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Supplementary Methods, Tables 1-3 from Dissecting the Role of Matrix Metalloproteinases (MMP) and Integrin αvβ3 in Angiogenesis In vitro: Absence of Hemopexin C Domain Bioactivity, but Membrane-Type 1-MMP and αvβ3 Are Critical

Supplementary Figure 1 from CYR61 and αVβ5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma

Supplementary Figure 2 from CYR61 and αVβ5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma

Supplementary Figure 3 from CYR61 and αVβ5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin αvβ3–mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti–membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin αvβ3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin αvβ3. Moreover, no specific binding of pro-MMP-2 to integrin αvβ3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin αvβ3, and this in a PEX-independent manner. Likewise, integrin αvβ3–expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and αvβ3- dependent but MMP-2 independent and does not support a role for PEX in αvβ3 integrin binding or in modulating angiogenesis in this system.

Supplementary Methods and Materials from CYR61 and αVβ5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma

Supplementary Table 1 from CYR61 and αVβ5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma

In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society developed a series of 10 webinars to address the issues involved. The webinars were delivered by speakers with both academic and commercial backgrounds. This report highlights the problems, and offers solutions to help the scientific community appropriately identify the right antibodies and to validate them for their research and development projects. Despite the various solutions proposed here, they must be applied on a case-by-case basis. Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly. The various approaches discussed here all have caveats, so a combination of solutions must be considered.

// Michael Weller 1 , Louis Burt Nabors 2 , Thierry Gorlia 3 , Henning Leske 4 , Elisabeth Rushing 4 , Pierre Bady 5, 6, 7 , Christine Hicking 8 , James Perry 9 , Yong-Kil Hong 10 , Patrick Roth 1 , Wolfgang Wick 11, 12 , Simon L. Goodman 8 , Monika E. Hegi 7 , Martin Picard 8 , Holger Moch 13 , Josef Straub 8 , Roger Stupp 14 1 Department of Neurology, University Hospital Zurich and University of Zurich, Zurich, Switzerland 2 University of Alabama at Birmingham, Birmingham, AL, USA 3 EORTC Data Centre, Brussels, Belgium 4 Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland 5 Department of Education and Research, University of Lausanne, Lausanne, Switzerland 6 SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland 7 Department of Clinical Neurosciences, University Hospital Lausanne, Lausanne, Switzerland 8 Department of Translational and Biomarkers Research, Oncology, Merck KGaA, Darmstadt, Germany 9 Sunnybrook Health Sciences Centre, Toronto, ON, Canada 10 The Catholic University of Korea, Seoul St. Mary’s Hospital, Seoul, Korea 11 Neurology Clinic, University of Heidelberg, Heidelberg, Germany 12 Clinical Cooperation Unit (CCU) Neurooncology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany 13 Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland 14 Department of Oncology, University Hospital Zurich, Zurich, Switzerland Correspondence to: Michael Weller, e-mail: michael.weller@usz.ch Keywords: glioblastoma, integrin, pSmad, TGF-β, biomarker Received: January 03, 2016 Accepted: January 29, 2016 Published: February 22, 2016 ABSTRACT Integrins αvβ3 and αvβ5 regulate angiogenesis and invasiveness in cancer, potentially by modulating activation of the transforming growth factor (TGF)-β pathway. The randomized phase III CENTRIC and phase II CORE trials explored the integrin inhibitor cilengitide in patients with newly diagnosed glioblastoma with versus without O 6 -methylguanine DNA methyltransferase ( MGMT ) promoter methylation. These trials failed to meet their primary endpoints. Immunohistochemistry was used to assess the levels of the target integrins of cilengitide, αvβ3 and αvβ5 integrins, of αvβ8 and of their putative target, phosphorylation of SMAD2, in tumor tissues from CENTRIC (n=274) and CORE (n=224). αvβ3 and αvβ5 expression correlated well in tumor and endothelial cells, but showed little association with αvβ8 or pSMAD2 levels. In CENTRIC, there was no interaction between the biomarkers and treatment for prediction of outcome. In CORE, higher αvβ3 levels in tumor cells were associated with improved progression-free survival by central review and with improved overall survival in patients treated with cilengitide. Integrins αvβ3, αvβ5 and αvβ8 are differentially expressed in glioblastoma. Integrin levels do not correlate with the activation level of the canonical TGF-β pathway. αvβ3 integrin expression may predict benefit from integrin inhibition in patients with glioblastoma lacking MGMT promoter methylation.

