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Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular βI domain, but not the hybrid or I-EGF2 domain of active β1 integrins, promotes their FAK-regulated clustering into tensin 1-containing fibrillar adhesions and impairs their endocytosis. In this regard, the βI domain-dependent clustering of active β1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by β1 integrin conformational activation alone., (© 2022 Mana et al.)

The dynamic integrin-mediated adhesion of endothelial cells (ECs) to the surrounding ECM is fundamental for angiogenesis both in physiological and pathological conditions, such as embryonic development and cancer progression. The dynamics of EC-to-ECM adhesions relies on the regulation of the conformational activation and trafficking of integrins. Here, we reveal that oncogenic transcription factor EB (TFEB), a known regulator of lysosomal biogenesis and metabolism, also controls a transcriptional program that influences the turnover of ECM adhesions in ECs by regulating cholesterol metabolism. We show that TFEB favors ECM adhesion turnover by promoting the transcription of genes that drive the synthesis of cholesterol, which promotes the aggregation of caveolin-1, and the caveolin-dependent endocytosis of integrin β1. These findings suggest that TFEB might represent a novel target for the pharmacological control of pathological angiogenesis and bring new insights in the mechanism sustaining TFEB control of endocytosis., (© 2022. The Author(s).)

The NMDARs are key players in both physiological and pathologic synaptic plasticity because of their involvement in many aspects of neuronal transmission as well as learning and memory. The contribution in these events of different types of GluN2A-interacting proteins is still unclear. The p140Cap scaffold protein acts as a hub for postsynaptic complexes relevant to psychiatric and neurologic disorders and regulates synaptic functions, such as the stabilization of mature dendritic spine, memory consolidation, LTP, and LTD. Here we demonstrate that p140Cap directly binds the GluN2A subunit of NMDAR and modulates GluN2A-associated molecular network. Indeed, in p140Cap KO male mice, GluN2A is less associated with PSD95 both in ex vivo synaptosomes and in cultured hippocampal neurons, and p140Cap expression in KO neurons can rescue GluN2A and PSD95 colocalization. p140Cap is crucial in the recruitment of GluN2A-containing NMDARs and, consequently, in regulating NMDARs' intrinsic properties. p140Cap is associated to synaptic lipid-raft (LR) and to soluble postsynaptic membranes, and GluN2A and PSD95 are less recruited into synaptic LR of p140Cap KO male mice. Gated-stimulated emission depletion microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in LR in an activity-dependent fashion. In the synaptic compartment, p140Cap influences the association between GluN2A and PSD95 and modulates GluN2A enrichment into LR. Overall, such increase in these membrane domains rich in signaling molecules results in improved signal transduction efficiency. SIGNIFICANCE STATEMENT Here we originally show that the adaptor protein p140Cap directly binds the GluN2A subunit of NMDAR and modulates the GluN2A-associated molecular network. Moreover, we show, for the first time, that p140Cap also associates to synaptic lipid rafts and controls the selective recruitment of GluN2A and PSD95 to this specific compartment. Finally, gated-stimulated emission depletion microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in lipid rafts in an activity-dependent fashion. Overall, our findings provide the molecular and functional dissection of p140Cap as a new active member of a highly dynamic synaptic network involved in memory consolidation, LTP, and LTD, which are known to be altered in neurologic and psychiatric disorders., (Copyright © 2022 the authors.)

Epithelial-mesenchymal transition (EMT) is a complex and pivotal process involved in organogenesis and is related to several pathological processes, including cancer and fibrosis. During heart development, EMT mediates the conversion of epicardial cells into vascular smooth muscle cells and cardiac interstitial fibroblasts. Here, we show that the oncogenic transcription factor EB (TFEB) is a key regulator of EMT in epicardial cells and that its genetic overexpression in mouse epicardium is lethal due to heart defects linked to impaired EMT. TFEB specifically orchestrates the EMT-promoting function of transforming growth factor (TGF) β, and this effect results from activated transcription of thymine-guanine-interacting factor (TGIF)1, a TGFβ/Smad pathway repressor. The Tgif1 promoter is activated by TFEB, and in vitro and in vivo findings demonstrate its increased expression when Tfeb is overexpressed. Furthermore, Tfeb overexpression in vitro prevents TGFβ-induced EMT, and this effect is abolished by Tgif1 silencing. Tfeb loss of function, similar to that of Tgif1, sensitizes cells to TGFβ, inducing an EMT response to low doses of TGFβ. Together, our findings reveal an unexpected function of TFEB in regulating EMT, which might provide insights into injured heart repair and control of cancer progression., (© 2022. The Author(s).)

