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β-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing β-galactosyl moieties from β-galactosylceramide and β-galactosylsphingosine. Previous observations have shown that GALC may exert pro-oncogenic functions in melanoma and Galc silencing, leading to decreased oncogenic activity in murine B16 melanoma cells. The tumor-driving BRAF(V600E) mutation is present in approximately 50% of human melanomas and represents a major therapeutic target. However, such mutation is missing in melanoma B16 cells. Thus, to assess the impact of GALC in human melanoma in a more relevant BRAF-mutated background, we investigated the effect of GALC overexpression on the proteomic landscape of A2058 and A375 human melanoma cells harboring the BRAF(V600E) mutation. The results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrate that significant differences exist in the protein landscape expressed under identical cell culture conditions by A2058 and A375 human melanoma cells, both harboring the same BRAF(V600E)-activating mutation. GALC overexpression resulted in a stronger impact on the proteomic profile of A375 cells when compared to A2058 cells (261 upregulated and 184 downregulated proteins versus 36 and 14 proteins for the two cell types, respectively). Among them, 25 proteins appeared to be upregulated in both A2058-upGALC and A375-upGALC cells, whereas two proteins were significantly downregulated in both GALC-overexpressing cell types. These proteins appear to be involved in melanoma biology, tumor invasion and metastatic dissemination, tumor immune escape, mitochondrial antioxidant activity, endoplasmic reticulum stress responses, autophagy, and/or apoptosis. Notably, analysis of the expression of the corresponding genes in human skin cutaneous melanoma samples (TCGA, Firehose Legacy) using the cBioPortal for Cancer Genomics platform demonstrated a positive correlation between GALC expression and the expression levels of 14 out of the 27 genes investigated, thus supporting the proteomic findings. Overall, these data indicate for the first time that the expression of the lysosomal sphingolipid-metabolizing enzyme GALC may exert a pro-oncogenic impact on the proteomic landscape in BRAF-mutated human melanoma.

The tumor microenvironment (TME) is comprised of different cellular components, such as immune and stromal cells, which co-operate in unison to promote tumor progression and metastasis. In the last decade, there has been an increasing focus on one specific component of the TME, the stromal component, often referred to as Cancer-Associated Fibroblasts (CAF). CAF modulate the immune response and alter the composition of the extracellular matrix with a decisive impact on the response to immunotherapies and conventional chemotherapy. The most recent publications based on single-cell analysis have underlined CAF heterogeneity and the unique plasticity that strongly impact the TME. In this review, we focus not only on the characterization of CAF based on the most recent findings, but also on their impact on the immune system. We also discuss clinical trials and preclinical studies where targeting CAF revealed controversial results. Therefore, future efforts should focus on understanding the functional properties of individual subtypes of CAF, taking into consideration the peculiarities of each pathological context.

Colorectal cancer (CRC) cells are traditionally considered unresponsive to TGFβ due to mutations in the receptors and/or downstream signaling molecules. TGFβ influences CRC cells only indirectly via stromal cells, such as cancer-associated fibroblasts. However, CRC cell ability to directly respond to TGFβ currently remains unexplored. This represents a missed opportunity for diagnostic and therapeutic interventions. Methods: We examined whether cancer cells from primary CRC and liver metastases respond to TGFβ by inducing TGFβ-induced protein ig-h3 (TGFBI) expression, and the contribution of canonical and non-canonical TGFβ signaling pathways to this effect. We then investigated in vitro and in vivo TGFBI impact on metastasis formation and angiogenesis. Using patient serum samples and an orthotopic mouse model of CRC liver metastases we assessed the diagnostic/tumor targeting value of novel antibodies against TGFBI. Results: Metastatic CRC cells, such as circulating tumor cells, directly respond to TGFβ. These cells were characterized by the absence of TGFβ receptor mutations and the frequent presence of p53 mutations. The pro-tumorigenic program orchestrated by TGFβ in CRC cells was mediated through TGFBI, the expression of which was positively regulated by non-canonical TGFβ signaling cascades. TGFBI inhibition was sufficient to significantly reduce liver metastasis formation in vivo. Moreover, TGFBI pro-tumorigenic function was linked to its ability to stimulate angiogenesis. TGFBI levels were higher in serum samples from untreated patients with CRC than in patients who were receiving chemotherapy. A radiolabeled anti-TGFBI antibody selectively targeted metastatic lesions in vivo, underscoring its diagnostic and therapeutic potential. Conclusions: TGFβ signaling in CRC cells directly contributes to their metastatic potential and stromal cell-independence. Proteins downstream of activated TGFβ, such as TGFBI, represent novel diagnostic and therapeutic targets for more specific anti-metastatic therapies., SCOPUS: ar.j, info:eu-repo/semantics/published

UniPR129, an L-β-homotryptophan conjugate of the secondary bile acid lithocholic acid (LCA), acts as an effective protein-protein interaction (PPI) inhibitor of the Eph–ephrin system but suffers from a poor oral bioavailability in mice. To improve UniPR129 bioavailability, a metabolic soft spot, i.e., the 3α-hydroxyl group on the LCA steroidal ring, was functionalized to 3-hydroxyimine. In vitro metabolism of UniPR129 and 3-hydroxyimine derivative UniPR500 was compared in mouse liver subcellular fractions, and main metabolites were profiled by high resolution (HR-MS) and tandem (MS/MS) mass spectrometry. In mouse liver microsomes (MLM), UniPR129 was converted into several metabolites: M1 derived from the oxidation of the 3-hydroxy group to 3-oxo, M2–M7, mono-hydroxylated metabolites, M8–M10, di-hydroxylated metabolites, and M11, a mono-hydroxylated metabolite of M1. Phase II reactions were only minor routes of in vitro biotransformation. UniPR500 shared several metabolic pathways with parent UniPR129, but it showed higher stability in MLM, with a half-life (t1/2) of 60.4 min, if compared to a t1/2 = 16.8 min for UniPR129. When orally administered to mice at the same dose, UniPR500 showed an increased systemic exposure, maintaining an in vitro valuable pharmacological profile as an EphA2 receptor antagonist and an overall improvement in its physico-chemical profile (solubility, lipophilicity), if compared to UniPR129. The present work highlights an effective strategy for the pharmacokinetic optimization of aminoacid conjugates of bile acids as small molecule Eph–ephrin antagonists.

Prostate cancer (PCa) is a leading cause of cancer mortality in the male population commonly treated with androgen deprivation therapy (ADT) and relapsing as aggressive and androgen-independent castration-resistant prostate cancer (CRPC). In PCa the FGF/FGFR family of growth factors and receptors represents a relevant mediator of cancer growth, tumor-stroma interaction, and a driver of resistance and relapse to ADT. In the present work, we validate the therapeutic efficacy the FDA-approved FGFR inhibitor pemigatinib, in an integrated platform consisting of human and murine PCa cells, and the transgenic multistage TRAMP model of PCa that recapitulates both androgen-dependent and CRPC settings. Our results show for the first time that pemigatinib causes intracellular stress and cell death in PCa cells and prevents tumor growth in vivo and in the multistage model. In addition, the combination of pemigatinib with enzalutamide resulted in long-lasting tumor inhibition and prevention of CRPC relapse in TRAMP mice. These data are confirmed by the implementation of a stochastic mathematical model and in silico simulation. Pemigatinib represents a promising FDA-approved FGFR inhibitor for the treatment of PCa and CRPC alone and in combination with enzalutamide.

Cancer-associated fibroblasts (CAF) are important constituents of the tumor microenvironment (TME) and are major drivers of tumorigenesis. Yet, therapies aiming at eliminating CAF have failed to cure patients. This setback has raised questions regarding whether CAF exclusively favour cancer progression, or if they may also assume tumor-suppressor functions. In the present study, we used proteomics and single cell RNA-sequencing analysis to examine the CAF landscape in hepatocellular carcinoma (HCC). We thereby unveil three major CAF populations in HCC, one of which specifically expressing the prolargin protein. This CAF subpopulation (further termed as CAF_Port) shared a strong transcriptomic signature with portal liver fibroblasts. We further show that CAF_Port deposit prolargin in the TME and that its levels are lower in tumors as compared to the peritumoral region. Mechanistically, aggressive cancer cells degraded prolargin using matrix metalloprotease activity. Survival analysis of 188 patients revealed that high prolargin protein levels correlate with good patient outcome (HR = 0.37; p = 0.01). In vivo, co-injection of cancer cells with fibroblasts silenced for prolargin, led to faster tumor development (5-fold; p = 0.01), mainly due to stronger angiogenesis. Using protein-protein interaction study and structural modelling, we further demonstrate that prolargin binds and inhibits the activity of several pro-agiogenic proteins, including hepatocyte and fibroblast growth factors. In conclusion, prolargin is angiogenesis modulator and CAF-derived tumor suppressor in HCC. Stabilizing prolargin levels in the CAF_Port subpopulation may revert their tumor-antagonizing properties, warranting exploration in further pre-clinical studies.

