The small molecule ephrin receptor inhibitor, GLPG1790, Reduces renewal capabilities of cancer stem cells, showing anti-tumour efficacy on preclinical glioblastoma models

Therapies against glioblastoma (GBM) show a high percentage of failure associated with the survival of glioma stem cells (GSCs) that repopulate treated tumours. Forced differentiation of GSCs is a promising new approach in cancer treatment. Erythropoietin-producing hepatocellular (Eph) receptors drive tumourigenicity and stemness in GBM. We tested GLPG1790, a first small molecule with inhibition activity versus inhibitor of various Eph receptor kinases, in preclinical GBM models using in vitro and in vivo assays. GLPG1790 rapidly and persistently inhibited Ephrin-A1-mediated phosphorylation of Tyr588 and Ser897, completely blocking EphA2 receptor signalling. Similarly, this compound blocks the ephrin B2-mediated EphA3 and EphB4 tyrosine phosphorylation. This resulted in anti-glioma effects. GLPG1790 down-modulated the expression of mesenchymal markers CD44, Sox2, nestin, octamer-binding transcription factor 3/4 (Oct3/4), Nanog, CD90, and CD105, and up-regulated that of glial fibrillary acidic protein (GFAP) and pro-neural/neuronal markers, βIII tubulin, and neurofilaments. GLPG1790 reduced tumour growth in vivo. These effects were larger compared to radiation therapy (RT; U251 and T98G xenografts) and smaller than those of temozolomide (TMZ; U251 and U87MG cell models). By contrast, GLPG1790 showed effects that were higher than Radiotherapy (RT) and similar to Temozolomide (TMZ) in orthotopic U87MG and CSCs-5 models in terms of disease-free survival (DFS) and overall survival (OS). Further experiments were necessary to study possible interactions with radio- and chemotherapy. GLPG1790 demonstrated anti-tumor effects regulating both the differentiative status of Glioma Initiating Cells (GICs) and the quality of tumor microenvironment, translating into efficacy in aggressive GBM mouse models. Significant common molecular targets to radio and chemo therapy supported the combination use of GLPG1790 in ameliorative antiglioma therapy

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype, induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines

A multiparameter approach to monitor disease activity in collagen-induced arthritis

Introduction: Disease severity in collagen-induced arthritis (CIA) is commonly assessed by clinical scoring of paw swelling and histological examination of joints. Although this is an accurate approach, it is also labour-intensive and the application of less invasive and less time-consuming methods is of great interest. However, it is still unclear which of these methods represents the most discriminating measure of disease activity. Methods: We undertook a comparative analysis in which different measurements of inflammation and tissue damage in CIA were studied on an individual mouse level. We compared the current gold standard methods - clinical scoring and histological examination - with alternative methods based on scoring of X-ray or micro-computed tomography (CT) images and investigated the significance of systemically expressed proteins, involved in CIA pathogenesis, that have potential as biomarkers. Results: Linear regression analysis revealed a marked association of serum matrix metalloproteinase (MMP)-3 levels with all features of CIA including inflammation, cartilage destruction and bone erosions. This association was improved by combined detection of MMP-3 and anti-collagen IgG2a antibody concentrations. In addition, combined analysis of both X-ray and micro-CT images was found to be predictive for cartilage and bone damage. Most remarkably, validation analysis using an independent data set proved that variations in disease severity, induced by different therapies, could be accurately represented by predicted values based on the proposed parameters. Conclusions: Our analyses revealed that clinical scoring, combined with serum MMP-3, anti-collagen IgG2a measurement and scoring of X-ray and micro-CT images, yields a comprehensive insight into the different aspects of disease activity in CIA

Ezrin Regulates E-Cadherin-dependent Adherens Junction Assembly through Rac1 Activation

Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in ezrin T567D-expressing cells, compared with cells expressing wild-type ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in ezrin T567D-producing cells. In confluent cells, ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane

Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression

Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express b-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone to mammary epithelial cells induced b- casein production. We asked whether recombinant nidogen-1 alone could signal directly for b-casein. Nidogen-1 did not induce b-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate b-casein expression. Addition of full length nidogen-1 to the mixed cultures had no effect on b-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on b-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells

Genetic depletion and pharmacological targeting of alpha v integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models

Introduction: Increased expression of alpha v integrins is frequently associated with tumor cell adhesion, migration, invasion and metastasis, and correlates with poor prognosis in breast cancer. However, the mechanism by which alpha v integrins can enhance breast cancer progression is still largely unclear. The effects of therapeutic targeting of alpha v integrins in breast cancer also have yet to be investigated. Methods: We knocked down alpha v integrin in MDA-MB-231 and MCF10A-M4 breast cancer cells, or treated these cells with the alpha v antagonist GLPG0187. The effects of alpha v integrin depletion on mesenchymal markers, transforming growth factor-beta (TGF-beta)/Smad signaling and TGF-beta-induced target gene expression were analyzed in MDA-MB-231 cells by RNA analysis or Western blotting. The function of alpha v integrin on breast cancer cell migration was investigated by transwell assay in vitro, and its effect on breast cancer progression was assessed by both zebrafish and mouse xenografts in vivo. In the mouse model, GLPG0187 was administered separately, or in combination with the standard-of-care anti-resorptive agent zoledronate and the chemotherapeutic drug paclitaxel, to study the effects of combinational treatments on breast cancer metastasis. Results: Genetic interference and pharmacological targeting of alpha v integrin with GLPG0187 in different breast cancer cell lines inhibited invasion and metastasis in the zebrafish or mouse xenograft model. Depletion of alpha v integrin in MDA-MB-231 cells inhibited the expression of mesenchymal markers and the TGF-beta/Smad response. TGF-beta induced alpha v integrin mRNA expression and alpha v integrin was required for TGF-beta-induced breast cancer cell migration. Moreover, treatment of MDA-MB-231 cells with non-peptide RGD antagonist GLPG0187 decreased TGF-beta signaling. In the mouse xenografts GLPG0187 inhibited the progression of bone metastasis. Maximum efficacy of inhibition of bone metastasis was achieved when GLPG0187 was combined with the standard-of-care metastatic breast cancer treatments. Conclusion: These findings show that alpha v integrin is required for efficient TGF-beta/Smad signaling and TGF-beta-induced breast cancer cell migration, and for maintaining a mesenchymal phenotype of the breast cancer cells. Our results also provide evidence that targeting alpha v integrin could be an effective therapeutic approach for treatment of breast cancer tumors and/or metastases that overexpress alpha v integrin

Characterization of BCE-1, a Transcriptional Enhancer Regulated by Prolactin and Extracellular Matrix and Modulated by the State of Histone Acetylation

We have previously described a 160-bp enhancer (BCE-1) in the bovine β-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-β and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin- and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-β but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones