Tyrosine residues at the carboxyl terminus of Vav1 play an important role in regulation of its biological activity.

The guanine nucleotide exchange factor (GEF) Vav1 is an essential signal transducer protein in the hematopoietic system, where it is expressed physiologically. It is also involved in several human malignancies. Tyrosine phosphorylation at the Vav1 amino terminus plays a central role in regulating its activity; however, the role of carboxyl terminal tyrosine residues is unknown. We found that mutation of either Tyr-826 (Y826F) or Tyr-841 (Y841F) to phenylalanine led to loss of Vav1 GEF activity. When these Vav1 mutants were ectopically expressed in pancreatic cancer cells lacking Vav1, they failed to induce growth in agar, indicating loss of transforming potential. Furthermore, although Y841F had no effect on Vav1-stimulated nuclear factor of activated T cells (NFAT) activity, Y826F doubled NFAT activity when compared with Vav1, suggesting that Tyr-826 mediates an autoinhibitory effect on NFAT activity. SH2 profiling revealed that Shc, Csk, Abl, and Sap associate with Tyr-826, whereas SH2-B, Src, Brk, GTPase-activating protein, and phospholipase C-gamma associate with Tyr-841. Although the mutations in the Tyr-826 and Tyr-841 did not affect the binding of the carboxyl SH3 of Vav1 to other proteins, binding to several of the proteins identified by the SH2 profiling was lost. Of interest is Csk, which associates with wild-type Vav1 and Y841F, yet it fails to associate with Y826F, suggesting that loss of binding between Y826F and Csk might relieve an autoinhibitory effect, leading to increased NFAT. Our data indicate that GEF activity is critical for the function of Vav1 as a transforming protein but not for NFAT stimulation. The association of Vav1 with other proteins, detected by SH2 profiling, might affect other Vav1-dependent activities, such as NFAT stimulation.

The haematopoietic specific signal transducer Vav1 is aberrantly expressed in lung cancer and plays a role in tumourigenesis.

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.

The association of Sam68 with Vav1 contributes to tumorigenesis.

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.

Osteopontin is an oncogenic Vav1- but not wild-type Vav1-responsive gene: implications for fibroblast transformation.

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.

Vav1 promotes lung cancer growth by instigating tumor-microenvironment cross-talk via growth factor secretion.

Vav1 is a signal transducer that functions as a scaffold protein and a regulator of cytoskeleton organization in the hematopoietic system, where it is exclusively expressed. Recently, Vav1 was shown to be involved in diverse human cancers, including lung cancer. We demonstrate that lung cancer cells that abnormally express Vav1 secrete growth factors in a Vav1-dependent manner. Transcriptome analysis demonstrated that Vav1 depletion results in a marked reduction in the expression of colony-stimulating-factor-1 (CSF1), a hematopoietic growth factor. The association between Vav1 expression and CSF1 was further supported by signal transduction experiments, supporting involvement of Vav1 in regulating lung cancer secretome. Blocking of ERK phosphorylation, led to a decrease in CSF1 transcription, thus suggesting a role for ERK, a downstream effector of Vav1, in CSF1 expression. CSF1-silenced cells exhibited reduced focus formation, proliferation abilities, and growth in NOD/SCID mice. CSF1-silenced H358 cells resulted in significantly smaller tumors, showing increased fibrosis and a decrease in tumor infiltrating macrophages. Finally, immunohistochemical analysis of primary human lung tumors revealed a positive correlation between Vav1 and CSF1 expression, which was associated with tumor grade. Additional results presented herein suggest a potential cross-talk between cancer cells and the microenvironment controlled by CSF1/Vav1 signaling pathways.

Guanine nucleotide exchange factors for RhoGTPases: good therapeutic targets for cancer therapy?

Rho guanosine triphosphatases (GTPases) are a family of small proteins which function as molecular switches in a variety of signaling pathways following stimulation of cell surface receptors. RhoGTPases regulate numerous cellular processes including cytoskeleton organization, gene transcription, cell proliferation, migration, growth and cell survival. Because of their central role in regulating processes that are dysregulated in cancer, it seems reasonable that defects in the RhoGTPase pathway may be involved in the development of cancer. RhoGTPase activity is regulated by a number of protein families: guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine nucleotide-dissociation inhibitors (GDIs). This review discusses the participation of RhoGTPases and their regulators, especially GEFs in human cancers. In particular, we focus on the involvement of the RhoGTPase GEF, Vav1, a hematopoietic specific signal transducer which is involved in human neuroblastoma, pancreatic ductal carcinoma and lung cancer. Finally, we summarize recent advances in the design and application of a number of molecules that specifically target individual RhoGTPases or their regulators or effectors, and discuss their potential for cancer therapy.