Vav1 Expression Research Articles

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.

Estrogen induces Vav1 expression in human breast cancer cells.

Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17β-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be α form, not β. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ERα might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

High nuclear level of Vav1 is a positive prognostic factor in early invasive breast tumors: a role in modulating genes related to the efficiency of metastatic process.

Vav1 is one of the signalling proteins normally restricted to hematopoietic cells that results ectopically expressed in solid tumors, including breast cancer. By immunohistochemical analysis on TMAs containing invasive breast tumor from patients without lymph node involvement, we have found that Vav1 is expressed in almost all investigated cancers and shows a peculiar localization inside the nucleus of tumor cells. High amounts of nuclear Vav1 are positively correlated with low incidence of relapse, regardless phenotype and molecular subtype of breast neoplasia. In particular, Kaplan-Meier plots showed an elevated risk of distant metastasis in patients with low Vav1 expression compared with patients with high Vav1 expression in their tumors. Experiments performed with breast tumor-derived cells indicated that Vav1 negatively modulates their invasiveness in vitro and their metastatic efficiency in vivo, possibly by affecting the expression of genes involved in invasion and/or metastasis of breast tumors. Since the high heterogeneity of breast tumors makes difficult to predict the evolution of early breast neoplasias, the evaluation of nuclear Vav1 levels may help in the characterization and management of early breast cancer patients. In particular, Vav1 may serve as a prognostic biomarker and a target for new therapies aimed to prevent breast cancer progression.

Quantitative phosphoproteomics reveals novel Vav1-dependent pathways in T cell receptor signaling (IRM10P.736)

Abstract Vav1, a Rac/Rho guanine nucleotide exchange factor, is a critical component of the T cell receptor (TCR) signaling cascade and is rapidly tyrosine phosphorylated in response to T cell activation. Previous studies using Vav1 knockout models have determined its importance in TCR-dependent signaling. However, a wide-scale characterization of Vav1-dependent phosphorylation events is still lacking. Using quantitative mass spectrometry, a phosphoproteomic analysis of 668 tyrosine phosphorylation sites was performed on Vav1-deficient T cells across four time points of TCR stimulation. Compared to controls, cells lacking Vav1 expression showed statistically significant decreases in phosphorylation on actin cytoskeletal signaling proteins, supporting the role of Vav1 in cytoskeletal reorganization. Interestingly, loss of Vav1 led to perturbations in phosphorylation on signaling molecules proximal to the TCR. Furthermore, proteins implicated in negative feedback pathways, including CBL and PAG, showed altered phosphorylation profiles. Effects further downstream of the TCR were also observed in Vav1-deficient cells, notably, hyperphosphorylation of inhibitory sites on various cyclin dependent kinases, suggesting cell cycle arrest. This data demonstrates novel signaling networks regulated by Vav1 and support the pleiotropic functions of Vav1 downstream of the TCR.

Vav1 is ectopically expressed in breast tumors in which reduces the efficiency of the metastatic process

Vav1, normally restricted to hematopoietic cells, results ectopically expressed in solid tumors, including breast cancer (Sebban et al., 2013) in which, contrarily to other neoplasias, seems to be higher in tumors from patients who remained diseasefree than in patients who developed recurrence (Lane et al., 2008). The significance of Vav1 expression in breast tumors was evaluated by immunohistochemical analysis on TMAs containing invasive breast tumors from patients without lymph node involvement. Our findings indicate that Vav1 is expressed in almost all investigated cancers and shows a peculiar localization inside the nucleus of tumor cells. High amounts of nuclear Vav1 positively correlates with low incidence of relapse, regardless phenotype and molecular subtype of the neoplasia. Experiments performed with breast tumor-derived cells showing different morphology, immunoprofile and invasive properties indicated that Vav1 negatively modulates their invasiveness in vitro and their metastatic efficiency in vivo, possibly by affecting the expression of genes involved in invasion and/or metastasis of breast cancer. Since the high heterogeneity of breast tumors makes difficult to predict the evolution of early neoplasias, the evaluation of nuclear Vav1 levels may help in profiling and management of early breast cancer patients. In addition, Vav1 may serve as a target for new therapies designed to prevent breast cancer progression.

