Vav family

VRK2 is a Ser-Thr kinase and has two isoforms, cytosolic and nuclear. The full-length VRK2 cytosolic isoform (A) inhibits MAPK signaling by a direct interaction with the corresponding JIP1 or KSR1 scaffold proteins. As a consequence, high levels of VRK2A inhibit in a dose-dependent manner signals mediated by the TAK1-MKK7-JNK in response to hypoxia or interleukin, and the signal of the ERBB2-RAS-RAF-MEK-ERK pathway. Downregulation of VRK2A relieves this inhibition and allows signal transmission in response to stimuli initiated in receptor-tyrosine kinases (RTK), such as ERBB2. Thus, in breast cancer VRK2A and ERBB2 present an inverse correlation.Peer Reviewe

Vav proteins maintain epithelial traits in breast cancer cells using miR-200c-dependent and independent mechanisms

The bidirectional regulation of epithelial–mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients.Fil: Lorenzo Martín, L. Francisco. Universidad de Salamanca; España. Consejo Superior de Investigaciones Científicas; España. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; EspañaFil: Citterio, Carmen. Consejo Superior de Investigaciones Científicas; España. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; EspañaFil: Menacho Márquez, Mauricio Ariel. Universidad de Salamanca; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentina. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; EspañaFil: Conde, Javier. Consejo Superior de Investigaciones Científicas; España. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; EspañaFil: Larive, Romain M.. Consejo Superior de Investigaciones Científicas; España. Institut Des Biomolécules Max Mousseron; FranciaFil: Rodríguez Fdez, Sonia. Consejo Superior de Investigaciones Científicas; EspañaFil: García Escudero, Ramón. Consejo Superior de Investigaciones Científicas; España. Universidad de Salamanca; EspañaFil: Robles Valero, Javier. Universidad de Salamanca; España. Consejo Superior de Investigaciones Científicas; España. Centro de Investigación del Cáncer; España. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; EspañaFil: Cuadrado, Myriam. Universidad de Salamanca; España. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Fernández Pisonero, Isabel. Universidad de Salamanca; España. Consejo Superior de Investigaciones Científicas; España. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; EspañaFil: Dosil, Mercedes. Universidad de Salamanca; España. Consejo Superior de Investigaciones Científicas; España. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; EspañaFil: Sevilla, María A.. Universidad de Salamanca; EspañaFil: Montero, María J.. Universidad de Salamanca; EspañaFil: Fernández Salguero, Pedro. Universidad de Extremadura; EspañaFil: Paramio, Jesús M.. Universidad de Salamanca; España. Consejo Superior de Investigaciones Científicas; España. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; EspañaFil: Bustelo, Xosé R.. Universidad de Salamanca; España. Consejo Superior de Investigaciones Científicas; Españ

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug’s molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach “Mutagenesis to Uncover Targets by deep Sequencing, or “MUTseq”, and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.(

Vav2 pharmaco-mimetic mice reveal the therapeutic value and caveats of the catalytic inactivation of a Rho exchange factor

The current paradigm holds that the inhibition of Rho guanosine nucleotide exchange factors (GEFs), the enzymes that stimulate Rho GTPases, can be a valuable therapeutic strategy to treat Rho-dependent tumors. However, formal validation of this idea using in vivo models is still missing. In this context, it is worth remembering that many Rho GEFs can mediate both catalysis-dependent and independent responses, thus raising the possibility that the inhibition of their catalytic activities might not be sufficient per se to block tumorigenic processes. On the other hand, the inhibition of these enzymes can trigger collateral side effects that could preclude the practical implementation of anti-GEF therapies. To address those issues, we have generated mouse models to mimic the effect of the systemic application of an inhibitor for the catalytic activity of the Rho GEF Vav2 at the organismal level. Our results indicate that lowering the catalytic activity of Vav2 below specific thresholds is sufficient to block skin tumor initiation, promotion, and progression. They also reveal that the negative side effects typically induced by the loss of Vav2 can be bypassed depending on the overall level of Vav2 inhibition achieved in vivo. These data underscore the pros and cons of anti-Rho GEF therapies for cancer treatment. They also support the idea that Vav2 could represent a viable drug target.XRB is supported by grants from Worldwide Cancer Research (14-1248), the Castilla-León Government (CSI252P18, CLC-2017-01), the Spanish Ministry of Science and Innovation (MSI) (RTI2018-096481-B-I00), and the Spanish Association against Cancer (GC16173472GARC). XRB’s institution is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Castilla-León autonomous government (CLC-2017-01). SF, SR-F, and LFL-M contracts have been supported by funding from the MSI (SF, BES-2010-031386; SR-F, BES-2013-063573), the Spanish Ministry of Universities (LFL-M, FPU13/02923), and the CLC-2017-01 grant (SR-F and LFL-M). JR-V has been supported by the CIBERONC and, currently, by the Spanish Association against Cancer. Both Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fund.Peer reviewe