The biological literature reverberates with the inadequacies of commercial research-tool antibodies. The scientific community spends some $2 billion per year on such reagents. Excellent accessible scientific platforms exist for reliably making, validating and using antibodies, yet the laboratory end-user reality is somehow depressing – because they often “don’t work”. This experience is due to a bizarre and variegated spectrum of causes including: inadequately identified antibodies; inappropriate user and supplier validation; poor user training; and overloaded publishers. Colourful as this may appear, the outcomes for the community are uniformly grim, including badly damaged scientific careers, wasted public funding, and contaminated literature. As antibodies are amongst the most important of everyday reagents in cell biology and biochemistry, I have tried here to gently suggest a few possible solutions, including: a move towards using recombinant antibodies; obligatory unique identification of antibodies, their immunogens, and their producers; centralized international banking of standard antibodies and their ligands; routine, accessible open-source documentation of user experience with antibodies; and antibody-user certification.

Transforming growth factor-β is a central mediator of the malignant phenotype of glioblastoma, the most common and malignant form of intrinsic brain tumours. Transforming growth factor-β promotes invasiveness and angiogenesis, maintains cancer cell stemness and induces profound immunosuppression in the host. Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation, differentiation and motility, and may be involved in the activation of transforming growth factor-β. We report that αvβ3, αvβ5 and αvβ8 integrins are broadly expressed not only in glioblastoma blood vessels but also in tumour cells. Exposure to αv, β3 or β5 neutralizing antibodies, RNA interference-mediated integrin gene silencing or pharmacological integrin inhibition using the cyclic RGD peptide EMD 121974 (cilengitide) results in reduced phosphorylation of Smad2 in most glioma cell lines, including glioma-initiating cell lines and reduced transforming growth factor-β-mediated reporter gene activity, coinciding with reduced transforming growth factor-β protein levels in the supernatant. Time course experiments indicated that the loss of transforming growth factor-β bioactivity due to integrin inhibition likely results from two distinct mechanisms: an early effect on activation of preformed inactive protein, and second, major effect on transforming growth factor-β gene transcription as confirmed by decreased activity of the transforming growth factor-β gene promoter and decreased transforming growth factor-β(1) and transforming growth factor-β(2) messenger RNA expression levels. In vivo, EMD 121974 (cilengitide), which is currently in late clinical development as an antiangiogenic agent in newly diagnosed glioblastoma, was a weak antagonist of pSmad2 phosphorylation. These results validate integrin inhibition as a promising strategy not only to inhibit angiogenesis, but also to block transforming growth factor-β-controlled features of malignancy including invasiveness, stemness and immunosuppression in human glioblastoma.

Integrins are transmembrane receptors that are central to the biology of many human pathologies. Classically mediating cell-extracellular matrix and cell-cell interaction, and with an emerging role as local activators of TGFβ, they influence cancer, fibrosis, thrombosis and inflammation. Their ligand binding and some regulatory sites are extracellular and sensitive to pharmacological intervention, as proven by the clinical success of seven drugs targeting them. The six drugs on the market in 2016 generated revenues of some US$3.5 billion, mainly from inhibitors of α4-series integrins. In this review we examine the current developments in integrin therapeutics, especially in cancer, and comment on the health economic implications of these developments.

Integrins are pivotal in cancer biology and are putative candidates for cancer therapy. The investigation of integrins has been hampered by the lack of antibodies suitable for formalin-fixed and paraffin-embedded (FFPE) tissue specimens. Here, we validated monoclonal rabbit antibodies (RabMAbs) against integrins αvβ3 (EM22703) and αvβ5 (EM09902) with murine monoclonal antibodies (MuMabs) LM609 (against αvβ3) and P1F6 (against αvβ5), in immunohistochemistry. Immunostaining was performed on sections of matching unfixed, cryoconserved (CC) and FFPE tissue from 19 colorectal, 20 lung, 17 breast, and 9 ovarian carcinomas. Sections were stained with LM609 and P1F6 and compared with the immunoreactions of the RabMAbs. The degree of concordance was assigned for staining patterns and intensity. Concordance between MuMAbs and RabMAbs ranged from weak, for anti-αvβ5 antibodies, to nearly complete for anti-αvβ3 antibodies. We confirmed that MuMAbs LM609 and P1F6 bound very weakly in FFPE tissue and no staining was seen. By contrast, RabMAbs EM22703 and EM09902 generally showed a high degree of agreement in staining patterns of CC and FFPE tissue. In summary, the RabMAbs had overlapping staining patterns that were generally more intense for CC when compared with FFPE material. This study suggests that EM22703 and EM09902 staining closely matches the staining of standard MuMAbs and also does so in archival FFPE tissue.

