Vav1 Mutations Identified in Human Cancers Confirm its Classification as an Oncogene

Introduction: The activity of Vav1 as a mutant oncogene in human tumors has remained questionable for decades. Although mutants of Vav1 were recently identified in human cancers of various origins, the functional activity of these mutants is not fully studied. Vav1 is physiologically active as a GDP/GTP nucleotide exchange factor (GEF) in the hematopoietic system. In this study, we addressed the contribution of several cancer-identified Vav1 mutants to tumorigenic processes.

Material and method: We introduced several amino-acid substitutions at residues identified in human lung cancer as follows: glutamic acid (position 59; calponin homology region) to lysine (E59K); aspartic acid (position 517; C1 domain) to glutamic acid (D517E); and leucine (position 801; carboxySH3) to proline (L801P). The biochemical and transforming activities of these mutants were tested following transfection into NIH3T3 cells.

Results and Discussion: Among the mutants produced, E59K generated a truncated protein, which preserved its expected size once cells are incubated with MG132, a specific proteasome inhibitor. E59K, D517E and oncVav1 are active as GEF towards Rho/RacGTPases, albeit E59K exhibited the uppermost activity. This result was illustrated by Pymol, a computer software for molecular visualization, that predicts its increased activity as a GEF. This activity is also manifested in changes in cytoskeleton organization indicative of transformation. Analysis of protein stability using cycloheximide decay assay revealed that D517E mutant protein is more stable than the other mutants, thus explaining its increased expression and activity. Furthermore, NIH3T3 cells expressing E59K, D517E and oncVav1 mutants exhibit increased cell proliferation, elevated number of transformed foci and increased number of generated tumors in NOD/Scid mice. Of note is the fact that tumors generated by E59K exhibit the most aggressive phenotype among the mutant proteins used in this study, reminiscent of epithelial morphology.

Conclusion: Our results convincingly attest to the transforming potential of the Vav1 mutants, E59K and D517E, thus providing compelling evidence that Vav1 mutants can act as “real” oncogenes in human cancer.