In metazoans, cell adhesion to the extracellular matrix (ECM) drives the development, functioning, and repair of different tissues, organs, and systems. Disruption or dysregulation of cell-to-ECM adhesion promote the initiation and progression of several diseases, such as bleeding, immune disorders and cancer. Integrins are major ECM transmembrane receptors, whose function depends on both allosteric changes and exo-endocytic traffic, which carries them to and from the plasma membrane. In apico-basally polarized cells, asymmetric adhesion to the ECM is maintained by continuous targeting of the plasma membrane by vesicles coming from the trans Golgi network and carrying ECM proteins. Active integrin-bound ECM is indeed endocytosed and replaced by the exocytosis of fresh ECM. Such vesicular traffic is finely driven by the teamwork of microtubules (MTs) and their associated kinesin and dynein motors. Here, we review the main cytoskeletal actors involved in the control of the spatiotemporal distribution of active integrins and their ECM ligands, highlighting the key role of the synchronous (ant)agonistic cooperation between MT motors transporting vesicular cargoes, in the same or in opposite direction, in the regulation of traffic logistics, and the establishment of epithelial and endothelial cell polarity., Competing Interests: Declaration of Competing Interest All authors declare that they have no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The formation of a functional blood vessel network relies on the ability of endothelial cells (ECs) to dynamically rearrange their adhesive contacts in response to blood flow and guidance cues, such as vascular endothelial growth factor-A (VEGF-A) and class 3 semaphorins (SEMA3s). Neuropilin 1 (NRP1) is essential for blood vessel development, independently of its ligands VEGF-A and SEMA3, through poorly understood mechanisms. Grounding on unbiased proteomic analysis, we report here that NRP1 acts as an endocytic chaperone primarily for adhesion receptors on the surface of unstimulated ECs. NRP1 localizes at adherens junctions (AJs) where, interacting with VE-cadherin, promotes its basal internalization-dependent turnover and favors vascular permeability initiated by histamine in both cultured ECs and mice. We identify a splice variant of tryptophanyl-tRNA synthetase (mini-WARS) as an unconventionally secreted extracellular inhibitory ligand of NRP1 that, by stabilizing it at the AJs, slows down both VE-cadherin turnover and histamine-elicited endothelial leakage. Thus, our work shows a role for NRP1 as a major regulator of AJs plasticity and reveals how mini-WARS acts as a physiological NRP1 inhibitory ligand in the control of VE-cadherin endocytic turnover and vascular permeability., (© 2022. The Author(s).)

Dynamic modulation of endothelial cell-to-cell and cell-to-extracellular matrix (ECM) adhesion is essential for blood vessel patterning and functioning. Yet the molecular mechanisms involved in this process have not been completely deciphered. We identify the adhesion G protein-coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of endothelial cell (EC) adhesion and barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM contacts, signals through cAMP/Rap1, and inhibits focal adhesion (FA) formation and nuclear localization of YAP/TAZ transcriptional regulators, while promoting tight junction (TJ) assembly. ECs also express an endogenous LPHN2 ligand, fibronectin leucine-rich transmembrane 2 (FLRT2), that prevents ECM-elicited EC behaviors in an LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive YAP/TAZ pathway, and lack proper intercellular TJs. Consistently, blood vessels are hyperpermeable, and intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination., (© 2021 Camillo et al.)

