RESUMEN

In this study, we investigated the mechanistic role of the long non-coding RNA (lncRNA) AC092171.4 in hepatocellular carcinoma (HCC). AC092171.4 was significantly upregulated in HCC tumor tissues compared to normal liver tissues. HCC patients with high AC092171.4 expression showed poorer overall survival (OS) and disease-free survival (DFS) than those with low AC092171.4 expression. In vitro cell proliferation, migration and invasiveness were all higher in AC092171.4-overexpressing HCC cells, but lower in AC092171.4-silenced HCC cells, than in controls. Balb/c nude mice injected with AC092171.4-silenced HCC cells had smaller xenograft tumors, which showed less growth and pulmonary metastasis than control tumors. Bioinformatics analyses and dual luciferase reporter assays confirmed that AC092171.4 binds directly to miR-1271, which targets the 3'UTR of GRB2 mRNA. AC092171.4 expression correlates negatively with miR1271 expression and correlates positively with GRB2 mRNA expression in HCC tissues from patients. HCC cells co-transfected with miR-1271 mimics and sh-AC092171.4 show less proliferation, migration, invasiveness, GRB2 protein, and epithelial to mesencyhmal transition (EMT) than sh-AC092171.4-transfected HCC cells. These findings demonstrate that AC092171.4 promotes growth and progression of HCC by sponging miR-1271 and upregulating GRB2. This makes AC092171.4 a potential prognostic indicator and therapeutic target for HCC patients.

Asunto(s)

Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteína Adaptadora GRB2/genética , Regulación Neoplásica de la Expresión Génica/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Regiones no Traducidas 3' , Adulto , Animales , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Proteína Adaptadora GRB2/biosíntesis , Humanos , Neoplasias Hepáticas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/metabolismo , Persona de Mediana Edad , Pronóstico , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Drug resistance remains a major problem in the treatment of conventional chemotherapeutic agents in breast cancers. Owing to heterogeneity and complexity of chemoresistance mechanisms, most efforts that focus on a single pathway were unsuccessful, and exploring novel personalized therapeutics becomes urgent. By a system approach, we identified that microRNA-27b-3p (miR-27b), a miRNA deleted in breast cancer tissues and cell lines, has a master role in sensitizing breast cancer cells to a broad spectrum of anticancer drugs in vitro and in vivo. Mechanistic analysis indicated that miR-27b enhanced responses to PTX by directly targeting CBLB and GRB2 to inactivate both PI3K/Akt and MAPK/Erk signaling pathways. Further, miR-27b was identified as a promising molecular biomarker in chemoresistance, clinicopathological features, and prognosis for breast cancer patients. In conclusion, we propose that combinational use of miR-27b and chemotherapeutic agents might be a promising therapeutic strategy to increase long-term drug responses in breast cancers.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proteína Adaptadora GRB2/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Regulación hacia Abajo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Femenino , Proteína Adaptadora GRB2/biosíntesis , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Paclitaxel/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-cbl/biosíntesis

RESUMEN

The adapter protein growth factor receptor-bound 2 (GRB2) is essential for various basic cellular functions by mediating the regulation of receptor tyrosine kinase (RTK) signaling, however, little is known about GRB2 expression in esophageal squamous cell carcinoma (ESCC). We sought to characterize GRB2 expression and its relationship with clinicopathological parameters and prognostic significance in ESCC patients. Here, it was presented that GRB2 was overexpressed in cytoplasm in 58.1% (100/172) of ESCC cases by immunohistochemistry. Survival analysis demonstrated overexpression of GRB2 protein was significantly related to poor prognosis of ESCC patients (P = 0.021). Furthermore, overexpression of GRB2 was significantly associated with the lymph node metastases. In addition, subgroup analysis according to lymph node metastasis revealed a shorter disease-free survival (DFS) in the ESCC patients with GRB2 overexpression than the patients with GRB2 low-expression (Means for DFS months: 33.8 versus 52.1). Finally, the significant difference between overexpression of GRB2 and poor survival rates exhibited in univariate analysis (P = 0.022) and multivariate Cox analysis (close to significance, P = 0.065), demonstrated that GRB2 was an independent factor in prognosis of ESCC patients. In conclusion, GRB2 expression status could be as a positive biomarker of ESCC progression and lymph node metastasis.

Asunto(s)

Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Proteína Adaptadora GRB2/biosíntesis , Adulto , Anciano , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Supervivencia sin Enfermedad , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidad , Carcinoma de Células Escamosas de Esófago , Femenino , Proteína Adaptadora GRB2/análisis , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Regulación hacia Arriba

RESUMEN

BACKGROUND: Deregulation of receptor tyrosine kinases (RTK) contributes to the initiation and progression of intestinal-derived epithelial cancers, including colorectal cancer (CRC). However, the roles of the proximal signaling molecules engaged by RTKs in different oncogenic functions of CRC remain unclear. METHODS: Herein, the functional impact of expressing variant forms of the oncogenic Met receptor (Tpr-Met) that selectively recruit the adaptor proteins Grb2 or Shc was investigated in a model derived from normal intestinal epithelial cells (IEC-6). An RNA interference (RNAi) approach was used to define the requirement of Grb2 or Shc in Tpr-Met-transformed IEC-6 cells. Since Grb2 and Shc couple RTKs to the activation of the Ras/MEK/Erk and PI3K/Akt pathways, Erk and Akt phosphorylation/activation states were monitored in transformed IEC-6 cells, and a pharmacological approach was employed to provide insights into the roles of these pathways in oncogenic processes evoked by activated Met, and downstream of Grb2 and Shc. RESULTS: We show, for the first time, that constitutive activation of either Grb2 or Shc signals in IEC-6 cells, promotes morphological transformation associated with down-regulation of E-cadherin, as well as increased cell growth, loss of growth contact inhibition, anchorage-independent growth, and resistance to serum deprivation and anoikis. Oncogenic activation of Met was revealed to induce morphological transformation, E-cadherin down-regulation, and protection against anoikis by mechanisms dependent on Grb2, while Shc was shown to be partly required for enhanced cell growth. The coupling of activated Met to the Ras/MEK/Erk and PI3K/Akt pathways, and the sustained engagement of Grb2 or Shc in IECs, was shown to trigger negative feedback, limiting the extent of activation of these pathways. Nonetheless, morphological alterations and E-cadherin down-regulation induced by the oncogenic Tpr-Met, and by Grb2 or Shc signals, were blocked by MEK, but not PI3K, inhibitors while the enhanced growth and resistance to anoikis induced by Tpr-Met were nearly abolished by co-treatment with both inhibitors. CONCLUSION: Overall, these results identify Grb2 and Shc as central signaling effectors of Met-driven progression of intestinal epithelial-derived cancers. Notably, they suggest that Grb2 may represent a promising target for the design of novel CRC therapies.

Asunto(s)

Neoplasias Colorrectales/genética , Proteína Adaptadora GRB2/biosíntesis , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Adaptadoras de la Señalización Shc/biosíntesis , Cadherinas/metabolismo , Línea Celular , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Células Epiteliales/metabolismo , Proteína Adaptadora GRB2/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patología , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal/genética

RESUMEN

Understanding the regulation of cardiomyocyte growth is crucial for the management of adverse ventricular remodeling and heart failure. MicroRNA-378 (miR-378) is a newly described member of the cardiac-enriched miRNAs, which is expressed only in cardiac myocytes and not in cardiac fibroblasts. We have previously shown that miR-378 regulates cardiac growth during the postnatal period by direct targeting of IGF1R (Knezevic, I., Patel, A., Sundaresan, N. R., Gupta, M. P., Solaro, R. J., Nagalingam, R. S., and Gupta, M. (2012) J. Biol. Chem. 287, 12913-12926). Here, we report that miR-378 is an endogenous negative regulator of cardiac hypertrophy, and its levels are down-regulated during hypertrophic growth of the heart and during heart failure. In primary cultures of cardiomyocytes, overexpression of miR-378 blocked phenylephrine (PE)-stimulated Ras activity and also prevented activation of two major growth-promoting signaling pathways, PI3K-AKT and Raf1-MEK1-ERK1/2, acting downstream of Ras signaling. Overexpression of miR-378 suppressed PE-induced phosphorylation of S6 ribosomal kinase, pERK1/2, pAKT, pGSK-3ß, and nuclear accumulation of NFAT. There was also suppression of the fetal gene program that was induced by PE. Experiments carried out to delineate the mechanism behind the suppression of Ras, led us to identify Grb2, an upstream component of Ras signaling, as a bona fide direct target of miR-378-mediated regulation. Deficiency of miR-378 alone was sufficient to induce fetal gene expression, which was prevented by knocking down Grb2 expression and blocking Ras activation, thus suggesting that miR-378 interferes with Ras activation by targeting Grb2. Our study demonstrates that miR-378 is an endogenous negative regulator of Ras signaling and cardiac hypertrophy and its deficiency contributes to the development of cardiac hypertrophy.

Asunto(s)

Cardiomegalia/metabolismo , Sistema de Señalización de MAP Quinasas , MicroARNs/metabolismo , Proteínas Musculares/metabolismo , Proteínas ras/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/efectos adversos , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/genética , Cardiomegalia/patología , Células Cultivadas , Proteína Adaptadora GRB2/biosíntesis , Proteína Adaptadora GRB2/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , MicroARNs/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Musculares/genética , Fenilefrina/efectos adversos , Fenilefrina/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-raf , Ratas , Ratas Sprague-Dawley , Proteínas ras/genética

RESUMEN

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways(1-3). Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus(4-6). Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

Asunto(s)

Clonación Molecular/métodos , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Membrana Celular/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Proteína Adaptadora GRB2/biosíntesis , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Transducción de Señal , Transfección/métodos

RESUMEN

Growth factor receptor-bound protein 2 is an adaptor molecule that mediates B-cell receptor (BCR) signaling pathways, but the expression of growth factor receptor-bound protein 2 in lymphoma tissues has not been reported. We sought to characterize growth factor receptor-bound protein 2 protein expression in reactive tonsillar tissues and lymphoma tissues obtained from diagnostic biopsies of classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and 20 low-grade B-cell lymphomas. Growth factor receptor-bound protein 2 expression was assessed in tissues by immunohistochemistry and in lymphoma cell lines by immunoblotting. In reactive lymphoid tissues, growth factor receptor-bound protein 2 was expressed in the cytoplasm of B-cells and histiocytes but not T-cells. Strong, cytoplasmic growth factor receptor-bound protein 2 expression was seen in the neoplastic cells of follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, splenic marginal zone lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, and nodular lymphocyte predominant Hodgkin lymphoma. In contrast, only 10% of the classical Hodgkin lymphomas showed growth factor receptor-bound protein 2 expression in the neoplastic cells. Growth factor receptor-bound protein 2 protein expression was detected by Western blotting in all lymphoma cell lines tested with higher levels in primary mediastinal large B-cell lymphoma compared with classical Hodgkin lymphoma cell lines. These findings support a role for growth factor receptor-bound protein 2 in the diagnostically challenging workup of classical Hodgkin lymphoma versus primary mediastinal large B-cell lymphoma and warrant further studies to evaluate the biologic significance of growth factor receptor-bound protein 2 in the pathogenesis of classical Hodgkin lymphoma.

Asunto(s)

Biomarcadores de Tumor/análisis , Proteína Adaptadora GRB2/biosíntesis , Enfermedad de Hodgkin/diagnóstico , Linfoma de Células B Grandes Difuso/diagnóstico , Neoplasias del Mediastino/diagnóstico , Western Blotting , Diagnóstico Diferencial , Enfermedad de Hodgkin/metabolismo , Humanos , Inmunohistoquímica , Linfoma de Células B Grandes Difuso/metabolismo , Neoplasias del Mediastino/metabolismo , Análisis de Matrices Tisulares

RESUMEN

Calorie restriction (CR) alleviates insulin resistance and has a beneficial effect on numerous metabolic disorders, yet the underlying mechanism has not been fully elucidated. In the present study, we found that CR of mice (60% of the diet consumption compared with ad libitum mice) reduces the expression levels of Grb2 in skeletal muscle, an insulin target tissue that accounts for 85% of insulin-stimulated blood glucose clearance. To determine whether Grb2 downregulation contributes to increased insulin sensitivity in the regulation of glucose metabolism, we generated C(2)C(12) cell lines in which the expression of Grb2 is suppressed by RNA interference. Suppressing Grb2 expression in C(2)C(12) myoblasts enhances insulin-stimulated insulin receptor substrate (IRS)-1, tyrosine phosphorylation, and Akt phosphorylation, which is associated with decreased IRS-1 serine phosphorylation at residues 307, 612, and 636/639. In addition, reducing Grb2 expression levels increased insulin-stimulated glucose uptake in C(2)C(12) myotubes. Reduced IRS-1 serine phosphorylation is also found in Grb2(+/-) heterozygous knockout mice, which is associated with enhanced insulin signaling and resistance to high-fat diet-induced glucose and insulin intolerance. All together, our results suggested that reducing the expression levels of Grb2 provides a mechanism by which CR increases insulin sensitivity in vivo.

Asunto(s)

Restricción Calórica , Proteína Adaptadora GRB2/biosíntesis , Resistencia a la Insulina/fisiología , Músculo Esquelético/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Western Blotting , Regulación hacia Abajo , Proteína Adaptadora GRB2/metabolismo , Prueba de Tolerancia a la Glucosa , Inmunoprecipitación , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/enzimología , Proteína Oncogénica v-akt/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismo

RESUMEN

We have identified a series of novel non-peptide compounds that activate the thrombopoietin-dependent cell line Ba/F3-huMPL. The compounds stimulated proliferation of Ba/F3-huMPL in the absence of other growth factors, but did not promote proliferation of the thrombopoietin-independent parent cell line Ba/F3. The thrombopoietin-mimetic compounds elicited signal-transduction responses comparable with recombinant human thrombopoietin, such as tyrosine phosphorylation of the thrombopoietin receptor, JAK (Janus kinase) 2, Tyk2 (tyrosine kinase 2), STAT (signal transducer and activator of transcription) 3, STAT5, MAPKs (mitogen-activated protein kinases), PLCgamma (phospholipase Cgamma), Grb2 (growth-factor-receptor-bound protein 2), Shc (Src homology and collagen homology), Vav, Cbl and SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and increased the number of CD41(+) cells (megakaryocyte lineage) in cultures of human CD34(+) bone-marrow cells (haematopoietic stem cells). These findings suggest that this series of compounds are novel agonists of the human thrombopoietin receptor and are possible lead compounds for the generation of anti-thrombocytopaenia drugs.

Asunto(s)

Materiales Biomiméticos/farmacología , Células de la Médula Ósea/metabolismo , Receptores de Trombopoyetina/agonistas , Transducción de Señal/efectos de los fármacos , Trombopoyesis/efectos de los fármacos , Trombopoyetina/farmacología , Animales , Células de la Médula Ósea/citología , Línea Celular , Proteína Adaptadora GRB2/biosíntesis , Humanos , Ratones , Fosfolipasa C gamma/biosíntesis , Proteínas Quinasas/biosíntesis , Proteína Tirosina Fosfatasa no Receptora Tipo 11/biosíntesis , Proteínas Proto-Oncogénicas c-cbl/biosíntesis , Proteínas Proto-Oncogénicas c-vav/biosíntesis , Receptores de Trombopoyetina/metabolismo , Factor de Transcripción STAT3/biosíntesis , Factor de Transcripción STAT5/biosíntesis , Proteínas Adaptadoras de la Señalización Shc/biosíntesis

RESUMEN

BACKGROUND: Keratinocyte growth factor (KGF) has been shown to induce breast cancer metastasis in animal models. cDNA microarrays have revealed that KGF increased Wilms tumor 1 (WT1) and focal adhesion kinase (FAK) expression in breast cancer cells. The role of WT1 and FAK in KGF signaling was investigated. MATERIALS AND METHODS: A cell culture wounding model was used to study the effects of WT1 and FAK down-regulation on KGF-induced proliferation and motility in breast cancer cells. RESULTS: WT1 down-regulation inhibited KGF-mediated proliferation and motility of breast cancer cells, while FAK down-regulation inhibited proliferation, but had no significant effect on cell motility. WT1 down-regulation, but not FAK down-regulation, led to Erk1,2 inactivation. CONCLUSION: KGF-mediated signaling employs WT1 and FAK to regulate breast cancer cell proliferation and motility and may represent therapeutic targets for the prevention of breast cancer progression.

Asunto(s)

Neoplasias de la Mama/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Movimiento Celular/fisiología , Regulación hacia Abajo , Activación Enzimática , Factor 7 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 7 de Crecimiento de Fibroblastos/farmacología , Proteína-Tirosina Quinasas de Adhesión Focal/biosíntesis , Proteína Adaptadora GRB2/biosíntesis , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Nucleares/biosíntesis , Fosforilación , Factores de Empalme de ARN , Proteínas Recombinantes/farmacología , Transducción de Señal

RESUMEN

Metastasis-associated genomic alterations have been recognized to play a critical role in tumor metastasis. Primary and metastatic tumor cells in mice and tumors in a patient were studied by cDNA array analysis. Selected genes were determined by RT-PCR and immunohistochemistry. Pathways on changed genes were statistically analyzed. The function of Grb2 was determined by in vitro wound assay. Nodal metastatic cells had a stronger ability of growth and metastasis than primary tumor cells. A total of 376 genes showed a different expression between primary and metastatic cells. The expression of Grb2 and genes in the Grb2-mediated pathways was significantly elevated in the metastases. Elevated levels of Grb2 expression in metastases were related to the distant metastasis of colorectal carcinoma. Blocking the Grb2-SH2 domain signaling transduction inhibited cell motility. Metastasis-associated genes identified by cDNA and tissue microarrays provide potentially valuable information on the metastasis of colorectal tumors. Overexpression of Grb2 may contribute to tumor growth, invasiveness and metastasis.

Asunto(s)

Carcinoma/metabolismo , Carcinoma/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteína Adaptadora GRB2/biosíntesis , Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia , Animales , ADN Complementario/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Trasplante de Neoplasias , Transducción de Señal

RESUMEN

There is evidence that the insulin-like growth factor-I (IGF-I) receptor is required for transformation by a variety of viral and cellular oncogenes in a mouse embryo fibroblast model. To further investigate the IGF-I receptor signaling pathways that are required for the permissive effect of the receptor on transformation by SV40 T antigen, we established three independent fibroblast cell lines each from wild-type and IGF-I receptor null embryos (R-). We transfected the wild-type and R- cell lines with an SV40 T antigen plasmid and selected three clones from each cell line that expressed T antigen. As in previous reports, none of the cloned R- cell lines expressing T antigen were transformed as measured by the ability to form large colonies in soft agar. However, with further passage, all three T antigen-expressing clones from one of the R- cell lines (R(-)3) formed large colonies in soft agar and the transformation of these T antigen-expressing clones was confirmed by tumorigenesis experiments in immunodeficient mice. DNA microarray analysis comparing gene expression between early passage and late passage R(-)3/T antigen clones showed, among other changes, an increase in the expression of ErbB-3 mRNA in the late passage clones. Also, the expression of ErbB-3 protein was dramatically increased in the late passage R(-)3/T antigen clones. We conclude that late passage IGF-I receptor null mouse embryo fibroblasts can be transformed by SV40 T antigen, and that ErbB-3 may play a role in permitting transformation by T antigen.

Asunto(s)

Antígenos Transformadores de Poliomavirus/fisiología , Transformación Celular Neoplásica/metabolismo , Fibroblastos/fisiología , Receptor IGF Tipo 1/deficiencia , Animales , Antígenos Transformadores de Poliomavirus/genética , Procesos de Crecimiento Celular/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , ADN/biosíntesis , Embrión de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/patología , Quinasa 1 de Adhesión Focal/metabolismo , Proteína Adaptadora GRB2/biosíntesis , Proteína Adaptadora GRB2/genética , Genotipo , Proteínas Sustrato del Receptor de Insulina , Ligandos , Ratones , Ratones Noqueados , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Fosforilación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor ErbB-3/biosíntesis , Receptor ErbB-3/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transfección

RESUMEN

BACKGROUND: The important metastatic potential of lung cancers is directly correlated with cell adhesion. Cell-extracellular matrix interactions occur in specialized structures termed focal adhesion (FA) complexes. Our aims were to investigate: (i) the expression of the major FA components in three lung cancer cell lines (non metastatic: A549, or metastatic: Calu-1 and H460), (ii) the modifications of the FA complex occurring when apoptosis was induced by Vinorelbine in the A549 cells. MATERIALS AND METHODS: The FA complex was characterized by flow cytometry, immunocytochemical staining and Western blot. RESULTS: The expressions of alpha3, betsaP, paxillin, p-paxillin and Grb2 varied depending on the histological type of the tumor. In apoptotic cells, the expressions of the PYK2, p-p38, PI3K and Grb2 adhesion proteins were increased. CONCLUSION: Our data suggest that these adhesion proteins may be implicated in the transduction of death signals.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Adhesiones Focales/fisiología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Apoptosis/fisiología , Línea Celular Tumoral , Citometría de Flujo , Quinasa 2 de Adhesión Focal/biosíntesis , Proteína-Tirosina Quinasas de Adhesión Focal/biosíntesis , Proteína Adaptadora GRB2/biosíntesis , Humanos , Inmunohistoquímica , Integrina alfa3/biosíntesis , Integrina beta1/biosíntesis , Paxillin/biosíntesis , Fosfatidilinositol 3-Quinasas/biosíntesis