EGFR-dependent pancreatic carcinoma cell metastasis through Rap1 activation.

Tyrosine kinase receptors have an essential role in various aspects of tumor progression. In particular, epidermal growth factor receptor (EGFR) and its ligands have been implicated in the growth and dissemination of a wide array of human carcinomas. Here, we describe an EGFR-mediated signaling pathway that regulates human pancreatic carcinoma cell invasion and metastasis, yet does not influence the growth of primary tumors. In fact, ligation/activation of EGFR induces Src-dependent phosphorylation of two critical tyrosine residues of p130CAS, leading to the assembly of a Crk-associated substrate (CAS)/Nck1 complex that promotes Ras-associated protein-1 (Rap1) signaling. Importantly, GTP loading of Rap1 is specifically required for pancreatic carcinoma cell migration on vitronectin but not on collagen. Furthermore, Rap1 activation is required for EGFR-mediated metastasis in vivo without impacting primary tumor growth. These findings identify a molecular pathway that promotes the invasive/metastatic properties of human pancreatic carcinomas driven by EGFR.

Oncogenic Ras/Src cooperativity in pancreatic neoplasia.

Pancreas cancer is one of the most lethal malignancies and is characterized by activating mutations of Kras, present in 95% of patients. More than 60% of pancreatic cancers also display increased c-Src activity, which is associated with poor prognosis. Although loss of tumor suppressor function (for example, p16, p53, Smad4) combined with oncogenic Kras signaling has been shown to accelerate pancreatic duct carcinogenesis, it is unclear whether elevated Src activity contributes to Kras-dependent tumorigenesis or is simply a biomarker of disease progression. Here, we demonstrate that in the context of oncogenic Kras, activation of c-Src through deletion of C-terminal Src kinase (CSK) results in the development of invasive pancreatic ductal adenocarcinoma (PDA) by 5-8 weeks. In contrast, deletion of CSK alone fails to induce neoplasia, while oncogenic Kras expression yields PDA at low frequency after a latency of 12 months. Analysis of cell lines derived from Ras/Src-induced PDA's indicates that oncogenic Ras/Src cooperativity may lead to genomic instability, yet Ras/Src-driven tumor cells remain dependent on Src signaling and as such, Src inhibition suppresses growth of Ras/Src-driven tumors. These findings demonstrate that oncogenic Ras/Src cooperate to accelerate PDA onset and support further studies of Src-directed therapies in pancreatic cancer.

Regulation of angiogenesis: apoptotic cues from the ECM.

The extracellular matrix (ECM) acts both as a physical scaffold for cells and as a repository for growth factors. Moreover, ECM structure and physical-chemical properties convey precise information to cells that profoundly influences their biology by interactions with cell surface receptors termed integrins. During angiogenesis, the perivascular ECM plays a critical role in determining the proliferative, invasive and survival responses of the local vascular cells to the angiogenic growth factors. Dynamic changes in both the ECM and the local vascular cells act in concert to regulate new blood vessel growth. The digestion of ECM components by proteolysis is critical for the invasive capacity of endothelial cells, but also creates ECM fragments, which antagonize the mechanosensory function of integrins, and can be apoptogenic. Here, we discuss the roles of integrins in modulating cellular responses to a changing ECM, in particular the regulation of survival and invasion among invasive endothelial cells.

Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors.

Elevated focal adhesion kinase (FAK) expression occurs in advanced cancers, yet a signaling role for FAK in tumor progression remains undefined. Here, we suppressed FAK activity in 4T1 breast carcinoma cells resulting in reduced FAK Y925 phosphorylation, Grb2 adaptor protein binding to FAK, and signaling to mitogen-activated protein (MAP) kinase (MAPK). Loss of a FAK-Grb2-MAPK linkage did not affect 4T1 cell proliferation or survival in culture, yet FAK inhibition reduced vascular endothelial growth factor (VEGF) expression and resulted in small avascular tumors in mice. This FAK-Grb2-MAPK linkage was essential in promoting angiogenesis as reconstitution experiments using Src-transformed FAK-null fibroblasts revealed that point mutations affecting FAK catalytic activity (R454) or Y925 phosphorylation (F925) disrupted the ability of FAK to promote MAPK- and VEGF-associated tumor growth. Notably, in both FAK-inhibited 4T1 and Src-transformed FAK-null cells, constitutively activated (CA) mitogen-activated protein kinase kinase 1 (MEK1) restored VEGF production and CA-MEK1 or added VEGF rescued tumor growth and angiogenesis. These studies provide the first biological support for Y925 FAK phosphorylation and define a novel role for FAK activity in promoting a MAPK-associated angiogenic switch during tumor progression.

Integrins and angiogenesis.

The growth of new blood vessels is a dynamic yet highly regulated process that depends on coordinated signaling by growth factor and cell adhesion receptors. As part of the molecular program regulating angiogenesis, endothelial cells acquire a proliferative and invasive phenotype but also show increased susceptibility to apoptotic stimuli. Integrins are the principle adhesion receptors used by endothelial cells to interact with their extracellular microenvironment, and integrin-mediated interactions play a critical role in regulating cell proliferation, migration, and survival. Alterations in the repertoire and?or activity of integrins, as well as the availability and structural property of their ligands, regulate the vascular cell during the growth or repair of blood vessels.

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 beta 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 beta 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.

Adhesion events in angiogenesis.

Recent work from several laboratories indicates that the coordination of endothelial cell adhesion events with growth factor receptor inputs regulates endothelial cell responses during angiogenesis. Analyses of the signaling pathways downstream of integrins, cadherins and growth-factor receptors are providing an insight into the molecular basis of known anti-angiogenic strategies, as well as into the design of novel therapies.

Alpha(v)beta3 and alpha(v)beta5 integrin expression in glioma periphery.

OBJECTIVE: This study analyzed the expression of integrins alpha(v)beta3 and alpha(v)beta5 in glioma tissue and focused on the periphery of high-grade gliomas. METHODS: The analysis was performed with Western blot, immunohistochemistry, and immunofluorescence, by use of two monoclonal antibodies able to recognize the functional integrin heterodimer. The expression of integrin-related ligands and growth factors also was studied. Sections from the tumor periphery were classified as either tumor periphery (light tumor infiltrate or scant visible cells) or peritumor (heavy tumor infiltration). RESULTS: Our data on glioma tissues demonstrated that both integrins were expressed in glioma cells and vasculature and their expression correlated with the histological grade. Alpha(v)beta3 expression was prominent in astrocytic tumors. Both integrins were markers of tumor vasculature, particularly of endothelial proliferation. A high-grade glioma periphery demonstrated a prominent expression of integrin alpha(v)beta3. Cells demonstrating alpha(v)beta3 positivity were identified as tumor astrocytes and endothelial cells by double imaging. The same cells were surrounded by some alpha(v)beta3 ligands and co-localized fibroblast growth factor 2. Matrix metalloproteinase 2 also was found to be co-localized with alpha(v)beta3 in the same cells. Alpha(v)beta3 expression was more relevant in tumor astrocytes. Alpha(v)beta3 integrin and vascular endothelial growth factor expression increased from the periphery to the tumor center. CONCLUSION: Our data support the role of integrins alpha(v)beta3 and alpha(v)beta5 in glioma-associated angiogenesis. In addition, they suggest a role for integrin alpha(v)beta3 in neoangiogenesis and cell migration in high-grade glioma periphery.

Identification of a novel class of small-molecule antiangiogenic agents through the screening of combinatorial libraries which function by inhibiting the binding and localization of proteinase MMP2 to integrin alpha(V)beta(3).

The process of new blood vessel growth from existing vasculature, known as angiogenesis, is critical to several pathological conditions, most notably cancer. Both MMP2, which degrades the extracellular matrix (ECM), and integrin alpha(V)beta(3), which contributes to endothelial cell attachment to the ECM, are critically involved in this process. Recent findings have shown that MMP2 is localized in an active form on the surface of invasive endothelial cells based on its ability to directly bind integrin alpha(V)beta(3), suggesting that disrupting this protein--protein interaction may represent a new target for the development of angiogenesis inhibitors. The screening of small molecule libraries led to the identification of compounds which disrupt the MMP2--alpha(V)beta(3) interaction in an in vitro binding assay. A prototypical inhibitor was further found to prevent the degradation of the protein matrix without directly inhibiting MMP2 activity or disrupting the binding of alpha(V)beta(3) to its classical ECM ligand, vitronectin. The synthesis and screening of analogues and substructures of this lead compound allowed the identification of requisite structural features for inhibition of MMP2 binding to alpha(V)beta(3). This led to the synthesis of a more water-soluble derivative which maintains the in vitro biological properties and has potent antiangiogenic and antitumor activity in vivo, validating the target as one useful for therapeutic intervention.

Integrin alpha(v)beta1 is an adenovirus coreceptor.

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.

Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging.

Viral vectors based on lentiviruses, such as the human immunodeficiency virus, are able to transduce a broad spectrum of nondividing cells in vivo. This ability of lentiviral vectors makes them an attractive vehicle for gene transfer into the liver. In order to determine the requirements for efficient lentiviral gene transfer, we used a fluorescence imaging system, which allows the detection of cells and tissues that express fluorescent reporter genes (e.g., green fluorescence protein) in the living animal. We show that the latest generation of lentiviral vectors efficiently transduces the murine liver. Further analysis demonstrated that neither cell-cycle activation nor division of liver cells is a prerequisite for lentiviral gene transfer in vivo.

Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke.

Vascular endothelial growth factor (VEGF), an angiogenic factor produced in response to ischemic injury, promotes vascular permeability (VP). Evidence is provided that Src kinase regulates VEGF-mediated VP in the brain following stroke and that suppression of Src activity decreases VP thereby minimizing brain injury. Mice lacking pp60c-src are resistant to VEGF-induced VP and show decreased infarct volumes after stroke whereas mice deficient in pp59c-fyn, another Src family member, have normal VEGF-mediated VP and infarct size. Systemic application of a Src-inhibitor given up to six hours following stroke suppressed VP protecting wild-type mice from ischemia-induced brain damage without influencing VEGF expression. This was associated with reduced edema, improved cerebral perfusion and decreased infarct volume 24 hours after injury as measured by magnetic resonance imaging and histological analysis. Thus, Src represents a key intermediate and novel therapeutic target in the pathophysiology of cerebral ischemia where it appears to regulate neuronal damage by influencing VEGF-mediated VP.

Disruption of matrix metalloproteinase 2 binding to integrin alpha vbeta 3 by an organic molecule inhibits angiogenesis and tumor growth in vivo.

Matrix metalloproteinase 2 (MMP2) can associate with integrin alpha(v)beta3 on the surface of endothelial cells, thereby promoting vascular invasion. Here, we describe an organic molecule (TSRI265) selected for its ability to bind to integrin alphav(v)beta3 and block alpha(v)beta3 interaction with MMP2. Although disrupting alpha(v)beta3/MMP2 complex formation, TSRI265 has no effect on alpha(v)beta3 binding to its extracellular matrix ligand vitronectin and does not influence MMP2 activation or catalytic activity directly. However, TSRI265 acts as a potent antiangiogenic agent and thereby blocks tumor growth in vivo. These findings suggest that activated MMP2 does not facilitate vascular invasion during angiogenesis unless it forms a complex with alpha(v)beta(3) on the endothelial cell surface. By disrupting endothelial cell invasion without broadly suppressing cell adhesion or MMP function, the use of compounds such as TSRI265 may provide a novel therapeutic approach for diseases associated with uncontrolled angiogenesis.

Preferential susceptibility of brain tumors to the antiangiogenic effects of an alpha(v) integrin antagonist.

OBJECTIVE: Brain tumors are highly angiogenic, and their growth and spread depend on the generation of new blood vessels. We examined the effect of the cyclic peptide antagonist pentapeptide EMD 121974, an antiangiogenic agent, on orthotopic and heterotopic brain tumor growth. METHODS: The human brain tumor cell lines DAOY (medulloblastoma) and U87 MG (glioblastoma) were injected into either the forebrain (orthotopic) or the subcutis (heterotopic) of nude mice, and daily systemic treatment with the active peptide was initiated after tumors were established. RESULTS: All control animals with orthotopic brain tumors and that received the inactive peptide EMD 135981 daily died as a result of tumor progression within 4 to 6 weeks; tumors measured 3 to 5 mm in diameter. In contrast, mice with orthotopic tumors that were treated daily with the active peptide survived for more than 16 weeks, and histological examination of the brains after 4, 8, and 12 weeks showed either no tumors or microscopic residual tumors. The growth of these brain tumor cells injected simultaneously or separately into the subcutis of nude mice (heterotopic model) was not affected by the active peptide, suggesting that the brain environment is a critical determinant of brain tumor susceptibility to growth inhibition by this pentapeptide. CONCLUSION: The cyclic pentapeptide EMD 121974 may become a treatment option specific to brain tumors. Because of its antiangiogenic effect, its use may be especially indicated after tumors are removed surgically.

beta1 integrin antagonism on adherent, differentiated human neuroblastoma cells triggers an apoptotic signaling pathway.

Integrin receptors mediate several functions including prevention of matrix detachment-induced apoptosis (anoikis) of several adherent cell types. We report here that antagonists of beta1 integrins trigger an apoptotic signaling pathway in adherent differentiated LAN-5 human neuroblastoma cells, a cell line which represents a model system for the study of human neurons. The pathway is characterized by cytochrome c release into the cytoplasm, and activation of caspase-9 and caspase-3, 4-6h after treatment; cleavage products of caspase-8 and caspase-2 were not detectable in the cells. Coordinate inactivation of cell survival pathways, including cleavage of focal adhesion kinase, decreased expression of protein kinase B, and reduced phosphorylation of the pro-apoptotic protein, Bad, also characterized the signaling pathway. These events occurred in adherent cells; DNA fragmentation and detachment followed as late events 18-24h after addition of beta1 integrin antagonists. zDEVD-fmk, an irreversible inhibitor of caspase-3-like enzymes, and cytochalasin D, an actin depolymerizing agent, blocked caspase-3 cleavage and delayed cell death. In contrast to these results, undifferentiated, adherent and dividing LAN-5 cells did not die in response to beta1 integrin antagonists. These studies identify a distinct apoptotic pathway which is triggered by antagonists of beta1 integrins on differentiated adherent neuronal cells.

Alpha(v)beta3 and alpha(v)beta5 integrin expression in meningiomas.

OBJECTIVE: Integrins are emerging as alternative receptors capable of mediating several biological functions, such as cell-matrix and cell-cell adhesion, cell migration, signal transduction, and angiogenesis. Two alpha(v) integrins, i.e., alpha(v)beta3 and alpha(v)beta5, play critical roles in mediating these activities, particularly in tumors. No data are available on the expression of these integrins in meningiomas. METHODS: Using Western blot and immunohistochemical analyses with LM609 and PG32, two monoclonal antibodies capable of recognizing the functional integrin heterodimer, we evaluated the expression of alpha(v)beta3 and alpha(v)beta5 integrins in a series of 34 meningiomas of different histological subtypes and grades. We studied their expression in tumor cells and vasculature, as well as the expression of their related angiogenic factors (fibroblast growth factor 2 and vascular endothelial growth factor) and the alpha(v)beta3 ligand vitronectin. RESULTS: Alpha(v)beta3 and alpha(v)beta5 integrins were expressed by neoplastic vasculature and cells. Alpha(v)beta3 and alpha(v)beta5 expression was associated and correlated with that of their respective growth factors (fibroblast growth factor 2 and vascular endothelial growth factor) and microvessel counts and densities. Alpha(v)beta3 was more strongly expressed than alpha(v)beta5 in two cases of histologically benign meningiomas with aggressive clinical behavior. Alpha(v)beta3 expression was associated with that of its related ligand vitronectin and was also evident in small vessels of brain tissue closely surrounding meningiomas. CONCLUSION: Our data demonstrate the expression of alpha(v)beta3 and alpha(v)beta5 integrins in meningioma cells and vasculature. Our findings suggest a role for both of these integrins, and particularly alpha(v)beta3, in meningioma angiogenesis.

Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2.

Modulation of the balance between pro- and antiangiogenic factors holds great promise for the treatment of a broad spectrum of human disease ranging from ischemic heart disease to cancer. This requires both the identification of angiogenic regulators and their efficient delivery to target organs. Here, we demonstrate the use of a noncatalytic fragment of matrix metalloproteinase 2 (termed PEX) delivered by lentiviral vectors in different angiogenesis models. Transduction of human endothelial cells with PEX virus suppressed endothelial invasion and formation of capillary-like structures without affecting chemotaxis in vitro. Lentiviral delivery of PEX blocked basic fibroblast growth factor-induced matrix metalloproteinase 2 activation and angiogenesis on chicken chorioallantoic membranes. PEX expression also inhibited tumor-induced angiogenesis and tumor growth in a nude mouse model. Thus, our study shows that lentiviral vectors can deliver sufficient quantities of antiangiogenic substances to achieve therapeutic effects in vivo.

Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin alphavbeta3.

Angiogenesis plays a central role in the growth and metastasis of cancers. Strategies aimed at interfering with tumor blood supply offer promise for new cancer therapies. Vitaxin (an anti-alphavbeta3 antibody) interferes with blood vessel formation by inducing apoptosis in newly generated endothelial cells. This Phase I study evaluates the safety and pharmacokinetics of Vitaxin in humans with cancer. Eligible patients demonstrated progressive tumors with stage IV disease and an Eastern Cooperative Oncology Group performance status < or =2. Treatment consisted of six weekly infusions of Vitaxin. Escalating doses from 0.1 and 4.0 mg/kg/week were evaluated based on the expectation that plasma levels would bracket the effective in vitro concentration. Escalation beyond 4 mg/kg/week was limited by drug availability. Adverse events were assessed weekly. Pharmacokinetics were performed weekly through week 9. Clinical response was assessed at week 9. Of 17 patients treated, 14 were evaluable for response. Treatment was well tolerated with little or no toxicity. The most common side effect was infusion-related fever, which could be controlled with prophylactic antipyretics. Doses > or =1 mg/kg/week produced plasma concentrations sufficient to saturate the alphavbeta3 receptor in vitro (25 microg/ml). Vitaxin demonstrated a half-life in excess of 5 days at higher doses with no accumulation over 6 weeks of therapy. One patient demonstrated a partial response, and seven patients demonstrated stable disease. Three patients received Vitaxin beyond the first cycle of therapy. Each of these patients demonstrated disease stabilization that in one case lasted 22 months. At the doses and schedule studied, Vitaxin appears safe and potentially active, suggesting that vascular integrin alphavbeta3 represents a clinically relevant antiangiogenic target for prolonged cancer therapy.

Role of alpha v integrins during angiogenesis.

Angiogenesis depends on specific molecular interactions between vascular cells and components of the extracellular matrix. This review focuses on the recent advances in the understanding of the mechanism of action of integrins and integrin antagonists during angiogenesis. For example, angiogenesis induced with vascular endothelial growth/permeability factor but not with basic fibroblast growth factor (bFGF) depends on integrin avb5 and Src kinase activity. In contrast, bFGF-induced angiogenesis requires integrin avb3 and functions independently of Src. Recent studies document a role for integrins and growth factor regulation of Src family kinases during angiogenesis. We also discuss the effect of av integrin antagonists on angiogenesis during tumor growth, inflammatory disease, and retinopathy and summarize recent clinical progress in using av integrin antagonists.