Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction. Chen, M; Zhang, Y; Yu, VC; Chong, YS; Yoshioka, T; Ge, R Cell death and differentiation
21
797-810
2014
Show Abstract
Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (Kd=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent. | | 24464222
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New blocking antibodies impede adhesion, migration and survival of ovarian cancer cells, highlighting MFGE8 as a potential therapeutic target of human ovarian carcinoma. Tibaldi, L; Leyman, S; Nicolas, A; Notebaert, S; Dewulf, M; Ngo, TH; Zuany-Amorim, C; Amzallag, N; Bernard-Pierrot, I; Sastre-Garau, X; Théry, C PloS one
8
e72708
2013
Show Abstract
Milk Fat Globule--EGF--factor VIII (MFGE8), also called lactadherin, is a secreted protein, which binds extracellularly to phosphatidylserine and to αvβ3 and αvβ5 integrins. On human and mouse cells expressing these integrins, such as endothelial cells, phagocytes and some tumors, MFGE8/lactadherin has been shown to promote survival, epithelial to mesenchymal transition and phagocytosis. A protumoral function of MFGE8 has consequently been documented for a few types of human cancers, including melanoma, a subtype of breast cancers, and bladder carcinoma. Inhibiting the functions of MFGE8 could thus represent a new type of therapy for human cancers. Here, we show by immunohistochemistry on a collection of human ovarian cancers that MFGE8 is overexpressed in 45% of these tumors, and we confirm that it is specifically overexpressed in the triple-negative subtype of human breast cancers. We have established new in vitro assays to measure the effect of MFGE8 on survival, adhesion and migration of human ovarian and triple-negative breast cancer cell lines. Using these assays, we could identify new MFGE8-specific monoclonal antibodies, which efficiently blocked these three tumor-promoting effects of MFGE8. Our results suggest future use of MFGE8-blocking antibodies as new anti-cancer therapeutics in subgroups of ovarian carcinoma, and triple-negative breast carcinoma patients. | Fluorescence Activated Cell Sorting (FACS) | 23977342
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Dual inhibition of αV integrins and Src kinase activity as a combination therapy strategy for colorectal cancer. Jia, J; Starodub, A; Cushman, I; Liu, Y; Marshall, DJ; Hurwitz, HI; Nixon, AB Anti-cancer drugs
24
237-50
2013
Show Abstract
Both Src and αV integrins are important for tumor growth and angiogenesis. They are interconnected and responsible for important features of the tumor phenotype including invasiveness, metastasis, angiogenesis, and resistance to apoptosis. This study examines whether combinational inhibition of both integrin and Src pathways would exert greater antiangiogenesis and antitumor effects than either pathway alone. Using in-vitro cell culture systems, the activity of CNTO95 (Intetumumab), an αV integrin inhibitor, and dasatinib, an Src inhibitor, on proliferation, adhesion, and migration was evaluated in colon cancer cell lines, HCT-116 and RKO, as well as HUVEC cells. The antiangiogenic effect of this combinatory regimen was also tested using an in-vitro tubular network formation assay. The effects of CNTO95 and dasatinib on the activation of Src and integrin pathway signal transduction were also determined by western blotting. The combination of CNTO95 plus dasatinib inhibited adhesion, migration, and paxillin phosphorylation in both HCT-116 and RKO cells. CNTO95 and dasatinib also led to increased apoptosis of HCT-116 cells; however, similar effects were not observed in RKO cells. In addition, dual treatment of CNTO95 and dasatinib exerted enhanced effects on HUVEC cell proliferation, invasion, tubular network formation, and paxillin phosphorylation. In conclusion, our results suggest that concurrent inhibition of both the integrin and the Src pathways exert more pronounced antiangiogenic and antitumor effects than with either pathway being inhibited alone. | | 23275294
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Synstatin: a selective inhibitor of the syndecan-1-coupled IGF1R-αvβ3 integrin complex in tumorigenesis and angiogenesis. Rapraeger, Alan C FEBS J., (2013)
2013
Show Abstract
The syndecans are a family of heparan-sulfate-decorated cell surface proteoglycans, matrix receptors with roles in cell adhesion and growth factor signaling. Their heparan sulfate chains recognize "heparin-binding" motifs ubiquitously present in the extracellular matrix (ECM), providing the means for syndecans to constitutively bind and cluster to sites of cell-matrix adhesion. Emerging evidence suggests that specialized docking sites in the syndecan extracellular domains may serve to localize other receptors to these sites as well, including integrins and growth factor receptor tyrosine kinases. A prototypic example of this mechanism is the capture of the αvβ3 integrin and insulin-like growth factor-1 receptor (IGF1R) by syndecan-1 (Sdc1) - forming a ternary receptor complex in which signaling downstream of IGF1R activates the integrin. This Sdc1-coupled ternary receptor complex is especially prevalent on tumor cells and activated endothelial cells undergoing angiogenesis, reflecting the upregulated expression of αvβ3 integrin in such cells. As such, much effort has focused on developing therapeutics that target this integrin in various cancers. Along these lines, the site in the Sdc1 ectodomain responsible for capture and activation of the αvβ3 or αvβ5 integrins by IGF1R can be mimicked by a short peptide called "synstatin" (SSTN), which competitively displaces the integrin and IGF1R kinase from the syndecan and inactivates the complex. This review summarizes our current knowledge of the Sdc1-coupled ternary receptor complex and the efficacy of SSTN as an emerging therapeutic to target this signaling mechanism. © 2013 The Authors Journal compilation © 2013 FEBS. | | 23375101
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Assessment of the integrin alpha5beta1 antagonist JSM6427 in proliferative vitreoretinopathy using in vitro assays and a rabbit model of retinal detachment. Zahn, G; Volk, K; Lewis, GP; Vossmeyer, D; Stragies, R; Heier, JS; Daniel, PE; Adamis, AP; Chapin, EA; Fisher, SK; Holz, FG; Löffler, KU; Knolle, J Investigative ophthalmology & visual science
51
1028-35
2010
Show Abstract
To explore the role of integrin alpha5beta1 in proliferative vitreoretinopathy (PVR) pathogenesis by evaluating the expression alpha5beta1 on ARPE-19 cells and patient proliferative membranes, quantifying the inhibitory effects of JSM6427 (a small molecule alpha5beta1 inhibitor) on ARPE-19 cell adhesion and migration, and assessing the therapeutic potential of JSM6427 in a rabbit retinal detachment model.Expression of alpha5beta1 was evaluated on activated ARPE-19 cells by flow cytometry and on PVR membranes by immunohistochemistry. ARPE-19 cells were used in fibronectin-dependent adhesion and migration assays with various concentrations of JSM6427; IC(50) was calculated. In the rabbit model, eyes were intravitreally injected with vehicle or JSM6427 on day 0 or 1 after retinal detachment; BrdU was administered intravitreally on day 3, and retinal tissues were harvested on day 3 (4 hours later) or 7. Retinal scarring, cellular proliferation, and inflammatory responses were quantified, and retinal morphology was analyzed in retinal sections.Activated ARPE-19 cells and PVR membranes expressed high levels of alpha5beta1; expression was low in control eyes. JSM6427 provided a dose-dependent blockade of ARPE-19 cell adhesion to fibronectin (IC(50), 7.1 +/- 2.5 microM) and inhibition of migration (IC(50), 6.0 +/- 4.5 microM). In the rabbit model, intravitreal injection of JSM6427 provided significant inhibition of proliferation of retinal cells (Müller cells, microglia, and macrophages) on days 3 and 7 after detachment and inhibition of inflammatory response and retinal scarring on day 7 after detachment.JSM6427 is a promising treatment for PVR, with data suggesting that inhibition of alpha5beta1-fibronectin interactions addresses multiple pathways involving retinal pigment epithelial, glial, and inflammatory cells. | | 19815730
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Fluctuations of cytoskeleton-bound microbeads--the effect of bead-receptor binding dynamics. C Metzner,C Raupach,C T Mierke,B Fabry Journal of physics. Condensed matter : an Institute of Physics journal
22
2010
Show Abstract
The cytoskeleton (CSK) of living cells is a crosslinked fiber network, subject to ongoing biochemical remodeling processes that can be visualized by tracking the spontaneous motion of CSK-bound microbeads. The bead motion is characterized by anomalous diffusion with a power-law time evolution of the mean square displacement (MSD), and can be described as a stochastic transport process with apparent diffusivity D and power-law exponent β: MSD ∼ D (t/t(0))(β). Here we studied whether D and β change with the time that has passed after the initial bead-cell contact, and whether they are sensitive to bead coating (fibronectin, integrin antibodies, poly-L-lysine, albumin) and bead size (0.5-4.5 µm). The measurements are interpreted in the framework of a simple model that describes the bead as an overdamped particle coupled to the fluctuating CSK network by an elastic spring. The viscous damping coefficient characterizes the degree of bead internalization into the cell, and the spring constant characterizes the strength of the binding of the bead to the CSK. The model predicts distinctive signatures of the MSD that change with time as the bead couples more tightly to the CSK and becomes internalized. Experimental data show that the transition from the unbound to the tightly bound state occurs in an all-or-nothing manner. The time point of this transition shows considerable variability between individual cells (2-30 min) and depends on the bead size and bead coating. On average, this transition occurs later for smaller beads and beads coated with ligands that trigger the formation of adhesion complexes (fibronectin, integrin antibodies). Once the bead is linked to the CSK, however, the ligand type and bead size have little effect on the MSD. On longer timescales of several hours after bead addition, smaller beads are internalized into the cell more readily, leading to characteristic changes in the MSD that are consistent with increased viscous damping by the cytoplasm and reduced binding strength. | | 21386432
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Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP-dependent proteolysis in 3-dimensional collagen matrices. Stratman, AN; Saunders, WB; Sacharidou, A; Koh, W; Fisher, KE; Zawieja, DC; Davis, MJ; Davis, GE Blood
114
237-47
2009
Show Abstract
Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly. Full Text Article | | 19339693
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Integrin alphaVbeta6 is a high-affinity receptor for coxsackievirus A9. Heikkilä, O; Susi, P; Stanway, G; Hyypiä, T The Journal of general virology
90
197-204
2009
Show Abstract
Coxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins alphaVbeta3 and alphaVbeta6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins alphaVbeta3 and alphaVbeta6, whereas alphaVbeta6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized alphaVbeta3 and alphaVbeta6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against alphaV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble alphaVbeta6, but not alphaVbeta3, bound to immobilized CAV9. Similarly, only soluble alphaVbeta6 blocked virus infectivity. These data suggest that CAV9 binding to alphaVbeta6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined. | | 19088289
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Src, PKCalpha, and PKCdelta are required for alphavbeta3 integrin-mediated metastatic melanoma invasion. Putnam, Andrew J, et al. Cell Commun. Signal, 7: 10 (2009)
2009
Show Abstract
ABSTRACT: BACKGROUND: Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor alphavbeta3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis. RESULTS: Two melanoma cell lines C8161.9 and M14 both express high levels of alphavbeta3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an alphavbeta3-depenent manner. Elevated levels of PKCalpha and PKCdelta, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed alphavbeta3-dependent invasion. Furthermore, over expression of Src or PKCalpha and PKCdelta was sufficient to confer alphavbeta3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKCalpha expression, but not PKCdelta, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKCalpha restored focal adhesion formation and partially restored stress fiber formation, while loss of PKCdelta primarily restored stress fibers. CONCLUSION: The misregulated expression of PKCalpha and PKCdelta and elevated Src activity in metastatic melanoma cells is required for efficient alphavbeta3-mediated invasion. PKCalpha and Src enhance alphavbeta3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKCalpha influences focal adhesion formation, while PKCdelta controls stress fibers. | | 19400942
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Anti-integrin monoclonal antibody CNTO 95 enhances the therapeutic efficacy of fractionated radiation therapy in vivo. Ning, S; Nemeth, JA; Hanson, RL; Forsythe, K; Knox, SJ Molecular cancer therapeutics
7
1569-78
2008
Show Abstract
Selective targeting of up-regulated integrins on tumor cells is a novel antiangiogenesis strategy for treating solid tumors. CNTO 95 is a fully human anti-alpha(v) integrin monoclonal antibody and has shown antitumor activity when used as a single agent in preclinical studies. We previously showed that radiation combined with an integrin alpha(v)beta(3) antagonist cRGD peptide increased the therapeutic efficacy of radiation in preclinical tumor models. We hypothesized that the combination of radiation and CNTO 95 would synergistically enhance the efficacy of radiation therapy. The in vitro studies showed that CNTO 95 radiosensitized and induced apoptosis in M21 cells in vitronectin-coated dishes. In mice bearing established human cancer xenograft tumors, CNTO 95 alone had only a moderate effect on tumor growth. The combined therapy of CNTO 95 and fractionated radiation significantly inhibited tumor growth and produced the longer tumor growth delay time in multiple tumor models. Maintenance dosing of CNTO 95 following irradiation contributed to efficacy and was important for continued inhibition of tumor regrowth. Immunohistochemistry studies showed that the combined use of CNTO 95 and radiation reduced the alpha(v) integrin and vascular endothelial growth factor receptor expression and the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. CNTO 95 alone and in combination with radiation did not produce any obvious signs of systemic toxicity. These results show that CNTO 95 can potentiate the efficacy of fractionated radiation therapy in a variety of human cancer xenograft tumor types in nude mice. These findings are very promising and may have high translational relevance for the treatment of patients with solid tumors. | | 18566227
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