Role of the oncogene Vav2 in lung tumorigenesis

Abstract

Vav proteins are a family of guanosine nucleotide exchange factors (GEFs) for the Rho family of GTPases. They regulate the exchange of GDP for GTP and the subsequent activation of Rho GTPases. These proteins control in turn a variety of cellular processes which include proliferation, migration or gene transcription, among others. Because of the relevance of these biological processes, Rho signaling is usually altered in human tumors. One of this disruptions is the alteration of Rho regulators, such as GEFs. Little is known about the role of the member of the Vav family Vav2 in tumorigenesis. It has been associated to invasiveness in metastatic melanoma, to initiation and promotion of skin tumors and to head and neck squamous cell carcinoma, as well as to the control of a lung metastasis-specific transcriptional program in breast cancer cells, together with Vav3. In order to expand the number of tumors in which Vav2 is implicated and shows an active role, the aim of this work is the analysis of the function of Vav2 in lung tumors, one of the main causes of cancer death in developed countries. A mouse model of lung urethane-induced carcinogenesis was used for this purpose. Vav2-/- mice showed a decrease in the incidence and multiplexity of lung tumors when compared with control mice, indicating an oncogenic role of Vav2 in the initiation stages of lung tumor development. This protection against tumor development was in part explained by the increase in cell apoptosis upon damage induction in Vav2-/- mice. To further analyze and characterize this phenotype, in vitro studies were carried out with a murine cell line derived from a KRas-mutant lung adenocarcinoma and the use of shRNAs to deplete Vav2. These studies showed that Vav2 deficient cells had defects in cell proliferation and cell survival upon DNA damage induction, similarly to the in vivo observations. Cell survival deficiencies were not due to an ineffective activation of any of the three canonical GTPases RhoA, Rac1 and Cdc42 by Vav2, as its overexpression didn’t rescue the phenotype. In fact, RhoA and Cdc42 had a pro-apoptotic effect that exacerbated the Vav2-deficienct phenotype. Neither Vav3, another member of the Vav family which is also ubiquitously expressed, could reduce the apoptosis levels. All the contrary, it acted in vitro as a pro-apoptotic protein capable of counteracting the resistance conferred by Vav2 in control cells. All these results bring light into the complex and tissue-specific role of Vav2 in lung tumorigenesis, but also opens more questions to be addressed in a future. It could not be determined if the observed effects were catalytically-dependent, or if they lie in the adaptor function of Vav2. If it were the first case, three GTPases were discarded as targets of Vav2 in lung tumorigenesis, but the actual ‘effector’ GTPase remains elusive. Finally, a different Vav2-knockdown/knockout cell model based on the CRISPR-Cas9 technology was tried. First steps of experiment design and guide RNA and Cas9 delivery were carried out, but the validation of the generated cell clones still remains a challenge due to the difficulties of analyzing Vav2 endogenous protein levels.