Targeting of PDGF-C/NRP-1 autocrine loop as a new strategy for counteracting the invasiveness of melanoma resistant to braf inhibitors

: Melanoma resistance to BRAF inhibitors (BRAFi) is often accompanied by a switch from a proliferative to an invasive phenotype. Therefore, the identification of signaling molecules involved in the development of metastatic properties by resistant melanoma cells is of primary importance. We have previously demonstrated that activation of neuropilin-1 (NRP-1) by platelet-derived growth factor (PDGF)-C confers melanoma cells with an invasive behavior similar to that of BRAFi resistant tumors. Aims of the present study were to evaluate the role of PDGF-C/NRP-1 autocrine loop in the acquisition of an invasive and BRAFi-resistant phenotype by melanoma cells and the effect of its inhibition on drug resistance and extracellular matrix (ECM) invasion. Furthermore, we investigated whether PDGF-C serum levels were differentially modulated by drug treatment in metastatic melanoma patients responsive or refractory to BRAFi as single agents or in combination with MEK inhibitors (MEKi). The results indicated that human melanoma cells resistant to BRAFi express higher levels of PDGF-C and NRP-1 as compared to their susceptible counterparts. Overexpression occurs early during development of drug resistance and contributes to the invasive properties of resistant cells. Accordingly, silencing of NRP-1 or PDGF-C reduces tumor cell invasiveness. Analysis of PDGF-C in the serum collected from patients treated with BRAFi or BRAFi+MEKi, showed that in responders PDGF-C levels decrease after treatment and raise again at tumor progression. Conversely, in non-responders treatment does not affect PDGF-C serum levels. Thus, blockade of NRP-1 activation by PDGF-C might represent a new therapeutic approach to counteract the invasiveness of BRAFi-resistant melanoma

Proteínas de membrana implicadas en la activación de neutrofilos humanos : su estudio mediante anticuerpos monoclonales

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 3-10-198

Intracellular location of T200 and Mo1 glycoproteins in human neutrophils

6 p.-5 fig.-1 tab.Mo1 (CD11b), a glycoprotein heterodimer that is involved in cellular adhesion processes and functions as the C3bi receptor of human myeloid cells, and T200 (CD45), a panleukocyte glycoprotein family whose function is still not well understood, increased their expression in the plasma membrane of human neutrophils after exposure to various stimuli which induce degranulation, such as formylmethionylleucylphenylalanine or calcium ionophore A23187. This increment in the expression of both molecules shows a good correlation with the release to the extracellular environment of gelatinase, a marker for an intracellular organelle named "tertiary granule" (Mollinedo, F., and Schneider, D. L. (1984) J. Biol. Chem. 259, 7143-7150). Flow cytometry studies indicate that at least 50% of the total Mo1 and T200 molecules are located in intracellular organelles. Furthermore, the subcellular distribution of Mo1 and T200 glycoproteins in resting human neutrophils was investigated by immunoprecipitation of the radiolabeled membrane proteins obtained from the distinct subcellular fractions. Both Mo1 and T200 were mainly localized in tertiary or specific intracellular granules, which were resolved from the azurophilic granules as well as from the cell membrane fraction. These findings suggest that the mobilization of intracellular Mo1 and T200 to the plasma membrane may regulate early events occurring upon neutrophil activation.This research was supported in part by Grant 424 from the Consejo Superior de Investigaciones Cientificas and Grant 87/1587 from the Fondo de Investigaciones Sanitarias de la Seguridad Social.Peer reviewe

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl- phenylalanine-triggered respiratory burst

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C- mediated signal transduction mechanisms coupled to the induction of the respiratory burst

The VEGFs/VEGFRs system in Alzheimer’s and Parkinson’s diseases: Pathophysiological roles and therapeutic implications

The vascular endothelial growth factors (VEGFs) and their cognate receptors (VEGFRs), besides their well-known involvement in physiological angiogenesis/lymphangiogenesis and in diseases associated to pathological vessel formation, play multifaceted functions in the central nervous system (CNS). In addition to shaping brain development, by controlling cerebral vasculogenesis and regulating neurogenesis as well as astrocyte differentiation, the VEGFs/VEGFRs axis exerts essential functions in the adult brain both in physiological and pathological contexts. In this article, after describing the physiological VEGFs/VEGFRs functions in the CNS, we focus on the VEGFs/VEGFRs involvement in neurodegenerative diseases by reviewing the current literature on the rather complex VEGFs/VEGFRs contribution to the pathogenic mechanisms of Alzheimer’s (AD) and Parkinson’s (PD) diseases. Thereafter, based on the outcome of VEGFs/VEGFRs targeting in animal models of AD and PD, we discuss the factual relevance of pharmacological VEGFs/VEGFRs modulation as a novel and potential disease-modifying approach for these neurodegenerative pathologies. Specific VEGFRs targeting, aimed at selective VEGFR-1 inhibition, while preserving VEGFR-2 signal transduction, appears as a promising strategy to hit the molecular mechanisms underlying AD pathology. Moreover, therapeutic VEGFs-based approaches can be proposed for PD treatment, with the aim of fine-tuning their brain levels to amplify neurotrophic/neuroprotective effects while limiting an excessive impact on vascular permeability

Progressive Increase in Telomerase Activity From Benign Melanocytic Conditions to Malignant Melanoma

The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR) in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction—based assay (TRAP). High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastasess = 54.5, lymph node metastasess = 56.5). Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9). Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions

The Anti-Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1) D16F7 Monoclonal Antibody Inhibits Melanoma Adhesion to Soluble VEGFR-1 and Tissue Invasion in Response to Placenta Growth Factor

Simple Summary Melanoma is an aggressive cancer type with a high tendency to spread to distant body sites, including bones. Despite the availability of effective therapies, many patients still do not fully benefit from treatment. The aim of our study was to evaluate the therapeutic potential of inhibiting the activation of the vascular endothelial growth factor receptor (VEGFR-1) by placenta growth factor (PlGF) using an investigational anti-VEGFR-1 monoclonal antibody (D16F7 mAb). The VEGFR-1 receptor is expressed by endothelial cells of blood vessels that nourish the tumor, protumoral macrophages and melanoma cells. Results indicate that PlGF stimulates the ability of melanoma to infiltrate the surrounding tissues and that treatment with D16F7 mAb counteracts melanoma properties, which contribute to tumor spread, reducing the invasiveness of the tumor and its tropism toward bone tissue. Therefore, blockade of VEGFR-1 stimulation by PlGF represents a suitable strategy to restrain the metastatic potential of melanoma. Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family involved in tumor-associated angiogenesis and melanoma invasion of the extra-cellular matrix (ECM) through activation of membrane VEGF receptor 1 (VEGFR-1). A soluble VEGFR-1 (sVEGFR-1) form is released in the ECM, where it sequesters proangiogenic factors and stimulates endothelial or tumor cell adhesion and chemotaxis through interaction with alpha 5 beta 1 integrin. The anti-VEGFR-1 monoclonal antibody (D16F7 mAb) inhibits VEGF-A or PlGF-mediated signal transduction without affecting ligand interaction, thus preserving sVEGFR-1 decoy function. The aim of this study was to investigate whether D16F7 mAb hampers melanoma spread by in vitro analysis of cell adhesion to sVEGFR-1, ECM invasion, transmigration through an endothelial cell monolayer and in vivo evaluation of tumor infiltrative potential in a syngeneic murine model. Results indicate that D16F7 mAb significantly inhibits melanoma adhesion to sVEGFR-1 and ECM invasion, as well as transmigration in response to PlGF. Moreover, treatment of melanoma-bearing mice with the anti-VEGFR-1 mAb not only inhibits tumor growth but also induces a significant reduction in bone infiltration associated with a decrease in PlGF-positive melanoma cells. Furthermore, D16F7 mAb reduces PlGF production by melanoma cells. Therefore, blockade of PLGF/VEGFR-1 signaling represents a suitable strategy to counteract the metastatic potential of melanoma

The Anti–Vascular Endothelial Growth Factor Receptor-1 Monoclonal Antibody D16F7 Inhibits Glioma Growth and Angiogenesis In Vivo

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Targeting the PI3K/AKT/mTOR pathway overcomes the stimulating effect of dabrafenib on the invasive behavior of melanoma cells with acquired resistance to the BRAF inhibitor

BRAF inhibitors (BRAFi) have proven clinical benefits in patients with BRAF-mutant melanoma. However acquired resistance eventually arises. The effects of BRAFi on melanoma cell proliferation and survival have been extensively studied, and several mechanisms involved in acquired resistance to the growth suppressive activity of these drugs have been identified. Much less is known about the impact of BRAFi, and in particular of dabrafenib, on the invasive potential of melanoma cells. In the present study, the BRAF-mutant human melanoma cell line A375 and its dabrafenib-resistant subline A375R were analyzed for invasive capacity, expression of vascular endothelial growth factor receptor (VEGFR)-2 and secretion of VEGF-A and matrix metalloproteinase (MMP)-9, under basal conditions or in response to dabrafenib. The consequences of inhibiting the PI3K/AKT/mTOR pathway on A375R cell responses to dabrafenib were also evaluated. We found that A375R cells were more invasive and secreted higher levels of VEGF-A and MMP-9 as compared with A375 cells. Dabrafenib reduced invasiveness, VEGFR-2 expression and VEGF-A secretion in A375 cells, whereas it increased invasiveness, VEGF-A and MMP-9 release in A375R cells. In these latter cells, the stimulating effects of dabrafenib on the invasive capacity were markedly impaired by the anti-VEGFA antibody bevacizumab, or by AKT1 silencing. A375R cells were not cross-resistant to the PI3K/mTOR inhibitor GSK2126458A. Moreover, this inhibitor given in combination with dabrafenib efficiently counteracted the stimulating effects of the BRAFi on invasiveness and VEGF-A and MMP-9 secretion. Our data demonstrate that melanoma cells with acquired resistance to dabrafenib possess a more invasive phenotype which is further stimulated by exposure to the drug. Substantial evidence indicates that continuing BRAFi therapy beyond progression produces a clinical benefit. Our results suggest that after the development of resistance, a regimen combining BRAFi with bevacizumab or with inhibitors of the PI3K/AKT/mTOR pathway might be more effective than BRAFi monotherapy