RESUMO
Lung cancer is the leading cause of cancer death worldwide. The spectrum of aberrations affecting signalling pathways in lung cancer pathogenesis has not been fully elucidated. Physiological expression of Vav1 is restricted to the haematopoietic system, where its best-known function is as a GDP/GTP nucleotide exchange factor for Rho/RacGTPases, an activity strictly controlled by tyrosine phosphorylation downstream of cell surface receptors. Here we find Vav1 expression in 42% of 78 lung cancer cell lines analysed. Moreover, immunohistochemical analysis of primary human lung cancer tissue samples revealed Vav1 expression in 26/59 malignant samples, including adenocarcinoma, squamous cell carcinoma and bronchioloalveolar carcinoma. Stronger Vav1 staining was associated with larger tumour size. siRNA-mediated knockdown of Vav1 in lung cancer cells reduced proliferation in agar and tumour growth in nude mice, while control siRNA had no effect, suggesting that Vav1 plays a critical role in the tumorigenicity of lung cancer cells. Vav1 is tyrosine-phosphorylated in lung cancer cells following activation by the growth factors EGF and TGFalpha, suggesting its participation in signalling events in these cells. Depletion of Vav1 reduced Rac-GTP activation and decreased expression of TGFalpha, an autocrine growth factor. These data suggest that Vav1 plays a role in the neoplastic process in lung cancer, identifying it as a potential therapeutic target for lung cancer therapy.
Assuntos
Carcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Hematopoese/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais/genética , Animais , Carcinoma/patologia , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-vav/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator de Crescimento Transformador alfa/genética , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Vav1 functions in the hematopoietic system as a specific GDP/GTP nucleotide exchange factor regulated by tyrosine phosphorylation. An intact C-terminal SH3 domain of Vav1 (Vav1SH3C) was shown to be necessary for Vav1-induced transformation, yet the associating protein(s) necessary for this activity have not yet been identified. Using a proteomics approach, we identified Sam68 as a Vav1SH3C-associating protein. Sam68 (Src-associated in mitosis of 68 kD) belongs to the heteronuclear ribonucleoprotein particle K (hnRNP-K) homology (KH) domain family of RNA-binding proteins. The Vav1/Sam68 interaction was observed in vitro and in vivo. Mutants of Vav1SH3C previously shown to lose their transforming potential did not associate with Sam68. Co-expression of Vav1 and Sam68 in Jurkat T cells led to increased localization of Vav1 in the nucleus and changes in cell morphology. We then tested the contribution of Sam68 to known functions of Vav1, such as focus-forming in NIH3T3 fibroblasts and NFAT stimulation in T cells. Co-expression of oncogenic Vav1 with Sam68 in NIH3T3 fibroblasts resulted in a dose-dependent increase in foci, yet no further enhancement of NFAT activity was observed in Jurkat T cells, as compared to cells overexpressing only Vav1 or Sam68. Our results strongly suggest that Sam68 contributes to transformation by oncogenic Vav1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Animais , Núcleo Celular/metabolismo , Forma Celular , Citoplasma/metabolismo , Humanos , Células Jurkat , Camundongos , Mutação , Fatores de Transcrição NFATC/metabolismo , Células NIH 3T3 , Ligação Proteica , Transporte Proteico , Proteômica , Proteínas Proto-Oncogênicas c-vav/química , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Recombinantes de Fusão/metabolismo , Domínios de Homologia de srcRESUMO
Mammalian wild-type Vav1 (wtVav1) encodes a specific GDP/GTP nucleotide exchange factor that is exclusively expressed in the hematopoietic system. Despite numerous studies, the mechanism underlying transformation of fibroblasts by oncogenic Vav1 (oncVav1) is not well defined. We identified osteopontin, a marker for tumor aggressiveness, as an oncVav1-inducible gene. Osteopontin is highly expressed in oncVav1-transformed NIH3T3 cells (NIH/oncVav1) but is barely detected in NIH3T3 expressing wtVav1 (NIH/wtVav1) even following epidermal growth factor stimulation, which normally induces osteopontin. Depleting oncVav1 in NIH/oncVav1 using small interfering RNA led to a considerable decrease in osteopontin, whereas reducing osteopontin expression did not affect oncVav1 expression, suggesting that oncVav1 operates upstream of osteopontin. Vav1-depleted NIH/oncVav1 cells, but not osteopontin-depleted NIH/oncVav1 cells, exhibited impaired extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase phosphorylation. Inhibition of ERK phosphorylation in NIH/oncVav1 cells led to a decrease in osteopontin expression, implying that the elevated osteopontin expression in these cells is dependent on ERK phosphorylation. Vav1-depleted or osteopontin-depleted NIH/oncVav1 cells lost their tumorigenic properties as judged by the soft agar and invasion assays, although loss of osteopontin expression had a less dramatic effect. Suppression of Vav1 expression in NIH/oncVav1 cells led to reversion to "normal" morphology, whereas when only osteopontin expression was diminished cells retained their transformed morphology. This work strongly supports a role for oncVav1 as a master oncogene and provides clues to the molecular mechanism underlying oncVav1 transformation.