β-galactosylceramidase (GALC) is a lysosomal enzyme that removes β-galactose from β-galactosylceramide, leading to the formation of the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth by acting as an oncosuppressive or oncogenic enzyme depending on the different experimental approaches, in vitro versus in vivo observations, preclinical versus clinical findings, and tumor type investigated. This review will recapitulate and discuss the contrasting experimental evidence related to the impact of GALC on the biological behavior of cancer and stromal cells and its contribution to tumor progression.

p17 matrix protein released by HIV+ cells interacts with leukocytes heparan sulfate proteoglycans (HSPGs), CXCR1 and CXCR2 exerting different cytokine-like activities that contribute to AIDS pathogenesis. Since the bioactive form of several cytokines is represented by dimers/oligomers and oligomerization is promoted by binding to heparin or HSPGs, here we evaluated if heparin/HSPGs also promote p17 oligomerization. Heparin favours p17 dimer, trimer and tetramer assembly, in a time- and biphasic dose-dependent way. Heparin-induced p17 oligomerization is of electrostatic nature, being it prevented by NaCl, by removing negative sulfated groups of heparin and by neutralizing positive lysine residues in the p17 N-terminus. A new computational protocol has been implemented to study heparin chains up to 24-mer accommodating a p17 dimer. Molecular dynamics show that, in the presence of heparin, two p17 molecules undergo conformational modifications creating a continuous “electropositive channel” in which heparin sulfated groups interact with p17 basic amino acids, promoting its dimerization. At the cell surface, HSPGs induce p17 oligomerization, as demonstrated by using B-lymphoblastoid Namalwa cells overexpressing the HSPG Syndecan-1. Also, HSPGs on the surface of BJAB and Raji human B-lymphoblastoid cells are required to p17 to induce ERK1/2 activation, suggesting that HS-induced oligomerization plays a role in p17-induced lymphoid dysregulation during AIDS.

Hepatocellular carcinoma (HCC) is a difficult to cure primary liver cancer. Recent breakthroughs in cancer treatment highlight the importance of the tumor microenvironment (TME) and its ability to promote or suppress tumor development. While understanding of this dual behaviour is expanding for immune cells, little is known for other stromal cells, and notably cancer-associated fibroblasts (CAF). Here, we unveil a novel CAF tumor-antagonizing protein, prolargin, and unravel its regulation and mechanism of action in human HCC. Employing proteomics and single cell RNA sequencing data analysis we investigated prolargin expression and the cell of origin in human HCC. The significance for clinical outcome was examined in a cohort of HCC patients (N=188), while prolargin function was studied in vivo using orthotopic models. Biochemical studies along with structural modelling/docking were employed to elucidate the mechanism of action. Our findings show that the expression of prolargin is confined to portal fibroblast-derived CAF. Prolargin is secreted in the TME where its quantity positively correlated with patient outcome (HR=0.37; p=0.01). Aggressive HCC cells reduce prolargin levels mainly through matrix metalloprotease 3 (MMP3) activity. In vivo, tumors with lower prolargin expression displayed faster progression (5-fold; p=0.01) and stronger angiogenesis. Models of prolargin structure revealed a solenoid (horseshoe) folding, favouring its binding and inhibition of several growth factors, except vascular endothelial growth factor (VEGF). MMP-inhibition (batimastat) combined with VEGFR targeting (sorafenib) in vivo demonstrated superior tumor control compared to sorafenib treatment alone. Prolargin-expressing fibroblasts have tumorantagonizing properties and stabilizing prolargin expression should be considered as therapeutic strategy for HCC.

Angiogenesis, the formation of new blood vessels from preexisting ones, is crucial for tumor growth and metastatization, and is considered a promising therapeutic target. Unfortunately, drugs directed against a specific proangiogenic growth factor or receptor turned out to be of limited benefit for oncology patients, likely due to the high biochemical redundancy of the neovascularization process. In this scenario, multitarget compounds that are able to simultaneously tackle different proangiogenic pathways are eagerly awaited. UniPR1331 is a 3β-hydroxy-Δ5-cholenic acid derivative, which is already known to inhibit Eph–ephrin interaction. Here, we employed an analysis pipeline consisting of molecular modeling and simulation, surface plasmon resonance spectrometry, biochemical assays, and endothelial cell models to demonstrate that UniPR1331 directly interacts with the vascular endothelial growth factor receptor 2 (VEGFR2) too. The binding of UniPR1331 to VEGFR2 prevents its interaction with the natural ligand vascular endothelial growth factor and subsequent autophosphorylation, signal transduction, and in vitro proangiogenic activation of endothelial cells. In vivo, UniPR1331 inhibits tumor cell-driven angiogenesis in zebrafish. Taken together, these data shed light on the pleiotropic pharmacological effect of UniPR1331, and point to Δ5-cholenic acid as a promising molecular scaffold for the development of multitarget antiangiogenic compounds.

Eph/ephrin system is an emerging target for cancer therapy but the lack of potent, stable and orally bioavailable compounds is impairing the development of the field. Since 2009 our research group has been devoted to the discovery and development of small molecules targeting Eph/ephrin system and our research culminated with the synthesis of UniPR129, a potent but problematic Eph/ephrin antagonist. Herein, we describe the in vitro pharmacological properties of two derivatives (UniPR139 and UniPR502) stemmed from structure of UniPR129. These two compounds acted as competitive and reversible antagonists of all Eph receptors reducing both ephrin-A1 and -B1 binding to EphAs and EphBs receptors in the low micromolar range. The compounds acted as antagonists inhibiting ephrin-A1-dependent EphA2 activation and UniPR139 exerted an anti-angiogenic effect, inhibiting HUVEC tube formation in vitro and VEGF-induced vessel formation in the chick chorioallantoic membrane assay. Finally, the oral bioavailability of UniPR139 represents a step forward in the search of molecules targeting the Eph/ephrin system and offers a new pharmacological tool useful for future in vivo studies.

Vascular endothelial growth factor receptor-2 (VEGFR2) is the main pro-angiogenic receptor expressed by endothelial cells (ECs). Using surface plasmon resonance, immunoprecipitation, enzymatic digestion, immunofluorescence and cross-linking experiments with specific sugar-binding lectins, we demonstrated that VEGFR2 bears both α,1-fucose and α(2,6)-linked sialic acid (NeuAc). However, only the latter is required for VEGF binding to VEGFR2 and consequent VEGF-dependent VEGFR2 activation and motogenic response in ECs. Notably, downregulation of β-galactoside α(2,6)-sialyltransferase expression by short hairpin RNA transduction inhibits VEGFR2 α(2,6) sialylation that is paralleled by an increase of β-galactoside α(2,3)-sialyltransferase expression. This results in an ex-novo α(2,3)-NeuAc sialylation of the receptor that functionally replaces the lacking α(2,6)-NeuAc, thus allowing VEGF/VEGFR2 interaction. In keeping with the role of VEGFR2 sialylation in angiogenesis, the α(2,6)-NeuAc-binding lectin Sambucus nigra (SNA) prevents VEGF-dependent VEGFR2 autophosphorylation and EC motility, proliferation and motogenesis. In addition, SNA exerts a VEGF-antagonist activity in tridimensional angiogenesis models in vitro and in the chick-embryo chorioallantoic membrane neovascularization assay and mouse matrigel plug assay in vivo. In conclusion, VEGFR2-associated NeuAc plays an important role in modulating VEGF/VEGFR2 interaction, EC pro-angiogenic activation and neovessel formation. VEGFR2 sialylation may represent a target for the treatment of angiogenesis-dependent diseases.

Primary cilium drives the left-right asymmetry process during embryonic development. Moreover, its dysregulation contributes to cancer progression by affecting various signaling pathways. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system modulates primary cilium length and plays a pivotal role in embryogenesis and tumor growth. Here, we investigated the impact of the natural FGF trap long-pentraxin 3 (PTX3) on the determination of primary cilium extension in zebrafish embryo and cancer cells. The results demonstrate that down modulation of the PTX3 orthologue ptx3b causes the shortening of primary cilium in zebrafish embryo in a FGF-dependent manner, leading to defects in the left-right asymmetry determination. Conversely, PTX3 upregulation causes the elongation of primary cilium in FGF-dependent cancer cells. Previous observations have identified the PTX3-derived small molecule NSC12 as an orally available FGF trap with anticancer effects on FGF-dependent tumors. In keeping with the non-redundant role of the FGF/FGR system in primary cilium length determination, NSC12 induces the elongation of primary cilium in FGF-dependent tumor cells, thus acting as a ciliogenic anticancer molecule in vitro and in vivo. Together, these findings demonstrate the ability of the natural FGF trap PTX3 to exert a modulatory effect on primary cilium in embryonic development and cancer. Moreover, they set the basis for the design of novel ciliogenic drugs with potential implications for the therapy of FGF-dependent tumors.

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase &beta, galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31&minus, /VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease.

Chemokine receptor CXCR4, its ligand stromal cell-derived factor-1 (CXCL12) and the decoy receptor atypical chemokine receptor 3 (ACKR3, also named CXCR7), are involved in the guidance of migrating cells in different anatomical districts. Here, we investigated the role of the ACKR3 zebrafish ortholog ackr3b in the vascularization process during embryonic development. Bioinformatics and functional analyses confirmed that ackr3b is a CXCL12-binding ortholog of human ACKR3. ackr3b is transcribed in the endoderm of zebrafish embryos during epiboly and is expressed in a wide range of tissues during somitogenesis, including central nervous system and somites. Between 18 somite and 26 h-post fertilization stages, the broad somitic expression of ackr3b becomes restricted to the basal part of the somites. After ackr3b knockdown, intersomitic vessels (ISVs) lose the correct direction of migration and are characterized by the presence of aberrant sprouts and ectopic filopodia protrusions, showing downregulation of the tip/stalk cell marker hlx1. In addition, ackr3b morphants show significant alterations of lateral dorsal aortae formation. In keeping with a role for ackr3b in endothelial cell guidance, CXCL12 gradient generated by ACKR3 expression in CHO cell transfectants guides human endothelial cell migration in an in vitro cell co-culture chemotaxis assay. Our results demonstrate that ackr3b plays a non-redundant role in the guidance of sprouting endothelial cells during vascular development in zebrafish. Moreover, ACKR3 scavenging activity generates guidance cues for the directional migration of CXCR4-expressing human endothelial cells in response to CXCL12.

