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Supplementary Table 1, Figures 1-3 from EphB4 Promotes Site-Specific Metastatic Tumor Cell Dissemination by Interacting with Endothelial Cell–Expressed EphrinB2

Aberrant activation in fibroblast growth factor signaling has been implicated in the development of various cancers, including squamous cell lung cancer, squamous cell head and neck carcinoma, colorectal and bladder cancer. Thus, fibroblast growth factor receptors (FGFRs) present promising targets for novel cancer therapeutics. Here, we evaluated the activity of a novel pan‐FGFR inhibitor, rogaratinib, in biochemical, cellular and in vivo efficacy studies in a variety of preclinical cancer models. In vitro kinase activity assays demonstrate that rogaratinib potently and selectively inhibits the activity of FGFRs 1, 2, 3 and 4. In line with this, rogaratinib reduced proliferation in FGFR‐addicted cancer cell lines of various cancer types including lung, breast, colon and bladder cancer. FGFR and ERK phosphorylation interruption by rogaratinib treatment in several FGFR‐amplified cell lines suggests that the anti‐proliferative effects are mediated by FGFR/ERK pathway inhibition. Furthermore, rogaratinib exhibited strong in vivo efficacy in several cell line‐ and patient‐derived xenograft models characterized by FGFR overexpression. The observed efficacy of rogaratinib strongly correlated with FGFR mRNA expression levels. These promising results warrant further development of rogaratinib and clinical trials are currently ongoing (ClinicalTrials.gov Identifiers: NCT01976741, NCT03410693, NCT03473756)., What's new? Deregulated fibroblast growth factor receptor (FGFR) signaling is involved in tumorigenesis and cancer progression. Here, the authors report on a novel pan‐FGFR inhibitor, rogaratinib, that potently and highly selectively prevents the activity of FGFRs 1, 2, 3, and 4. Rogaratinib inhibits cell proliferation in various FGFR‐addicted cancers in vitro, including colon, lung, and bladder cancer. Rogaratinib also exhibits strong in vivo efficacy in several cell line‐ and patient‐derived xenograft models characterized by FGFR mRNA overexpression with good tolerability. Altogether, these data warrant the further development of rogaratinib for treatment of cancers with FGFR alterations, and clinical trials are currently ongoing.

Rogaratinib (BAY 1163877) is a highly potent and selective small-molecule pan-fibroblast growth factor receptor (FGFR) inhibitor (FGFR1-4) for oral application currently being investigated in phase 1 clinical trials for the treatment of cancer. In this publication, we report its discovery by de novo structure-based design and medicinal chemistry optimization together with its pharmacokinetic profile.

To improve tumor selectivity of cytotoxic agents we designed small molecule drug conjugates leveraging two independent mechanisms of targeted delivery by utilizing an integrin binder for tumor homing and release of the active drug by proteases present in tumor stroma. Integrins are transmembrane receptors that mediate cell-extracellular matrix and cell-cell interaction. αvβ3 integrins in the tumor microenvironment are involved in critical events for tumor progression, cytotoxic therapy resistance and metastasis including angiogenesis, matrix remodeling and the recruitment of immune and inflammatory cells. Proteases in tumor stoma such as neutrophil elastase also contribute to cancer progression by enhancing tumor evasion and metastasis. The expectation is that small molecule drug conjugates could have better efficacy for solid tumors than antibody drug conjugates due to better tissue penetration. Following this rationale the small molecule drug conjugate is composed of a peptidomimetic αvβ3 integrin antagonist linked to a cytotoxic camptothecin derivative via a linker susceptible to cleavage by neutrophil elastase. Imaging studies with the αvβ3 antagonist conjugated with an IR-800 dye demonstrated efficient tumor targeting to 786-0 renal adenocarcinoma tumors, as compared with a weakly binding epimer used as a control conjugate. The targeted conjugate is highly stable in rat plasma and shows strong cytotoxic activity only in the presence of neutrophil elastase. The payload displays high cellular permeability and in contrast to SN38, the active metabolite of irinotecan, it is not a substrate of efflux transporters. The conjugate is highly efficacious in vivo effecting tumor regressions in SW480 (colon cancer), MX1 (breast cancer) and NCI-H69 (lung cancer) xenograft models with T/C ratios of 0.1, 0.03 and 0.06 (p Citation Format: Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Charlotte Kopitz, Melanie Heroult, Dmitry Zubov, Joerg Willuda, Thomas Schlange, Antje Kahnert, Raquel Izumi, Ahmed Hamdy. A novel small molecule drug conjugate -avb3 integrin antagonist linked to a cytotoxic camptothecin derivative- for the treatment of multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1314.

The Digital Transformation is heavily impacting the way corporations are conducting their R&D and innovation activities, particularly in the most traditional industrial sectors. We explore how an o...

Objectives Pleiotrophin is a heparin-binding growth factor expressed in embryonic but not mature cartilage, suggesting a role in cartilage development. Elucidation of the molecular changes observed during the remodelling process in osteoarthritis is of paramount importance. This study aimed to investigate serum pleiotrophin levels and expression of pleiotrophin and its receptor protein tyrosine phosphatase beta/zeta in the cartilage and subchondral bone of osteoarthritis patients. Methods Serum samples derived from 16 osteoarthritis patients and 18 healthy donors. Pleiotrophin and receptor protein tyrosine phosphatase beta/zeta in the cartilage and subchondral bone were studied in 29 patients who had undergone total knee or hip replacement for primary osteoarthritis and in 10 control patients without macroscopic osteoarthritis changes. Results Serum pleiotrophin levels and expression of pleiotrophin in chondrocytes and subchondral bone osteocytes significantly increased in osteoarthritis patients graded Ahlback II to III. Receptor protein tyrosine phosphatase beta/zeta was mainly detected in the subchondral bone osteocytes of patients with moderate osteoarthritis and as disease severity increased, in the osteocytes and bone lining cells of the distant trabeculae. Conclusions These data render pleiotrophin and receptor protein tyrosine phosphatase beta/zeta promising candidates for further studies towards developing targeted therapeutic schemes for osteoarthritis.

Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 (‘BAY’) triggers death of BRAFV600E melanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a chain of events in which: (i) CI inhibition induces mPTP opening and Δψ depolarization, that (ii) stimulate autophagosome formation, mitophagy and an associated ROS increase, leading to (iii) activation of combined necroptotic/ferroptotic cell death.

Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A165). NRP1 is also the receptor for the CendR peptides, a class of cell- and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A165, a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.

The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4–expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. Mol Cancer Res; 8(10); 1297–309. ©2010 AACR.

A specific splice variant of the CD44 cell- surface protein family, CD44v6, has been shown to act as a coreceptor for the receptor tyrosine kinase c-Met on epithelial cells. Here we show that also on endothelial cells (ECs), the activity of c-Met is dependent on CD44v6. Furthermore, another receptor tyrosine kinase, VEGFR-2, is also regulated by CD44v6. The CD44v6 ectodomain and a small peptide mimicking a specific extracellular motif of CD44v6 or a CD44v6-specific antibody prevent CD44v6-mediated receptor activation. This indicates that the extracellular part of CD44v6 is required for interaction with c-Met or VEGFR-2. In the cytoplasm, signaling by activated c-Met and VEGFR-2 requires association of the CD44 carboxy-terminus with ezrin that couples CD44v6 to the cytoskeleton. CD44v6 controls EC migration, sprouting, and tubule formation induced by hepatocyte growth factor (HGF) or VEGF-A. In vivo the development of blood vessels from grafted EC spheroids and angiogenesis in tumors is impaired by CD44v6 blocking reagents, suggesting that the coreceptor function of CD44v6 for c-Met and VEGFR-2 is a promising target to block angiogenesis in pathologic conditions.

The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. Here, we show that R-spondin 3(Rspo3), a member of a novel family of secreted proteins in vertebrates that activate Wnt/β-catenin signaling, plays a key role in these processes. In Xenopus embryos, morpholino antisense knockdown of Rspo3 induces vascular defects because Rspo3 is essential for regulating the balance between angioblast and blood cell specification. In mice, targeted disruption of Rspo3 leads to embryonic lethality caused by vascular defects. Specifically in the placenta, remodeling of the vascular plexus is impaired. In human endothelial cells, R-spondin signaling promotes proliferation and sprouting angiogenesis in vitro, indicating that Rspo3 can regulate endothelial cells directly. We show that vascular endothelial growth factor is an immediate early response gene and a mediator of R-spondin signaling. The results identify Rspo3 as a novel, evolutionarily conserved angiogenic factor in embryogenesis.

Heparin affin regulatory peptide (HARP) is an heparin-binding molecule involved in the regulation of cell proliferation and differentiation. Here, we report that HARP inhibited the biological activity induced by the 165-amino-acid form of vascular endothelial growth factor (VEGF165) on human umbilical vein endothelial cells. Endothelial-cell proliferation induced by VEGF165 showed about 50% inhibition in the presence of HARP in a concentration of 3 nM. In similar range of concentrations, HARP blocked tube formation induced by VEGF165 in three-dimensional angiogenesis assay. In vivo studies showed that HARP inhibited the VEGF165-induced Matrigel trade mark infiltration of endothelial cells. We then investigated the mechanisms of this inhibition and shown that HARP inhibited the binding of 125I-VEGF165 to the VEGF receptors of endothelial cells. Additional studies using VEGF soluble receptors indicated that binding of 125I-VEGF165 to kinase insert domain-containing receptor and neuropilin receptor was inhibited by HARP, but conversely the binding of 125I-VEGF165 to fms-like tyrosine kinase I receptor was unaffected. A competitive affinity-binding assay demonstrated that HARP interacted directly with VEGF165 with a dissociation coefficient of 1.38 nM. Binding assay using deletion mutants of HARP revealed that the thrombospondin type-1 repeats domains were involved in this interaction. These data demonstrate for the first time that the angiogenic factor HARP can also negatively regulates the angiogenic activity of VEGF165.

Heparin affin regulatory peptide (HARP) and midkine (MK) are growth factors, expressed in carcinomas, neuroblastomas and gliomas. In this study, we measured the levels of HARP and MK in plasma samples from 77 cancer patients. The patients had advanced tumors with loco-regional (n=18) or metastatic (n=49) diseases and 10 patients have their diseases limited to the primary site. HARP and MK plasma concentrations were significantly higher in all of these different subgroups of cancer patients (P

Background Numerous studies have demonstrated that functional mitochondria are required for tumorigenesis, suggesting that mitochondrial oxidative phosphorylation (OXPHOS) might be a potential target for cancer therapy. In this study, we investigated the effects of BAY 87-2243, a small molecule that inhibits the first OXPHOS enzyme (complex I), in melanoma in vitro and in vivo. Results BAY 87-2243 decreased mitochondrial oxygen consumption and induced partial depolarization of the mitochondrial membrane potential. This was associated with increased reactive oxygen species (ROS) levels, lowering of total cellular ATP levels, activation of AMP-activated protein kinase (AMPK), and reduced cell viability. The latter was rescued by the antioxidant vitamin E and high extracellular glucose levels (25 mM), indicating the involvement of ROS-induced cell death and a dependence on glycolysis for cell survival upon BAY 87-2243 treatment. BAY 87-2243 significantly reduced tumor growth in various BRAF mutant melanoma mouse xenografts and patient-derived melanoma mouse models. Furthermore, we provide evidence that inhibition of mutated BRAF using the specific small molecule inhibitor vemurafenib increased the OXPHOS dependency of BRAF mutant melanoma cells. As a consequence, the combination of both inhibitors augmented the anti-tumor effect of BAY 87-2243 in a BRAF mutant melanoma mouse xenograft model. Conclusions Taken together, our results suggest that complex I inhibition has potential clinical applications as a single agent in melanoma and also might be efficacious in combination with BRAF inhibitors in the treatment of patients with BRAF mutant melanoma. Electronic supplementary material The online version of this article (doi:10.1186/s40170-015-0138-0) contains supplementary material, which is available to authorized users.