The International Antibody Validation meetings offer a welcome British forum for discussing this important topic, which is existentially crucial for the biological sciences community. Now in its 6th year, the biennial meeting is organized by Andrew Chalmers (University of Bath; CiteAb), this year with Carly Dix (Astra Zeneca). The organizers gathered some 100 members of industry and academia, producers and users, for a day and a half to describe their efforts to ensure that their antibodies have the desired specificity and selectively for well-defined molecular targets. The meeting is largely available as WebCasts (http://www.antibodyvalidation.co.uk/past-events/2018).

The aim of this study was to investigate the expression of integrins, their ligands, and integrin signaling-related molecules in a cohort of human primary non-small cell lung cancers (NSCLC). Formalin-fixed and paraffin-embedded tissue samples from 215 NSCLC were immunohistochemically stained using antibodies directed against αvβ3, αvβ5, αvβ6, αvβ8, αv, osteopontin, fibronectin, vitronectin, epidermal growth factor (EGFR), vascular endothelial growth factor receptor (VEGFR), and Ki67. Immunostaining of tumor, stroma, and endothelial cells was evaluated separately by quantity and intensity (tumor cells) or intensity (stroma and endothelial cells) expressed in an immunoreactivity score. We studied correlations between the staining patterns of the different markers and of marker expression with clinicopathological data and patient survival. In the majority of NSCLC, each marker was expressed in at least one tumor component. As expected, αv and αv integrin heterodimers were significantly co-expressed, as were integrins and EGFR. Vitronectin was expressed significantly more often in smaller (T-category) and in well-differentiated tumors; Ki67 index was higher in larger (T-category) and in poorly differentiated tumors. No significant correlation was found between any marker expression and gender, venous invasion, lymph vessel invasion, lymph node metastasis, or survival. Although integrin expression does not seem to be associated with indicators of progression of NSCLC, the expression of αvβ3 in 89 % and αvβ5 in 100 % of NSCLC is novel and merits to be further investigated.

To determine whether metastasis to brain is associated with altered expression patterns of integrins, we investigated the expression of αvβ3, αvβ5, αvβ6 and αvβ8 integrins in primary malignancies and metastases to brain of breast, lung and renal carcinomas and in malignant melanoma. Inhibitors of αv integrins are currently in clinical trials for glioblastoma. The role of integrins in the process of brain metastasis from other human tumors is unknown. Immunohistochemistry with novel integrin subtype specific rabbit monoclonal antibodies was performed on tissue microarrays of archival material of surgical biopsies taken from primary tumors and brain metastases. Integrin αvβ3 expression was increased in brain metastases compared to primary tumors of breast adenocarcinoma, non-small cell lung cancer, renal clear cell cancer and malignant cutaneous melanoma (all p

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)-α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.

Integrins are a large family of molecules that are central regulators in multicellular biology. They orchestrate cell-cell and cell-extracellular matrix (ECM) adhesive interactions from embryonic development to mature tissue function. Diverse human pathologies involve integrin adhesion, including thrombotic diseases, inflammation, cancer, fibrosis and infectious diseases. Integrins are exciting pharmacological targets because they are exposed on the cell surface and are sensitive to pharmacological blockade, but the scale of current efforts involving integrin therapeutics continues to surprise. Several therapeutics targeting integrins are effective drugs: five have been approved for use in clinic, with combined sales of over $1.5 billion in 2010 (based on company reports from that year). We gathered information from three major drug-trial databases and found that ∼260 anti-integrin drugs have entered clinical trials. Here we overview integrins as drug targets and focus on cancer.

