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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)., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.) more...

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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...

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., (Copyright © 2016, American Association for the Advancement of Science.) more...

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. more...

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. AACR. more...

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. more...

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 (DeltaC-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 DeltaC-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 DeltaC-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. more...

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. VEGF(165) 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 VEGF(165) 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. more...

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. more...

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. more...

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. more...

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<0.05 in all cases), when compared to healthy controls (n=30). Neither HARP nor MK levels were significantly different between patients with loco-regional and metastatic tumors (P=0.203 and 0.242, respectively). Moreover, a strong correlation between the elevations of the plasma levels of these two proteins (r2=0.546) in these cancer patients was found. Measurements of these secreted angiogenic growth factors may be useful for evaluation of cancer diagnosis. more...

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. more...

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., (Copyright 1999 Academic Press.) more...