9 +/- 19%, p = 0.005), albumin (34.1 +/- 5.9 vs. 30.5 +/- 7.2 g/L, p = 0.04) and Child-Pugh (6.7 +/- 1.8 vs. 7.7 +/- 2.3, p = 0.04). After multivariate analysis, only PT was associated with positive SRS (p = 0.028). Immunohistochemistry was positive for SSTR2s in 6/7 tumors (SRS uptake in 5/6 cases). METHODS: SRS was performed prior treatment, with images at 4, 24 and 48 h. For seven tumors, SSTR2 subtype was detected immunohistochemically. CONCLUSIONS: In advanced hepatocarcinoma, we report SRS uptake in 35.7% of livers and 41.2% of metastatic sites. SRS value in screening patients for somatostatin analogue treatment remains to be assessed.
Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Receptores de Somatostatina/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , França , Humanos , Imuno-Histoquímica , Radioisótopos de Índio/farmacocinética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Cintilografia/métodos , Compostos Radiofarmacêuticos/farmacocinética , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Somatostatina/farmacocinética , Resultado do TratamentoRESUMO
Juvenile hormone (JH) controls insect development, metamorphosis and reproduction. In insect hemolymph a significant proportion of JH is bound to juvenile hormone binding protein (JHBP), which serves as a carrier supplying the hormone to the target tissues. To shed some light on JHBP passage within insect tissues, the interaction of this carrier with other proteins from Galleria mellonella (Lepidoptera) was investigated. Our studies revealed the presence of JHBP within the tracheal epithelium and fat body cells in both the membrane and cytoplasmic sections. We found that the interaction between JHBP and membrane proteins occurs with saturation kinetics and is specific and reversible. ATP synthase was indicated as a JHBP membrane binding protein based upon SPR-BIA and MS analysis. It was found that in G. mellonella fat body, this enzyme is present in mitochondrial fraction, plasma membranes and cytosol as well. In the model system containing bovine F(1) ATP synthase and JHBP, the interaction between these two components occurs with K(d)=0.86 nM. In hemolymph we detected JHBP binding to apolipophorin, arylphorin and hexamerin. These results provide the first demonstration of the physical interaction of JHBP with membrane and hemolymph proteins which can be involved in JHBP molecule traffic.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Insetos/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Transporte Proteico/fisiologia , Animais , Apolipoproteínas/metabolismo , Corpo Adiposo/química , Corpo Adiposo/ultraestrutura , Hemolinfa/metabolismo , Hormônios Juvenis/metabolismo , Proteínas de Membrana/metabolismo , Mariposas , Ressonância de Plasmônio de SuperfícieRESUMO
Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival sphingosine-1-phosphate (S1P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, sphingosine kinase-1 (SphK1), a tumor-associated protein overexpressed in many cancers, favors survival through S1P production, and inhibitors of SphK1 are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S1P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S1P ratio, associated with a high SphK1 activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S1P ratio, by using pharmacologic (SphK1 inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK1 activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S1P ratio, by up-regulating SphK1 activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug.
