PO-86 Cancer cells modify the microenvironment: RGS5, a novel endothelial protein of the abnormal tumor vasculature

Abstract

s / Thrombosis Research 129, Supplement 1 (2012) S155–S194 S193 PO-86 Cancer cells modify the microenvironment: RGS5, a novel endothelial protein of the abnormal tumor vasculature S. Figini1, A. Silini1, C. Ghilardi1, R. Fruscio2, R. Giavazzi1, M.R. Bani1 1Laboratory of Biology and Treatment of Metastases, Department of Oncology, MarioNegri Institute for Pharmacological Research, Milan; 2Clinic of Obstetrics and Gynecology, San Gerardo Hospital, Monza, Italy Background and Aim: An ever-increasing body of evidence suggests that cancer cells are able to surmount environmental controls by the host and modify the surroundings to promote local tumor growth, invasion and metastasis, and to affect drug response. Vascular endothelial growth factor (VEGF) -which overproduction is associated with poor prognosisis thought to play a decisive role in remodeling the environment. Molecules expressed by the tumor microenvironment provide opportunities for the development of innovative therapeutic strategies; thus our purpose was to analyze, in an in vivo setting, the transcriptional features of the tumor microenvironment (stroma) in reaction to the stimuli provided by the cancer cells. Groundwork: Stroma was micro-dissected from 1A9-VS1 (high VEGF) and 1A9-VAS3 (low VEGF) human ovarian carcinoma xenografts and hybridized to GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix). The VEGF produced by the cancer cells triggered the modification of the tumor environment: regulator of G protein signaling-5 (RGS5) was listed among the transcripts over-expressed by the stroma of 1A9-VS1. Methods: In this study, RGS5 expression was assayed by qRT-PCR and immunofluorescence. Tumor xenografts were obtained by subcutaneous injection of cancer cells in nude mice. Human ovarian carcinoma specimens were collected at time of the first surgery. Endothelial cells (EC) were isolated as described. Results: We show elevated RGS5 associated to the abnormal vasculature developing in 1A9-VS1 ovarian carcinoma xenografts, in comparison to the poorly angiogenic 1A9-VAS3. In addition, we show RGS5 protein associated to blood vessels of human melanoma and renal carcinoma xenografts, but not to the vasculature of healthy mouse tissues. We prove for the first time that RGS5 protein co-localizes predominantly with the endothelium expressing platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) and also with perivascular/mural cells expressing either platelet-derived growth factor receptor-beta (PDGFR-β) or alpha smooth muscle actin (αSMA), though to a much lesser extent. We demonstrate Rgs5 transcript expression by EC isolated from human ovarian carcinomas; such expression being elicited by a milieu of pro-angiogenic factors such as VEGF, FGF-2 and EGF (known to be abundant within the tumor microenvironment). At last, we provide in vivo/ex vivo evidence of RGS5 protein in the vasculature of ovarian carcinoma clinical specimens and its absence in human ovaries. Conclusions: RGS5 expression by the cancer vasculature -triggered and retained by the proangiogenic microenvironmentsupports its exploitation as a novel biomarker and opens the path to explore new possibilities of therapeutic intervention aimed at affecting tumor microenvironment (by the dual targeting of endothelial cells and mural cells comprising the blood vessels). PO-87 Inhibition of FGF-2 angiogenic activity by novel small molecules mimetic of thrombospondin-1 (TSP-1) C. Foglieni1, R. Torella2, A. Bugatti3, K. Pagano4, L. Ragona4, D. Ribatti5, M. Rusnati3, M. Presta3, R. Giavazzi1, G. Colombo2, G. Taraboletti1 1Department of Oncology, Mario Negri Institute for Pharmacological Research, Bergamo; 2Istituto di Chimica del Riconoscimento Molecolare, CNR, Milan; 3Department of Biomedical Sciences and Biotechnology, University of Brescia; 4Istituto per lo Studio delle Macromolecole, CNR, Milan; 5Department of Human Anatomy and Histology, University of Bari, Italy The development of the tumor vasculature is a fundamental step in tumor progression. This process is triggered by tumorand host-derived angiogenic factors, among which fibroblast growth factor-2 (FGF-2) plays a crucial role. FGF-2 is sequestered by TSP-1, a major endogenous inhibitor of angiogenesis, suggesting that the FGF-2-binding site of TSP-1 represents a model to design new antiangiogenic agents. In previous works we have