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This corrects the article DOI: 10.1038/nature24042.
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Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.
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Peso Corporal/fisiologia , Tronco Encefálico/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Animais , Tronco Encefálico/citologia , Tronco Encefálico/efeitos dos fármacos , Núcleo Central da Amígdala/citologia , Núcleo Central da Amígdala/fisiologia , Ingestão de Alimentos/fisiologia , Metabolismo Energético/fisiologia , Comportamento Alimentar , Feminino , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/deficiência , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/farmacologia , Homeostase , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Núcleos Parabraquiais/citologia , Núcleos Parabraquiais/fisiologia , Estresse PsicológicoRESUMO
We recently reported that resistance to PD-1 blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). Thus, we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. In this study, we report that LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist mAb, elicits myeloid inflammation and allogeneic T-cell responses by binding to LAIR1 and blocking collagen engagement. Furthermore, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T-cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic.
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Antineoplásicos , Colágeno , Inibidores de Checkpoint Imunológico , Receptores Imunológicos , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Colágeno/metabolismo , Modelos Animais de Doenças , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Antineoplásicos/uso terapêuticoRESUMO
Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.
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Antígenos CD , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Glicoproteínas de Membrana , Células Mieloides , Receptores Imunológicos , Microambiente Tumoral , Receptores Imunológicos/metabolismo , Animais , Humanos , Camundongos , Microambiente Tumoral/imunologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Glicoproteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismoRESUMO
Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.
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Fibronectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Receptores Imunológicos/metabolismo , Diferenciação Celular , Linhagem Celular , HumanosRESUMO
Connective tissue growth factor (CTGF/CCN2) is thought to play a role in normal wound repair and bone remodeling, but also promotes fibrosis in several disease processes including diabetic nephropathy, sclerodoma and pancreatitis. A contribution to desmoplasia associated with pancreatic cancer progression has also been proposed. CTGF is induced by TGFbeta in diverse cell types, but TGFbeta receptor mediated signaling is impaired in pancreatic cancers and cell lines, usually due to DPC4/Smad4 mutations which arise during the later stages of intraepithelial neoplastic progression. Therefore, in order to define signaling pathways that mediate basal and TGFbeta-induced CTGF expression in normal and transformed cells, we compared CTGF gene regulation in pancreatic cancer cells and fibroblasts by measuring the effects of small molecule inhibitors and dominant negative mutants of signaling proteins on CTGF promoter reporter activity, message, and protein expression. We determined that the previously identified TEF-1 cis element is essential for CTGF promoter reporter activity in pancreatic cancer cell lines. Whereas p38 mediated CTGF induction by TGFbeta in fibroblasts, MEK/ERK signaling mediated TGFbeta-induced CTGF expression in pancreatic cancer cells and was also responsible for basal CTGF expression in pancreatic cancer cell lines with defective Smad signaling. Since activating Ras mutations occur in the earliest stages of pancreatic cancer, CTGF may be induced independent of Smad4 in pancreatic cancer cells.
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Regulação Neoplásica da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Células 3T3 , Animais , Western Blotting , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta , Proteínas Smad/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismoRESUMO
PURPOSE: Mucinous cancers and signet ring carcinomas are distinct classes of colon cancers characterized by their production of copious quantities of intestinal goblet cell mucin, MUC2. Deletion of transcription factor HATH1 ablates the biogenesis of goblet cells in developing mouse intestine, and forced expression of HATH1 results in elevated expression of MUC2 in colon cancer cells. The aim of this study was to assess the possible role of HATH1 in the development of mucinous cancers and signet ring carcinomas. EXPERIMENTAL DESIGN: Immunohistochemistry and confocal microscopy was used to examine HATH1 expression and subcellular distribution in normal colon and small intestine, mucinous cancers, signet ring carcinomas, and nonmucinous cancers and in precursor lesions, including hyperplastic polyps, serrated adenomas, tubular adenomas, and villous adenomas. We also analyzed the transactivation of MUC2 promoter/reporter constructs by a HATH1 expression vector. RESULTS: HATH1 expression transactivated MUC2 promoter/reporter constructs, an activity that was significantly inhibited by mutation of putative HATH1-binding sites. HATH1 was expressed in the nuclei of goblet cells and in the cytoplasm and nuclei of enteroendocrine cells of the colon. In the small intestine, only cytoplasmic expression of HATH1 in enteroendocrine cells was detected. HATH1 was found to be strongly expressed in the nuclei of hyperplastic polyps, serrated adenomas, villous adenomas, mucinous cancers, and signet ring carcinomas but repressed in nonmucinous cancers and tubular adenomas. CONCLUSIONS: This study confirms the importance of HATH1 for the development of intestinal secretory cells. The results further suggest that HATH1 is an important factor in the up-regulation of MUC2 expression that occurs in mucinous cancers and signet ring carcinomas. In addition, the expression of HATH1 in hyperplastic polyps, serrated adenomas, and villous adenomas lends support to the hypothesis that these neoplasms are frequent precursors in mucinous cancer and signet ring carcinoma development.
