Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor κB-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-Kinase activity in breast cancer cells

Tumor growth and metastasis are multifaceted processes that mainly involve cell adhesion, proteolytic degradation of the extracellular matrix, and cell migration. Syk is a member of a tyrosine kinase family that is expressed mostly in hematopoietic cells. Syk is expressed in cell lines of epithelial origin, but its function in these cells remains unknown. Here we report that Syk is expressed in MCF-7 cells but not in MDA-MB-231 cells. The overexpression of wild type Syk kinase but not kinase-negative Syk suppressed cell motility and inhibited the activation of phosphatidylinositol (PI) 3'-kinase in MDA-MB-231 cells. In contrast, when Syk-specific antisense S-oligonucleotide but not the sense S-oligonucleotide was transfected to MCF-7 cells the level of PI 3'-kinase activity as well as cell motility were increased. The MDA-MB-231 cells transfected with wild type Syk cDNA followed by treatment with piceatannol, a Syk inhibitor, enhanced cell motility and PI 3'-kinase activity. Pervanadate, a phosphotyrosine phosphatase inhibitor, induced PI 3'-kinase activity and stimulated the interaction between the inhibitor of nuclear factor κBα (IκBα) and the p85α domain of PI 3'-kinase through tyrosine phosphorylation of the IκBα, which ultimately resulted in nuclear factor κB (NFκB) activation. Pervanadate had no effect on the activation of Syk in these cells. However, Syk suppressed the NFκB transcriptional activation and interaction between IκBα and PI 3'-kinase by inhibiting the tyrosine phosphorylation of IκBα. Syk, PI 3'-kinase inhibitors, and NFκB inhibitory peptide inhibited urokinase type plasminogen activator (uPA) secretion and cell motility in these cells. To our knowledge, this is the first report that Syk suppresses the cell motility and inhibits the PI 3'-kinase activity and uPA secretion by blocking NFκB activity through tyrosine phosphorylation of IκBα. These data further demonstrate a functional molecular link between Syk-regulated PI 3'-kinase activity and NFκB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells

Tyrosine kinase, p56<SUP>lck</SUP>-induced cell motility, and urokinase-type plasminogen activator secretion involve activation of epidermal growth factor receptor/extracellular signal regulated kinase pathways

We have recently reported that tyrosine kinase, p56lck regulates cell motility and nuclear factor &#954;B-mediated secretion of urokinase-type plasminogen activator (uPA) through tyrosine phosphorylation of I&#954;B&#945; following hypoxia/reoxygenation (Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 52598-52612). However, the role of hypoxia/reoxygenation (H/R) on ERK1/2-mediated uPA secretion and cell motility and the involvement of p56lck and EGF receptor in these processes in breast cancer cells is not well defined. We provide here evidence that H/R induces Lck kinase activity and Lck-dependent tyrosine phosphorylation of EGF receptor in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. H/R also stimulates MEK-1 and ERK1/2 phosphorylations, and H/R-induced phosphorylations were suppressed by the dominant negative form of Lck (DN Lck, K273R) as well as pharmacological inhibitors of EGF receptor and Lck indicating that EGF receptors and Lck are involved in these processes. Transfection of these cells with wild type Lck or Lck F505 (Y505F) but not with Lck F394 (Y394F) induced phosphorylations of EGF receptor followed by MEK-1 and ERK1/2, suggesting that Lck is upstream of EGF receptor and Tyr-394 of Lck is crucial for these processes. H/R also induced uPA secretion and cell motility in these cells. DN Lck and inhibitors of Lck, EGF receptor, and MEK-1 suppressed H/R-induced uPA secretion and cell motility. To our knowledge, this is the first report that p56lck in presence of H/R regulates MEK-1-dependent ERK1/2 phosphorylation and uPA secretion through tyrosine phosphorylation of EGF receptor, and it further demonstrates that all of these signaling molecules ultimately control the motility of breast cancer cells

Integrin signaling is critical for pathological angiogenesis

The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in tissue remodeling upon injury. However, the molecular events underlying this complex process are not well understood and numerous issues remain controversial, including the regulatory function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in angiogenesis, we generated knock-in mice that express a mutant β3 integrin unable to undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed that neovascularization is impaired in mutant β3 knock-in mice. In an ex vivo angiogenesis assay, mutant β3 knock-in endothelial cells did not form complete capillaries in response to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine phosphorylation in mutant β3 knock-in cells resulted in impaired adhesion, spreading, and migration of endothelial cells. At the molecular level, VEGF stimulated complex formation between VEGF receptor-2 and β3 integrin in wild-type but not in mutant β3 knock-in endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in cells expressing mutant β3 compared to wild type, leading to impaired integrin activation in these cells. These findings provide novel mechanistic insights into the role of integrin–VEGF axis in pathological angiogenesis

The angiogenic response is dictated by β3 integrin on bone marrow–derived cells

Angiogenesis is dependent on the coordinated action of numerous cell types. A key adhesion molecule expressed by these cells is the αvβ3 integrin. Here, we show that although this receptor is present on most vascular and blood cells, the key regulatory function in tumor and wound angiogenesis is performed by β3 integrin on bone marrow–derived cells (BMDCs) recruited to sites of neovascularization. Using knockin mice expressing functionally stunted β3 integrin, we show that bone marrow transplantation rescues impaired angiogenesis in these mice by normalizing BMDC recruitment. We demonstrate that αvβ3 integrin enhances BMDC recruitment and retention at angiogenic sites by mediating cellular adhesion and transmigration of BMDCs through the endothelial monolayer but not their release from the bone niche. Thus, β3 integrin has the potential to control processes such as tumor growth and wound healing by regulating BMDC recruitment to sites undergoing pathological and adaptive angiogenesis