identified the FGF-2-binding aminoacidic sequence of TSP-1, and used it as a model to develop non-peptidic, TSP-1-mimetic angiostatic small molecules. To this end, we used a pharmacophore-based computational screening of the NCI 3D database, identifying 3 active molecules. Among them, sm27 showed the best activity in terms of affinity for FGF-2 and antiangiogenic activity (Colombo et al, J Biol Chem, 285: 8733, 2010). Aim of this study was to develop second-generation angiostatic molecules selected for similarity with sm27. Analogs of sm27 were selected using the Tanimoto coefficient as a measure of functional and structural similarity. A threshold of 0.7 was defined in the screening of the NCI database, using the program InstantJChem. 41 molecules matched our search criteria and 25 were obtained from the Developmental Therapeutics Program, NCI, NIH (Rockville, MD). After an initial screening, 8 compounds resulted more active than sm27 and were selected for further analysis. We first investigated their effect on the formation of the FGF-2 receptor complex (FGF-2/FGFR-1/heparan sulfate proteoglycans), using ELISA-based binding experiments and SPR analysis on Biacore. Next, we investigated the inhibitory potential of the small molecules by analyzing their effect on FGF-2 binding to endothelial cells, FGF-2-induced endothelial cell proliferation and angiogenesis induced by FGF-2 in the chicken chorioallantoic membrane assays. This study allowed us to select 8 small molecules with improved antiangiogenic activity compared to the original molecule sm27, laying the basis for further studies in the development of compounds inhibitors of angiogenesis. PO-88 Role of the FGF-2-binding fragment of thrombospondin-1 (TSP-1) in tumor progression A. Resovi1, P. Borsotti1, S. Bonifacio1, P. Oliva2, V. Castiglioni3, R. Giavazzi2, G. Taraboletti1 1Tumor Angiogenesis Unit, Department of Oncology, Mario Negri Institute for Pharmacological Research, Bergamo; 2Laboratory of Biology and Treatment of Metastasis, Department of Oncology, Mario Negri Institute for Pharmacological Research, Milano; 3Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, Facolta di Medicina Veterinaria, Universita di Milano, Italy New blood vessels formed by angiogenesis supply oxygen and nutrients to solid tumors and provide a gateway for metastatic cells to enter the bloodstream and disseminate to distant organs. Thrombospondin-1 (TSP1), a large, modular glycoprotein, is a major endogenous inhibitors of angiogenesis. TSP-1 inhibits angiogenesis through different mechanisms, including the direct binding to and sequestration of angiogenic factors such as FGF-2. Our previous studies identified the FGF-2 recognition site of TSP-1 in the “type III repeats” (TS3R) (Int J Biochem Cell Biol 2008;40:700). Aim of this study was to assess the biological relevance of this domain in angiogenesis and tumor progression. To address this point, a line of human ovarian carcinoma 1A9 was engineered to express the whole TSP-1 protein, the entire TS3R domain, the N-terminal part of TS3R containing the FGF-2 binding fragment, or, as a control, the C-terminal part of TS3R that lacks the FGF-2 binding sequence. The secretion of the proteins of interest by the transfected lines was confirmed by Western blot analysis of the conditioned medium. No relevant differences between the lines was observed, in terms of proliferation and invasive properties, indicating that the FGF-2 binding fragments does not have autocrine effects on these tumor cells. In contrast, the presence of the domain altered the angiogenic properties of cancer cells. The conditioned medium of parental 1A9 tumor cells stimulated in vitro the proliferation and motility of endothelial cells, whereas supernatants of 1A9 cells expressing TSP-1 or the FGF-2 binding fragment did not stimulate, but actually inhibited endothelial cell proliferation and motility. Each transfected cell line was implanted subcutaneously into nude mice, and tumor take and growth rate were evaluated. The expression of the entire TSP-1 protein or the FGF-2 binding fragments strongly inhibited tumor growth compared with the control lines. Our study shows that the FGF-2-binding fragment of TSP-1 has a relevant antineoplastic activity in vivo. This finding contributes to clarify the role and mechanism of action of TSP-1 in tumor progression and set the basis for future studies aimed to develop anti-angiogenic and anti-cancer therapies based on this TSP-1 fragment.