Enhancing integrin function by VEGF/neuropilin signaling: Implications for tumor biology

This review advances the hypothesis that the ability of integrins to engage their extracellular matrix ligands and signal can be regulated in tumor cells by vascular endothelial growth factor (VEGF), a major angiogenic factor that also has direct effects on the function of tumor cells. More specifically, we will discuss how neuropilins (NRPs), a distinct class of VEGF receptors, enable the function of specific integrins that contribute to tumor initiation and progression.     

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.     

Molecular pathway for cancer metastasis to bone

The molecular mechanism leading to the cancer metastasis to bone is poorly understood but yet determines prognosis and therapy. Here, we define a new molecular pathway that may account for the extraordinarily high osteotropism of prostate cancer. By using SPARC (secreted protein, acidic and rich in cysteine)-deficient mice and recombinant SPARC, we demonstrated that SPARC selectively supports the migration of highly metastatic relative to less metastatic prostate cancer cell lines to bone. Increased migration to SPARC can be traced to the activation of integrins alphaVbeta3 and alphaVbeta5 on tumor cells. Such activation is induced by an autocrine vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-2 loop on the tumor cells, which also supports the growth and proliferation of prostate cancer cells. A consequence of SPARC recognition by alphaVbeta5 is enhanced VEGF production. Thus, prostate cancer cells expressing VEGF/VEGFR-2 will activate alphaVbeta3 and alphaVbeta5 on their surface and use these integrins to migrate toward SPARC in bone. Within the bone environment, SPARC engagement of these integrins will stimulate growth of the tumor and further production of VEGF to support neoangiogenesis, thereby favoring the development of the metastatic tumor. Supporting this model, activated integrins were found to colocalize with VEGFR-2 in tissue samples of metastatic prostate tumors from patients.     

Integrin regulation by vascular endothelial growth factor in human brain microvascular endothelial cells: role of alpha6beta1 integrin in angiogenesis

The precise role of vascular endothelial growth factor (VEGF) in regulating integrins in brain microvascular endothelial cells is unknown. Here, we analyzed VEGF effects on integrin expression and activation in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and ribonuclease (RNase) protection assays, we observed that VEGF up-regulated the mRNA expression of alpha(6) integrin in HBMECs. VEGF significantly increased alpha(6)beta(1) integrin expression, but not alpha(6)beta(4) integrin expression in these cells. Specific down-regulation of alpha(6) integrin expression by small interfering RNA (siRNA) oligonucleotides inhibited both the capillary morphogenesis of HBMECs and their adhesion and migration. Additionally, VEGF treatment resulted in activation of alpha(6)beta(1) integrins in HBMECs. Functional blocking of alpha(6) integrin with its specific antibody inhibited the VEGF-induced adhesion and migration as well as in vivo angiogenesis, and markedly suppressed tumor angiogenesis and breast carcinoma growth in vivo. Thus, VEGF can modulate angiogenesis via increased expression and activation of alpha(6)beta(1) integrins, which may promote VEGF-driven tumor angiogenesis in vivo.     

Endothelial cell survival and apoptosis in the tumor vasculature

Angiogenesis is essential for the growth and metastasis of solid tumors. The balance of endothelial cell (EC) proliferation and apoptosis is a major determinant in tumor angiogenesis. Recently, several studies demonstrated that numerous angiogenic factors not only induce angiogenesis but also function as EC survival factors. Vascular endothelial growth factor (VEGF), a potent angiogenic factor, is also an EC survival factor in embryonic vasculogenesis and tumor angiogenesis. VEGF activates specific intracellular survival pathways in ECs including Bcl-2, A1, IAP, Akt, and Erk. Integrins may function as EC survival factors by preventing anoikis by enhancing binding to the extracellular matrix. In addition, integrins may function in concert with VEGF to promote EC survival. Angiopoietin-1 (Ang-1) has recently been shown to stabilize EC networks by binding to the EC-specific tyrosine kinase receptor Tie-2. Pericytes also function as EC survival factors, by cell-cell contact, secretion of survival factors, or both. Targeting any of the above mechanisms for EC survival may provide novel antineoplastic strategies.     

Integrin (alpha 6 beta 4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells

We define a novel mechanism by which integrins regulate growth factor expression and the survival of carcinoma cells. Specifically, we demonstrate that the alpha 6 beta 4 integrin enhances vascular endothelial growth factor (VEGF) translation in breast carcinoma cells. The mechanism involves the ability of this integrin to stimulate the phosphorylation and inactivation of 4E-binding protein (4E-BP1), a translational repressor that inhibits the function of eukaryotic translation initiation factor 4E (eIF-4E). The regulation of 4E-BP1 phosphorylation by alpha 6 beta 4 derives from the ability of this integrin to activate the PI-3K-Akt pathway and, consequently, the rapamycin-sensitive kinase mTOR that can phosphorylate 4E-BP1. Importantly, we show that this alpha 6 beta 4-dependent regulation of VEGF translation plays an important role in the survival of metastatic breast carcinoma cells by sustaining a VEGF autocrine signaling pathway that involves activation of PI-3K and Akt. These findings reveal that integrin-mediated activation of PI-3K-Akt is amplified by integrin-stimulated VEGF expression and they provide a mechanism that substantiates the reported role of alpha 6 beta 4 in carcinoma progression.     

Hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor VEGF, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability

Hantaviruses infect human endothelial cells and cause two vascular permeability-based diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Hantavirus infection alone does not permeabilize endothelial cell monolayers. However, pathogenic hantaviruses inhibit the function of alphav beta3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional beta3 integrins that normally regulate permeabilizing vascular endothelial growth factor (VEGF) responses. Here we show that pathogenic Hantaan, Andes, and New York-1 hantaviruses dramatically enhance the permeability of endothelial cells in response to VEGF, while the nonpathogenic hantaviruses Prospect Hill and Tula have no effect on endothelial cell permeability. Pathogenic hantaviruses directed endothelial cell permeability 2 to 3 days postinfection, coincident with pathogenic hantavirus inhibition of alphav beta3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to alphav beta3 integrin-deficient cells, specifically enhance VEGF-directed permeabilizing responses. Using the hantavirus permeability assay we further demonstrate that the endothelial-cell-specific growth factor angiopoietin 1 (Ang-1) and the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) inhibit hantavirus directed endothelial cell permeability at physiologic concentrations. These results demonstrate the utility of a hantavirus permeability assay and rationalize the testing of Ang-1, S1P, and antibodies to VEGFR2 as potential hantavirus therapeutics. The central importance of beta3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering beta3 or VEGF responses may be a common feature of additional viral hemorrhagic diseases. As a result, our findings provide a potential mechanism for vascular leakage after infection by pathogenic hantaviruses and the means to inhibit hantavirus-directed endothelial cell permeability that may be applicable to additional vascular leak syndromes.     

The soluble form of the cancer-associated L1 cell adhesion molecule is a pro-angiogenic factor

A soluble form of the L1 cell adhesion molecule (sL1) is released from various tumor cells and can be found in serum and ascites fluid of uterine and ovarian carcinoma patients. sL1 is a ligand for several Arg-Gly-Asp (RGD)-binding integrins and can be deposited in the extracellular matrix. In this study we describe a novel function of this physiologically relevant form of L1 as a pro-angiogenic factor. We demonstrated that the anti-L1 monoclonal antibody (mAb) chCE7 binds near or to the sixth Ig-like domain of human L1 which contains a single RGD sequence. mAb chCE7 inhibited the RGD-dependent adhesion of ovarian carcinoma cells to sL1 and reversed the sL1-induced proliferation, matrigel invasion and tube formation of bovine aortic endothelial (BAE) cells. A combination of sL1 with vascular endothelial growth factor-A (VEGF-A₁₆₅), which is an important angiogenic inducer in tumors, strongly potentiated VEGF receptor-2 tyrosine phosphorylation in BAE cells. Chick chorioallantoic membrane (CAM) assays revealed the pro-angiogenic potency of sL1 in vivo which could be abolished by chCE7. These results indicate an important role of released L1 in tumor angiogenesis and represent a novel function of antibody chCE7 in tumor therapy.     

A mechanism for modulation of cellular responses to VEGF: activation of the integrins

Many similarities exist in the cellular responses elicited by VEGF and governed by integrins. Here, we identify a basis for these interrelationships: VEGF activates integrins. VEGF enhanced cell adhesion, migration, soluble ligand binding, and adenovirus gene transfer mediated by alphavbeta3 and also activated other integrins, alphavbeta5, alpha5beta1, and alpha2beta1, involved in angiogenesis. Certain tumor cells exhibited high spontaneous adhesion and migration, which were attributable to a VEGF-dependent autocrine/paracrine activation of integrins. This activation was mediated by the VEGFR2 receptor and regulated via phosphatidylinositol-3-kinase, Akt, and the PTEN signaling axis. Thus, integrin activation provides a mechanism for VEGF to induce a broad spectrum of cellular responses.     