One hallmark of cancer is the accelerated metabolism, high energy requirements, and increased glucose uptake by the tumor cells, the latter being the first and rate-limiting step for glucose metabolism. Glucose transport into the tumor cell is mediated by facilitative high-affinity glucose transporter (GLUT) proteins. Among the 14 GLUT proteins, expression of GLUT1 in normal organs is nearly exclusively restricted to the blood brain barrier, while other GLUTs are also expressed in a wide variety of vital organs such as liver and heart. Interestingly, GLUT1 expression is highly regulated by hypoxia-inducible factor (HIF)-1α, a key driver of tumor progression. In line with this finding, GLUT1 over-expression was found to be associated with tumor progression and poor overall survival in various tumor indications. Consequently, GLUT1 represents a potential target for cancer treatment. Therefore, we have developed a highly-selective GLUT1 inhibitor, namely BAY-876, with selectivity over GLUT2, 3, and 4 of 4700-, 800-, and 135-fold, respectively. We here show for the first time the pharmacological characterization of BAY-876, comprising inhibition of glucose-uptake, anti-proliferative activity in vitro, and anti-tumor efficacy in vivo in models of different tumor indications in monotherapy as well as first results on the combinability of BAY-876. Furthermore, at the therapeutic dose, BAY-876 treatment did not show any relevant finding on the behavior of treated mice in the Irwin test, assuming no or only minor effects on brain function. In conclusion, BAY-876 is the first GLUT1-selective inhibitor which reduces glucose uptake and growth of tumor cells with sufficient tolerability at the efficacious dose in preclinical models. Citation Format: Charlotte Kopitz, Luisella Toschi, Carolyn Algire, Mélanie Héroult, Anna-Lena Frisk, Kirstin Meyer, Arndt Schmitz, Eleni Lagkadinou, Heike Petrul, Iring Heisler, Roland Neuhaus, Bernd Buchmann, Herbert Himmel, Marcus Bauser, Andrea Haegebarth, Karl Ziegelbauer. Pharmacological characterization of BAY-876, a novel highly selective inhibitor of glucose transporter (GLUT)-1 in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4746.

Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFR1-4) and activating downstream signaling pathways. Alterations in FGFR encoding genes are frequently observed in a variety of solid tumors including lung, gastric, breast and urothelial cancer. Therefore, targeting FGFRs using selective FGFR inhibitors is an attractive therapeutic approach to treat cancer patients. BAY 1163877 is an orally active, highly potent and selective small molecule FGFR-1, -2 and -3 kinase inhibitor. We disclose for the very first time its discovery and chemical structure. BAY 1163877 was derived from a de novo structure-based design approach and medicinal chemistry optimization. Data on the structure activity relationship and the pharmacokinetic profile of the benzothiophenyl-pyrrolotriazine structure class will be presented. Based on its favorable preclinical profile, BAY 1163877 is currently being investigated in a Phase 1 clinical trial (NCT01976741). Citation Format: Marie-Pierre L. Collin, Mario Lobell, Walter Huebsch, Dirk Brohm, Mélanie Héroult, Klemens Lustig, Sylvia Gruenewald, Ulf Boemer, Rolf Jautelat, Holger Hess-Stump, Stefan Jaroch, Michael Brands, Karl Ziegelbauer. Discovery of BAY 1163877 - A pan-FGFR inhibitor: De novo structure-based design and lead optimization of benzothiophenyl-pyrrolotriazines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4332.

Objective— To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Methods and Results— Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain–containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. Conclusion— Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell–expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

The vascular endothelium is a crucial interface that controls the recruitment of circulating leukocytes. Based on the luminal expression of the ephrin-B2 ligand by endothelial cells (ECs) and the expression of EphB receptors (EphBRs) by circulating monocytes, we hypothesized that EphBR-ephrinB interactions are involved in monocyte adhesion. Adhesion experiments with monocytic cells were performed on ECs that overexpressed either full-length ephrin-B2 or cytoplasmically truncated ephrin-B2 (ΔC-ephrin-B2). Atomic force microscopy confirmed similar adhesive strengths of EphBR-expressing J774 cells to ECs that either overexpressed full-length ephrin-B2 or truncated ΔC-ephrin-B2 (1-minute interaction). Yet, adhesion experiments under static or flow conditions for 30 minutes demonstrated the preferential adhesion of monocytic cells to ECs that overexpressed full-length ephrin-B2 but not to ΔC-ephrin-B2 or to ECs that had been mock transduced. Adhesion was blocked by ephrin-B2-specific and EphBR-specific antibodies. Correspondingly, adhesion of EphB4-receptor-overexpressing monocytes to ephrin-B2-positive ECs was further augmented. Trafficking experiments of cell-surface molecules revealed that, prior to internalization, the resulting EphB4-receptor–ephrin-B2 complex translocated from the luminal surface to inter-endothelial junctions. Lastly, full-length ephrin-B2 in ECs was also involved in monocyte transmigration. Collectively, our study identifies a role of EphBR-ephrinB interactions as a new step in the cascade of events leading to monocyte adhesion and transmigration through the vascular endothelium.

Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF165 binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF165 binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1.

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

Pleiotrophin (PTN), is a heparin-dependent growth factor involved in angiogenesis and tumor growth. PTN contains a thrombospondin repeat-I (TSR-I) motif in its two beta-sheet domains that are involved in its binding to heparin and its neurite outgrowth activity. Based on the importance of the binding of PTN to heparin in its dimerization and biological activities, we have designed two synthetic peptides, P(13-39) and P(65-97) corresponding to a part of the N-terminal and C-terminal TSR-I motif of PTN, respectively. P(65-97) inhibited the mitogenic, tumorigenic and angiogenic activities of PTN, as well as the mitogenic and an angiogenic activity of fibroblast growth factor-2 (FGF-2). However, P(65-97) had no effect on the mitogenic activity of epidermal growth factor, which does not bind heparin. P(65-97) but not P(13-39) inhibited the binding of PTN and to a lesser extent of FGF-2 to heparin using an immunoassay and an optical biosensor assay and bound directly to heparin with a K(d) of 120 nM. These findings suggest that P(65-97), containing amino acids 65-97 of the TSR-I motif of the C-terminal domain of PTN, inhibits the activities of PTN and FGF-2 by virtue of its ability to bind heparin very effectively and so compete with the growth factors for their polysaccharide co-receptor.

Malignant cells are known for their accelerated metabolism, high energy requirements, and increased glucose uptake. They are characterized by high rates of glycolysis and metabolize glucose into lactate even under aerobic conditions, a hallmark of cancer known as the Warburg effect. Transport of glucose across the plasma membrane is the first and rate-limiting step for glucose metabolism and is mediated among others by facilitative glucose transporter (GLUT) proteins. Among the 14 GLUT proteins, GLUT1 and to a lesser degree GLUT3 are up regulated in many tumors. GLUT1 is also a prime target for the transcription factor hypoxia-inducible factor (HIF)-1α. Close relationships between GLUT1 expression, tumor development and progression, as well as poor overall survival have been described for several tumor entities. We have developed a GLUT1-selective, potent small molecule inhibitor, BAY-GLUT1, which inhibits both glucose uptake and ATP generation in GLUT1-expressing DLD-1 tumor cells in a glucose competitive way with IC50s of 4 and 3 nM, respectively. Selectivity was tested in either GLUT1-deficient DLD-1 cells or GLUT2/4-overexpressing recombinant CHO cells. In this study we report on the metabolic reaction of two adenocarcinoma cell lines, HeLa-MaTu (cervix) and DLD-1 (colon), to BAY-GLUT1 treatment. The two cell lines were treated with 30 nM BAY-GLUT1 for 6 and 24 hours under normoxic conditions and investigated by Metanomics Health's untargeted, broad metabolite profiling platform (MxP® Broad Profiling) to identify metabolic mechanisms explaining the differential susceptibility of cancer cells towards BAY-GLUT1. Hereby, two types of mass spectrometry (gas chromatography-mass spectrometry and liquid chromatography-MS/MS) analysis were applied to evaluate levels of 215 intracellular metabolites covering major metabolic pathways. Both inhibitor-treated cell lines showed dramatic decreases of glucose-6-phosphate and other glycolysis-related metabolites indicating efficient reduction in glycolytic activity. Subsequently, levels of several TCA cycle intermediates were also reduced. Our metabolic findings might suggest that DLD-1 preferentially uses glutamate and its precursor metabolites proline and 5-oxoproline to replenish the TCA cycle at the level of alpha-ketoglutarate. Interestingly, due to a gain-of-function mutation of isocitrate dehydrogenase, DLD-1 cells are able to reduce alpha-ketoglutarate into 2-hydroxyglutaric acid which might play an important role in controlling stability of transcription factor HIF-1α and in inducing metabolic reprogramming. Understanding the metabolic pathway changes upon GLUT inhibition could help to identify predictive conditions and/or biomarkers for treatment response. Citation Format: Sylvia Gruenewald, Ulrike Rennefahrt, Sandra G. Maldonado, Alexander Walter, Heike Petrul, Mélanie Héroult, Iring Heisler, Maria Quanz, Patrick Steigemann, Bernd Buchmann, Andrea Haegebarth. Metabolic responses in cancer cells with differential susceptibility to GLUT1 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1164. doi:10.1158/1538-7445.AM2015-1164

Eph receptors comprise the largest family of receptor tyrosine kinases. They are classified into an A family and a B family on the basis of the characteristic properties of the corresponding ephrin ligands which are either GPI-anchored peripheral membrane molecules (A class ephrins) or transmembrane molecules (B class ephrins). Eph receptors and ephrin ligands were originally identified as neuronal pathfinding molecules. Yet, gene targeting experiments in mice have identified the EphB/ephrinB system as critical and rate-limiting determinant of arterio-venous differentiation during embryonic vascular development. Identification of vascular EphB/ephrinB functions has in the last few years stimulated two emerging fields of vascular biology research, namely (1) the molecular analysis of the structural and functional mechanisms of arterio-venous differentiation, and (2) the molecular study of the commonalities between vascular and neuronal guidance and patterning mechanisms. This review summarizes the current understanding of vascular Eph receptor and ephrin ligand functions and provides an overview of emerging roles of the Eph/ephrin system in controlling tumor and vascular functions during tumorigenesis and tumor progression.

HARP (heparin affin regulatory peptide) is a heparin binding growth factor implicated in cellular growth and differentiation. Previously, HARP had been localized in the human mammary, in both alveolar epithelial and myoepithelial cells although HARP mRNAs were only expressed by myoepithelial cells [J. Histochem. Cytochem. 45 (1997) 1]. In the present study, we demonstrate that HARP is secreted in human mature milk with concentrations ranging from 17.68+/-6.4ng/ml in mature milk to 59.9+/-11.22ng/ml in colostrum. In vitro, HARP was found to be mitogenic on human mammary epithelial and myoepithelial cell lines and correlated with the expression of its high affinity receptor tyrosine kinase ALK (anaplastic lymphoma kinase). In vivo, ALK is expressed in both mammary epithelial and myoepithelial cells, suggesting that HARP could act in vivo as a paracrine and autocrine growth factor in the regulation of the mammary gland development and its homeostatic maintenance during pregnancy and lactation.

Heparin-affin regulatory peptide (HARP) and Midkine (MK) belong to a family of growth/differentiation factors that have a high affinity for heparin. The involvement of these molecules in various proliferative diseases prompted us to develop an assay for measuring the concentrations of these factors in biological fluids and culture media. This report describes an immunoassay that uses only commercially available materials, based on the high affinity of certain molecules for heparin. It consists of adsorbing heparin-BSA covalent complexes to microtiter plate wells and to quantify the heparin bound HARP or MK by using appropriate antibody. The method is specific and measures concentrations ranging from 40-1200 pg/mL HARP and from 25-1200 pg/mL MK and various parameters are investigated. The within-assay coefficient of variation was less than 5% for both assays. The method was checked by measuring the concentrations of these growth factors in the sera of healthy humans and in patients with cancer. As previously reported, we confirmed that the serum concentrations of MK are higher in patients with tumours (n = 139) than in controls (n = 19). The synthesis of HARP and MK by various cells in culture was also analysed.