An interactive crosstalk between tumor and stroma cells is essential for metastatic melanoma progression. We evidenced that ESDN/DCBLD2/CLCP1 plays a crucial role in endothelial cells during the spread of melanoma. Precisely, increased extravasation and metastasis formation were revealed in ESDN-null mice injected with melanoma cells, even if the primary tumor growth, vessel permeability, and angiogenesis were not enhanced. Interestingly, improved adhesion of melanoma cells to ESDN-depleted endothelial cells was observed, due to the presence of higher levels of E-selectin transcripts/proteins in ESDN-defective cells. In accordance with these results, anticorrelation was observed between ESDN and E-selectin in human endothelial cells. Most importantly, our data revealed that cimetidine, an E-selectin inhibitor, was able to block cell adhesion, extravasation, and metastasis formation in ESDN-null mice, underlying a major role of ESDN in E-selectin transcription upregulation, which according to our data, may presumably be linked to STAT3. Based on our results, we propose a protective role for ESDN during the spread of melanoma and reveal its therapeutic potential., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Integrin-mediated adhesion of cells to the extracellular matrix (ECM) is crucial for the physiological development and functioning of tissues but is pathologically disrupted in cancer. Indeed, abnormal regulation of integrin receptors and ECM ligands allows cancer cells to break down tissue borders, breach into blood and lymphatic vessels, and survive traveling in suspension through body fluids or residing in metabolically or pharmacologically hostile environments. Different molecular and cellular mechanisms responsible for the modulation of integrin adhesive function or mechanochemical signaling are altered and participate in cancer. Cancer development and progression are also bolstered by dysfunctionalities of integrin-mediated ECM adhesion occurring both in tumor cells and in elements of the surrounding tumor microenvironment, such as vascular cells, cancer-associated fibroblasts, and immune cells. Mounting evidence suggests that integrin inhibitors may be effectively exploited to overcome resistance to standard-of-care anti-cancer therapies., Competing Interests: The authors declare that they have no competing interests.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2021 Serini G et al.)

We introduce a simple physical picture to explain the process of molecular sorting, whereby specific proteins are concentrated and distilled into submicrometric lipid vesicles in eukaryotic cells. To this purpose, we formulate a model based on the coupling of spontaneous molecular aggregation with vesicle nucleation. Its implications are studied by means of a phenomenological theory describing the diffusion of molecules toward multiple sorting centers that grow due to molecule absorption and are extracted when they reach a sufficiently large size. The predictions of the theory are compared with numerical simulations of a lattice-gas realization of the model and with experimental observations. The efficiency of the distillation process is found to be optimal for intermediate aggregation rates, where the density of sorted molecules is minimal and the process obeys simple scaling laws. Quantitative measures of endocytic sorting performed in primary endothelial cells are compatible with the hypothesis that these optimal conditions are realized in living cells.

In endothelial cells (ECs), the onset of apicobasal polarity is primarily regulated by the interaction of integrins with the surrounding extracellular matrix (ECM). ECs secrete and polymerize fibronectin (FN), a unique, permissive substrate that allows for vascular morphogenesis and lumen formation. We previously identified a signaling pathway that, under the control of the adhesion site adaptor protein PPFIA1, integrates the polarized secretion of freshly synthesized FN with the recycling of conformationally active α5β1 integrin, the main FN receptor in ECs. To characterize the functional role of PPFIA1-dependent signaling in ECs, we set up a Transwell-based assay to quantify the polarized secretion of ECM proteins. To this aim, we allowed ECs to form a confluent monolayer on the Transwell membrane and checked its integrity by measuring transendothelial electric resistance and controlling the stability of tight junctions over time by fluorescent confocal microscope analysis. Finally, we quantified apical and basolateral FN secretion in control and PPFIA1-silenced EC culture medium by western blot analysis coupled to spike-in normalization.

In endothelial cells, trafficking of active α5β1 integrins and polarized fibronectin secretion are important for vascular morphogenesis. A new study unveils how the endothelial small GTPase RHOJ, by repressing trafficking of active α5β1 integrins, controls fibronectin polymerization and in vivo angiogenesis., Competing Interests: Declaration of Interests G.S. is co-inventor of a patent application on “Non-natural semaphorins 3 and their medical use” (international application no. PCT/EP2016/053750), co-cofounder and shareholder of SeaGull Therapeutics SAS that is developing mutant semaphorins for clinical applications. D.V. declares that she has no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Various species of non-coding RNAs (ncRNAs) are enriched in specific subcellular compartments, but the mechanisms orchestrating their localization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked to distal axons by hitchhiking primarily on late endosomes/lysosomes. Upon exposure to the axon guidance cue semaphorin 3A (Sema3A), pre-miRNAs are processed specifically within axons into newly generated miRNAs, one of which, in turn, silences the basal translation of tubulin beta 3 class III (TUBB3), but not amyloid beta precursor protein (APP). At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly generated miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodeling of subcellular compartments., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Spatiotemporal control of integrin-mediated cell adhesion to the extracellular matrix (ECM) is critical for physiological and pathological events in multicellular organisms, such as embryonic development, angiogenesis, platelet aggregation, leukocytes extravasation, and cancer cell metastatic dissemination. Regulation of integrin adhesive function and signaling relies on the modulation of both conformation and traffic. Indeed, integrins exist in a dynamic equilibrium between a bent/closed (inactive) and an extended/open (active) conformation, respectively endowed with low and high affinity for ECM ligands. Increasing evidence proves that, differently to what hypothesized in the past, detachment from the ECM and conformational inactivation are not mandatory for integrin to get endocytosed and trafficked. Specific transmembrane and cytosolic proteins involved in the control of ECM proteolytic fragment-bound active integrin internalization and recycling exist. In the complex masterplan that governs cell behavior, active integrin traffic is key to the turnover of ECM polymers and adhesion sites, the polarized secretion of endogenous ECM proteins and modifying enzymes, the propagation of motility and survival endosomal signals, and the control of cell metabolism., (© 2020 The Author(s).)

Cadherins are a major family of cell-cell adhesive receptors, which are implicated in development, tissue homeostasis, and cancer. Here, we show a novel mechanism of post-translational regulation of E-cadherin in cancer cells by an intramembrane protease of the Rhomboid family, RHBDL2, which leads to the shedding of E-cadherin extracellular domain. In addition, our data indicate that RHBDL2 mediates a similar activity on VE-cadherin, which is selectively expressed by endothelial cells. We show that RHBDL2 promotes cell migration, which is consistent with its ability to interfere with the functional role of cadherins as negative regulators of motility; moreover, the two players appear to lie in the same functional pathway. Importantly, we show that RHBDL2 expression is induced by the inflammatory chemokine TNFα. The E-cadherin extracellular domain is known to be released by metalloproteases (MMPs); however, here, we provide evidence of a novel MMP-independent, TNFα inducible, E-cadherin processing mechanism that is mediated by RHBDL2. Thus, the intramembrane protease RHBDL2 is a novel regulator of cadherins promoting cell motility.

Vascular normalizing strategies, aimed at ameliorating blood vessel perfusion and lessening tissue hypoxia, are treatments that may improve the outcome of cancer patients. Secreted class 3 semaphorins (SEMA3), which are thought to directly bind neuropilin (NRP) co-receptors that, in turn, associate with and elicit plexin (PLXN) receptor signaling, are effective normalizing agents of the cancer vasculature. Yet, SEMA3A was also reported to trigger adverse side effects via NRP1. We rationally designed and generated a safe, parenterally deliverable, and NRP1-independent SEMA3A point mutant isoform that, unlike its wild-type counterpart, binds PLXNA4 with nanomolar affinity and has much greater biochemical and biological activities in cultured endothelial cells. In vivo, when parenterally administered in mouse models of pancreatic cancer, the NRP1-independent SEMA3A point mutant successfully normalized the vasculature, inhibited tumor growth, curbed metastatic dissemination, and effectively improved the supply and anticancer activity of chemotherapy. Mutant SEMA3A also inhibited retinal neovascularization in a mouse model of age-related macular degeneration. In summary, mutant SEMA3A is a vascular normalizing agent that can be exploited to treat cancer and, potentially, other diseases characterized by pathological angiogenesis., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objective: Molecular pathways governing blood vessel patterning are vital to vertebrate development. Because of their ability to counteract proangiogenic factors, antiangiogenic secreted Sema3 (class 3 semaphorins) control embryonic vascular morphogenesis. However, if and how Sema3 may play a role in the control of extraembryonic vascular development is presently unknown., Approach and Results: By characterizing genetically modified mice, here, we show that surprisingly Sema3F acts instead as a selective extraembryonic, but not intraembryonic proangiogenic cue. Both in vivo and in vitro, in visceral yolk sac epithelial cells, Sema3F signals to inhibit the phosphorylation-dependent degradation of Myc, a transcription factor that drives the expression of proangiogenic genes, such as the microRNA cluster 17/92. In Sema3f -null yolk sacs, the transcription of Myc-regulated microRNA 17/92 cluster members is impaired, and the synthesis of Myc and microRNA 17/92 foremost antiangiogenic target Thbs1 (thrombospondin 1) is increased, whereas Vegf (vascular endothelial growth factor) signaling is inhibited in yolk sac endothelial cells. Consistently, exogenous recombinant Sema3F inhibits the phosphorylation-dependent degradation of Myc and the synthesis of Thbs1 in mouse F9 teratocarcinoma stem cells that were in vitro differentiated in visceral yolk sac epithelial cells. Sema3f -/- mice placentas are also highly anemic and abnormally vascularized., Conclusions: Sema3F functions as an unconventional Sema3 that promotes extraembryonic angiogenesis by inhibiting the Myc-regulated synthesis of Thbs1 in visceral yolk sac epithelial cells., (© 2017 The Authors.)

Endothelial cell adhesion and migration are critical steps of the angiogenic process, whose dysfunction is associated with tumor growth and metastasis. The TRPM8 channel has recently been proposed to play a protective role in prostate cancer by impairing cell motility. However, the mechanisms by which it could influence vascular behavior are unknown. Here, we reveal a novel non-channel function for TRPM8 that unexpectedly acts as a Rap1 GTPase inhibitor, thereby inhibiting endothelial cell motility, independently of pore function. TRPM8 retains Rap1 intracellularly through direct protein-protein interaction, thus preventing its cytoplasm-plasma membrane trafficking. In turn, this mechanism impairs the activation of a major inside-out signaling pathway that triggers the conformational activation of integrin and, consequently, cell adhesion, migration, in vitro endothelial tube formation, and spheroid sprouting. Our results bring to light a novel, pore-independent molecular mechanism by which endogenous TRPM8 expression inhibits Rap1 GTPase and thus plays a critical role in the behavior of vascular endothelial cells by inhibiting migration., (© 2017 Genova et al.)

Basolateral polymerization of cellular fibronectin (FN) into a meshwork drives endothelial cell (EC) polarity and vascular remodelling. However, mechanisms coordinating α5β1 integrin-mediated extracellular FN endocytosis and exocytosis of newly synthesized FN remain elusive. Here we show that, on Rab21-elicited internalization, FN-bound/active α5β1 is recycled to the EC surface. We identify a pathway, comprising the regulators of post-Golgi carrier formation PI4KB and AP-1A, the small GTPase Rab11B, the surface tyrosine phosphatase receptor PTPRF and its adaptor PPFIA1, which we propose acts as a funnel combining FN secretion and recycling of active α5β1 integrin from the trans-Golgi network (TGN) to the EC surface, thus allowing FN fibrillogenesis. In this framework, PPFIA1 interacts with active α5β1 integrin and localizes close to EC adhesions where post-Golgi carriers are targeted. We show that PPFIA1 is required for FN polymerization-dependent vascular morphogenesis, both in vitro and in the developing zebrafish embryo.

Secreted class 3 semaphorins (Sema3), which signal through holoreceptor complexes that are formed by different subunits, such as neuropilins (Nrps), proteoglycans, and plexins, were initially characterized as fundamental regulators of axon guidance during embryogenesis. Subsequently, Sema3A, Sema3C, Sema3D, and Sema3E were discovered to play crucial roles in cardiovascular development, mainly acting through Nrp1 and Plexin D1, which funnels the signal of multiple Sema3 in vascular endothelial cells. Mechanistically, Sema3 proteins control cardiovascular patterning through the enzymatic GTPase-activating-protein activity of the cytodomain of Plexin D1, which negatively regulates the function of Rap1, a small GTPase that is well-known for its ability to drive vascular morphogenesis and to elicit the conformational activation of integrin adhesion receptors.

The molecular mechanisms orchestrating peritoneal and hematogenous metastases of ovarian cancer cells are assumed to be distinct. We studied the p90RSK family of serine/threonine kinases that lie downstream the RAS-ERK/MAPK pathway and modulate a variety of cellular processes including cell proliferation, survival, motility and invasiveness. We found the RSK1 and RSK2 isoforms expressed in a number of human ovarian cancer cell lines, where they played redundant roles in sustaining in vitro motility and invasiveness. In vivo, silencing of both RSK1 and RSK2 almost abrogated short-term and long-term metastatic engraftment of ovarian cancer cells in the peritoneum. In addition, RSK1/RSK2 silenced cells failed to colonize the lungs after intravenous injection and to form hematogenous metastasis from subcutaneous xenografts. RSK1/RSK2 suppression resulted in lessened ovarian cancer cell spreading on endogenous fibronectin (FN). Mechanistically, RSK1/RSK2 knockdown diminished FN transcription, α5β1 integrin activation and TGF-β1 translation. Reduced endogenous FN deposition and TGF-β1 secretion depended on the lack of activating phosphorylation of the transcription/translation factor YB-1 by p90RSK. Altogether data show how p90RSK activates a self-reinforcing cell autonomous pro-adhesive circuit necessary for metastatic seeding of ovarian cancer cells. Thus, p90RSK inhibitors might hinder both the hematogenous and the peritoneal metastatic spread of human ovarian cancer.

Angiogenesis is one of the major mechanisms controlling tumor proliferation and metastatic spreading. Targeting of pro-angiogenic factors and their downstream effectors represents an appealing therapeutic option in the treatment of different cancer types. Linifanib (ABT-869) is a novel tyrosine-kinase inhibitor (TKI) inhibitor and its anti-angiogenic activity has been explored in numerous clinical trials. Here, we review preclinical development of linifanib focusing on its pharmacodynamic and pharmacokinetic characteristics and briefly summarize its evaluation in clinical trials. Linifanib selectively targets VEGFR and PDGFR and has low off-target inhibitory activity. Preclinical and early-phase trials have been showing promising efficacy results However, although signals of anti-tumor activity have been proven in some malignancies, linifanib late-phase development has been facing some challenges due to limited efficacy and increased toxicities. New strategies aimed at finding biomarkers of response and minimizing toxicities are needed to allow the further development of a promising compound.

Purpose: MET, the high-affinity receptor for hepatocyte growth factor, is frequently deregulated in human cancer. Tivantinib (ARQ197; Arqule), a staurosporine derivative that binds to the dephosphorylated MET kinase in vitro, is being tested clinically as a highly selective MET inhibitor. However, the mechanism of action of tivantinib is still unclear., Experimental Design: The activity of tivantinib was analyzed in multiple cellular models, including: cells displaying c-MET gene amplification, strictly 'addicted' to MET signaling; cells with normal c-MET gene copy number, not dependent on MET for growth; cells not expressing MET; somatic knockout cells in which the ATP-binding cleft of MET, where tivantinib binds, was deleted by homologous recombination; and a cell system 'poisoned' by MET kinase hyperactivation, where cells die unless cultured in the presence of a specific MET inhibitor., Results: Tivantinib displayed cytotoxic activity independently of c-MET gene copy number and regardless of the presence or absence of MET. In both wild-type and isogenic knockout cells, tivantinib perturbed microtubule dynamics, induced G2/M arrest, and promoted apoptosis. Tivantinib did not rescue survival of cells 'poisoned' by MET kinase hyperactivation, but further incremented cell death. In all cell models analyzed, tivantinib did not inhibit HGF-dependent or -independent MET tyrosine autophosphorylation., Conclusions: We conclude that tivantinib displays cytotoxic activity via molecular mechanisms that are independent from its ability to bind MET. This notion has a relevant impact on the interpretation of clinical results, on the design of future clinical trials, and on the selection of patients receiving tivantinib treatment., (©2013 AACR.)