Assuntos
Ceramidas/metabolismo , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Lisofosfolipídeos/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias Pancreáticas/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleotídeo Redutases/antagonistas & inibidores , Esfingosina/metabolismo , Células Tumorais Cultivadas , GencitabinaRESUMO
Since its discovery three decades ago as an inhibitor of GH release from the pituitary gland, somatostatin has attracted much attention because of its functional role in the regulation of a wide variety of physiological functions in the brain, pituitary, pancreas, gastrointestinal tract, adrenals, thyroid, kidney and immune system. In addition to its negative role in the control of endocrine and exocrine secretions, somatostatin and analogs also exert inhibitory effects on the proliferation and survival of normal and tumor cells. Over the past 15 years, studies have begun to reveal some of the molecular mechanisms underlying the antitumor activity of somatostatin. This review covers the present knowledge in the antitumor effect of somatostatin and analogs and discusses the perspectives of novel clinical strategies based on somatostatin receptor sst2 gene transfer therapy.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Somatostatina/farmacologia , Animais , Antineoplásicos Hormonais/uso terapêutico , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Hormônio do Crescimento Humano/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Receptores de Somatostatina/agonistas , Transdução de Sinais/efeitos dos fármacos , Somatostatina/uso terapêuticoRESUMO
Somatostatin is a neuropeptide that inhibits exocrine and endocrine secretions of several hormones and negatively regulates cell proliferation. These events are mediated through somatostatin engagement on one of five G protein-coupled receptors named SSTR1 to STTR5. Somatostatin binding to SSTR2 mediates predominantly antisecretory and antiproliferative effects; two important biological activities in the gastroenteropancreatic endocrine and exocrine system. Herein we demonstrate novel regulatory sequences for human (h) SSTR2 transcription. By genomic DNA sequence analysis, we reveal two CpG islands located 3.8 kb upstream from the transcription start site. We identify a novel transcription start site and a promoter region within one of these CpG islands. We demonstrate that two epigenetic modifications, DNA methylation and histone acetylation, regulate the activation of hSSTR2 upstream promoter. Furthermore, we show that the transcription from this upstream promoter region directly correlates to hSSTR2 mRNA expression in various human cell lines. A combined treatment of a demethylating agent, 5-aza-2-deoxycytidine and a histone deacetylase inhibitor, trichostatin A, leads to increased expression of hSSTR2 mRNA in cell lines in which the CpG island is methylated. The epigenetic regulation of this promoter region results in differential expression of hSSTR2 mRNA in human cell lines. This study reveals the existence of a novel upstream promoter for the hSSTR2 gene that is regulated by epigenetic modifications, suggesting for complex control of the hSSTR2 transcription.
Assuntos
Cromossomos Humanos Par 17 , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Receptores de Somatostatina/genética , Sequência de Bases , Cromatina/genética , Cromatina/ultraestrutura , Metilação de DNA , Fosfatos de Dinucleosídeos , Éxons , Genes Reporter , Humanos , Luciferases/genética , Dados de Sequência Molecular , Plasmídeos , Deleção de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
PURPOSE: Well-differentiated metastatic endocrine carcinomas are difficult to manage because of variable disease outcome. New prognostic factors are required. These tumors overexpress somatostatin receptors (sst), implying the use of somatostatin analogs for tumor localization by somatostatin receptor scintigraphy using indium-111-pentetreotide ((111)In-pentetreotide) and for medical treatment. The aim of the present study was to evaluate the correlation between (111)In-pentetreotide scintigraphy, sst receptor expression, and prognosis. PATIENTS AND METHODS: Between 1994 and 2002, 48 consecutive patients with well-differentiated endocrine carcinomas and a negative (111)In-pentetreotide scintigraphy were retrospectively paired according to sex, age, and tumor localization with 50 patients with well-differentiated endocrine carcinomas and a positive tracer uptake at (111)In-pentetreotide scintigraphy. Overall survival and expression of sst1 to sst5 receptors by immunohistochemistry were assessed. RESULTS: The lack of tracer uptake at the (111)In-pentetreotide scintigraphy seemed to be a poor prognostic factor (P = .007) for overall survival by Kaplan-Meier test and in multivariate analysis; age and absence of clinical secretory syndrome also seemed to be poor prognostic factors. The tracer uptake (positive (111)In-pentetreotide scintigraphy) correlated with the tumor expression of somatostatin receptor sst2 (P < .001) but not with that of sst1, sst3, sst4, or sst5. In a bivariate analysis, lack of sst2 expression also significantly correlated with poor prognosis. CONCLUSION: We demonstrate the prognostic value of (111)In-pentetreotide scintigraphy in well-differentiated malignant endocrine tumors. In these tumors, sst2 somatostatin receptor expression correlates with both tracer uptake and a better prognosis.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias das Glândulas Endócrinas/diagnóstico por imagem , Neoplasias das Glândulas Endócrinas/metabolismo , Radioisótopos de Índio , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Adulto , Idoso , Neoplasias das Glândulas Endócrinas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Valor Preditivo dos Testes , Prognóstico , Cintilografia , Estudos RetrospectivosRESUMO
The receptor-like phosphotyrosine phosphatase eta (PTPeta) is an important intracellular effector of the cytostatic action of SST. Here we characterize, in Chinese hamster ovary-k1 cells, the intracellular pathway that from somatostatin receptor 1 (SSTR1), leads to the activation of PTPeta and that involves, in a multimeric complex and sequential activation, the tyrosine kinases Janus kinase (JAK) 2 and Src, and the cytosolic phosphotyrosine phosphatase SHP-2. We show that inhibitors of JAK2 and Src and dominant-negative mutants of SHP-2 and Src abolished the SSTR1-mediated PTPeta activation, suggesting that all these effectors participate in the activation of PTPeta. In basal conditions, JAK2 forms a multimeric complex with SHP-2, Src and PTPeta. In response to SST, JAK2 is activated in a G protein-dependent manner, dissociates from and phosphorylates SHP-2, increasing its activity. Subsequently, SHP-2 dissociates from Src, dephosphorylates the Src inhibitory tyrosine-529, and causes an autocatalytical increase of the phosphorylation of Src tyrosine 418, located inside its kinase activation loop. Active Src, in turn, controls the activity of PTPeta, via a direct interaction and phosphorylation of the phosphatase. These data for the first time depict an intracellular pathway involving a precise sequence of interactions and cross-activation among tyrosine phosphatases and kinases acting upstream of PTPeta. In particular the sequential activation of JAK2, SHP-2, and Src conveys the molecular signaling from SSTR1 to the activation of this phosphatase that is responsible for the final biological effects of SST.