Eph/ephrin interactions and their bidirectional signaling are integral part of the complex communication system between β-cells, essential for glucose homeostasis. Indeed, Eph/ephrin system was shown to be directly involved in the glucose-stimulated insulin secretion (GSIS) process occurring in the pancreatic islets. Here we tested the Eph antagonist UniPR500 as GSIS enhancer. UniPR500 was validated as EphA5-ephrin-A5 inhibitor in vitro and its efficacy as GSIS enhancer was assessed on EndoC-βH1 cells. The selectivity of UniPR500 was evaluated by testing this compound on a panel of well-known molecular targets responsible for the regulation of glucose homeostasis. Plasmatic levels of UniPR500 were measured by HPLC/MS approach after oral administration. Finally, UniPR500 was tested as hypoglycemic agent in healthy mice, in a non-genetic mouse model of insulin resistance (IR) and in a non-genetic mouse model of type 1 diabetes (T1D). The compound is an orally bioavailable and selective Eph antagonist, able to increase GSIS from EndoC-βH1 cells. When tested in vivo UniPR500 showed to improve glucose tolerance in healthy and IR mice. As expected by a GSIS enhancer acting on healthy β-cells, UniPR500 was ineffective when tested on a non-genetic mouse model of type 1 diabetes, where pancreatic function was severely compromised. In conclusion our findings suggest that Eph targeting is a new and valuable pharmacological strategy in the search of new hypoglycemic agents.

The possible resistance of influenza virus against existing antiviral drugs calls for new therapeutic concepts. One appealing strategy is to inhibit virus entry, in particular at the stage of internalization. This requires a better understanding of virus-host interactions during the entry process, including the role of receptor tyrosine kinases (RTKs). To search for cellular targets, we evaluated a panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 displayed robust anti-influenza A and B virus activity and was selected for mechanistic investigations. Ki8751 efficiently disrupted the endocytic process of influenza virus in different cell lines carrying platelet-derived growth factor receptor β (PDGFRβ), an RTK that is known to act at GM3 ganglioside-positive lipid rafts. The more efficient virus entry in CHO-K1 cells compared to the wild-type ancestor (CHO-wt) cells indicated a positive effect of GM3, which is abundant in CHO-K1 but not in CHO-wt cells. Entering virus localized to GM3-positive lipid rafts and the PDGFRβ-containing endosomal compartment. PDGFRβ/GM3-dependent virus internalization involved PDGFRβ phosphorylation, which was potently inhibited by Ki8751, and desialylation of activated PDGFRβ by the viral neuraminidase. Virus uptake coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRβ signalling pathway for cell penetration, providing an opportunity for host cell-targeting antiviral intervention. ispartof: JOURNAL OF GENERAL VIROLOGY vol:100 issue:4 pages:583-601 ispartof: location:England status: published

Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. A second putative FGF2 binding sequence was also identified in repeat 11C of both TSPs. Computational docking analysis predicted that both the TSP-2 and TSP-1-derived heptapeptides interacted with FGF2 with comparable binding properties. Accordingly, small molecules based on the TSP-1 active sequence blocked TSP-2/FGF2 interaction. Binding of TSP-2 to FGF2 impaired the growth factor ability to interact with its cellular receptors, since TSP-2-derived fragments prevented the binding of FGF2 to both heparin (used as a structural analog of heparan sulfate proteoglycans) and FGFR-1. These findings identify TSP-2 as a new FGF2 ligand that shares with TSP-1 the same molecular requirements for interaction with the growth factor and a comparable capacity to block FGF2 interaction with proangiogenic receptors. These features likely contribute to TSP-2 antiangiogenic and antineoplastic activity, providing the rationale for future therapeutic applications.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue tumor classified in two major subtypes namely embryonal and alveolar, which have distinctive histopathological and genetic signatures and worse outcomes in the presence of metastases. Here, in order to evaluate the role of Caveolin-1 (Cav-1) in embryonal RMS dissemination, we employed an experimental in vivo metastasis assay using immunodeficient NOD/SCID mice. We found that the intravenous injection of human RD cells engineered for Cav-1 overexpression promoted the formation of lung metastases compared to parental cells. The arisen metastases were isolated and cultured in vitro to establish two derivative lines that showed greater metastatic capacity, as detected by performing in vivo metastasis and tumor spheroid invasion assays. Compared to parental cells, all metastatic lines were characterized by an increase in cell proliferation, migration and invasiveness that were downregulated by synthetic inhibition of Erk pathway. The metastatic cells showed a marked cell apoptosis induced by nutrient deprivation and consistent loss of differentiation characterized by depletion of MyoD and Myogenin factors. Furthermore, they showed marked changes in cell size, a re-organization of the three-dimensional cytoskeleton characterized by an increased actin stress fiber content, and increased adhesion and angiogenic properties. Collectively, these data provide new insights into Cav-1-driven metastatic process of embryonal RMS through cooperation of the Erk signaling pathway. Furthermore, our derivative metastatic lines represent useful tools for identifying genes or molecular pathways that regulate the metastatic progression of embryonal RMS.

