VAV2 orchestrates the interplay between regenerative proliferation and ribogenesis in both keratinocytes and oral squamous cell carcinoma.

VAV2 is an activator of RHO GTPases that promotes and maintains regenerative proliferation-like states in normal keratinocytes and oral squamous cell carcinoma (OSCC) cells. Here, we demonstrate that VAV2 also regulates ribosome biogenesis in those cells, a program associated with poor prognosis of human papilloma virus-negative (HPV-) OSCC patients. Mechanistically, VAV2 regulates this process in a catalysis-dependent manner using a conserved pathway comprising the RAC1 and RHOA GTPases, the PAK and ROCK family kinases, and the c-MYC and YAP/TAZ transcription factors. This pathway directly promotes RNA polymerase I activity and synthesis of 47S pre-rRNA precursors. This process is further consolidated by the upregulation of ribosome biogenesis factors and the acquisition of the YAP/TAZ-dependent undifferentiated cell state. Finally, we show that RNA polymerase I is a therapeutic Achilles' heel for both keratinocytes and OSCC patient-derived cells endowed with high VAV2 catalytic activity. Collectively, these findings highlight the therapeutic potential of modulating VAV2 and the ribosome biogenesis pathways in both preneoplastic and late progression stages of OSCC.

Characterization of mutant versions of the R-RAS2/TC21 GTPase found in tumors.

The R-RAS2 GTP hydrolase (GTPase) (also known as TC21) has been traditionally considered quite similar to classical RAS proteins at the regulatory and signaling levels. Recently, a long-tail hotspot mutation targeting the R-RAS2/TC21 Gln72 residue (Q72L) was identified as a potent oncogenic driver. Additional point mutations were also found in other tumors at low frequencies. Despite this, little information is available regarding the transforming role of these mutant versions and their relevance for the tumorigenic properties of already-transformed cancer cells. Here, we report that many of the RRAS2 mutations found in human cancers are highly transforming when expressed in immortalized cell lines. Moreover, the expression of endogenous R-RAS2Q72L is important for maintaining optimal levels of PI3K and ERK activities as well as for the adhesion, invasiveness, proliferation, and mitochondrial respiration of ovarian and breast cancer cell lines. Endogenous R-RAS2Q72L also regulates gene expression programs linked to both cell adhesion and inflammatory/immune-related responses. Endogenous R-RAS2Q72L is also quite relevant for the in vivo tumorigenic activity of these cells. This dependency is observed even though these cancer cell lines bear concurrent gain-of-function mutations in genes encoding RAS signaling elements. Finally, we show that endogenous R-RAS2, unlike the case of classical RAS proteins, specifically localizes in focal adhesions. Collectively, these results indicate that gain-of-function mutations of R-RAS2/TC21 play roles in tumor initiation and maintenance that are not fully redundant with those regulated by classical RAS oncoproteins.

Characterization of the spectrum of trivalent VAV1-mutation-driven tumours using a gene-edited mouse model.

Mutations in the VAV1 guanine nucleotide exchange factor 1 have been recently found in peripheral T cell lymphoma and nonsmall-cell lung cancer (NSCLC). To understand their pathogenic potential, we generated a gene-edited mouse model that expresses a VAV1 mutant protein that recapitulates the signalling alterations present in the VAV1 mutant subclass most frequently found in tumours. We could not detect any overt tumourigenic process in those mice. However, the concurrent elimination of the Trp53 tumour suppressor gene in them drives T cell lymphomagenesis. This process represents an exacerbation of the normal functions that wild-type VAV1 plays in follicular helper T cells. We also found that, in combination with the Kras oncogene, the VAV1 mutant version favours progression of NSCLC. These data indicate that VAV1 mutations play critical, although highly cell-type-specific, roles in tumourigenesis. They also indicate that such functions are contingent on the mutational landscape of the tumours involved.

The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function.

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.

A hotspot mutation targeting the R-RAS2 GTPase acts as a potent oncogenic driver in a wide spectrum of tumors.

A missense change in RRAS2 (Gln72 to Leu), analogous to the Gln61-to-Leu mutation of RAS oncoproteins, has been identified as a long-tail hotspot mutation in cancer and Noonan syndrome. However, the relevance of this mutation for in vivo tumorigenesis remains understudied. Here we show, using an inducible knockin mouse model, that R-Ras2Q72L triggers rapid development of a wide spectrum of tumors when somatically expressed in adult tissues. These tumors show limited overlap with those originated by classical Ras oncogenes. R-Ras2Q72L-driven tumors can be classified into different subtypes according to therapeutic susceptibility. Importantly, the most relevant R-Ras2Q72L-driven tumors are dependent on mTORC1 but independent of phosphatidylinositol 3-kinase-, MEK-, and Ral guanosine diphosphate (GDP) dissociation stimulator. This pharmacological vulnerability is due to the extensive rewiring by R-Ras2Q72L of pathways that orthogonally stimulate mTORC1 signaling. These findings demonstrate that RRAS2Q72L is a bona fide oncogenic driver and unveil therapeutic strategies for patients with cancer and Noonan syndrome bearing RRAS2 mutations.

Efficient fractionation and analysis of ribosome assembly intermediates in human cells.

Biochemical studies of the human ribosome synthesis pathway have been hindered by technical difficulties in obtaining intact preribosomal complexes from internal regions of the nucleolus. Here we provide a detailed description of an extraction method that enables efficient detection, isolation, and characterization of nucleolar preribosomes containing large pre-rRNA species. The three-step Preribosome Sequential Extraction (PSE) protocol preserves the integrity of early preribosomal complexes and yields preparations amenable to biochemical analyses from low amounts of starting material. We validate this procedure through the detection of specific trans-acting factors and pre-rRNAs in the extracted preribosomes using affinity matrix pull-downs and sedimentation assays. In addition, we describe the application of the PSE method for monitoring cellular levels of ribosome-free 5S RNP complexes as an indicator of ribosome biogenesis stress. Our optimized experimental procedures will facilitate studies of human ribosome biogenesis in normal, mutant and stressed-cell scenarios, including the characterization of candidate ribosome biogenesis factors, preribosome interactors under specific physiological conditions or effects of drugs on ribosome maturation.

New Functions of Vav Family Proteins in Cardiovascular Biology, Skeletal Muscle, and the Nervous System.

Vav proteins act as tyrosine phosphorylation-regulated guanosine nucleotide exchange factors for Rho GTPases and as molecular scaffolds. In mammals, this family of signaling proteins is composed of three members (Vav1, Vav2, Vav3) that work downstream of protein tyrosine kinases in a wide variety of cellular processes. Recent work with genetically modified mouse models has revealed that these proteins play key signaling roles in vascular smooth and skeletal muscle cells, specific neuronal subtypes, and glia cells. These functions, in turn, ensure the proper regulation of blood pressure levels, skeletal muscle mass, axonal wiring, and fiber myelination events as well as systemic metabolic balance. The study of these mice has also led to the discovery of new physiological interconnection among tissues that contribute to the ontogeny and progression of different pathologies such as, for example, hypertension, cardiovascular disease, and metabolic syndrome. Here, we provide an integrated view of all these new Vav family-dependent signaling and physiological functions.

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.

Identification of distinct maturation steps involved in human 40S ribosomal subunit biosynthesis.

Technical problems intrinsic to the purification of preribosome intermediates have limited our understanding of ribosome biosynthesis in humans. Addressing this issue is important given the implication of this biological process in human disease. Here we report a preribosome purification and tagging strategy that overcomes some of the existing technical difficulties. Using these tools, we find that the pre-40S precursors go through two distinct maturation phases inside the nucleolus and follow a regulatory step that precedes late maturation in the cytoplasm. This regulatory step entails the intertwined actions of both PARN (a metazoan-specific ribonuclease) and RRP12 (a phylogenetically conserved 40S biogenesis factor that has acquired additional functional features in higher eukaryotes). Together, these results demonstrate the usefulness of this purification method for the dissection of ribosome biogenesis in human cells. They also identify distinct maturation stages and metazoan-specific regulatory mechanisms involved in the generation of the human 40S ribosomal subunit.

Pol5 is an essential ribosome biogenesis factor required for 60S ribosomal subunit maturation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, more than 250 trans-acting factors are involved in the maturation of 40S and 60S ribosomal subunits. The expression of most of these factors is transcriptionally coregulated to ensure correct ribosome production under a wide variety of environmental and intracellular conditions. Here, we identified the essential nucleolar Pol5 protein as a novel trans-acting factor required for the synthesis of 60S ribosomal subunits. Pol5 weakly and/or transiently associates with early to medium pre-60S ribosomal particles. Depletion of and temperature-sensitive mutations in Pol5 result in a deficiency of 60S ribosomal subunits and accumulation of half-mer polysomes. Both processing of 27SB pre-rRNA to mature 25S rRNA and release of pre-60S ribosomal particles from the nucle(ol)us to the cytoplasm are impaired in the Pol5-depleted strain. Moreover, we identified the genes encoding ribosomal proteins uL23 and eL27A as multicopy suppressors of the slow growth of a temperature-sensitive pol5 mutant. These results suggest that Pol5 could function in ensuring the correct folding of 25S rRNA domain III; thus, favoring the correct assembly of these two ribosomal proteins at their respective binding sites into medium pre-60S ribosomal particles. Pol5 is homologous to the human tumor suppressor Myb-binding protein 1A (MYBBP1A). However, expression of MYBBP1A failed to complement the lethal phenotype of a pol5 null mutant strain though interfered with 60S ribosomal subunit biogenesis.

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.

Ribosome biogenesis and cancer: basic and translational challenges.

Increasing evidence suggests that alterations in ribosome biogenesis (RiBi) confer competitive advantages to cancer cells. This has led to the discovery of regulatory layers mediated by signaling proteins, oncoproteins, and tumor suppressors whose deregulation leads to increased RiBi rates in cancer cells. In addition to boosting protein synthesis, these alterations probably contribute to shape the protumorigenic proteome of cancer cells. Mutations negatively affecting RiBi are also unexpectedly found in some spontaneous and ribosomopathy-associated tumors. The advantages provided by these genetic lesions to cancer cells remain unsettled as yet. Efforts are being made nowadays to exploit RiBi-associated vulnerabilities and tumor suppressor pathways to design new therapeutic avenues. In this review, we will summarize the main developments and pending challenges in this research area.

Focal accumulation of preribosomes outside the nucleolus during metaphase-anaphase in budding yeast.

Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud.

Rrp12 and the Exportin Crm1 participate in late assembly events in the nucleolus during 40S ribosomal subunit biogenesis.

During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes.

Contribution of the R-Ras2 GTP-binding protein to primary breast tumorigenesis and late-stage metastatic disease.

R-Ras2 is a transforming GTPase that shares downstream effectors with Ras subfamily proteins. However, little information exists about the function of this protein in tumorigenesis and its signalling overlap with classical Ras GTPases. Here we show, by combining loss- and gain-of-function studies in breast cancer cells, mammary epithelial cells and mouse models, that endogenous R-Ras2 has a role in both primary breast tumorigenesis and the late metastatic steps of cancer cells in the lung parenchyma. R-Ras2 drives tumorigenesis in a phosphatidylinostiol-3 kinase (PI3K)-dependent and signalling autonomous manner. By contrast, its prometastatic role requires other priming oncogenic signals and the engagement of several downstream elements. R-Ras2 function is required even in cancer cells exhibiting constitutive activation of classical Ras proteins, indicating that these GTPases are not functionally redundant. Our results also suggest that application of long-term R-Ras2 therapies will result in the development of compensatory mechanisms in breast tumours.

Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes.

The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled pre-ribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA.

Ribosome synthesis-unrelated functions of the preribosomal factor Rrp12 in cell cycle progression and the DNA damage response.

Given the high metabolic cost required to generate ribosomes, it has been assumed that proteins involved in ribosome synthesis might establish functional cross talk with other intracellular processes to efficiently couple ribosome production and cell growth. However, such interconnections have remained elusive due to the difficulty in separating the intra- and extraribosomal roles of ribosome biogenesis factors. Using a yeast functional screen, I have discovered that Rrp12, a conserved protein involved in ribosome maturation and export, plays roles in the cell cycle and the DNA damage response. These results indicate that Rrp12 participates in a karyopherin Kap121-dependent import route that is crucial for nuclear sequestration of ribonucleotide reductase subunits and, thereby, ensures the proper kinetics of deoxyribonucleotide production during the cell cycle. Within this route, Rrp12 acts as a cofactor important for the full functionality of Kap121. This activity is mechanistically different from the known roles of Rrp12 in ribosome biogenesis. I propose that the functional duality of Rrp12 may couple the control of ribosome production to the regulation of other cellular processes during cell cycle progression.

Strategies for isolating constitutively active and dominant-negative pheromone receptor mutants in yeast.

Mating pheromone receptors of the yeast Saccharomyces cerevisiae are useful models for the study of G protein-coupled receptors. The mating pheromone receptors, Ste2 and Ste3, are not essential for viability so they can be readily targeted for analysis by a variety of genetic approaches. This chapter will describe methods for identification of two kinds of mutants that have been very informative about the mechanisms of receptor signaling: constitutively active mutants and dominant-negative mutants. Interestingly, these distinct types of mutants have revealed complementary information. Constitutive signaling is caused by mutations that are thought to weaken interactions between the seven transmembrane domains (TMDs), whereas the dominant-negative mutants apparently stabilize contacts between TMDs and lock receptors in the off conformation. In support of these conclusions, certain combinations of constitutively active and dominant-negative mutants restore nearly normal signaling properties.

The 90S preribosome is a multimodular structure that is assembled through a hierarchical mechanism.

The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture.

Control of lymphocyte shape and the chemotactic response by the GTP exchange factor Vav.

Rho GTPases control many facets of cell polarity and migration; namely, the reorganization of the cellular cytoskeleton to extracellular stimuli. Rho GTPases are activated by GTP exchange factors (GEFs), which induce guanosine diphosphate (GDP) release and the stabilization of the nucleotide-free state. Thus, the role of GEFs in the regulation of the cellular response to extracellular cues during cell migration is a critical step of this process. In this report, we have analyzed the activation and subcellular localization of the hematopoietic GEF Vav in human peripheral blood lymphocytes stimulated with the chemokine stromal cell-derived factor-1 (SDF-1alpha). We show a robust activation of Vav and its redistribution to motility-associated subcellular structures, and we provide biochemical evidence of the recruitment of Vav to the membrane of SDF-1alpha-activated human lymphocytes, where it transiently interacts with the SDF-1alpha receptor CXCR4. Overexpression of a dominant negative form of Vav abolished lymphocyte polarization, actin polymerization, and migration. SDF-1alpha-mediated cell polarization and migration also were impaired by overexpression of an active, oncogenic Vav, although the mechanism appears to be different. Together, our data postulate a pivotal role for Vav in the transmission of the migratory signal through the chemokine receptor CXCR4.