Gene Expression of Neuropilin-1 and Its Receptors,VEGF/Semaphorin 3a,in Normal and Cancer Cells

Extracellular domains of the transmembrane glycoprotein, neuropilin-1 (Np1), specifically bind an array of factors and co-receptors including class-3 semaphorins (Sema3a), vascular endothelial growth factor (VEGF), hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-β 1 (TGF-β1), and fibroblast growth factor2 (FGF2). Np1 may have a role in immune response, tumor cell growth, and angiogenesis, but its relative expression in comparison to its co-primary receptors, VEGF and Sema3a, is not known. In this study we determined the mRNA expression of Np1 and its co-receptors, VEGF and Sema3a, and the ratio of VEGF/Sema3a in different human and rodent cell lines. Expression of Np1, VEGF and Sema3a is very low in cells derived from normal tissues, but these proteins are highly expressed in tumor-derived cells. Furthermore, the ratio of VEGF/Sema3a is highly variable in different tumor cells. The elevated mRNA expression of Np1 and its putative receptors in tumor cells suggests a role for these proteins in tumor cell migration and angiogenesis. As different tumor cells exhibit varying VEGF/Sema3a ratios, it appears that cancer cells show differential response to angiogenic factors. These results bring to light the individual variation among the cancer-related genes, Np1, VEGF, and Sema3a, and provide an important impetus for the possible personalized therapeutic approaches for cancer patients.     

Inflammatory cytokines synergize with the HIV-1 Tat protein to promote angiogenesis and Kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin.

The Tat protein of HIV-1, a transactivator of viral gene expression, is released by acutely infected T cells and, in this form, exerts angiogenic activities. These have linked the protein to the pathogenesis of Kaposi's sarcoma (KS), a vascular tumor frequent and aggressive in HIV-1-infected individuals (AIDS-KS). In this study, we show that a combination of the same inflammatory cytokines increased in KS lesions, namely IL-1 beta, TNF-alpha, and IFN-gamma, synergizes with Tat to promote in nude mice the development of angioproliferative KS-like lesions that are not observed with each factor alone. Inflammatory cytokines induce the tissue expression of both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), two angiogenic molecules highly produced in primary KS lesions. However, bFGF, but not VEGF, synergizes with Tat in vivo and induces endothelial cells to migrate, to adhere, and to grow in response to Tat in vitro. Tat angiogenic effects correlate with the expression of the alpha v beta 3 integrin that is induced by bFGF and binds the arginine-glycine-aspartic acid (RGD) region of Tat. In contrast, no correlation is observed with the expression of alpha v beta 5, which is promoted by VEGF and binds Tat basic region. Finally, KS lesion formation induced by bFGF and Tat in nude mice is blocked by antagonists of RGD-binding integrins. Because alpha v beta 3 is an RGD-binding integrin that is highly expressed in primary KS lesions, where it colocalizes with extracellular Tat on vessels and spindle cells, these results suggest that alpha v beta 3 competitors may represent a new strategy for the treatment of AIDS-KS.     

Autocrine regulation of glioblastoma cell-cycle progression,viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and progression of malignant brain tumors. Given the significance of tumor microenvironment in general, and the established role of paracrine VEGF signaling in glioblastoma (GBM) biology in particular, we explored the potential autocrine control of human astrocytoma behavior by VEGF. Using a range of cell and molecular biology approaches to study a panel of astrocytoma (grade III and IV/GBM)-derived cell lines and a series of clinical specimens from low- and high-grade astrocytomas, we show that co-expression of VEGF and VEGF receptors (VEGFRs) occurs commonly in astrocytoma cells. We found VEGF secretion and VEGF-induced biological effects (modulation of cell cycle progression and enhanced viability of glioblastoma cells) to function in an autocrine manner. Morevover, we demonstrated that the autocrine VEGF signaling is mediated via VEGFR2 (KDR), and involves co-activation of the c-Raf/MAPK, PI3K/Akt and PLC/PKC pathways. Blockade of VEGFR2 by the selective inhibitor (SU1498) abrogated the VEGF-mediated enhancement of astrocytoma cell growth and viability under unperturbed culture conditions. In addition, such interference with VEGF-VEGFR2 signaling potentiated the ionizing radiation-induced tumor cell death. In clinical specimens, both VEGFRs and VEGF were co-expressed in astroglial tumor cells, and higher VEGF expression correlated with tumor progression, thereby supporting the relevance of functional VEGF-VEGFR signaling in vivo. Overall, our results are consistent with a potential autocrine role of the VEGF-VEGFR2 (KDR) interplay as a factor contributing to malignant astrocytoma growth and radioresistance, thereby supporting the candidacy of this signaling cascade as a therapeutic target, possibly in combination with radiotherapy.     

Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that has been shown to act as an endothelial cell mitogen as well as a vascular permeability factor. Several recent reports have also implicated VEGF as a major survival factor for endothelial cells during angiogenesis and vasculogenesis along with other growth factors such as bFGF and angiopoietin-1. VEGF has been shown to mediate this additional function, at least in part through the induction of bcl-2 and the activation of the PI3 kinase-Akt/PKB signaling pathway. We report here that VEGF can also mediate the induction/upregulation of members of a newly discovered family of antiapoptotic proteins, namely the Inhibitors of Apoptosis (IAP), in vascular endothelial cells. We show that VEGF(165) leads to the induction of XIAP (2.9-fold) and survivin (19.1-fold) protein in human umbilical vein endothelial cells (HUVECs). In contrast, bFGF had little effect on XIAP expression, but produced approximately a 10-fold induction on survivin. VEGF-dependent upregulation of survivin could be prevented by cell cycle arrest in the G1 and S phases. These findings implicate that the survival and mitotic functions of VEGF in an angiogenic context may be more intrinsically related than previously anticipated. Moreover, they also raise the possibility of therapeutically targeting XIAP or survivin in antiangiogenic therapy as a means of suppressing tumor growth, in addition to directly targeting tumor cells which express these survival proteins.     

Betulinic acid inhibits the expression of hypoxia-inducible factor 1α and vascular endothelial growth factor in human endometrial adenocarcinoma cells

Although betulinic acid (BA) is known to induce apoptosis and antiangiogenic response in tumor cells, the underlying mechanism of its action is unknown. Deregulation of tissue collagen metabolism is one of the consequences of neoplastic transformation. The final step of collagen degradation is mediated by prolidase [E.C.3.4.13.9] which may play a role in angiogenesis. The formation of new blood vessels is regulated by the hypoxia-inducible factor 1 (HIF-1). The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of vascular endothelial cell growth factor (VEGF). Since BA evokes anticancer activity, its effect on collagen biosynthesis, HIF-1α and VEGF expressions, as well as prolidase activity and expression was studied in cultured endometrial adenocarcinoma (EA) cells. It was found that BA inhibits collagen biosynthesis in EA cells (5[³H] proline incorporation assay). It was accompanied by a parallel decrease in prolidase activity and expression and decrease in expressions of α₁ and α₂ integrins, HIF-1α, and VEGF (western immunoblot analysis) in cultured human EA cells. The data suggest that BA may have anti-angiogenic potential by inhibition of prolidase, HIF-1α and VEGF expressions, and inhibition of collagen biosynthesis.     

Hypoxia Inducible Factors-Mediated Inhibition of Cancer by GM-CSF: A Mathematical Model

Under hypoxia, tumor cells, and tumor-associated macrophages produce VEGF (vascular endothelial growth factor), a signaling molecule that induces angiogenesis. The same macrophages, when treated with GM-CSF (granulocyte/macrophage colony-stimulating factor), produce sVEGFR-1 (soluble VEGF receptor-1), a soluble protein that binds with VEGF and inactivates its function. The production of VEGF by macrophages is regulated by HIF-1α (hypoxia inducible factor-1α), and the production of sVEGFR-1 is mediated by HIF-2α. Recent experiments measured the effect of inhibiting tumor growth by GM-CSF treatment in mice with HIF-1α-deficient or HIF-2α-deficient macrophages. In the present paper, we represent these experiments by a mathematical model based on a system of partial differential equations. We show that the model simulations agree with the above experiments. The model can then be used to suggest strategies for inhibiting tumor growth. For example, the model qualitatively predicts the extent to which GM-CSF treatment in combination with a small molecule inhibitor that stabilizes HIF-2α will reduce tumor volume and angiogenesis.