The aim of this study was to investigate the effect of inhibiting avb3/avb5 integrins by cilengitide in experimentally induced breast cancer bone metastases using noninvasive imaging techniques. For this purpose, nude rats bearing established breast cancer bone metastases were treated with cilengitide, a small molecule inhibitor of avb3 and avb5 integrins (75 mg/kg, five days per week; n 5 12 rats) and compared to vehicle-treated control rats (n 5 12). In a longitudinal study, conventional magnetic resonance imaging (MRI) and flat panel volumetric computed tomography were used to assess the volume of the soft tissue tumor and osteolysis, respectively, and dynamic contrast-enhanced (DCE-) MRI was performed to determine functional parameters of the tumor vasculature reflecting blood volume and blood vessel permeability. In rats treated with cilengitide, VCT and MRI showed that osteolytic lesions and the respective bone metastatic soft tissue tumors progressed more slowly than in vehicle-treated controls. DCE-MRI indicated a decrease in blood volume and an increase in vessel permeability and immunohistology revealed increased numbers of immature vessels in cilengitide-treated rats compared to vehicle controls. In conclusion, treatment of experimental breast cancer bone metastases with cilengitide resulted in pronounced antiresorptive and antitumor effects, suggesting that avb3/avb5 inhibition may be a promising therapeutic approach for bone metastases. Bone metastases occur frequently in many human malignancies including breast, prostate and lung carcinoma. The stimulation of osteoclasts by tumor cells proliferating within the bone marrow is a feature of the pathogenesis of bone metastases, and both the tumor and the bone microenvironment

We determined the crystal structure of 1TM-alphaVbeta3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the alphaV and beta3 subunits. 1TM-alphaVbeta3 is more compact and less active in solution when compared with DeltaTM-alphaVbeta3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the alpha-beta interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length alphaVbeta3 on live cells yields a donor-membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2-thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state.

We investigated whether cilengitide could amplify the antitumor effects of radiotherapy in an orthotopic rat glioma xenograft model. Cilengitide is a specific inhibitor of alphav series integrins, and acts as an antiangiogenic. U251 human glioma cells express alphavbeta3 and alphavbeta5 integrins. We used in vitro assays of adhesion and growth of tumor and endothelial cells to evaluate cytotoxicity and the potential for cilengitide to enhance radiation toxicity. Treatment was then evaluated in an orthotopic model to evaluate synergy with therapeutic radiation in vivo. In vitro, cilengitide blocked cell adhesion, but did not influence the effects of radiation on U251 cells; cilengitide strongly amplified radiation effects on endothelial cell survival. In vivo, radiotherapy prolonged the survival of U251 tumor-bearing rats from 50 to over 110 days. Cotreatment with cilengitide and radiation dramatically amplified the effects of radiation, producing survival over 200 days and triggering an enhanced apoptotic response and suppression of tumor growth by histology at necropsy. Signaling pathways activated in the tumor included NFkappab, a documented mediator of cellular response to radiation. Because cilengitide has a short plasma half-life (t((1/2)) approximately 20 min), antiangiogenic scheduling typically uses daily injections. We found that a single dose of cilengitide (4 mg/kg) given between 4 and 12 hr prior to radiation was sufficient to produce the same effect. Our results demonstrate that blockade of alphav integrins mediates an unanticipated rapid potentiation of radiation, and suggests possible clinical translation for glioma therapy.

Abundant preclinical and indirect clinical data have for several decades convincingly supported the notion that anti-angiogenesis is an effective strategy for the inhibition of tumor growth. The recent success achieved in patients with metastatic colon carcinoma using a neutralizing antibody directed against vascular endothelial growth factor (VEGF) has translated preclinical optimism into a clinical reality.With this transformation in the field of angiogenesis has come a need for reliable surrogate markers. A surrogate marker by definition serves as a substitute for the underlying process in question, and in the case of angiogenesis, microvessel density (usually in so-called "hot-spots") has until now been the most widely used parameter. However, this parameter is more akin to a static "snap-shot" and does not lend itself either to the dynamic in situ assessment of the status of the tumor microvasculature or to the molecular factors that regulate its growth and involution. This has led to an acute need for developing circulating and imaging markers of angiogenesis that can be monitored in vivo at repeated intervals in large number of patients with a variety of tumors in a non-invasive manner. Such markers of angiogenesis are the subject of this review.

Radiotherapy is widely used to treat human cancer. Patients locally recurring after radiotherapy, however, have increased risk of metastatic progression and poor prognosis. The clinical management of postradiation recurrences remains an unresolved issue. Tumors growing in preirradiated tissues have an increased fraction of hypoxic cells and are more metastatic, a condition known as tumor bed effect. The transcription factor hypoxia inducible factor (HIF)-1 promotes invasion and metastasis of hypoxic tumors, but its role in the tumor bed effect has not been reported. Here, we show that tumor cells derived from SCCVII and HCT116 tumors growing in a preirradiated bed, or selected in vitro through repeated cycles of severe hypoxia, retain invasive and metastatic capacities when returned to normoxia. HIF activity, although facilitating metastatic spreading of tumors growing in a preirradiated bed, is not essential. Through gene expression profiling and gain- and loss-of-function experiments, we identified the matricellular protein CYR61 and αVβ5 integrin as proteins cooperating to mediate these effects. The anti-αV integrin monoclonal antibody 17E6 and the small molecular αVβ3/αVβ5 integrin inhibitor EMD121974 suppressed invasion and metastasis induced by CYR61 and attenuated metastasis of tumors growing within a preirradiated field. These results represent a conceptual advance to the understanding of the tumor bed effect and identify CYR61 and αVβ5 integrin as proteins that cooperate to mediate metastasis. They also identify αV integrin inhibition as a potential therapeutic approach for preventing metastasis in patients at risk for postradiation recurrences. [Cancer Res 2008;68(18):7323–31]

Background & Aims: Integrin αvβ6 is highly expressed on certain activated epithelia, where it mediates attachment to fibronectin and serves as coreceptor for the activation of latent transforming growth factor (TGF)-β1. Because its role in liver fibrosis is unknown, we studied αvβ6 function in vitro and explored the antifibrotic potential of the specific αvβ6 antagonist EMD527040. Methods: Experimental liver fibrosis was studied in rats after bile duct ligation (BDL) and in Mdr2(abcb4)−/− mice. Different doses of EMD527040 were given to rats from week 2 to 6 after BDL and to Mdr2−/− mice from week 4 to 8. Liver collagen was quantified, and expression of αvβ6 and fibrosis-related transcripts was determined by quantitative reverse-transcription polymerase chain reaction. αvβ6-expressing cells, bile duct proliferation, and apoptosis were assessed histologically. The effect of EMD527040 on cholangiocyte adhesion, proliferation, apoptosis, and TGF-β1 activation was studied in vitro. Results: αvβ6 was highly expressed on proliferating bile duct epithelia in fibrosis, with 100-fold increased transcript levels in advanced fibrosis. EMD527040 attenuated bile ductular proliferation and peribiliary collagen deposition by 40%–50%, induced down-regulation of fibrogenic and up-regulation of fibrolytic genes, and improved liver architecture and function. In vitro αvβ6 inhibition reduced activated cholangiocyte proliferation, their adhesion to fibronectin, and endogenous activation of TGF-β1 by 50% but did not affect bile duct apoptosis. Conclusions: Integrin αvβ6 is strongly up-regulated in proliferating bile duct epithelia and drives fibrogenesis via adhesion to fibronectin and auto/paracrine TGF-β1 activation. Pharmacologic inhibition of αvβ6 potently inhibits the progression of primary and secondary biliary fibrosis.

Activated hepatic stellate cells (HSC) play a central role in the development of liver fibrosis. Platelet-derived growth factor (PDGF)-BB and the integrin alphavbeta3 mediate mesenchymal cell migration and proliferation. However, their contribution and interaction during fibrogenic activation of HSC remains unclear. To this aim we investigated if PDFGF-BB and alphavbeta3 interact, and how far small molecular inhibitors of alphavbeta3 modulate PDGF-BB and serum-induced migration, proliferation and fibrogenic activation of HSC.Rat and human HSC were subjected to migration and proliferation assays in the presence or absence of a peptide or a nonpeptide alphavbeta3 inhibitor. Activation of mitogen-activated protein kinases (ERK1/2, p38), Akt, focal adhesion kinase (FAK), paxillin and beta3 integrin was evaluated by phospho-specific Western blotting. Fibrosis related transcripts were determined by quantitative real-time PCR.PDGF-BB-stimulated HSC migration which was blocked dose-dependently by the alphavbeta3 antagonists, with complete inhibition at 10(-6)M. alphavbeta3 blockage did not affect cell viability or proliferation, while it decreased phosphorylation of FAK, paxillin, beta3 integrin and p38, but not of ERK1/2 or Akt. alphavbeta3 inhibition led to downregulation of certain profibrogenic transcripts, while it upregulated fibrolytic MMP-13 mRNA.Inhibition of integrin alphavbeta3 leads to abrogation of migration of HSC stimulated with PDGF-BB and to an antifibrogenic gene expression pattern.

International audience; Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.Oncogene (2007) 26, 5722-5732; doi:10.1038/sj.onc.1210354; published online 19 March 2007.

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.

Integrins are alphabeta heterodimeric cell surface receptors that mediate transmembrane signaling by binding extracellular and cytoplasmic ligands. The ectodomain of integrin alphaVbeta3 crystallizes in a bent, genuflexed conformation considered to be inactive (unable to bind physiological ligands in solution) unless it is fully extended by activating stimuli. We generated a stable, soluble complex of the Mn(2+)-bound alphaVbeta3 ectodomain with a fragment of fibronectin (FN) containing type III domains 7 to 10 and the EDB domain (FN7-EDB-10). Transmission electron microscopy and single particle image analysis were used to determine the three-dimensional structure of this complex. Most alphaVbeta3 particles, whether unliganded or FN-bound, displayed compact, triangular shapes. A difference map comparing ligand-free and FN-bound alphaVbeta3 revealed density that could accommodate the RGD-containing FN10 in proximity to the ligand-binding site of beta3, with FN9 just adjacent to the synergy site binding region of alphaV. We conclude that the ectodomain of alphaVbeta3 manifests a bent conformation that is capable of stably binding a physiological ligand in solution.

Aims To analyse the expression of alphavbeta6, an epithelial integrin involved in wound healing and tumorigenesis, in various human carcinoma types. Methods and results A new monoclonal antibody to the human beta6 subunit, 5C4, was used to locate alphavbeta6 in 157 cancers of gastroenteropancreatic and 21 of lung origin. The data were validated by analysis of alphavbeta6 extracted from histological sections. Alphavbeta6 integrin showed strongest expression in 34 pancreatic ductal adenocarcinomas (mean score 2.88 +/- 0.52), followed by 24 intestinal-type gastric carcinomas (1.45 +/- 1.06) and eight lung adenocarcinomas (1.37 +/- 1.1). Moderate expression was found in 31 diffuse-type gastric carcinomas (0.94 +/- 0.83), seven duodenal adenocarcinomas (0.8 +/- 1.34) and 26 colorectal adenocarcinomas (0.76 +/- 0.71). Little alphavbeta6 was seen in seven liver cell carcinomas and six neuroendocrine tumours. Well-differentiated carcinomas expressed more beta6 than poorly differentiated tumours. Peritumoral epithelial tissues where alphavbeta6-expressing tumours arose also expressed alphavbeta6. There was no correlation between expression of alphavbeta6 and its ligands tenascin and fibronectin in pancreatic and gastric carcinomas. Spheroid formation by pancreatic carcinoma cell lines led to alphavbeta6 up-regulation, but appeared independent of classical ligand binding to alphavbeta6. Conclusions Our findings indicate that: (i) alphavbeta6 is overexpressed in pancreatic adenocarcinomas; (ii) alphavbeta6-positive carcinomas originate from alphavbeta6-expressing tissues; (iii) alphavbeta6 expression in tumours seems to be regulated independently from that of its ligands tenascin and fibronectin; and (iv) in-vitro overexpression of alphavbeta6 in pancreatic carcinoma cell lines accompanies spheroid formation.

Although angiogenesis is believed to require cell-extracellular matrix interactions which are mediated in part via integrins alphav beta 3 and alphav beta 5, a formal demonstration that alphav beta 3 and alphav beta 5 are involved in endothelial-cell invasion and capillary-like tube formation is still required. This has arisen from the cellular complexities which occur in vivo and the difficulty in finding appropriate in vitro model systems. Here we have used a three-dimensional assay which employs bovine aortic and microvascular endothelial cells, to show that alphav beta 3 and alphav beta 5 regulate angiogenesis in vitro. We cloned and characterized 350-450 bp regions of the bovine homologues of alphav, beta 3 and beta 5, covering much of the beta -propeller and A-domain regions, and show that they are >95% identical to their human orthologues. We used cyclic peptides EMD 121974, 85189 and 66203, which selectively inhibit alphav beta 3 and alphav beta 5, but not gpIIbIIIa or alpha5 beta 1, to probe in vitro angiogenesis induced by angiogenic cytokines in three-dimensional fibrin or collagen gels. We found that these peptides are potent inhibitors of endothelial cell invasion and differentiation induced by vascular endothelial growth factor-A or fibroblast growth factor-2 but do not affect the unstimulated cells in 3D culture. Inhibition was greatest when cells were grown on fibrin, but also occurred on collagen I which is not a recognized ligand for alphav beta 3. These findings demonstrate the requirement for endothelial cell alphav beta 3 and alphav beta 5 integrins during angiogenesis in vitro, and are in accord with the proposed therapeutic application of alphav beta 3 and alphav beta 5 antagonists.

Integrins are αβ heterodimeric cell-surface receptors that are vital to the survival and function of nucleated cells. They recognize aspartic-acid- or a glutamic-acid-based sequence motifs in structurally diverse ligands. Integrin recognition of most ligands is divalent cation dependent and conformationally sensitive. In addition to this common property, there is an underlying binding specificity between integrins and ligands for which there has been no structural basis. The recently reported crystal structures of the extracellular segment of an integrin in its unliganded state and in complex with a prototypical Arg–Gly–Asp (RGD) ligand have provided an atomic basis for cation-mediated binding of aspartic-acid-based ligands to integrins. They also serve as a basis for modelling other integrins in complex with larger physiologic ligands. These models provide new insights into the molecular basis for ligand binding specificity in integrins and its regulation by activation-driven tertiary and quaternary changes.

The structural basis for the divalent cation-dependent binding of heterodimeric alphabeta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alphaV and beta3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in betaA, the ligand-binding domain of beta3; in the complex, betaA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alphaV relative to beta3.

Background We investigated the expression of two αv integrins, αvβ3 and αvβ5, in gastric cancer (GC) by testing the following hypotheses: that these molecules are expressed in GC; that they are implicated in GC biology; that they help to distinguish between the two major histological subtypes of GC, according to Laurén; and that they are prognostically relevant. Methods Formalin-fixed and paraffin-embedded tissue samples from 482 GC samples were stained immunohistochemically using rabbit monoclonal antibodies directed against αvβ3 (EM22703) and αvβ5 (EM09902). Immunostaining of tumor, stroma, and endothelial cells was evaluated separately by the quantity and intensity, generating an immunoreactivity score. The immunoreactivity score of both antibodies was correlated with clinicopathology data and patient survival. Results Each integrin was expressed in at least one tumor component in all GCs. Both were expressed significantly more often in the intestinal phenotype according to Laurén. Moreover, patients who grouped as “positive” for expression of αvβ3 on endothelial cells, and patients with an intestinal type GC, grouped as “negative” for expression of αvβ5 on stroma cells, had significantly longer survival. The expression of αvβ5 on stroma cells was confirmed to be an independent prognostic factor of intestinal-type GC. Conclusion The expression of αvβ3 and αvβ5 in at least one tumor component in all GC samples is an interesting new result that should form a basis for further investigations; for example, regarding selective integrin antagonists and the value of αvβ3 and αvβ5 as putative prognostic biomarkers. Moreover, both markers might be helpful in the routine classification of GC subtypes. Electronic supplementary material The online version of this article (doi:10.1007/s10120-014-0435-2) contains supplementary material, which is available to authorized users.

The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.

Integrins are transmembranous adhesion molecules postulated to be involved in the brain metastatic cascade. We investigated the correlation of alpha v beta 3 (αvβ3), alpha v beta 5 (αvβ5) and alpha v beta 6 (αvβ6) integrin isoform expression with clinical characteristics including survival times in lung cancer patients with brain metastases (BM). All BM from lung cancer operated at our institution between 1990 and 2011, were identified; where available, primary tumors were retrieved as well. Immunohistochemical analysis for αvβ3, αvβ5 and αvβ6 integrin subunits was performed and correlated with Ki67 and hypoxia-inducible factor (HIF)-1α indexes. Clinical data including survival data were obtained by chart review. 191 BM specimens of 191 patients with histologically confirmed lung cancer (172 non-small cell lung cancer and 19 small cell lung cancer) were included. In 18 patients matched primary tumor samples were available. αvβ6 expression was commonly found on BM tumor cells (103/191; 53.9 %) and showed a significant association with low Ki67 proliferation indices (46 vs. 36 %, p = 0.001, Mann-Whitney U test) and favorable survival times (p = 0.020; log rank test) in patients with non-squamous NSCLC BM. αvβ5 expression was highly expressed on vascular structures (167/191; 87.4 %) and tumor stroma in BM (151/191; 79.1 %) and associated with high HIF-1α indices (60 vs. 90, p = 0.007, Mann-Whitney U test). αvβ3 expression was more frequently found on vascular structures in BM than in primary tumors (68.1 vs. 5.6 %; p = 0.645; Chi square test) and its expression in BM tumor cells correlated with low Ki67 indices (41 vs. 28 %; p = 0.046, Mann-Whitney U test). Expression of αv integrin subunits seem to be of pathobiological and clinical relevance in patients with NSCLC BM. Further investigations of their involvement in the brain metastatic cascade and their role as biomarkers are warranted.

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVβ3 bound to a physiologic ligand, the tenth type III RGD domain of wild-type fibronectin (wtFN10), or to a high-affinity mutant (hFN10) shown here to act as a pure antagonist. Comparison of these structures revealed a central π-π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the β3 subunit that blocked conformational changes triggered by wtFN10 and trapped hFN10-bound αVβ3 in an inactive conformation. Removing the Trp1496 or Tyr122 side chains or reorienting Trp1496 away from Tyr122 converted hFN10 into a partial agonist. These findings offer new insights into the mechanism of integrin activation and a basis for the design of RGD-based pure antagonists.

The synthesis of a series of RGD mimetics is described. All compounds consist of a central 2,5-disubstituted tetrahydrofuran core, a variable linker to a guanidino group, and a beta-amino alanine unit to mimic the carboxylic acid. Three types of linkers were investigated: a simple four-atom methylene chain (type A, compounds 14, 15, 16, and 17), a four-atom methylene chain with an additional chiral center, and a nitrogen substituent (type B, compounds 38, 39, and 40), and an amide linker of different length with an additional chiral center (type C, compounds 59, 60, 61, and 62). A variety of compounds were tested as potential integrin antagonists in a receptor binding assay (alphaIIbbeta3, alphavbeta3, and alphavbeta5). The relative and absolute configuration of the chiral centers at the THF ring had a pronounced effect on the binding activity and selectivity. Compound 14 proved to be a selective inhibitor of alphaIIbbeta3 (IC50=20nM), whereas compound 40 exhibited high activity for binding of alphaIIbbeta3 (IC50=67nM) and alphavbeta3 (IC50=52nM).

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.

Integrin alphavbeta3 (vitronectin receptor) has been implicated in human malignant melanoma progression and angiogenesis as a receptor that provides survival signals. However, little is known about the therapeutic potential of antagonists of alphavbeta3. In this report, we characterize the activities of 2 antagonists of alphavbeta3 integrins: a human specific monoclonal antibody (MAb), 17E6, and a cyclic RGD peptide that blocked cell adhesion and induced detachment of previously substrate-attached cells in vitro. In vivo, alphavbeta3 antagonists behaved as anti-tumor drugs in a dose- and time-dependent manner. Moreover, different therapeutic treatments proved to be effective even in the therapy of established macroscopic tumor masses, thus supporting the use of these antagonists in clinical therapy. Using a panel of 6 human melanomas and 5 carcinomas, MAb 17E6 efficiently blocked the in vivo tumor growth of melanomas expressing alphavbeta3 as xenografts but did not affect the alphavbeta3-negative (although alphav integrin-positive) tumors. This demonstrated that alphavbeta3 is a pivotal integrin for the growth of human melanomas. Furthermore, since MAb 17E6 does not recognize murine alphavbeta3, the effect is due only to the direct anti-tumor activity and not to the well-known anti-angiogenic activity of alphav-integrin antagonists. Taken together, our results confirm the essential role of alphavbeta3 integrin in the growth of human malignant melanoma in vivo and provide strong evidence of the therapeutic potential of alphav-integrin antagonists for the treatment of such tumors.

The synthesis of a series of RGD mimetics is described. All compounds have a 1,4:3,6-dianhydrohexitol core, a variable linker to a guanidino group, and a serine ether to mimic the carboxylic acid. Two types of linkers were combined with 1,4:3,6-dianhydro-D-sorbitol (1 – 4) and with 1,4:3,6-dianhydro-L-iditol (5). The five compounds were tested as potential integrin antagonists in a receptor binding assay (αIIbβ3, αvβ3, and αvβ5 type). Receptor binding activities in the μmol range were observed.

Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including alphav beta6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin alphav beta6. We compared the recognition profiles of recombinant alphav beta6 and alphav beta3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to alphav beta3 contained ubiquitous RGD sequences. However, on alphav beta6, in addition to RGD- containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to alphav beta6 in isolated receptor assays (IC50 = 20 nM), and in cell adhesion assays (IC50 = 50 microM). DLXXL peptides were highly specific inhibitors of alphav beta6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH2- and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with alphav beta6. The DLXXL sequence is present in several matrix components and in the beta chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin alphav beta6 which has been unrecognized previously.