Vav1 as a Central Regulator of Invadopodia Assembly

Invadopodia are protrusive structures used by tumor cells for degradation of the extracellular matrix to promote invasion [1]. Invadopodia formation and function are regulated by cytoskeletal-remodeling pathways and the oncogenic kinase Src. The guanine nucleotide exchange factor Vav1, which is an activator of Rho family GTPases, is ectopically expressed in many pancreatic cancers, where it promotes tumor cell survival and migration [2, 3]. We have now determined that Vav1 is also a potent regulator of matrix degradation by pancreatic tumor cells as depletion of Vav1 by siRNA-mediated knockdown inhibits the formation of invadopodia. This requires the exchange function of Vav1 toward the GTPase Cdc42, which is required for invadopodia assembly [4, 5]. In addition, we have determined that Src-mediated phosphorylation and activation of Vav1 are both required for, and, unexpectedly, sufficient for, invadopodia formation. Expression of Vav1 Y174F, which mimics its activated state, is a potent inducer of invadopodia formation through Cdc42, even in the absence of Src activation and phosphorylation of other Src substrates, such as cortactin. Thus, these data identify a novel mechanism by which Vav1 can enhance the tumorigenicity and invasive potential of cancer cells. These data suggest that Vav1 promotes the matrix-degrading processes underlying tumor cell migration and further, under conditions of ectopic Vav1 expression, that Vav1 is a central regulator and major driver of invasive matrix remodeling by pancreatic tumor cells.

VAV1 represses E-cadherin expression through the transactivation of Snail and Slug: a potential mechanism for aberrant epithelial to mesenchymal transition in human epithelial ovarian cancer

Ovarian cancer is the most lethal gynecological malignancy in the western world. Although patients with early-stage ovarian cancer generally have a good prognosis, approximately 20%-30% of patients will die of the disease, and 5-year recurrence rates are 25%-45%, highlighting the need for improved detection and treatment. We investigated the role of VAV1, a protein with guanine nucleotide exchange factor activity, which is associated with survival in patients with early-stage ovarian cancer (International of Obstetrics and Gynecology [FIGO] stages I and II). We analyzed 88 samples from patients with primary epithelial ovarian cancer, which were divided into FIGO stages I and II (n = 46), and III and IV (n = 42). Prognostic analysis revealed that upregulated VAV1 expression correlated significantly with poor prognosis in patients with early-stage epithelial ovarian cancer (P ≤ 0.05), but not with other clinicopathologic features. Stable overexpression of VAV1 in human high-grade serous ovarian cancer SKOV3 cells induced morphologic changes indicative of loss of intercellular adhesions and organized actin stress fibers. Western blotting and real-time reverse transcriptase-polymerase chain reaction demonstrated that these cells had downregulated E-cadherin protein and messenger RNA levels, respectively. This downregulation is associated with epithelial-mesenchymal transition (EMT) and invasive cancer. Furthermore, VAV1 overexpression in both SKOV3 and human ovarian surface epithelial cells demonstrated that its upregulation of an E-cadherin transcriptional repressor, Snail and Slug, was not confined to ovarian cancer cells. Conversely, knockdown of VAV1 by RNA interference reduced Snail and Slug. Our findings suggest that VAV1 may play a role in the EMT of ovarian cancer, and may serve as a potential therapeutic target.

Vav1 Fine Tunes p53 Control of Apoptosis versus Proliferation in Breast Cancer

Vav1 functions as a signal transducer protein in the hematopoietic system, where it is exclusively expressed. Vav1 was recently implicated in several human cancers, including lung, pancreatic and neuroblasoma. In this study, we analyzed the expression and function of Vav1 in human breast tumors and breast cancer cell lines. Immunohistochemical analysis of primary human breast carcinomas indicated that Vav1 is expressed in 62% of 65 tumors tested and is correlated positively with estrogen receptor expression. Based on published gene profiling of 50 breast cancer cell lines, several Vav1-expressing cell lines were identified. RT-PCR confirmed Vav1 mRNA expression in several of these cell lines, yet no detectable levels of Vav1 protein were observed due to cbl-c proteasomal degradation. We used two of these lines, MCF-7 (Vav1 mRNA negative) and AU565 (Vav1 mRNA positive), to explore the effect of Vav1 expression on breast cell phenotype and function. Vav1 expression had opposite effects on function in these two lines: it reduced proliferation and enhanced cell death in MCF-7 cells but enhanced proliferation in AU565 cells. Consistent with these findings, transcriptome analysis revealed an increase in expression of proliferation-related genes in Vav1-expressing AU565 cells compared to controls, and an increase in apoptosis-related genes in Vav1-expressing MCF-7 cells compared with controls. TUNEL and γ-H2AX foci assays confirmed that expression of Vav1 increased apoptosis in MCF-7 cells but not AU565 cells and shRNA experiments revealed that p53 is required for this pro-apoptotic effect of Vav1 in these cells. These results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer and the p53 dependence of its pro-apoptotic effect in breast cells.

Vav1 Deficiency Differentially Affects Engraftment of Fetal Hematopoietic Organs During Development Compared with Adult Marrow After Transplantation

Abstract 1212Rac GTPases function as molecular switches, which integrate extracellular signals that are critical for hematopoietic stem cell (HSC) engraftment and bone marrow repopulation during transplantation. We have recently reported that loss of the Vav1 protein, a known hematopoietic-specific activator of Rac, drastically reduces engraftment of adult HSCs without compromising steady-state hematopoiesis in adult mice (Sanchez-Aguilera et al., PNAS 2011). This suggests that HSC migration during ontogeny is unaffected by Vav1 loss and implies that GTPase regulation of HSC migration is distinctly different during embryonic migration versus (vs.) migration required during transplant-mediated engraftment. To verify this, we have examined the role of Vav1 in fetal hematopoietic cells. In the developing embryo, Vav1 expression is first detected at E11.5 and increases to E14.5 (Bustelo et al., Cell Growth Differ 1993). At this developmental age, fetal liver (FL) is the major hematopoietic organ in the embryo. We found no difference between Vav1−/− and WT animals in the number of immunophenotypically defined HSCs (LSK SLAM or c-Kit+CD34+) in the FL isolated from E13.5 embryo (4.4 +/− 0.6% LSK-SLAM in the WT group vs. 7.6 +/− 1.4 in the Vav1−/−group). Vav1−/− FL cells also formed normal numbers of hematopoietic progenitor colonies in methylcellulose (42.01 +/−3.2 in the WT group vs. 37.63 +/− 2.5 in the Vav1−/−group). We then compared the number of LSK SLAM cells in the BM of Vav1−/− newborn mice to WT, as well as their ability to form colonies in methylcellulose, and did not detect any difference between the two genotypes. Together, these data suggest that during hematopoietic development, the initial colonization of the FL and subsequent transition to BM hematopoiesis are not affected by deletion of Vav1. To determine whether Vav1 is required for engraftment of FL cells after transplantation, we performed competitive transplantation experiments. We found that Vav1−/− E13.5 FL cells failed to provide long term engraftment to lethally irradiated syngeneic recipients, as measured by peripheral blood (PB) chimerism at 4, 8 and 16 weeks after transplantation (an average of 0.08%, 0.42% and 0.12% donor-derived CD45.2 positive cells in the PB of Vav mice at the three time points analyzed vs. 42.1%, 49.5% and 66.35% in the WT group; p<0.0001, p=0.0049, p=0.0016, respectively). In addition, Vav1−/− FL cells failed to contribute to PB reconstitution in newborn syngeneic recipients that were sublethally irradiated or born from pregnant mothers conditioned with busulfan treatment. These data confirm the differential requirement for Vav1 during fetal physiological organ engraftment compared to HSC transplant. Next, we sought to determine the mechanisms underlying the lack of engraftment of Vav1−/− fetal cells during transplantation. Given that the Vav1-Rac axis has been shown to contribute to the activation of the JNK pathway (Crespo et al., Oncogene 1996), which is induced by pro-inflammatory cytokines (Kyaris et al., Nature 1994) produced during bone marrow ablation, we tested whether Vav1−/− cells were hypersensitive to pro-inflammatory cytokines. Vav1−/− cells produced normal numbers of progenitor-derived colonies in vitro in the presence of increasing doses of TNFα and IFNγ. Similarly, the lack of engraftment of Vav1−/−HSC was not rescued upon transplantation into W/Wv mice, which do not require any myelablation for effective engraftment of WT HSC. These results indicate that the lack of engraftment of Vav1 cells is not due to an increased sensitivity to pro-inflammatory cytokines.In conclusion, our data suggest that distinct mechanisms regulate the physiological migration of FL cells to the BM during development and their ability to reconstitute the hematopoietic system of recipients during transplantation. We believe that understanding the basis of these mechanisms is critical for transplant biology and regenerative medicine. Disclosures:Williams:bluebird bio: Consultancy; Wyeth:; Takara bio:.

581 CpG Methylation and Transcription Factor C-Myb Regulate Human Vav1 Expression in Hematopoietic and Cancer Cell Lines

581 CpG Methylation and Transcription Factor C-Myb Regulate Human Vav1 Expression in Hematopoietic and Cancer Cell Lines

Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5′ regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well.

Vav‐1 expression correlates with NFκB activation and CD40‐mediated cell death in diffuse large B‐cell lymphoma cell lines

Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy with a variable response to therapy. We have previously shown that DLBCL cell lines differ in their susceptibility to CD40-mediated cell death, and that resistance to CD40-targeted antibodies correlated with increased expression of markers of immature B-cell and absence of Vav-1 mRNA. We used gene expression profiling to investigate the mechanism of CD40 resistance in these cell lines, and found that resistance correlated with lack of Vav-1 and inability to activate NFκB upon CD40 ligation. Analysis of tissue microarrays of 213 DLBCL cases revealed that Vav-1 expression correlated with a higher proliferative index and the presence of the post-germinal centre marker Irf-4. Our results suggest that Vav-1 expression may be associated with activated B-cell DLBCL origin and higher proliferative activity, and indicate Vav-1 as a potential marker to identify tumours likely to respond to CD40-targeted therapies.

A Role for VAV1 in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

Multiple sclerosis, the most common cause of progressive neurological disability in young adults, is a chronic inflammatory disease. There is solid evidence for a genetic influence in multiple sclerosis, and deciphering the causative genes could reveal key pathways influencing the disease. A genome region on rat chromosome 9 regulates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Using interval-specific congenic rat lines and association of single-nucleotide polymorphisms with inflammatory phenotypes, we localized the gene of influence to Vav1, which codes for a signal-transducing protein in leukocytes. Analysis of seven human cohorts (12,735 individuals) demonstrated an association of rs2546133-rs2617822 haplotypes in the first VAV1 intron with multiple sclerosis (CA: odds ratio, 1.18; CG: odds ratio, 0.86; TG: odds ratio, 0.90). The risk CA haplotype also predisposed for higher VAV1 messenger RNA expression. VAV1 expression was increased in individuals with multiple sclerosis and correlated with tumor necrosis factor and interferon-gamma expression in peripheral blood and cerebrospinal fluid cells. We conclude that VAV1 plays a central role in controlling central nervous system immune-mediated disease and proinflammatory cytokine production critical for disease pathogenesis.

Vav1 and PU.1 are recruited to the CD11b promoter in APL-derived promyelocytes: Role of Vav1 in modulating PU.1-containing complexes during ATRA-induced differentiation

Vav1 plays an important role in the all- trans retinoic acid (ATRA)-induced completion of the differentiation program of acute promyelocytic leukemia (APL)-derived cells, in which it strengthens the drug effects and is involved in the regulation of maturation-related proteins, such as the CD11b surface antigen. In both myeloid and lymphoid cells, accumulating data attribute to the multidomain protein Vav1 a functional relevance in the control of gene expression, by direct interaction with chromatin remodeling and/or transcriptional proteins. The present study provides evidence that, in the APL-derived NB4 cell line, Vav1 and the transcription factor PU.1 cooperate in regulating the ATRA-induced CD11b expression. Both chromatin immunoprecipitation (ChIP) experiments and electrophoretic mobility shift assays (EMSA) indicate that Vav1 and PU.1 are recruited to CD11b promoter. Even if the two proteins may participate in diverse protein/DNA complexes, the amounts of complexes including PU.1 seem to be dependent on the interaction of this transcription factor with tyrosine-phosphorylated Vav1. The reported data suggest that the ATRA-induced increase of Vav1 expression and tyrosine phosphorylation may be involved in recruiting PU.1 to its consensus sequence on the CD11b promoter and, ultimately, in regulating CD11b expression during the late stages of neutrophil differentiation of APL-derived promyelocytes.

IDO inhibits T-cell function through suppressing Vav1 expression and activation

Purpose: Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolic enzyme, plays an important role in immune escape through suppressing T-cell function. Since Vav1 signaling pathway regulates T cell homeostasis, this study was designed to test the hypothesis that IDO induces T-cell immunosuppression through inhibiting Vav1 signaling.Results: We found that IDO produced by IDO stably expressing CHO cells significantly inhibited interleukin (IL)-2 expression and proliferative response in T cells and increased apoptosis of T cells. IDO suppressed Vav1 mRNA and protein production in T cells. Furthermore, IDO inhibited TCR activation-induced Vav1 phosphorylation, which represents Vav1’s activation state in T cells. These effects on T-cells induced by co-culture of CHO/IDO with T cells were attenuated by 1-MT.Materials and methods: Chinese hamster ovary (CHO) cells were stably transfected with human IDO (CHO/IDO). CD3+ T cells were isolated from human peripheral blood monouclear cells. After co-culture of CHO/IDO cells with T cells in the presence or absence of an anti-CD3 antibody to activate T cell receptor (TCR) and/or 1-methyl-L-tryptophan (1-MT) to inhibit IDO activity, T cell proliferation and apoptosis were determined. T cell total RNA and cellular protein samples were isolated for detecting Vav1 gene and protein expression and activation state.Conclusions: The inhibitory effects of IDO on T cell immune responses may be through downregulation of Vav1 protein expression and activation. These studies provide insight into understanding the mechanisms of immune escape induced by IDO and therapeutic application of IDO inhibitors for cancer treatment.

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.

Host deficiency in Vav2/3 guanine nucleotide exchange factors impairs tumor growth, survival, and angiogenesis in vivo.

Vav guanine nucleotide exchange factors modulate changes in cytoskeletal organization through activation of Rho, Rac, and Cdc42 small GTPases. Although Vav1 expression is restricted to the immune system, Vav2 and Vav3 are expressed in several tissues, including highly vascularized organs. Here, we provide the first evidence that Vav2 and Vav3 function within the tumor microenvironment to promote tumor growth, survival, and neovascularization. Host Vav2/3 deficiency reduced microvascular density, as well as tumor growth and/or survival, in transplanted B16 melanoma and Lewis lung carcinoma models in vivo. These defects were due in part to Vav2/3 deficiency in endothelial cells. Vav2/3-deficient endothelial cells displayed reduced migration in response to tumor cells in coculture migration assays, and failed to incorporate into tumor vessels and enhance tumor volume in tumor-endothelial cotransplantation experiments. These data suggest that Vav2/3 guanine nucleotide exchange factors play a critical role in host-mediated tumor progression and angiogenesis, particularly in tumor endothelium.

Vav1: A hematopoietic signal transduction molecule involved in human malignancies

Vav1 encodes a unique protein with several motifs known to play a role in tyrosine mediated signal transduction, including a DBL homology (DH) domain, a pleckstrin homology (PH) domain, a Src homology 2 (SH2) domain, and two Src homology 3 (SH3) domains. Physiological Vav1 expression is restricted to the hematopoietic system, where it functions primarily as a specific GDP/GTP nucleotide exchange factor (GEF), a function strictly regulated by tyrosine phosphorylation. In hematopoietic cells, Vav1 is phosphorylated following cell surface receptor activation, triggering re-organization of the cytoskeleton and regulation of other cellular functions including transcription, cytokine production, cell cycle progression, and Ca 2+ mobilization. Vav1 also functions as an adapter, facilitating interaction between other proteins. A truncated Vav1 was first isolated as an oncogene, and its wild-type form has recently been implicated in mammalian malignancies. These properties make Vav1 a promising target for new therapeutic approaches to organ transplantation and cancer therapy.

Vav1 Acidic Region Tyrosine 174 Is Required for the Formation of T Cell Receptor-induced Microclusters and Is Essential in T Cell Development and Activation

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.

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.