Assuntos
Proteínas Tirosina Fosfatases/química , Receptores de Somatostatina/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Citosol/enzimologia , Ativação Enzimática , Genes Dominantes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Janus Quinase 2/metabolismo , Toxina Pertussis/farmacologia , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
Phosphatidylinositol 3-kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein-coupled receptor (GPCR) sst2, has potent proapoptotic and anti-invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre-existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated-Y71 residue of a p85-binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH-terminal SH2 as direct interacting domains. Somatostatin-mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y71 residue. Mutating sst2-Y71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand-activated GPCR of PI3K-dependent survival pathways, which may be an important molecular target for antineoplastic therapy.
Assuntos
Fosfatidilinositol 3-Quinases/fisiologia , Receptores de Somatostatina/fisiologia , Somatostatina/fisiologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Ativação Enzimática , Feminino , Humanos , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Ligação Proteica , Receptores de Somatostatina/genética , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Transplante Heterólogo , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
AIM: To compare gene expression profiles of pancreatic adenocarcinoma tissue specimens, human pancreatic and colon adenocarcinoma and leukemia cell lines and normal pancreas samples in order to distinguish differentially expressed genes and to validate the differential expression of a subset of genes by quantitative real-time RT-PCR (RT-QPCR) in endoscopic ultrasound-guided fine needle aspiration (EUS-guided FNA) specimens. METHODS: Commercially dedicated cancer cDNA macroarrays (Atlas Human Cancer 1.2) containing 1176 genes were used. Different statistical approaches (hierarchical clustering, principal component analysis (PCA) and SAM) were used to analyze the expression data. RT-QPCR and immunohistochemical studies were used for validation of results. RESULTS: RT-QPCR validated the increased expression of LCN2 (lipocalin 2) and for the first time PLAT (tissue-type plasminogen activator or tPA) in malignant pancreas as compared with normal pancreas. Immunohistochemical analysis confirmed the increased expression of LCN2 protein localized in epithelial cells of ducts invaded by carcinoma. The analysis of PLAT and LCN2 transcripts in 12 samples obtained through EUS-guided FNA from patients with pancreatic adenocarcinoma showed significantly increased expression levels in comparison with those found in normal tissues, indicating that a sufficient amount of high quality RNA can be obtained with this technique. CONCLUSION: Expression profiling is a useful method to identify biomarkers and potential target genes. Molecular analysis of EUS-guided FNA samples in pancreatic cancer appears as a valuable strategy for the diagnosis of pancreatic adenocarcinomas.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pancreáticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Fase Aguda/análise , Proteínas de Fase Aguda/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Biópsia por Agulha Fina/métodos , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Endossonografia/métodos , Genes Neoplásicos/genética , Humanos , Queratina-7 , Queratinas/análise , Queratinas/genética , Leucemia/genética , Leucemia/patologia , Lipocalina-2 , Lipocalinas , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Prognóstico , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Reprodutibilidade dos Testes , Ativador de Plasminogênio Tecidual/análise , Ativador de Plasminogênio Tecidual/genéticaRESUMO
Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase Tipo I/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Animais , Células CHO , Linhagem Celular Tumoral , Proliferação de Células , Cricetinae , Dimerização , Humanos , Fosforilação , Isoformas de Proteínas , Proteínas Proto-Oncogênicas pp60(c-src)/química , Ratos , Proteínas RecombinantesRESUMO
