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The Vav family of proteins are guanine nucleotide exchange factors which have been shown to be deregulated in several types of human cancer. There are three members of the Vav family that have been identified which are members of the Dbl domain superfamily and have specificity towards Rho/Rac GTPases. The Vav family plays an important role in normal hematologic system development and homeostasis, and Vav1 is largely restricted to the hematologic system. While Vav1 was originally identified as a proto-oncogene, several recent studies have shown that Vav family deletion leads to the development of T-cell malignancies in mice. In addition, Vav1 has been shown to play a role in the ATRA-mediated differentiation of promyelocytic leukemia cells. In this concise review, the gene structure and normal function of Vav1, as well as a possible role for Vav1 in the development of hematologic and other malignancies is reviewed.
The Vav family of proteins, which consists of Vav1, 2, and 3, are intracellular signal transduction proteins which function, in part, as a guanine nucleotide exchange factor (GEF). Vav1 was first identified in the Barbacid laboratory in 1989 as part of a screen for oncogenes with the ability to transform NIH3T3 fibroblasts . Interestingly, the version of the Vav protein responsible for transformation was an N-terminal deletion that has not been identified in human cancers to date . Research into the structure and function of Vav1 has identified it as a member of the Dbl-homology (DH) domain superfamily, over 70 of which have been identified to date in humans. The Vav DH domain mediates GEF activity towards the Rho/Rac family of GTPases.
Examination of a pooled database of mRNA gene expression profiles of normal tissue samples from many independent laboratories reveals that Vav1 is largely restricted to blood, spleen, and to a lesser extent lung, supporting previous reports that Vav1 expression is limited to the hematopoietic system during normal development and homeostasis . Interestingly, data from the same database indicates that expression of Vav1 remains high in hematologic malignancies as a class.
There are two other members of the Vav family that have been identified, Vav2 and Vav3. These family members have a broader expression pattern and are not restricted to the hematopoietic system. Similar to Vav1, N-terminal deletion of Vav2 and 3 renders them oncogenic in vitro [4,5]. In the hematologic system, the Vav proteins can partially compensate for each other. Elsewhere, Vav2 has been suggested to mediate a role in cell movement by transducing signals from Afadin dilute domain-interacting protein (ADIP), which localizes to the leading edge of cells, to the GTPase Rac . Recent research has implicated Vav3 in the development of a β2 integrin dependent phagocytic synapse between apoptotic polymorphonuclear leukocytes and macrophages during wound healing . This review will focus on the role of Vav1 in the development of hematopoietic malignancies.
Examination of the gene structure of Vav1 reveals that it has 27 exons which span 77kb on chromosome 19 (19p12-12p13.2) . Careful research has identified several functional domains in the Vav1 protein (Figure 1): i.) A calponin homology domain which may function as an actin binding domain and is involved in calcium mobilization; ii.) An acidic domain; iii.) Dblhomology domain (DH) which catalyzes guanine nucleotide exchange towards Rho family GTPases (Rac1, cdc42, RhoG); iv.) Pleckstrin homology domain (PH) which mediates interaction with phospholipids [9,10] and the WD-40 repeats motif [11,12] and results in localization of Vav to the plasma membrane. This domain is required for the normal catalytic activity of the DH domain [13,14]; v.) Src homology domain which mediates protein-protein interactions .
N-terminal deletion oncogenic Vav1 is a consequence of the removal of the calponin homology and acidic domains. There are multiple sites of tyrosine phosphorylation which regulate Vav1 GEF activity; three N-terminal tyrosine residues located at 140, 160, and 174 within the acidic domain, and C-terminal tyrosine residues 826 and 841 [16,17]. Interestingly, when Lazer and colleagues  expressed the C-terminal Y826F and Y841F mutants in Vav1 null pancreatic cancer cells, their transforming ability was abrogated, suggesting that normally regulated GEF activity is required for pancreatic carcinogenesis.
Vav1 plays a critical role in the development and activation of T-cells, in part through activation of specific Rho/Rac GTPases by catalyzing the exchange of GTP for GDP (Figure 2). By activating Rac GTPase, Vav1 mediates downstream responses to Src and Syk family kinases signals following T-cell receptor stimulation and thus plays an important role in the transduction of extracellular signals. The guanine nucleotide exchange factor activity and specificity for Rac GTPases is mediated by the DH and PH domains. While many GEF family members are specific for one GTPase, Vav1 has been shown to be capable of activating Cdc42, Rac1, RhoG, and RhoA .
Vav1 plays an important role in the actin cytoskeletal reorganization following T-cell activation through the T-cell receptor (TCR). For example, it has been demonstrated that Vav1 null murine lymphocytes have inhibited actin polymerization and a lack of clustering of TCR into patches following TCR activation . T-cell interaction with antigen presenting cells requires cytoskeletal reorganization to create a functional immunological synapse. The catalytic GEF function of Vav1 has been shown to be required for transmitting signal from the TCR to the cytoskeleton. When the murine wild type Vav1 allele is replaced with a version of Vav1 that folds correctly, but has deficient GEF catalytic activity, actin cytoskeletal reorganization following TCR stimulation is abrogated . Following stimulation of the TCR, Vav1 is phosphorylated at the N-terminal tyrosine amino acids 140, 160, and 174 which upregulate GEF activity . Research in several different systems has demonstrated that tyrosine phosphorylation can also be induced by stimulation of the B-cell receptor, FcRI, cytokine receptors, integrins and growth factor receptors .
Activation of Vav1 via TCR stimulation has been shown to regulate transcription factor activity in T-cells, such as NFAT, and subsequent IL2 transcriptional activation. In addition studies of Vav null mice have provided evidence that Vav1 regulates the calcium, ERK-MAP kinase, and NF- κB pathways in B and T-cells [22,23].
Vav1 is required for normal T-cell development as it transduces the developmental signals received at the pre-TCR, and an absence of Vav1, leads to a block at developmental checkpoints, which is accentuated by further deletion of Vav2 and 3 . Deficiency of Vav1 alone is sufficient to cause arrest of positive and negative selection of CD4+CD8+ double positive thymocytes . Mice deficient for the entire Vav1/2/3 family have been generated and these animals do not develop mature B- and Tlymphocytes, and do not develop normal T-cell dependent and independent humoral responses following stimulation .
In further regard to the mature B-cell deficiency seen in mice lacking all three Vav proteins, it has been shown to be associated with the disruption of both constitutive NF-κB activity in resting naïve B-cells and decreased expression of NF-κB regulated anti-apoptotic genes, such as BCL-2, in mature B-cells, resulting in shortened B-cell lifespan . The role of Vav in NF- κB pathway signal transduction is also highlighted by the finding that Vav-deficient mice are unable to generate a neutrophil oxidative burst when stimulated by LPS, a Toll-like receptor (TLR) ligand whose binding to TLR activates NF- κB.
Vav1 and 3 have also been shown to play an important role in modulating the reactivity of platelets . Finally, Vav1 appears to assist in the perivascular localization of hematopoietic stem and progenitor cells near nestin+ mesenchymal stem cells . In sum, the Vav family, and in particular Vav1, play an important role in the normal development and maintenance of the hematopoietic and immunologic system in humans.
Given the established role of Vav1 in transducing extracellular activation and developmental signals, it is not surprising that this pathway has been implicated in a number of diverse human cancers. Abberrant Vav1 expression was demonstrated in a majority of human neuroblastomas examined by Hornstein et al. . Another study found that Vav1 is expressed in greater than 50% of pancreatic adenocarcinomas examined, and data was presented to suggest that Vav1 promoter demethylation was the mechanism . Importantly, the authors presented data that suggested Vav1 expression contributed to the neoplastic pancreatic phenotype and may be a potential prognostic biomarker for more aggressive disease. A recent report identified ectopic Vav1 expression in 26/59 of a variety of primary lung carcinomas including adenocarcinoma, squamous cell carcinoma, and bronchioloaveloar carcinoma . Interestingly, the authors show that siRNA knockdown of Vav1 expression in a lung cancer cell line reduced their potential to generate tumors when injected into nude mice. Thus, ectopic Vav1 expression has been linked to a number of human cancers, and preliminary evidence appears to support Vav1 as a possible pharmacologic target in these malignancies.
GEFs other than Vav have previously been shown to play a role in the development of hematologic malignancy . For example, the Rho specific GEF, BCR is frequently translocated in hematologic malignancies, often translocating proximally to the tyrosine kinase ABL to form the Philadelphia chromosome, the expression of which is both necessary and sufficient for the development of chronic myelogenous leukemia. Another RhoA specific GEF, LARG, has been identified as a fusion partner of the mixed lineage leukemia gene in acute myeloid leukemia, and some evidence has shown that this protein in excess amounts can contribute to neoplasia . Thus, there are multiple lines of evidence that deregulation of the Vav associated pathways of signalling can lead to the development of cancer.
Multiple studies have begun to define a role for Vav in the development and progression of hematological malignancies. Transgenic mice have been generated with Vav1, 2, 3, and Rasgrf2 null alleles individually and in combination, and as mentioned above, these animals have defects in T-cell development and activation. Somewhat unexpectedly, longitudinal studies of Vav1 null mice revealed an increased incidence of aggressive lymphoblastic-like T-cell leukemia/lymphomas . The latency to the development of this murine T-cell leukemia/ lymphoma decreased and the incidence increased when Rasgrf2, a GEF with specificity for Ras and Rho/Rac GTPases, was additionally deleted from the genome in these mice. Examination of the immunophenotypic characteristics reveal the T-cell leukemia/lymphoma to be similar to those that develop spontaneously in older mice.
This increased development of T-cell lymphoblastic- like tumors in Vav1 null mice was unexpected given previous data implicating Vav1 as a proto-oncogene; indeed, this result complicates predictions of possible efficacy of Vav1-targeted small molecule inhibitors on the development and progression of human cancer. Given that Vav1 plays a role in the normal selection of mature thymocytes during development, one possibility is that Vav1 may help to reduce the negative selection of T-cell clones with potentially neoplastic rearrangements of somatic DNA during development. Alternatively, Dumont et al.  have shown that deletion of both the Vav and Rac family in mice leads to upregulation of Notch signaling during early thymic development. The role of gain of function Notch1 alterations in the development of murine and human T-cell lymphoblastic lymphoma has been well established , and thus it is tempting to speculate that Vav and Rac signalling alterations lead to T-cell lymphoblastic leukemia/ lymphoma through deregulation of Notch signaling (Figure 3).
Interestingly, Berquam-Vrieze et al. describe their results with a novel forward genetic screen where they induce T-cell lymphoblastic leukemia/ lymphomas in mice by targeting mutational transposons to specific developmental stages of thymocytes . When they conditionally delete Vav from hematopoetic stem cells and examine the resultant T-cell leukemia for transposon mediated driver mutations, mutations in Notch1 were frequently found (72%), again emphasizing the connection between Vav signaling, Notch activation, and the development of T-cell malignancies. When the T-cell leukemia is induced in a later stage of T-cell development using an alternate transgene to Vav (Lck), the driver mutations are no longer found in Notch1.
The first human hematological malignancies that provided evidence of deregulated Vav1 was B-cell chronic lymphocytic leukemia (B-CLL). Prieto-Sanchez et al.  examined Vav1 protein levels and phosphorylation status in 118 unselected cases of hematologic neoplasms which included myeloproliferative neoplasms and a variety of B-cell malignancies. They found that Vav1 was phosphorylated and overexpressed in 10 of 14 cases of B-CLL with 13q deletion. Vav1 protein levels were not altered in any of the myeloproliferative neoplasms examined.
An expression profiling study by Hollmann et al.  revealed correlation between the expression of Vav1 and CD40-mediated apoptosis in diffuse large B-cell lymphoma cell lines. They found that Vav1 expression may have a role in the disparity between DLBCL cell lines sensitive to CD40 stimulation and those resistant to it. The cell lines resistant to CD40 induced apoptosis had reduced NF-κB activation and markedly reduced Vav1 levels. Interestingly, the authors found an association between increased Vav1 expression and a higher proliferative index, and the post-germinal center marker Irf-4. While it is unclear whether accentuation or reduction of Vav levels is the best approach to address the malignant B-cell phenotype, there is clear data in support of the role of Vav1 regulation in the survival of normal B-cells . In a malignant T-cell line, Yin, et. al.  has demonstrated a correlation between increased Vav1 expression and increased Bcl2 expression which is associated with decreased sensitivity to fas-mediated apoptosis, but to our knowledge, this has not been described in malignant B-cells.
In acute myeloid leukemia (AML), several studies have shown that Vav1 is required for ATRA induced differentiation of human promyelocytic leukemia cell lines to neutrophils as well as PMA induced maturation of these same cell lines to monocytes/macrophages [38-40]. Interestingly, evidence is presented that Vav1 is directly recruited to the promoter of CD11b, an integrin selectively expressed on the surface of mature granulocytes, and may participate in transcriptional regulation of CD11b during ATRA induced differentiation . However, there is no evidence that Vav1 has a tumor suppressor role in AML as repressed Vav1 expression has not been associated with AML in humans nor has it lead to the development AML in Vav-deficient mice.
The Vav family of proteins mediate signal transduction through tyrosine phosphorylation dependent guanine nucleotide exchange activity. Vav1 is expressed only in the immune system and plays a role in a diverse number cellular processes including TCR and BCR activation with concomitant cytoskeleton reorganization. While Vav1 was originally identified as an protooncogene capable of inducing transformation in NIH3T3 fibroblasts, recent data has identified a requirement for Vav for normal T-and B-cell development. In animals lacking the Vav family, not only do T-cells fail to normally develop, but there is an increased incidence of T-cell malignancies. While Vav remains an attractive pharmacologic target for a small molecule inhibitor of its enzymatic activity, more work is required to more precisely delineate the specific subtypes of human hematologic malignancies in which Vav1 deregulation is coupled to a neoplastic phenotype.
This study was partially supported by the grant IIRG-08-90524 from Alzhermer's Association (to DSW) and the NIH grant R21 AG029972 (to DSW).