Browsing by Author "Crespo, Piero"
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Item Cbl-b, a member of the Sli-1/c-Cbl protein family, inhibits Vav-mediated c-Jun N-terminal kinase activation.(Oncogene, 1997) Bustelo, Xose R; Crespo, Piero; López Barahona, Mónica; Gutkind, J Silvio; Barbacid, MarianoWe have used the yeast two-hybrid system to identify proteins that interact with Vav, a GDP/GTP exchange factor for the Rac-1 GTPase that plays an important role in cell signaling and oncogenic transformation. This experimental approach resulted in the isolation of Cbl-b, a signal transduction molecule highly related to the mammalian c-cbl proto-oncogene product and to the C. elegans Sli-1 protein, a negative regulator of the EGF-receptor-like Let23 protein. The interaction between Vav and Cbl-b requires the entire SH3-SH2-SH3 carboxy-terminal domain of Vav and a long stretch of proline-rich sequences present in the central region of Cbl-b. Stimulation of quiescent rodent fibroblasts with either epidermal or platelet-derived growth factors induces an increased affinity of Vav for Cbl-b and results in the subsequent formation of a Vav-dependent trimeric complex with the ligand-stimulated tyrosine kinase receptors. During this process, Vav, but not Cbl-b, becomes highly phosphorylated on tyrosine residues. Overexpression of Cbl-b inhibits the signal transduction pathway of Vav that leads to the stimulation of c-Jun N-terminal kinase. By contrast, expression of truncated Cbl-b proteins and of missense mutants analogous to those found in inactive Sli-1 proteins have no detectable effect on Vav activity. These results indicate that Vav and Cbl-b act coordinately in the first steps of tyrosine protein kinase receptor-mediated signaling and suggest that members of the Sli-1/Cbl family are also negative regulators of signal transduction in mammalian cells.Item Inhibiting ERK dimerization ameliorates BRAF‑driven anaplastic thyroid cancer.(Cellular and Molecular Life Sciences, 2022) Zaballos, Miguel A.; Acuña‑Ruiz, Adrián; Morante, Marta; Riesco Eizaguirre, Garcilaso; Crespo, Piero; Santisteban, PilarBackground RAS-to-ERK signaling is crucial for the onset and progression of advanced thyroid carcinoma, and blocking ERK dimerization provides a therapeutic beneft in several human carcinomas. Here we analyzed the efects of DEL-22379, a relatively specifc ERK dimerization inhibitor, on the activation of the RAS-to-ERK signaling cascade and on tumor-related processes in vitro and in vivo. Methods We used a panel of four human anaplastic thyroid carcinoma (ATC) cell lines harboring BRAF or RAS mutations to analyze ERK dynamics and tumor-specifc characteristics. We also assessed the impact of DEL-22379 on the transcriptional landscape of ATC cell lines using RNA-sequencing and evaluated its therapeutic efcacy in an orthotopic mouse model of ATC. Results DEL-22379 impaired upstream ERK activation in BRAF- but not RAS-mutant cells. Cell viability and metastasisrelated processes were attenuated by DEL-22379 treatment, but mostly in BRAF-mutant cells, whereas in vivo tumor growth and dissemination were strongly reduced for BRAF-mutant cells and mildly reduced for RAS-mutant cells. Transcriptomics analyses indicated that DEL-22379 modulated the transcriptional landscape of BRAF- and RAS-mutant cells in opposite directions. Conclusions Our fndings establish that BRAF- and RAS-mutant thyroid cells respond diferentially to DEL-22379, which cannot be explained by the previously described mechanism of action of the inhibitor. Nonetheless, DEL-22379 demonstrated signifcant anti-tumor efects against BRAF-mutant cells in vivo with an apparent lack of toxicity, making it an interesting candidate for the development of combinatorial treatments. Our data underscore the diferences elicited by the specifc driver mutation for thyroid cancer onset and progression, which should be considered for experimental and clinical approaches.Item Rac-1 dependent stimulation of the JNK/SAPK signaling pathway by Vav.(Oncogene, 1996) Crespo, Piero; Bustelo, Xose R; Aaronson, David S; Coso, Ornar A; López Barahona, Mónica; Barbacid, Mariano; Gutkind, J SilvioThe protein product of the human vav oncogene, Vav exhibits a number of structural motifs suggestive of a role in signal transduction pathways, including a leucine-rich region, a plekstrin homology (PH) domain, a cysteine-rich domain, two SH3 regions, an SH2 domain, and a central Dbl homology (DH) domain. However, the transforming pathway(s) activated by Vav has not yet been elucidated. Interestingly, DH domains are frequently found in guanine nucleotide-exchange factors for small GTP-binding proteins of the Ras and Rho families, and it has been recently shown that, whereas Ras controls the activation of mitogen activated kinases (MAPKs), two members of the Rho family of small GTPases, Rac 1 and Cdc42, regulate activity of stress activated protein kinases (SAPKs), also termed c-jun N-terminal kinases (JNKs). The structural similarity between Vav and other guanine nucleotide exchange factors for small GTP-binding proteins, together with the recent identification of biochemical routes specific for members of the Ras and Rho family of GTPases, prompted us to explore whether MAPK or JNK are downstream components of the Vav signaling pathways. Using the COS-7 cell transient expression system, we have found that neither Vav nor the product of the vav proto-oncogene, proto-Vav, can enhance the enzymatic activity of a coexpressed, epitope tagged MAPK. On the other hand, we have observed that, whereas proto-Vav can slightly elevate JNK/SAPK activity, oncogenic Vav potently activates JNK/SAPK to an extent comparable to that elicited by two guanine-nucleotide exchange factors for Rho family members, Dbl and Ost. We also show that point mutations in conserved residues within the cysteine rich and DH domains of Vav both prevent its ability to activate JNK/SAPK and render Vav oncogenically inactive. In addition, we found that coexpression of the Rac-1 N17 dominant inhibitory mutant dramatically diminishes JNK/SAPK stimulation by Vav, as well as reduces the focus-forming ability of Vav in NIH3T3 murine fibroblasts. Taken together, these findings provide the first evidence that Rac-1 and JNK are integral components of the Vav signaling pathway.Item RAS Subcellular Localization Inversely Regulates Thyroid Tumor Growth and Dissemination.(Cancers, 2020) García Ibáñez, Yaiza; Riesco Eizaguirre, Garcilaso; Santisteban, Pilar; Casar, Berta; Crespo, PieroRAS mutations are the second most common genetic alteration in thyroid tumors. However, the extent to which they are associated with the most aggressive phenotypes is still controversial. Regarding their malignancy, the majority of RAS mutant tumors are classified as undetermined, which complicates their clinical management and can lead to undesired under- or overtreatment. Using the chick embryo spontaneous metastasis model, we herein demonstrate that the aggressiveness of HRAS-transformed thyroid cells, as determined by the ability to extravasate and metastasize at distant organs, is orchestrated by HRAS subcellular localization. Remarkably, aggressiveness inversely correlates with tumor size. In this respect, we also show that RAS site-specific capacity to regulate tumor growth and dissemination is dependent on VEGF-B secretion. Furthermore, we have identified the acyl protein thioesterase APT-1 as a determinant of thyroid tumor growth versus dissemination. We show that alterations in APT-1 expression levels can dramatically a ect the behavior of thyroid tumors, based on its role as a regulator of HRAS sublocalization at distinct plasma membrane microdomains. In agreement, APT-1 emerges in thyroid cancer clinical samples as a prognostic factor. As such, APT-1 levels could serve as a biomarker that could help in the stratification of HRAS mutant thyroid tumors based on their aggressiveness.