The dynamic control of integrin-mediated cell adhesion to extracellular matrix proteins is crucial for several physiological and pathological phenomena as diverse as embryonic morphogenesis, muscle contraction, tissue repair, and cancer cell dissemination. On one hand, the intrinsic conformational plasticity of integrins, which can be bidirectionally modulated by their ligands and cytosolic adaptors in combination with physical forces, is a key regulatory parameter. On the other hand, endo-exocytic integrin traffic logistics represent an additional important mode of control. Herein, we highlight how these two apparently parallel and independent strategies for tuning integrin function appear instead to be indissolubly intermingled, as eukaryotic cells have evolved distinct molecular strategies and endosomal pathways to traffic ligand-bound and ligand-free integrins., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1.

In multicellular organisms, the execution of complex morphogenetic events, such as gastrulation or vascular morphogenesis, depends on the dynamic modulation of adhesion. Guidance cues, such as chemokines, growth factors, and semaphorins control the attachment of cells to extracellular matrix proteins by regulating the conformational activation of integrin receptors. The endo-exocytic traffic of integrins back and forth from the plasma membrane represents another crucial regulatory aspect in cell adhesion and motility. Recent work added an additional layer of complexity by indicating that distinct molecular machineries are required for trafficking active and inactive integrins.

The process of blood vessels formation and remodeling is highly regulated by a plethora of promoting and inhibiting signals that activate a large array of signaling cascades. The main molecular players of these signaling pathways are surface-localized receptors, which can transmit signals into the cytosol. Endocytosis and intracellular trafficking, by controlling protein receptor localization, distribution, and amount in space and time, can strongly impact on cell signaling outcomes. Recent work showed that, in vascular cells, integrin adhesive receptors undertake different intracellular routes, depending on their activation state, giving more complexity to the system. In addition, the endo-exocytic cycle of angiogenic growth factor receptors is also essential to integrate multiple signals and coordinate different cellular events., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The poor prognosis of most non small cell lung carcinomas is due to their ability to efficiently invade surrounding tissues and blood vessels, finally metastasizing to distant organs. Integrin mediated adhesive interaction with the surrounding extracellular matrix is a key limiting step in the regulation of the invasive properties of several cancer cell types. Here, we examine the rising evidences about the role that integrins can play in the physiopathology of non small cell lung carcinomas by regulating cell adhesion as well as the activation of growth factors and the traffic of their cognate receptors. Modulation of the signaling pathways controlled by integrins in lung cancer cells might offer the opportunity to design and develop new drugs that might be successfully combined with conventional chemotherapy and radiotherapy.

Acquisition of new genes encoding for extracellular matrix (ECM) proteins and their cognate integrin adhesive receptors, as well as secreted pro- and anti-angiogenic factors, proved to be essential for the development of functional vascular networks in the vertebrate embryo. There is now clear evidence that post-natal, pathological tissue neo-vascularization is crucial for cancer growth and therapy as well. Integrins are major ECM receptors that can exist in different functional states with respect to their affinity for ECM proteins. Regulation of integrin activation is crucial for their biological functions. In the embryo, the development of a properly patterned network of blood vessels relies upon the fine modulation of integrin activation by chemoattractant and chemorepulsive cues, such as angiogenic growth factors and semaphorins. Such a fine-tuning of endothelial integrin function is likely to be disrupted in cancer. Here, the vasculature is structurally and functionally abnormal and therefore inadequate for an efficient drug and oxygen delivery, which is a mandatory pre-requisite for successful chemotherapy and radiotherapy. It is thus important to identify the molecular mechanisms that regulate integrin function in normal ECs and which are altered in tumor ECs.

Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes alpha5beta1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with alpha5beta1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with alpha5beta1, and the associated motor myosin VI (Myo6) support active alpha5beta1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1. Nrp1 modulation of alpha5beta1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice., Competing Interests: Competing interests. The authors have declared that no competing interests exist.