The EphA2 receptor has been validated in animal models as new target for treating tumors depending on angiogenesis and vasculogenic mimicry. In the present work, we extended our current knowledge on structure-activity relationship (SAR) data of two related classes of antagonists of the EphA2 receptor, namely 5β-cholan-24-oic acids and 5β-cholan-24-oyl l-β-homotryptophan conjugates, with the aim to develop new antiangiogenic compounds able to efficiently prevent the formation of blood vessels. As a result of our exploration, we identified UniPR505, N-[3α-(Ethylcarbamoyl)oxy-5β-cholan-24-oyl]-l-β-homo-tryptophan (compound 14), as a submicromolar antagonist of the EphA2 receptor capable to block EphA2 phosphorylation and to inhibit neovascularization in a chorioallantoic membrane (CAM) assay.

Sialic acid is associated with glycoproteins and gangliosides of eukaryotic cells. It regulates various molecular interactions, being implicated in inflammation and cancer, where its expression is regulated by sialyltransferases and sialidases. Angiogenesis, the formation of new capillaries, takes place during inflammation and cancer, and represents the outcome of several interactions occurring at the endothelial surface among angiogenic growth factors, inhibitors, receptors, gangliosides and cell-adhesion molecules. Here, we elaborate on the evidences that many structures involved in angiogenesis are sialylated and that their interactions depend on sialic acid with implications in angiogenesis itself, inflammation and cancer. We also discuss the possibility to exploit sialic acid as a target for the development of novel antiangiogenic drugs.

Originally characterized as angiogenic factors, fibroblast growth factors (FGFs) are pleiotropic factors that exert autocrine and paracrine functions on tumor and stromal cells. Thus, they may represent key players in the complex crosstalk among angiogenesis, inflammation, tumor growth, and drug resistance, all contributing to tumor progression. Given the multiple activities of FGFs, inhibitors of the FGF/FGFR system may act as "two compartment" targeting drugs able to exert a deep impact on the growth of FGF/FGFR-driven tumors. To date, the discovery of drugs targeting the FGF/FGFR system has focused mainly on the development of selective and non-selective tyrosine kinase FGFR inhibitors. Recently, a different approach has been emerging, aimed at the development of extracellular "FGF ligand traps" able to bind and sequester FGFs, thus preventing their interaction with cognate signaling receptors. This approach is based on the identification of natural FGF ligands followed by the development of small molecule mimetics endowed with a significant FGF binding/neutralizing capacity. Aim of this review is to provide an overview of the role of the FGF/FGFR system in cancer and a comprehensive analysis of the process, based on the study of the FGF interactome, which has led to the identification and characterization of FGF ligand traps. This approach has allowed the development of promising FGF-targeting molecules with potential implications for the therapy of FGF-driven tumors.

Despite decades of antiviral drug research and development, viruses still remain a top global healthcare problem. Compared to eukaryotic cells, viruses are composed by a limited numbers of proteins that, nevertheless, set up multiple interactions with cellular components, allowing the virus to take control of the infected cell. Each virus/host interaction can be considered as a therapeutical target for new antiviral drugs but, unfortunately, the systematic study of a so huge number of interactions is time-consuming and expensive, calling for models overcoming these drawbacks. Surface plasmon resonance (SPR) is a label-free optical technique to study biomolecular interactions in real time by detecting reflected light from a prism-gold film interface. Launched 20 years ago, SPR has become a nearly irreplaceable technology for the study of biomolecular interactions. Accordingly, SPR is increasingly used in the field of virology, spanning from the study of biological interactions to the identification of putative antiviral drugs. From the literature available, SPR emerges as an ideal link between conventional biological experimentation and system biology studies functional to the identification of highly connected viral or host proteins that act as nodal points in virus life cycle and thus considerable as therapeutical targets for the development of innovative antiviral strategies.

PDR is characterized by neovascularization and a persistent grade of inflammation. N-formyl peptide receptors (FPRs) belong to a transmembrane G protein-coupled receptor superfamily able to modulate angiogenic and inflammatory responses. However, their involvement in PDR remains to be elucidated. Here, we examined FPR expression in human umbilical vein endothelial cells (HUVECs). We further assessed the capacity of vitreous fluid obtained from PDR patients after pars plana vitrectomy to induce pro-angiogenic/pro-inflammatory responses in endothelium and the contribution of FPR inhibitors to abrogate these responses.

AIMS/HYPOTHESIS: Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses. METHODS: Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous. RESULTS: PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45+ cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay. CONCLUSIONS/INTERPRETATION: This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR therapy.

The peptides N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) and BOC-Met-Leu-Phe (BOC1) are widely used antagonists of formyl peptide receptors (FPRs), BOC2 acting as an FPR1/FPR2 antagonist whereas BOC1 inhibits FPR1 only. Extensive investigations have been performed by using these FPR antagonists as a tool to assess the role of FPRs in physiological and pathological conditions. Based on previous observations from our laboratory, we assessed the possibility that BOC2 may exert also a direct inhibitory effect on the angiogenic activity of vascular endothelial growth factor-A (VEGF-A). Our data demonstrate that BOC2, but not BOC1, inhibits the angiogenic activity of heparin-binding VEGF-A165 with no effect on the activity of the non-heparin-binding VEGF-A121 isoform. Endothelial cell-based bioassays, surface plasmon resonance analysis, and computer modeling indicate that BOC2 may interact with the heparin-binding domain of VEGF-A165, thus competing for heparin interaction and preventing the binding of VEGF-A165 to tyrosine kinase receptor VEGFR2, its phosphorylation and downstream signaling. In addition, BOC2 inhibits the interaction of a variety of heparin-binding angiogenic growth factors with heparin, including fibroblast growth factor 2 (FGF2) whose angiogenic activity is blocked by the compound. Accordingly, BOC2 suppresses the angiogenic potential of human tumor cell lines that co-express VEGF-A and FGF2. Thus, BOC2 appears to act as a novel multi-heparin-binding growth factor antagonist. These findings caution about the interpretation of FPR-focusing experimental data obtained with this compound and set the basis for the design of novel BOC2-derived, FPR independent multi-target angiogenesis inhibitors.

Once released by HIV+ cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (Kd = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg→Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists.

Objective— Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P 1 ) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P 1 ortholog in vascular development in zebrafish. Methods and Results— zS1P 1 is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P 1 downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P 1 morphants are characterized by downregulation of vascular endothelial cadherin ( VE-cadherin ) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P 1 morphants. Finally, S1P 1 small interfering RNA transfection and the S1P 1 antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. Conclusion— The data demonstrate a nonredundant role of zS1P 1 in the regulation of venous endothelial barrier in zebrafish and identify a S1P 1 / VE-cadherin / EphB4a genetic pathway that controls venous vascular integrity.

Fibroblast growth factors (FGFs) are a family of pleiotropic factors produced by stromal and parenchymal tumor cells. Even though FGFs have been firstly characterized as angiogenic factors, they exert autocrine and paracrine functions not only on endothelial cells but also on tumor cells and other stromal components. Thus, the FGF/FGF receptor (FGFR) pathway may represent a key player in tumor growth by regulating the complex cross-talk between stromal and tumor compartments. The ligand dependent or independent activation of the FGF/FGFR system by gene upregulation, oncogenic mutation or amplification occurs in a variety of human tumors and is implicated in various key steps of tumor growth and progression. In addition, FGF/FGFR activation has been described as a mechanism of tumor escape in response to antiangiogenic/anti-VEGF therapies. Experimental and clinical evidences provide a compelling biologic rationale for the development of anti-FGF/FGFR targeting agents in cancer therapy. However, the development of drugs specifically targeting the FGF/FGFR pathway proved to be difficult, also due to the high redundancy and pleiotropic effects of FGF and FGFR family members. On the other hand, the possibility to develop "two-compartment" targeting agents endowed with both antiangiogenic and antitumor activities remains promising. Here we will review the preclinical and clinical approaches and potential therapeutics currently available to block the FGF/FGFR system in human cancer.

Once released by HIV(+) cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (K(d) = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg→Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists.

Sialic acid (NeuAc) is a major anion on endothelial cells (ECs) that regulates different biological processes including angiogenesis. NeuAc is present in the oligosaccharidic portion of integrins, receptors that interact with extracellular matrix components and growth factors regulating cell adhesion, migration, and proliferation. Tat is a cationic polypeptide that, once released by HIV-1(+) cells, accumulates in the extracellular matrix, promoting EC adhesion and proangiogenic activation by engaging α(v)β(3). By using two complementary approaches (NeuAc removal by neuraminidase or its masking by NeuAc-binding lectin from Maackia amurensis, MAA), we investigated the presence of NeuAc on endothelial α(v)β(3) and its role in Tat interaction, EC adhesion, and proangiogenic activation. α(v)β(3) immunoprecipitation with biotinylated MAA or Western blot analysis of neuraminidase-treated ECs demonstrated that NeuAc is associated with both the α(v) and the β(3) subunits. Surface plasmon resonance analysis demonstrated that the masking of α(v)β(3)-associated NeuAc by MAA prevents Tat/α(v)β(3) interaction. MAA and neuraminidase prevent α(v)β(3)-dependent EC adhesion to Tat, the consequent FAK and ERK1/2 phosphorylation, and EC proliferation, migration, and regeneration in a wound-healing assay. Finally, MAA inhibits Tat-induced neovascularization in the ex vivo human artery ring sprouting assay. The inhibitions are specific because the NeuAc-unrelated lectin from Ulex europaeus is ineffective on Tat. Also, MAA and neuraminidase affect only weakly integrin-dependent EC adhesion and proangiogenic activation by fibronectin. In conclusion, NeuAc is associated with endothelial α(v)β(3) and mediates Tat-dependent EC adhesion and proangiogenic activation. These data point to the possibility to target integrin glycosylation for the treatment of angiogenesis/AIDS-associated pathologies.

