Differential alphav integrin-mediated Ras-ERK signaling during two pathways of angiogenesis.

Antagonists of alphavbeta3 and alphavbeta5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these alphav integrins differentially contribute to sustained Ras-extracellular signal-related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or alphavbeta5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin alphavbeta3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-alphavbeta5 blocked upstream of Ras, whereas anti-alphavbeta3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/alphavbeta3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins alphavbeta3 and alphavbeta5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis

Re-thinking the preclinical development of GBM therapeutics.

International audienceGlioblastoma multiforme (GBM) remains one of the most incurable cancers. Although the genomic era has produced massive quantities of data in an attempt to characterize its molecular drivers 1, 2 , these advances have yet to be effectively translated into clinical impact. In fact, several studies have identified the presence of all three GBM molecular subtypes within a single patient tumor 3, 4 , illustrating the complexity of designing personalized medicine approaches. In addition to the strong intra/inter-tumoral heterogeneity, the inability of targeted therapies to achieve long-term remissions is likely a function of multiple complicating factors, including the presence of glioblastoma stem cells, redundant signaling pathways, the unique infiltrative nature of GBM cells, and difficulties associated with drug delivery across the blood-brain-barrier. New strategies to match patients to molecularly targeted therapies will also need to overcome these challenges imposed by the challenging GBM microenvironment within the brain

Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression.

Vascular cell adhesion molecule-1 (VCAM-1) plays important roles in development and inflammation. Tumor necrosis factor-α (TNF-α) and focal adhesion kinase (FAK) are key regulators of inflammatory and integrin-matrix signaling, respectively. Integrin costimulatory signals modulate inflammatory gene expression, but the important control points between these pathways remain unresolved. We report that pharmacological FAK inhibition prevented TNF-α-induced VCAM-1 expression within heart vessel-associated endothelial cells in vivo, and genetic or pharmacological FAK inhibition blocked VCAM-1 expression during development. FAK signaling facilitated TNF-α-induced, mitogen-activated protein kinase activation, and, surprisingly, FAK inhibition resulted in the loss of the GATA4 transcription factor required for TNF-α-induced VCAM-1 production. FAK inhibition also triggered FAK nuclear localization. In the nucleus, the FAK-FERM (band 4.1, ezrin, radixin, moesin homology) domain bound directly to GATA4 and enhanced its CHIP (C terminus of Hsp70-interacting protein) E3 ligase-dependent polyubiquitination and degradation. These studies reveal new developmental and anti-inflammatory roles for kinase-inhibited FAK in limiting VCAM-1 production via nuclear localization and promotion of GATA4 turnover

Regulation of Cell Contraction and Membrane Ruffling by Distinct Signals in Migratory Cells

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively

Kinase-independent role for CRAF-driving tumour radioresistance via CHK2

Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase

Induction of the Angiogenic Phenotype by Hox D3

Angiogenesis is characterized by distinct phenotypic changes in vascular endothelial cells (EC). Evidence is provided that the Hox D3 homeobox gene mediates conversion of endothelium from the resting to the angiogenic/invasive state. Stimulation of EC with basic fibroblast growth factor (bFGF) resulted in increased expression of Hox D3, integrin αvβ3, and the urokinase plasminogen activator (uPA). Hox D3 antisense blocked the ability of bFGF to induce uPA and integrin αvβ3 expression, yet had no effect on EC cell proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin αvβ3 and uPA. In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis

Differential αv integrin–mediated Ras-ERK signaling during two pathways of angiogenesis

Antagonists of αvβ3 and αvβ5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these αv integrins differentially contribute to sustained Ras-extracellular signal–related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or αvβ5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin αvβ3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-αvβ5 blocked upstream of Ras, whereas anti-αvβ3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/αvβ3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins αvβ3 and αvβ5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis

Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins

Integrin-mediated adhesion promotes cell survival in vitro, whereas integrin antagonists induce apoptosis of adherent cells in vivo. Here, we demonstrate that cells adherent within a three-dimensional extracellular matrix undergo apoptosis due to expression of unligated integrins, the β subunit cytoplasmic domain, or its membrane proximal sequence KLLITIHDRKEF. Integrin-mediated death requires initiator, but not stress, caspase activity and is distinct from anoikis, which is caused by the loss of adhesion per se. Surprisingly, unligated integrin or β integrin tails recruit caspase-8 to the membrane, where it becomes activated in a death receptor–independent manner. Integrin ligation disrupts this integrin–caspase containing complex and increases survival, revealing an unexpected role for integrins in the regulation of apoptosis and tissue remodeling

MicroRNA regulation of endothelial TREX1 reprograms the tumour microenvironment

Rather than targeting tumour cells directly, elements of the tumour microenvironment can be modulated to sensitize tumours to the effects of therapy. Here we report a unique mechanism by which ectopic microRNA-103 can manipulate tumour-associated endothelial cells to enhance tumour cell death. Using gain-and-loss of function approaches, we show that miR-103 exacerbates DNA damage and inhibits angiogenesis in vitro and in vivo. Local, systemic or vascular-targeted delivery of miR-103 in tumour-bearing mice decreased angiogenesis and tumour growth. Mechanistically, miR-103 regulation of its target gene TREX1 in endothelial cells governs the secretion of pro-inflammatory cytokines into the tumour microenvironment. Our data suggest that this inflammatory milieu may potentiate tumour cell death by supporting immune activation and inducing tumour expression of Fas and TRAIL receptors. Our findings reveal miR-mediated crosstalk between vasculature and tumour cells that can be exploited to improve the efficacy of chemotherapy and radiation.United States. National Institutes of Health (R00HL112962)United States. National Institutes of Health (R01 HL57900)Oregon Health & Science University. Knight Cancer Institute (2015-Dive-Knight-01