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Adenocarcinoma Mucinoso/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Colorretais/metabolismo , Adenocarcinoma Mucinoso/patologia , Adenocarcinoma Mucinoso/ultraestrutura , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Neoplasias Colorretais/ultraestrutura , Progressão da Doença , Feminino , Humanos , Pólipos Intestinais/patologia , Intestinos/citologia , Intestinos/ultraestrutura , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Mucina-2 , Mucinas/metabolismo , Regiões Promotoras Genéticas , Distribuição Tecidual , Ativação TranscricionalRESUMO
MUC5AC is a secretory mucin normally expressed by the surface mucous cells of the human stomach and in the bronchial tract. It is absent from normal pancreas, but de novo expression of this mucin occurs in early-stage pancreatic intraepithelial neoplasias and in the invasive ductal adenocarcinoma of the pancreas, prompting this study of MUC5AC gene regulation in pancreatic cancer cells. Promoter deletion constructs and EMSA studies revealed that transcription factors Sp1 and AP-1 are both involved in basal transcription of the MUC5AC gene. Phorbol 12-myrisate 13-acetate (PMA) increased MUC5AC mRNA expression and transcriptional activities of MUC5AC promoter-reporter deletion constructs containing AP-1 consensus sites. EMSA studies showed that Fos/Jun binding to putative AP-1 sites is increased by PMA treatment. Western blot analysis showed that ERK, JNK and p38 are all activated by PMA treatment in SW1990 cells. Inhibitors of mitogen-activated protein/extracellular signal regulated kinase (MEK), such as ERK inhibitor PD98059 and JNK inhibitors dicumarol and SP60015, but not p38 inhibitor SB203580, inhibited PMA-induced MUC5AC reporter activity. Our studies indicate that Sp1 is involved in basal MUC5AC promoter activity while AP-1 is involved in basal and PMA-induced MUC5AC promoter activation in pancreatic cancer cells. Furthermore, PMA-induced MUC5AC gene transcription appears to be mediated by activating Sp1, PKC/ERK/AP-1 and PKC/JNK/AP-1 pathways.
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Linhagem Celular Tumoral/metabolismo , Regulação Neoplásica da Expressão Gênica , Mucinas/metabolismo , Carcinoma Ductal Pancreático , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Luciferases/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mucina-5AC , Mucinas/biossíntese , Mucinas/genética , Neoplasias Pancreáticas , Regiões Promotoras Genéticas , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/análise , Fator de Transcrição Sp1/metabolismo , Acetato de Tetradecanoilforbol , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
The colonic epithelium contains three major types of mature cells, namely, absorptive, goblet, and enteroendocrine cells. These cells are maintained by a complex process of cell renewal involving progenitor and stem cells, and colon cancers develop when this process goes awry. Much is known about the genetic and epigenetic changes that occur in cancer; however, little is known as to the specific cell types involved in carcinogenesis. In this study, we expressed the SV40 Tag oncogene in the intestinal epithelium under the control of an intestinal trefoil factor (ITF) promoter. This caused tumor formation in the proximal colon with remarkable efficiency. ITFTag tumors were rapidly growing, multifocal, and invasive. ITFTag tumor cells express synaptophysin and contain dense core secretory granules, markers of neuroendocrine differentiation. The cell type involved in the early steps of ITFTag tumorigenesis was studied by examining partially transformed crypts that contained populations of both normal and dysplastic cells. The dysplastic cell population always expressed both Tag and synaptophysin. Cells expressing Tag alone were never observed; however, normal enteroendocrine cells expressing synaptophysin but not Tag were readily visualized. This suggests that ITFTag tumor cells originate from the enteroendocrine cell lineage following a transforming event that results in Tag expression. ITFTag tumors closely resemble human small cell carcinomas of the colon, suggesting the possibility that these tumors might be derived from the enteroendocrine cell lineage as well.
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Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Carcinoma de Células Pequenas/patologia , Neoplasias do Colo/patologia , Mucinas/genética , Proteínas Musculares/genética , Peptídeos/genética , Regiões Promotoras Genéticas/genética , Animais , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/ultraestrutura , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/ultraestrutura , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Camundongos , Camundongos Transgênicos , Oncogenes/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator Trefoil-2RESUMO
PURPOSE: The BRAF gene encodes a serine/threonine kinase and plays an important role in the mitogen-activated protein kinase signaling pathway. BRAF mutations in sporadic colorectal cancer with microsatellite instability (MSI) are more frequently detected than those in microsatellite stable cancer. In this study, we sought to compare the frequencies of BRAF mutations in sporadic colorectal cancer with MSI with those in hereditary nonpolyposis colorectal cancer (HNPCC). EXPERIMENTAL DESIGN: We analyzed BRAF mutations in 26 colorectal cancer cell lines, 80 sporadic colorectal cancers, and 20 tumors from HNPCC patients by DNA sequencing and sequence-specific PCR. The methylation status of the hMLH1 gene was measured by either sequencing or restriction enzyme digestion after NaHSO(3) treatment. RESULTS: We observed a strong correlation of BRAF mutation with hMLH1 promoter methylation. BRAF mutations were present in 13 of 15 (87%) of the colorectal cell lines and cancers with methylated hMLH1, whereas only 4 of 91 (4%) of the cell lines and cancers with unmethylated hMLH1 carried the mutations (P < 0.00001). Sixteen of 17 mutations were at residue 599 (V599E). A BRAF mutation was also identified at residue 463 (G463V) in one cell line. In addition, BRAF mutations were not found in any cancers or cell lines with K-ras mutations. In 20 MSI+ cancers from HNPCC patients, however, BRAF mutations were not detectable, including a subset of 9 tumors with negative hMLH1 immunostaining and methylated hMLH1. CONCLUSIONS: BRAF mutations are frequently present in sporadic colorectal cancer with methylated hMLH1, but not in HNPCC-related cancers. This discrepancy of BRAF mutations between sporadic MSI+ cancer and HNPCC might be used in a strategy for the detection of HNPCC families.
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Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Metilação de DNA , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Reparo do DNA , DNA de Neoplasias/genética , Genes ras/genética , Instabilidade Genômica , Humanos , Técnicas Imunoenzimáticas , Repetições de Microssatélites/genética , Proteína 1 Homóloga a MutL , Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas B-raf , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
The nucleic acid sequence of the human gene, MUC17, indicates that this mucin contains an SEA domain, a transmembrane domain, and putative N-glycosylation sites in the carboxyl terminus. Mucins that possess an SEA domain are usually proteolytically cleaved within that domain to yield two subunits, the smaller of which is associated with the surface membrane. Homogenates of ASPC-1 pancreatic cancer cells showed three main bands of immunoreactivity with alpha-SEA (a polyclonal antibody directed against a site downstream of the postulated cleavage site) after SDS-PAGE and Western blotting (38, 45, and 49 kDa). Experiments utilizing N-glycan specific hydrolases showed that the 38 kDa band contained high mannose glycans whereas the 45 and 49 kDa bands contained complex-type glycans. Only two smaller alpha-SEA reactive bands (30 and 32 kDa) were present after cells had been treated with the N-glycosylation inhibitor tunicamycin. Surface biotinylation studies showed that only the forms possessing complex-type N-glycans were localized to the cell surface. Both tunicamycin and brefeldin A, an inhibitor of protein transport, reduced surface localization. In summary, our results indicate that the surface localization of the smaller subunit of MUC17 is dependent on its N-glycosylation status.
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Glicosilação , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/química , Mucinas/biossíntese , Mucinas/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Anticorpos/química , Biotinilação , Western Blotting , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Humanos , Hidrolases/química , Imuno-Histoquímica , Dados de Sequência Molecular , Peptídeos/química , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/farmacologia , RNA/química , Coelhos , Tunicamicina/farmacologiaRESUMO
BACKGROUND: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, we developed a novel high-content, high-throughput cell cycle assay and profiled the effects of two distinct small molecule HSP90 inhibitors (XL888 and 17-AAG [17-allylamino-17-demethoxygeldanamycin]) in a large, genetically diverse panel of cancer cell lines. The cell cycle phenotypes of both inhibitors were strikingly similar and fell into three classes: accumulation in M-phase, G2-phase, or G1-phase. Accumulation in M-phase was the most prominent phenotype and notably, was also correlated with TP53 mutant status. We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines. CONCLUSIONS/SIGNIFICANCE: Our analysis of the cell cycle phenotypes induced by HSP90 inhibition in 25 cancer cell lines revealed that the phenotypic response was highly dependent on cellular genotype as well as on the concentration of HSP90 inhibitor and the time of treatment. M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53. We draw upon previous literature to suggest an integrated model that accounts for these varying observations.
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Compostos Azabicíclicos/farmacologia , Ciclo Celular , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Ácidos Ftálicos/farmacologia , Benzoquinonas/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Tempo , Quinase 1 Polo-LikeRESUMO
MUC2 is a major secretory mucin normally expressed by goblet cells of the intestine, but is aberrantly expressed in colonic neoplasia. Bile acids have been implicated in colorectal carcinogenesis and, therefore, we sought to determine the effects of bile acids on MUC2 expression and regulation in colon cancer cells. Since deoxycholic acid (DCA), a secondary bile acid, has been reported to be a potent mucin secretagogue and tumor promoter, DCA-treated HM3 colon cancer cells were analyzed using promoter-reporter assays of the 5' flanking region of the MUC2 gene. Chemical inhibitors, mutant reporter constructs and EMSA showed that DCA upregulates MUC2 transcription via multiple pathways involving activation of EGFR/PKC/Ras/Raf-1/MEK1/ERK/CREB, PI3/Akt/IkappaB/NF-kappaB and p38/MSK1/CREB while DCA induced MUC2 transcription is inhibited by JNK/c-Jun/AP-1 pathway. These results provide new insight into the complex molecular mechanisms involved in the regulation of mucin gene by bile acids in colon cancer cells that may contribute to further elucidation of colorectal carcinogenesis.
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Neoplasias do Colo/enzimologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ácido Desoxicólico/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas I-kappa B/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mucina-2/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/metabolismo , Região 5'-Flanqueadora , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/genética , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Mucina-2/genética , Mutação , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/genética , Transcrição Gênica , Transfecção , Proteínas ras/antagonistas & inibidores , Proteínas ras/genéticaRESUMO
Regional DNA hypermethylation and global DNA hypomethylation are 2 epigenetic alterations associated with colorectal cancers. However, their correlation with microsatellite instability (MSI) and chromosomal instability (CIN) in colorectal cancer, and their relationship with chromatin conformation and histone modification are not clear. In this study, we analyzed regional and global methylation in 16 cell lines and 64 primary colorectal cancers. We found that MSI and CIN are 2 alternative events in most cell lines and tumors. Furthermore, regional hypermethylation and global hypomethylation are also alternative events in most cases. We also observed a strong correlation between MSI and regional hypermethylation and between CIN and global hypomethylation. We further analyzed chromatin conformation and histone acetylation in cell lines with CIN or MSI. CIN cancers had open chromatin conformation and enriched histone acetylation in repetitive as well as in gene-specific regions. MSI cancers, on the other hand, had closed chromatin conformation and low levels of histone acetylation. After a MSI cell line was treated with 5-aza-2'-deoxycytidine or trichostatin A, the closed chromatin conformation became open, and histone acetylation was enriched. These observations support our hypothesis that in colorectal cancer, regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation, which might have a causal correlation with MSI and CIN, respectively.
Assuntos
Cromatina/metabolismo , Instabilidade Cromossômica , Neoplasias Colorretais/metabolismo , Metilação de DNA , Histonas/metabolismo , Repetições de Microssatélites , Acetilação , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , DNA de Neoplasias/metabolismo , Epigênese Genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Imunoprecipitação , Perda de Heterozigosidade , Conformação ProteicaRESUMO
The molecular mechanisms responsible for TNF-alpha-mediated MUC2 intestinal mucin up-regulation in HM3 colon adenocarcinoma cells were analyzed using promoter-reporter assays of the 5'-flanking region of the MUC2 gene. Chemical inhibitors, mutant reporter constructs, and EMSA confirmed I-kappaB/NF-kappaB pathway involvement. Wortmannin, LY294002 and dominant negative Akt, as well as dominant negative NF-kappaB-inducing kinase (NIK) inhibited MUC2 reporter transcription, indicating that both phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling pathway and NIK pathways mediate the effects of TNF-alpha. Wortmannin inhibited NF-kappaB binding and transcriptional activity without inhibiting NF-kappaB translocation to the nucleus, indicating that PI3K/Akt signaling activates NF-kappaB transcriptional activity directly. Our results demonstrate that TNF-alpha up-regulates MUC2 in human colon epithelial cells via several signaling pathways, involving both NIK and PI3K/Akt, which converge at the common IKK/I-kappaB/NF-kappaB pathway. TNF-alpha activated JNK, but JNK inhibitor SP600125 and dominant negative cJun consistently activated transcription, revealing a negative role for this signaling pathway. Thus TNF-alpha causes a net up-regulation of MUC2 gene expression in cultured colon cancer cells because NF-kappaB transcriptional activation of this gene is able to counter-balance the suppressive effects of the JNK pathway. However, the existence of this inhibitory JNK pathways suggests a mechanism whereby--in the absence of NF-kappaB activation--TNF-alpha production during inflammation in vivo could actually inhibit MUC2 production, giving rise to the defective mucosal protection which characterizes inflammatory bowel disease.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mucinas/genética , NF-kappa B/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Luciferases/metabolismo , Mucina-2 , Mucinas/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Quinase Induzida por NF-kappaBRESUMO
VIP exerts a spectrum of effects as a potent anti-inflammatory factor. In addition, VIP increases expression of MUC2, a major intestinal secretory mucin. We therefore investigated the effects of VIP on the promoter activity of the 5'-flanking region of the MUC2 gene. VIP activated MUC2 transcription in human colonic epithelial cells via cAMP signaling to ERK and p38. cAMP/Epac/Rap1/B-Raf signaling was not involved in MUC2 reporter activation. Furthermore, activation of MUC2 transcription was independent of many of the reported downstream effectors of G protein-coupled receptors, such as PKC, Ras, Raf, Src, calcium, and phosphoinositide 3-kinase. VIP induced cAMP response element-binding protein (CREB)/ATF1 phosphorylation, and this was prevented by treatment with inhibitors of either MEK or p38 and by PKA and MSK1 inhibitor H89. CREB/ATF1 and c-Jun were shown to bind to an oligonucleotide encompassing a distal, conserved CREB/AP1 site in the 5'-flanking region of the MUC2 gene, and this cis element was shown to mediate promoter reporter activation by VIP. This study has identified a new, functional cis element within the MUC2 promoter and also a new pathway regulating MUC2 expression, thus providing further insight into the molecular mechanism of VIP action in the colon. These findings are relevant to the normal biology of the colonic mucosa as well as to the development of VIP as a therapeutic agent for treatment of inflammatory bowel disease.
Assuntos
Fator 1 Ativador da Transcrição/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Mucinas/biossíntese , Mucinas/genética , Regulação para Cima/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/farmacologia , Linhagem Celular , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mucina-2 , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais , Transcrição Gênica , Peptídeo Intestinal Vasoativo/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
MUC5AC mucin is not expressed in normal pancreas but is expressed in tumors. Little is known about the mechanisms that lead to this atypical expression. In this study, we demonstrate that stimulation of adenylyl cyclase and the protein kinase A (PKA) pathway by forskolin and vasoactive intestinal peptide (VIP) increased MUC5AC antigen expression and release from pancreatic cancer cells. Stimulation of the PKA pathway also increased MUC5AC mRNA. When SW1990 pancreatic cancer cells were grown on porous membranes they released MUC5AC mucins apically in response to VIP (10(-7) M) applied to their basolateral surfaces. SW1990 cells, as have been reported for other pancreatic cancer cells, have high affinity (<10(-7) M) VIP receptors and low affinity (>10(-6) M) secretin receptors. We also showed that four antibodies (CLH2, 21M1, 45M1, and Nd2) react with MUC5AC antigen in different cellular compartments of both tissues and cultured cells. In conclusion, the PKA pathway may contribute to the up-regulation of MUC5AC expression seen in pancreatic tumors.
Assuntos
Colforsina/farmacologia , Fármacos Gastrointestinais/farmacologia , Mucinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Humanos , Mucina-5AC , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Membrane mucins have several functions in epithelial cells including cytoprotection, extravasation during metastases, maintenance of luminal structure, and signal transduction. In this paper we describe a large membrane mucin expressed in the normal intestine. This novel mucin, designated MUC17, contains an extended, repetitive extracellular glycosylation domain and a carboxyl terminus with two EGF-like domains, a SEA module domain, a transmembrane domain, and a cytoplasmic domain with potential serine and tyrosine phosphorylation sites. RNA blot analysis and in situ hybridization indicates that MUC17 is expressed in select pancreatic and colon cancer cell lines and in intestinal absorptive cells. Radiation hybrid mapping localized MUC17 to chromosome 7q22 where it resides in close proximity with three other membrane mucin genes, MUC3A, MUC3B, and MUC12. Thus, these membrane mucins reside together in a gene cluster, but are expressed in different tissues and are likely to have different functions as well.
Assuntos
Mucinas/biossíntese , Mucinas/genética , Sequência de Aminoácidos , Animais , Cromossomos Humanos Par 7 , Clonagem Molecular , Cricetinae , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Humanos , Hibridização In Situ , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Mucinas/química , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , Mapeamento de Híbridos Radioativos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Human intestinal mucin genes MUC3A and MUC3B are members of a membrane mucin gene family residing at chromosome 7q22. In this paper, we utilized genomic and cDNA cloning to elucidate the sequence of the 5'-region of the MUC3A gene including the gene promoter and the amino terminus coding sequence. Following its 21-residue signal peptide, the amino terminus of the mucin consists of a 233-residue Thr-, Ser-, and Pro-rich nonrepetitive sequence that is contiguous with its hypervariable domain of 375-residue repeats. RNase protection analysis and 5'-GeneRacer PCR indicated that MUC3A gene transcripts initiate from multiple start sites along a region spanning approximately 180 bases. The 5'-flanking region of the gene had promoter activity when fused to a luciferase reporter gene in all of the tested cell lines. This region contained binding sites for several transcription factors, including those implicated in the regulation of intestinal genes, but lacked a cognate TATA box. These features of the gene promoter may enable the gene to be expressed at variable levels in several cell types with different repertoires of transcription factors. We also utilized 5'-GeneRacer PCR to determine the sequence of the 5'-terminus of the MUC3B message. The amino termini of the MUC3A and MUC3B mucins are 91% conserved at the amino acid level. Thus, MUC3A and MUC3B have highly conserved amino and carboxyl termini, suggesting a recent duplication of the entire ancestral gene. It remains to be determined whether other members of the 7q22 membrane mucin gene family have amino-terminal domains similar to MUC3A and MUC3B.
Assuntos
Mucosa Intestinal/metabolismo , Mucinas/genética , Regiões Promotoras Genéticas , TATA Box , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar , Humanos , Dados de Sequência Molecular , Mucina-3 , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND & AIMS: It has recently been suggested that infiltrating adenocarcinoma of the pancreas arises from histologically well-defined precursor ductal lesions called pancreatic intraepithelial neoplasia (PanIN-1A, -1B, -2, and -3). This study examined alterations in the pattern and the level of expression of several mucin genes (MUC1, MUC2, MUC5AC, and MUC6) and mucin-associated tumor antigens (Nd2 and sialyl Tn) in these precursor lesions. METHODS: We examined 139 PanINs and 68 infiltrating ductal adenocarcinomas of the pancreas by using immunohistochemistry and in situ hybridization methods. RESULTS: Overexpression of MUC1, a pan-epithelial mucin, and MUC6, a pyloric-gland mucin, and de novo expression of MUC5AC, a gastric foveolar mucin, was observed in all stages of PanINs and invasive ductal adenocarcinoma. In contrast, the expression of mucin-associated carbohydrate antigen, sialyl Tn, was markedly increased only in PanlN-3 and invasive ductal adenocarcinoma. In addition, a decrease in the expression of these mucin-associated peptide and carbohydrate antigens was correlated with the degree of differentiation of the tumor. CONCLUSIONS: Expression of both gastric-foveolar and pyloric-gland mucin in PanINs is an early event, whereas sialyl Tn expression is a late event in the recently defined progression model of pancreatic carcinogenesis. This altered mucin gene expression provides new insight into the role of cell lineage-associated metaplasia in pancreatic carcinogenesis.