Objective— Heparan sulfate proteoglycans (HSPGs) modulate the interaction of proangiogenic heparin-binding vascular endothelial growth factors (VEGFs) with signaling VEGF receptor-2 (VEGFR2) and neuropilin coreceptors in endothelial cells (ECs). The bone morphogenic protein antagonist gremlin is a proangiogenic ligand of VEGFR2, distinct from canonical VEGFs. Here we investigated the role of HSPGs in VEGFR2 interaction, signaling, and proangiogenic capacity of gremlin in ECs. Methods and Results— Surface plasmon resonance demonstrated that gremlin binds heparin and heparan sulfate, but not other glycosaminoglycans, via N -, 2- O , and 6- O -sulfated groups of the polysaccharide. Accordingly, gremlin binds HSPGs of the EC surface and extracellular matrix. Gremlin/HSPG interaction is prevented by free heparin and heparan sulfate digestion or undersulfation following EC treatment with heparinase II or sodium chlorate. However, at variance with canonical heparin-binding VEGFs, gremlin does not interact with neuropilin-1 coreceptor. On the other hand, HSPGs mediate VEGFR2 engagement and autophosphorylation, extracellular signaling-regulated kinase 1/2 and p38 mitogen-activated protein kinase activation, and consequent proangiogenic responses of ECs to gremlin. On this basis, we evaluated the gremlin-antagonist activity of a panel of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. The results demonstrate that the highly N,O -sulfated derivative K5-N,OS(H) binds gremlin with high potency, thus inhibiting VEGFR2 interaction and angiogenic activity in vitro and in vivo. Conclusion— HSPGs act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2 and angiogenic activity. This has allowed the identification of the biotechnological K5-N,OS(H) as a novel angiostatic gremlin antagonist.

The transactivating factor (Tat) of HIV-1 is involved in AIDS progression and associated pathologies. Tat possesses a basic amino acid sequence implicated in heparan sulfate proteoglycan (HSPG)-mediated internalization, nuclear localization and transactivation by Tat and in the interaction of Tat with integrins and with the vascular endothelial growth factor receptor 2 (KDR) (kinase insert domain receptor). A BSA conjugate bearing an average of four copies of a peptide representing the basic domain/nuclear localization signal of Tat (BSA-Tat-NLS) inhibits transactivation by Tat exogenously added to cells but not by Tat endogenously produced after cell transfection with a tat cDNA, indicating that BSA-Tat-NLS does not interfere with Tat at an intracellular level. Surface plasmon resonance (SPR) experiments revealed that BSA-Tat-NLS binds to the HSPG analogue heparin. Accordingly, BSA-Tat-NLS binds to HSPGs of HL3T1 cell surface and inhibits HSPG-dependent Tat internalization. BSA-Tat-NLS retains its inhibitory potential when pre-incubated with HL3T1 cells before Tat administration, possibly by masking cell-surface HSPGs thus preventing Tat binding and internalization. SPR experiments revealed that BSA-Tat-NLS binds also to integrin alpha(v)beta(3) and KDR. Accordingly, it inhibits pro-angiogenic endothelial cell adhesion to Tat and motogenesis. In conclusion, BSA-Tat-NLS binds/masks three different cell-surface receptors of Tat inhibiting different biological activities. These data point to BSA-Tat-NLS as a prototype for the development of Tat-antagonists endowed with a multitargeted mechanism of action.

Polyanionic macromolecules are extremely abundant both in the extracellular environment and inside the cell, where they are readily accessible to many proteins for interactions that play a variety of biological roles. Among polyanions, heparin, heparan sulfate proteoglycans (HSPGs) and glycosphingolipids (GSLs) are widely distributed in biological fluids, at the cell membrane and inside the cell, where they are implicated in several physiological and/or pathological processes such as infectious diseases, angiogenesis and tumor growth. At a molecular level, these processes are mainly mediated by microbial proteins, cytokines and receptors that exert their functions by binding to HSPGs and/or GSLs, suggesting the possibility to use polyanionic antagonists as efficient drugs for the treatment of infectious diseases and cancer. Polysulfated (PS) or polysulfonated (PSN) compounds are a heterogeneous group of natural, semi-synthetic or synthetic molecules whose prototypes are heparin and suramin. Different structural features confer to PS/PSN compounds the capacity to bind and inhibit the biological activities of those same heparin-binding proteins implicated in infectious diseases and cancer. In this review we will discuss the state of the art and the possible future development of polyanionic drugs in the treatment of infectious diseases and cancer.