RESUMEN
ENDOGLIN/CD105 (ENG) is a transmembrane glycoprotein and an auxiliary unit of the transforming growth factor-ß (TGF-ß); receptor, expressed predominantly in vascular endothelium. Noteworthy, Eng mRNA expression has been reported also in Kit(+) interstitial cells of Cajal (ICC) in the mouse intestine. Gastrointestinal stromal tumours (GIST) are thought to derive from ICC. Here we have investigated Eng expression in the Kit(K641E) mouse GIST model, in human GIST and in the Ba/F3 cell model. In wild type (WT) mouse antrum, Eng immunoreactivity (-ir) was detected in CD34(+) /CD31(+) endothelium and in Kit(+) ICC. In Kit(K641E) mice, hyperplasia of Kit(+) cells made Eng-ir even more evident. Quantitative PCR confirmed the increased expression of Eng transcript in Kit(K641E) mice. On human GIST TMA, 26/49 cases stained positive for ENG. Strong ENG staining was associated with malignant and high-risk tumours. ENG negative cases were predominantly of the epithelioid type or harboured PDGFRA mutation. In vitro, Eng mRNA was up-regulated in Ba/F3 cell lines stably expressing various oncogenic Kit mutations (K641E, del559, del814). This effect appeared to be independent of Kit activation, as neither the stimulation of WT Kit by its ligand SCF, nor the inhibition of Kit autophosphorylation by imatinib mesylate in oncogenic mutants, altered Eng expression. Elevated Eng expression in Kit oncogenic mutants appeared rather to be indirectly mediated by DNA hypomethylation, because treatment with the demethylating agent 5-Aza/dC increased Eng mRNA expression in Kit(WT) cells. ENG expression in ICC and in GIST deserves further consideration as ENG is emerging as a potential target for cancer therapy.
Asunto(s)
Antígenos CD/metabolismo , Tumores del Estroma Gastrointestinal/metabolismo , Tracto Gastrointestinal/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Antígenos CD34/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacología , Benzamidas , Línea Celular Tumoral , Metilación de ADN , Decitabina , Endoglina , Endotelio Vascular , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/patología , Tracto Gastrointestinal/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Mesilato de Imatinib , Células Intersticiales de Cajal/metabolismo , Ratones , Ratones Transgénicos , Piperazinas/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Approximately 85% of GISTs harbor activating mutations of the KIT or PDGFRA receptor tyrosine kinases. PTEN and SHIP2 are major phosphatases that dephosphorylate PI(3,4,5)P(3), one of the intracellular signal pathways downstream of KIT. PTEN is an important tumor suppressor, whereas the involvement of SHIP2 in cancer has been proposed based essentially on cell line studies. We have used a mouse model of GIST, i.e. Kit(K641E) knock-in mice, resulting in the substitution of a Lys by Glu at position 641 of Kit. In homozygous Kit(K641E) mice, PTEN-immunoreactivity (ir) in antrum was found in the hyperplastic Kit-ir layer. The same localization was found for SHIP2. Western blot analysis in antrum showed a large increase in PTEN expression in Kit(K641E) homozygous mice as compared to wild type. In contrast, SHIP2 expression was not affected between the two genotypes. Erk1, but not PKB, phosphorylation appears to be upregulated in Kit(K641E) homozygous mice. In the human GIST882 imatinib sensitive cell line, both PTEN and SHIP2 were expressed and showed, in part, a nuclear localization. The upregulation of PTEN in antrum in Kit(K641E) mice might serve as a feedback mechanism to limit PI 3-kinase activation downstream of Kit in a context of oncogenic mutation.
Asunto(s)
Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Proto-Oncogénicas c-kit , Antro Pilórico/metabolismo , Transducción de Señal/genética , Animales , Benzamidas , Western Blotting , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/metabolismo , Tumores del Estroma Gastrointestinal/patología , Regulación Neoplásica de la Expresión Génica , Técnicas de Sustitución del Gen , Homocigoto , Humanos , Mesilato de Imatinib , Inositol Polifosfato 5-Fosfatasas , Ratones , Ratones Transgénicos , Mutación , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Antro Pilórico/patología , Pirimidinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
Gastrointestinal stromal tumors (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the KIT or PDGFRA receptor tyrosine kinases are present in the majority of GIST, leading to ligand-independent activation of the intracellular signal transduction pathways. We previously investigated the gene expression profile in the murine Kit(K641E) GIST model and identified Ntsr1 mRNA, encoding the Neurotensin receptor 1, amongst the upregulated genes. Here we characterized Ntsr1 mRNA and protein expression in the murine Kit(K641E) GIST model and in tissue microarrays of human GIST. Ntsr1 mRNA upregulation in Kit(K641E) animals was confirmed by quantitative PCR. Ntsr1 immunoreactivity was not detected in the Kit positive ICC of WT mice, but was present in the Kit positive hyperplasia of Kit(K641E) mice. In the normal human gut, NTSR1 immunoreactivity was detected in myenteric neurons but not in KIT positive ICC. Two independent tissue microarrays, including a total of 97 GIST, revealed NTSR1 immunoreactivity in all specimens, including the KIT negative GIST with PDGFRA mutation. NTSR1 immunoreactivity exhibited nuclear, cytoplasmic or mixed patterns, which might relate to variable levels of NTSR1 activation. As studies using radio-labeled NTSR1 ligand analogues for whole body tumor imaging and for targeted therapeutic interventions have already been reported, this study opens new perspectives for similar approaches in GIST.
Asunto(s)
Tumores del Estroma Gastrointestinal/genética , Células Intersticiales de Cajal/metabolismo , Receptores de Neurotensina/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Tumores del Estroma Gastrointestinal/metabolismo , Tumores del Estroma Gastrointestinal/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células Intersticiales de Cajal/patología , Masculino , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Neurotensina/metabolismoRESUMEN
Gastrointestinal stromal tumours (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the receptor tyrosine kinase KIT are present in most GIST. KIT K642E was originally identified in sporadic GIST and later found in the germ line of a familial GIST cohort. A mouse model harbouring a germline Kit K641E mutant was created to model familial GIST. The expression profile was investigated in the gastric antrum of the Kit(K641E) murine GIST model by microarray, quantitative PCR and immunofluorescence. Gja1/Cx43, Gpc6, Gpr133, Pacrg, Pde3a, Prkar2b, Prkcq/Pkce, Rasd2, Spry4 and Tpbg/5T4 were found to be up-regulated. The proteins encoded by Gja1/Cx43, Pde3a, Prkcq/Pkce were localized in Kit-ir ICC in wild-type and Kit(K641E) animals while Spry4 and Tpbg/5T4 were detected in Kit-ir cells only in Kit(K641E), but not in Kit(WT/WT) animals. Most up-regulated genes in this mouse model belong to the gene expression profile of human GIST but also to the profile of normal Kit(+) ICC in the mouse small intestine. Spry4 and Tpbg/5T4 may represent candidates for targeted therapeutic approaches in GIST with oncogenic KIT mutations.
Asunto(s)
Tumores del Estroma Gastrointestinal/genética , Células Intersticiales de Cajal/enzimología , Glicoproteínas de Membrana/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas c-kit/genética , Regulación hacia Arriba/genética , Sustitución de Aminoácidos/genética , Animales , Modelos Animales de Enfermedad , Tumores del Estroma Gastrointestinal/enzimología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células Intersticiales de Cajal/patología , Glicoproteínas de Membrana/metabolismo , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Expression of VP16-CREB, a constitutively active form of CREB, in hippocampal neurons of the CA1 region lowers the threshold for eliciting the late, persistent phase of long-term potentiation (L-LTP) in the Schaffer collateral pathway. This VP16-CREB-mediated L-LTP differs from the conventional late phase of LTP in not being dependent on new transcription. This finding suggests that in the transgenic mice the mRNA transcript(s) encoding the protein(s) necessary for this form of L-LTP might already be present in CA1 neurons in the basal condition. We used high-density oligonucleotide arrays to identify the mRNAs differentially expressed in the hippocampus of transgenic and wild-type mice. We then explored the contribution of the most prominent candidate genes revealed by our screening, namely prodynorphin, BDNF, and MHC class I molecules, to the facilitated LTP of VP16-CREB mice. We found that the overexpression of brain-derived neurotrophic factor accounts for an important component of this phenotype.