Paola Defilippi

Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2.

Interaction between integrin αvβ3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin‐mediated outside‐in signals co‐operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between αvβ3 integrin and tyrosine kinase vascular endothelial growth factor receptor‐2 (VEGFR‐2) in human endothelial cells. We report that tyrosine‐phosphorylated VEGFR‐2 co‐immunoprecipitated with β3 integrin subunit, but not with β1 or β5, from cells stimulated with VEGF‐A165. VEGFR‐2 phosphorylation and mitogenicity induced by VEGF‐A165 were enhanced in cells plated on the αvβ3 ligand, vitronectin, compared with cells plated on the α5β1 ligand, fibronectin or the α2β1 ligand, collagen. BV4 anti‐β3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR‐2; (ii) the activation of downstream transductor phosphoinositide 3‐OH kinase; and (iii) biological effects triggered by VEGF‐A165. These results indicate a new role for αvβ3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, αvβ3 integrin participates in the full activation of VEGFR‐2 triggered by VEGF‐A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.

Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival

Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, β1 or αv integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor‐transfected NIH‐3T3 cells. Use of a kinase‐negative EGF receptor mutant demonstrates that the integrin‐stimulated tyrosine phosphorylation is due to activation of the receptors intrinsic kinase activity. Integrin‐mediated EGF receptor activation leads to Erk‐1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant‐negative EGF receptor mutant. EGF receptor and Erk‐1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix‐mediated cell survival. Adhesion‐dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5×103 receptors per cell). These results demonstrate that integrin‐dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extracellular matrix.

Dynamic Microtubules Regulate Dendritic Spine Morphology and Synaptic Plasticity

Dendritic spines are the major sites of excitatory synaptic input, and their morphological changes have been linked to learning and memory processes. Here, we report that growing microtubule plus ends decorated by the microtubule tip-tracking protein EB3 enter spines and can modulate spine morphology. We describe p140Cap/SNIP, a regulator of Src tyrosine kinase, as an EB3 interacting partner that is predominantly localized to spines and enriched in the postsynaptic density. Inhibition of microtubule dynamics, or knockdown of either EB3 or p140Cap, modulates spine shape via regulation of the actin cytoskeleton. Fluorescence recovery after photobleaching revealed that EB3-binding is required for p140Cap accumulation within spines. In addition, we found that p140Cap interacts with Src substrate and F-actin-binding protein cortactin. We propose that EB3-labeled growing microtubule ends regulate the localization of p140Cap, control cortactin function, and modulate actin dynamics within dendritic spines, thus linking dynamic microtubules to spine changes and synaptic plasticity.

Integrin signalling adaptors: not only figurants in the cancer story

Current evidence highlights the ability of adaptor (or scaffold) proteins to create signalling platforms that drive cellular transformation upon integrin-dependent adhesion and growth factor receptor activation. The understanding of the biological effects that are regulated by these adaptors in tumours might be crucial for the identification of new targets and the development of innovative therapeutic strategies for human cancer. In this Review we discuss the relevance of adaptor proteins in signalling that originates from integrin-mediated cell–extracellular matrix (ECM) adhesion and growth factor stimulation in the context of cell transformation and tumour progression. We specifically underline the contribution of p130 Crk-associated substrate (p130CAS; also known as BCAR1), neural precursor cell expressed, developmentally down-regulated 9 (NEDD9; also known as HEF1), CRK and the integrin-linked kinase (ILK)–pinch–parvin (IPP) complex to cancer, along with the more recently identified p140 Cas-associated protein (p140CAP; also known as SRCIN1).

Extracellular matrix, integrins, and growth factors as tailors of the stem cell niche.

It is widely acknowledged that integrins, the major receptors for the extracellular matrix (ECM) proteins, exert an extensive crosstalk with many growth factor and cytokine receptors. Among them, growth factor receptors, such as the EGFR, MET, PDGFR and VEGFR, and the IL-3 receptor have been shown to be physically and functionally associated to integrins. The connection between integrins and other transmembrane receptors is bidirectional, integrins being essential for receptor signalling, and receptors being involved in regulation of integrin expression or activation. Moreover, there is accumulating evidence for direct binding of specific growth factors and morphogens to the ECM proteins, suggesting that ECM might spatially integrate different types of signals in a specific microenvironment, facilitating integrin/transmembrane receptors connection. These interactions are crucial in controlling a variety of cell behaviours including proliferation, survival and differentiation. The increasing interest for cell therapy in regenerative medicine has recently emphasized the role of cell-ECM adhesion as stem cell determinant. The relevance of ECM, integrins and growth factor receptor network in the establishment of stem cell niche, in maintenance of stem cells and in their differentiation will be analyzed in the present review.

Actin cytoskeleton organization in response to integrin-mediated adhesion

Cell matrix adhesion regulates actin cytoskeleton organization through distinct steps, from formation of filopodia and lamellipodia in the early phases of cell adhesion to organization of focal adhesions and stress fibers in fully adherent cells. In this review, we follow the events induced by integrin‐mediated adhesion, such as activation of GTPases Cdc42 and Rac and their effectors and their role in actin polymerization leading to formation of lamellipodia and filopodia and cell spreading. We also show that actin stress fiber and focal adhesion formation following adhesion requires cooperation between integrin‐mediated signaling and additional stimuli, including activation of PKC, Rho GTPases, and PTKs such as p125Fak and Src. Microsc. Res. Tech. 47:67–78, 1999.

p130Cas interacts with estrogen receptor alpha and modulates non-genomic estrogen signaling in breast cancer cells.

Steroid hormones bind to their receptors and trans-activate target genes. Rapid non-genomic action of steroid hormones has been proposed in addition to the one at the genomic level. Estrogen has been described to activate c-Src kinase and this activation has been shown to be responsible for estrogen-dependent mitogenicity. A major substrate of c-Src kinase activity is the cytoskeletal protein p130Cas, originally identified in v-Src-transformed cells. We show that in the human breast carcinoma T47D cells, upon estrogen treatment, p130Cas rapidly and transiently associates with the estrogen receptor α in a multi-molecular complex containing the c-Src kinase and the p85 subunit of PI 3-kinase. Association of p130Cas with the estrogen receptor α occurs within 3 minutes of estrogen treatment and is dependent on c-Src kinase activation. Transient overexpression of p130Cas in T47D cells increases estrogen-dependent Src kinase and Erk1/2 MAPKs activities and accelerates their kinetics of stimulation. A similar effect was detected on estrogen-dependent cyclin D1 expression, suggesting a role for p130Cas in regulating estrogen-dependent cell cycle progression. Double-stranded small RNA interference (siRNA) by silencing endogenous p130Cas protein, was sufficient to inhibit estrogen-dependent Erk1/2 MAPKs activity and cyclin D1 induction, demonstrating the requirement of p130Cas in such events. Therefore, our data show that the adaptor protein p130Cas associates with the estrogen receptor transducing complex, regulating estrogen-dependent activation of c-Src kinase and downstream signaling pathways.

Integrins and Signal Transduction

Integrin signaling has a critical function in organizing cells in tissues during both embryonic development and tissue repair. Following their binding to the extracellular ligands, the intracellular signaling pathways triggered by integrins are directed to two major functions: organization of the actin cytoskeleton and regulation of cell behaviour including survival, differentiation and growth. Basic research conducted in the past twelve years has lead to remarkable breakthroughs in this field. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors, cytosolic tyrosine kinases or growth factor and cytokine receptors. The purpose of this chapter is to highlight recent experimental and conceptual advances in integrin signaling with particular emphasis on the ability of integrins to regulate Fak/Src family kinases (SFKs) activation and the cross-talk with soluble growth factors receptors and cytokines.

β1 integrin controls EGFR signaling and tumorigenic properties of lung cancer cells

Lung cancer is the leading cause of cancer death worldwide. The epidermal growth factor receptor (EGFR) represents the main target for non-small cell lung cancer (NSCLC) therapy, as its overexpression or constitutive activation contributes to malignancy and correlates with poor prognosis. Our previous work demonstrated that in epithelial cells β1 integrin is required for propagating EGFR signaling from the plasma membrane to the nucleus. In this study, we silenced β1 integrin in human NSCLC A549 cells. The β1 integrin-silenced cells show a defective activation of the EGFR signaling cascade, leading to decreased in vitro proliferation, enhanced sensitivity to cisplatin and Gefitinib, impaired migration and invasive behavior. Inhibitory effects on tumor growth and on the EGFR pathway were also observed in in vivo experiments. Moreover, β1 integrin silencing increases the amount of EGFR on the cell surface, suggesting that β1 integrin is required for efficient constitutive EGFR turnover at the cell membrane. Although the rate of EGF internalization and recycling is not affected in silenced cells, EGFR signaling is recovered only by expression of the Rab-coupling protein RCP, indicating that β1 integrin sustains the endocytic machinery required for EGFR signaling. Overall, these results show that β1 integrin is an essential regulator of EGFR signaling and tumorigenic properties of lung cancer cells, and that its silencing might represent an adjuvant approach to anti-EGFR therapy.

p27kip1 controls cell morphology and motility by regulating microtubule-dependent lipid raft recycling

ABSTRACT p27kip1 (p27) is an inhibitor of cyclin/cyclin-dependent kinase complexes, whose nuclear loss indicates a poor prognosis in various solid tumors. When located in the cytoplasm, p27 binds Op18/stathmin (stathmin), a microtubule (MT)-destabilizing protein, and restrains its activity. This leads to MT stabilization, which negatively affects cell migration. Here, we demonstrate that this p27 function also influences morphology and motility of cells immersed in three-dimensional (3D)matrices. Cells lacking p27 display a decrease in MT stability, a rounded shape when immersed in 3D environments, and a mesenchymal-amoeboid conversion in their motility mode. Upon cell contact to extracellular matrix, the decreased MT stability observed in p27 null cells results in accelerated lipid raft trafficking and increased RhoA activity. Importantly, cell morphology, motility, MT network composition, and distribution of p27 null cells were rescued by the concomitant genetic ablation of Stathmin, implicating that the balanced expression of p27 and stathmin represents a crucial determinant for cytoskeletal organization and cellular behavior in 3D contexts.