Pancreatic cancer is one of the most aggressive and devastating human malignancies. The present study was conducted to determine whether in vivo sst2 gene transfer into human pancreatic tumors would impair tumor progression, and to characterize sst2 antitumoral bystander mechanisms. sst2 administration, using the synthetic vector PEI, strongly inhibited tumor progression of human pancreatic adenocarcinoma, in vivo. sst2 gene transfer induced intratumoral production of its ligand somatostatin. Disruption of this autocrine loop by RNA interference completely reversed sst2 antitumoral activity. Mice depleted of natural killer (NK) cells did not hamper sst2 tumor growth inhibition. However, microvessel density and vascular endothelial growth factor (VEGF) expression were markedly reduced in sst2-transfected tumors, whereas sst3 somatostatin receptor was upregulated. Depleting somatostatin by RNA interference completely abolished the sst2 inhibitory effect on VEGF expression and tumor angiogenesis, and sst2-induced sst3 expression in peripheral tumor vessels. We conclude that in vivo sst2 gene transfer elicited intratumoral somatostatin production and strongly impaired human pancreatic tumor growth. NK cells were not involved in this antitumoral bystander effect. VEGF and tumor vascularization were identified as novel targets for sst2-mediated antitumoral bystander effect. sst3 somatostatin receptor was upregulated in sst2-transfected tumors. Therefore, in vivo gene delivery of sst2 receptor to target the angiogenic process in pancreatic ductal adenocarcinoma might be a new therapeutic approach for treatment of pancreatic cancer in patients with unresectable disease.
Assuntos
Efeito Espectador , Carcinoma , Terapia Genética , Transplante de Neoplasias , Neoplasias Pancreáticas , Receptores de Somatostatina/metabolismo , Animais , Comunicação Autócrina/genética , Efeito Espectador/genética , Carcinoma/irrigação sanguínea , Carcinoma/metabolismo , Carcinoma/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Camundongos , Transplante de Neoplasias/métodos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Receptores de Somatostatina/genéticaRESUMO
Somatostatin is a neuropeptide that acts as an endogenous inhibitor of various cellular functions including endocrine and exocrine secretions and the proliferation of normal and tumour cells. Its action is mediated by a family of G-protein-coupled receptors (sst1-sst5) that are widely distributed in normal and tumour cells. Gastroenteropancreatic endocrine tumours express multiple somatostatin receptors, sst2 being clearly predominant. These receptors represent the molecular basis for the clinical use of somatostatin analogues in the treatment of endocrine tumours and their in vivo localisation. This review covers current knowledge in somatostatin receptor biology and signalling.
Assuntos
Neoplasias Gastrointestinais , Neoplasias Pancreáticas , Receptores de Somatostatina/fisiologia , Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/terapia , Humanos , Radioisótopos de Índio , MAP Quinase Quinase 2/fisiologia , Neovascularização Fisiológica/fisiologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Transdução de Sinais/fisiologia , Somatostatina/análogos & derivados , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Gap junctions are composed of connexins and are critical for the maintenance of the differentiated state. Consistently, connexin expression is impaired in most cancer cells, and forced expression of connexins following cDNA transfection reverses the tumor phenotype. We have found that the restoration of density inhibition of human pancreatic cancer cells by the antiproliferative somatostatin receptor 2 (sst2) is due to overexpression of endogenous connexins Cx26 and Cx43 and consequent formation of functional gap junctions. Immunoblotting along with protein metabolic labeling and mRNA monitoring revealed that connexin expression is enhanced at the level of translation but is not sensitive to the inhibition of cap-dependent translation initiation. Furthermore, we identified a new internal ribosome entry site (IRES) in the Cx26 mRNA. The activity of Cx26 IRES and that of the previously described Cx43 IRES are enhanced in density-inhibited cells. These data indicate that the restoration of functional gap junctions is likely a critical event in the antiproliferative action of the sst2 receptor. We further suggest that the existence of IRESes in connexin mRNAs permits connexin expression in density-inhibited or differentiated cells, where cap-dependent translation is generally reduced.