Integrin β3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2

Suppression of β3-integrin in mice triggers a neuropilin-1- dependent change in focal adhesion remodelling that can be targeted to block pathological angiogenesis

Anti-angiogenic treatments against αvβ3-integrin fail to block tumour growth in the long term, which suggests tumour vascular escape through αvβ3-integrin-independent mechanisms. Here, we show that suppression of β3-integrin leads to the activation of a neuropilin-1 (NRP1) dependent cell migration pathway in endothelial cells via a mechanism that depends on NRP1’s mobilisation away from mature focal adhesions following VEGF-stimulation. The simultaneous genetic targeting of both molecules significantly impairs paxillin-1 activation and focal adhesion remodelling in endothelial cells and therefore inhibits tumour angiogenesis and the growth of already established tumours. These findings provide a firm foundation for testing drugs against these molecules in combination to treat patients with advanced cancers

Latent KSHV Infection of Endothelial Cells Induces Integrin Beta3 to Activate Angiogenic Phenotypes

Kaposi's Sarcoma (KS), the most common tumor of AIDS patients, is a highly vascularized tumor supporting large amounts of angiogenesis. The main cell type of KS tumors is the spindle cell, a cell of endothelial origin, the primary cell type involved in angiogenesis. Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of KS and is likely involved in both tumor formation and the induction of angiogenesis. Integrins, and specifically integrin αVβ3, have known roles in both tumor induction and angiogenesis. αVβ3 is also important for KSHV infection as it has been shown to be involved in KSHV entry into cells. We found that during latent infection of endothelial cells KSHV induces the expression of integrin β3 leading to increased surface levels of αVβ3. Signaling molecules downstream of integrins, including FAK and Src, are activated during viral latency. Integrin activation by KSHV is necessary for the KSHV-associated upregulation of a number of angiogenic phenotypes during latent infection including adhesion and motility. Additionally, KSHV-infected cells become more reliant on αVβ3 for capillary like formation in three dimensional culture. KSHV induction of integrin β3, leading to induction of angiogenic and cancer cell phenotypes during latency, is likely to be important for KS tumor formation and potentially provides a novel target for treating KS tumors

Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response

Recent studies have shown that a carbohydrate-binding protein, galectin-3, is a novel pro-angiogenic molecule. The mechanism by which galectin-3 promotes angiogenesis remains unknown. We demonstrate here that galectin-3 is a mediator of vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-mediated angiogenic response. Angiogenesis assays revealed that galectin-3 inhibitors, β-lactose and dominant-negative galectin-3, reduce VEGF- and bFGF-mediated angiogenesis in vitro and that VEGF- and bFGF-mediated angiogenic response is reduced in galectin-3 knockdown cells and Gal3−/− animals. Integrin αvβ3 was identified as the major galectin-3–binding protein and anti-αv, -β3, and -αvβ3 integrin function-blocking antibodies significantly inhibited the galectin-3–induced angiogenesis. Furthermore, galectin-3 promoted the clustering of integrin αvβ3 and activated focal adhesion kinase. Knockdown of GnTV, an enzyme that synthesizes high-affinity glycan ligands for galectin-3, substantially reduced: (a) complex N-glycans on αvβ3 integrins and (b) VEGF- and bFGF-mediated angiogenesis. Collectively, these data suggest that galectin-3 modulates VEGF- and bFGF-mediated angiogenesis by binding via its carbohydrate recognition domain, to the GnTV synthesized N-glycans of integrin αvβ3, and subsequently activating the signaling pathways that promote the growth of new blood vessels. These findings have broad implications for developing novel, carbohydrate-based therapeutic agents for inhibition of angiogenesis

Vitronectin Increases Vascular Permeability by Promoting VE-Cadherin Internalization at Cell Junctions

Cross-talk between integrins and cadherins regulates cell function. We tested the hypothesis that vitronectin (VN), a multi-functional adhesion molecule present in the extracellular matrix and plasma, regulates vascular permeability via effects on VE-cadherin, a critical regulator of endothelial cell (EC) adhesion.Addition of multimeric VN (mult VN) significantly increased VE-cadherin internalization in human umbilical vein EC (HUVEC) monolayers. This effect was blocked by the anti-α(V)β(3) antibody, pharmacological inhibition and knockdown of Src kinase. In contrast to mult VN, monomeric VN did not trigger VE-cadherin internalization. In a modified Miles assay, VN deficiency impaired vascular endothelial growth factor-induced permeability. Furthermore, ischemia-induced enhancement of vascular permeability, expressed as the ratio of FITC-dextran leakage from the circulation into the ischemic and non-ischemic hindlimb muscle, was significantly greater in the WT mice than in the Vn(-/-) mice. Similarly, ischemia-mediated macrophage infiltration was significantly reduced in the Vn(-/-) mice vs. the WT controls. We evaluated changes in the multimerization of VN in ischemic tissue in a mouse hindlimb ischemia model. VN plays a previously unrecognized role in regulating endothelial permeability via conformational- and integrin-dependent effects on VE-cadherin trafficking.These results have important implications for the regulation of endothelial function and angiogenesis by VN under normal and pathological conditions

Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN