RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is characterised pathologically by a marked desmoplastic stromal reaction that significantly reduces the sensitivity and specificity of cytogenetic analysis. To identify genetic alterations that reflect the characteristics of the tumour in vivo, we screened a total of 23 microdissected PDAC tissue samples using array-based comparative genomic hybridisation (array CGH) with 1 Mb resolution. Highly stringent statistical analysis enabled us to define the regions of nonrandom genomic changes. We detected a total of 41 contiguous regions (>3.0 Mb) of copy number changes, such as a genetic gain at 7p22.2-p15.1 (26.0 Mb) and losses at 17p13.3-p11.2 (13.6 Mb), 18q21.2-q22.1 (12.0 Mb), 18q22.3-q23 (7.1 Mb) and 18q12.3-q21.2 (6.9 Mb). To validate our array CGH results, fluorescence in situ hybridisation was performed using four probes from those regions, showing that these genetic alterations were observed in 37-68% of a separate sample set of 19 PDAC cases. In particular, deletion of the SEC11L3 gene (18q21.32) was detected at a very high frequency (13 out of 19 cases; 68%) and in situ RNA hybridisation for this gene demonstrated a significant correlation between deletion and expression levels. It was further confirmed by reverse transcription-PCR that SEC11L3 mRNA was downregulated in 16 out of 16 PDAC tissues (100%). In conclusion, the combination of tissue microdissection and array CGH provided a valid data set that represents in vivo genetic changes in PDAC. Our results raise the possibility that the SEC11L3 gene may play a role as a tumour suppressor in this disease.
Assuntos
Hibridização de Ácido Nucleico , Neoplasias Pancreáticas/genética , Análise Serial de Tecidos , Idoso , Sequência de Bases , Linhagem Celular Tumoral , Mapeamento Cromossômico , Primers do DNA , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Pancreatic ductal adenocarcinoma is a devastating disease, characterized by a rapid progression and poor treatment response. Using gene expression profiling of pancreatic cancer tissues, we previously identified periostin as a potential diagnostic and therapeutic target. In this study, we report the overexpression of periostin in a larger set of pancreatic cancer tissues and show that although the periostin transcript is exclusively expressed in tumour cells, the protein product is only detected in the extracellular matrix adjacent to cancer cells. Using an enzyme-linked immunosorbent assay (ELISA) assay, we show significantly increased levels of periostin in the sera of pancreatic cancer patients compared to non-cancer controls. We demonstrate that periostin promotes the invasiveness of tumour cells by increasing the motility of cells without inducing expression of proteases, and enhances the survival of tumour cells exposed to hypoxic conditions. At the molecular level, we provide evidence that the alpha(6)beta(4) integrin complex acts as the cell receptor of periostin in pancreatic cancer cells and that interaction promotes phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT) though activation of the PI3 kinase pathway, but not the RAS/MEK/ERK pathway. These findings suggest an important role of periostin in pancreatic cancer and provide a rationale to study periostin for diagnostic and therapeutic applications.
Assuntos
Carcinoma Ductal Pancreático/patologia , Moléculas de Adesão Celular/fisiologia , Integrina beta4/metabolismo , Neoplasias Pancreáticas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Apoptose , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/metabolismo , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Hipóxia/metabolismo , Integrina alfa6beta4/metabolismo , Invasividade Neoplásica , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Hypoxia-inducible factor 1 (HIF-1) is a master regulator of oxygen homeostasis that controls angiogenesis, erythropoiesis, and glycolysis via transcriptional activation of target genes under hypoxic conditions. O(2)-dependent binding of the von Hippel-Lindau (VHL) tumor suppressor protein targets the HIF-1alpha subunit for ubiquitination and proteasomal degradation. The activity of the HIF-1alpha transactivation domains is also O(2) regulated by a previously undefined mechanism. Here, we report the identification of factor inhibiting HIF-1 (FIH-1), a protein that binds to HIF-1alpha and inhibits its transactivation function. In addition, we demonstrate that FIH-1 binds to VHL and that VHL also functions as a transcriptional corepressor that inhibits HIF-1alpha transactivation function by recruiting histone deacetylases. Involvement of VHL in association with FIH-1 provides a unifying mechanism for the modulation of HIF-1alpha protein stabilization and transcriptional activation in response to changes in cellular O(2) concentration.
Assuntos
Hipóxia Celular/fisiologia , Ligases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Células Cultivadas , Primers do DNA/química , Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Histona Desacetilases/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Técnicas In Vitro , Luciferases/metabolismo , Oxigenases de Função Mista , Dados de Sequência Molecular , Proteínas Nucleares/genética , Oxigênio/metabolismo , Reação em Cadeia da Polimerase , Conformação Proteica , Coelhos , Proteínas Repressoras/genética , Reticulócitos/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Transcrição Gênica , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator composed of HIF-1alpha and HIF-1beta subunits. Several dozen HIF-1 targets are known, including the gene encoding vascular endothelial growth factor (VEGF). Under hypoxic conditions, HIF-1alpha expression increases as a result of decreased ubiquitination and degradation. The tumor suppressors VHL (von Hippel-Lindau protein) and p53 target HIF-1alpha for ubiquitination such that their inactivation in tumor cells increases the half-life of HIF-1alpha. Increased phosphatidylinositol 3-kinase (PI3K) and AKT or decreased PTEN activity in prostate cancer cells also increases HIF-1alpha expression by an undefined mechanism. In breast cancer, increased activity of the HER2 (also known as neu) receptor tyrosine kinase is associated with increased tumor grade, chemotherapy resistance, and decreased patient survival. HER2 has also been implicated as an inducer of VEGF expression. Here we demonstrate that HER2 signaling induced by overexpression in mouse 3T3 cells or heregulin stimulation of human MCF-7 breast cancer cells results in increased HIF-1alpha protein and VEGF mRNA expression that is dependent upon activity of PI3K, AKT (also known as protein kinase B), and the downstream kinase FRAP (FKBP-rapamycin-associated protein). In contrast to other inducers of HIF-1 expression, heregulin stimulation does not affect the half-life of HIF-1alpha but instead stimulates HIF-1alpha synthesis in a rapamycin-dependent manner. The 5'-untranslated region of HIF-1alpha mRNA directs heregulin-inducible expression of a heterologous protein. These data provide a molecular basis for VEGF induction and tumor angiogenesis by heregulin-HER2 signaling and establish a novel mechanism for the regulation of HIF-1alpha expression.