Background Heparin affin regulatory peptide (HARP), also known as pleiotrophin (PTN) is a potent mitogen for various cell types and an angiogenic polypeptide implicated in tumour development.1–4 Objectives Since the progression of the pannus is associated with angiogenic processes and synovial membrane hypertrophy,5,6 the involvement of HARP in comparison with two other growth factors including MK and VEGF in rheumatoid pathologies was investigated. Methods Presence of HARP in human synovial fluids and sera from patients was investigated by immunoassay. The distribution of HARP mRNA and protein was studied by in situ hybridization and immunohistochemistry respectively. Results Comparative analysis showed that HARP as well as MK concentrations were significantly higher (p Conclusion These results suggest that HARP, in concert with other cytokines and growth factors, may play a role in the pathophysiology of acute and chronic synovitis in conditions such as crystal-induced arthritis and rheumatoid arthritis, respectively. References Li YS, Milner PG, Chauhan AK, Watson MA, Hoffman RM, Kodner CM, Milbrandt J, Deuel TF. Science 1990;250:1690–4 Courty J, Dauchel MC, Caruelle D, Pederiset M, Barritault D. Biochem Biophy Res Commun. 1991;180:145–51 Bernard-Pierrot I, Delbe J, Caruelle D, Barritault D, Courty J. J Biol Chem. in press Choudhuri R, Zhang HT, Donnini S, Ziche M, Bicknell R. Cancer Res. 1997;57:1814–19 Peacok DJ, Banquerigo ML, Brahn E. J Exp Med. 1992;175:1135–8 Koch AE. Ann Rheu Dis. 2000;59(Suppl 1):I65–71

HARP (heparin affin regulatory peptide) is a growth factor displaying high affinity for heparin. In the present work, we studied the ability of human recombinant HARP as well as its two terminal peptides (HARP residues 1–21 and residues 121–139) to promote angiogenesis. HARP stimulates endothelial cell tube formation on matrigel, collagen and fibrin gels, stimulates endothelial cell migration and induces angiogenesis in the in vivo chicken embryo chorioallantoic membrane assay. The two HARP peptides seem to be involved in most of the angiogenic effects of HARP. They both stimulate in vivo angiogenesis and in vitro endothelial cell migration and tube formation on matrigel. We conclude that HARP has an angiogenic activity when applied exogenously in several in vitro and in vivo models of angiogenesis and its NH 2 and COOH termini seem to play an important role.

HARP (Heparin Affin Regulatory Peptide) is a 18-kDa secreted protein displaying high affinity for heparin. It has neurite outgrowth-promoting activity, while there are conflicting results regarding its mitogenic activity. In the present work, we studied the effect of human recombinant HARP expressed in bacterial cells as well as two peptides (HARP residues 1–21 and residues 121–139) on the proliferation of three endothelial cell types derived from human umbilical vein (HUVEC), rat adrenal medulla (RAME), and bovine brain capillaries (BBC) either added as a soluble form in the cell culture medium or coated onto the culture plate. HARP added in a soluble form in the culture medium had no effect on the proliferation of BBC, HUVEC, and RAME cells. However, when immobilized onto the cell culture plate, HARP had a concentration-dependent mitogenic effect on both BBC cells and HUVEC. The peptides presented as soluble factor induced a significant concentration-dependent mitogenic effect on BBC cells but only a small effect on HUVEC and RAME cells. When they were immobilized onto the cell culture plate, the mitogenic effect was much greater. The most responsive cells were BBC that expressed and secreted in the culture medium the higher amounts of HARP.

Heparin affin regulatory peptide (HARP), also called pleiotrophin (PTN), is a secreted polypeptide which binds to heparin and plays a key role in cellular growth and differentiation. In order to assess the determinants potentially important to its biological activity, we tested the ability of HARP to oligomerize, a process involved in mitogenic activity of the heparin-binding fibroblast growth factor. Using dissuccinimidyl suberate cross-linking experiments and affinity chromatography, we report that human HARP forms noncovalent dimers. Dimerization is dependent on the presence of heparin or other sulfated glycosaminoglycans, as chlorate treatment of cells inhibits this process. In vitro, different glycosaminoglycans, such as dermatan sulfate and chondroitin sulfate-C, also induce a dimer assembly of HARP. The relevance of this process was supported by experiments demonstrating that HARP is secreted as a dimer in conditioned medium of NIH-3T3 cells that overexpressed this growth factor and is also associated to the cell surface or to the extracellular matrix.

Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP.