Inflammatory angiogenesis: A target for chemoprevention.

PL01-03

Recent clinical data have affirmed that angiogenesis, a process necessary for solid tumor growth and dissemination, is a key clinical target that will improve therapeutic outcomes. It is clear however that targeting angiogenesis prior to the angiogenic switch, the moment in which a hyperplastic foci gains the capacity to induce vascularization and therefore clinically relevant growth, would be the most efficient mechanism. In addition to angiogenesis, it has become increasingly clear that inflammation is a key component in cancer insurgence that can promote tumor angiogenesis and that these are tightly linked processes (1). We noted that angiogenesis is a common and key target of most chemopreventive molecules, where they most likely suppress the angiogenic switch in pre-malignant tumors, a concept we termed "Angioprevention" (2). We have shown that various molecules, ranging from flavonoids and antioxidants to retinoids, act in the tumor micro-environment inhibiting the recruitment and/or activation of endothelial cells. Further, most of these compounds appear to act upon phagocytes of the innate immune system and repress inflammation. N-acetyl-cysteine, the green tea flavonoid epigallocatechin-3-gallate (EGCG) the beer/hops derived chalcone Xanthohumol and the rotenoid deguelin all prevent angiogenesis in the Matrigel sponge angiogenesis assay in vivo and inhibit the growth of angiogenic tumors xenografts in nude mice (3-5). We analyzed the regulation of gene expression they exert in primary human umbilical endothelial cells (HUVEC) in culture with functional genomics. Expression profiles obtained through Affymetrix GeneChip arrays identified overlapping sets of genes regulated by anti-oxidants (3). The. In contrast, the ROS-producing synthetic retinoid 4-hydroxyfenretinide (4HPR), which also shows anti-angiogenic effects, induced members of the TGF-β ligand superfamily, which, at least in part, explains its anti-angiogenic activity (6). NAC and the flavonoids all suppressed the IκB/NF-κB signalling pathway even in the presence of NF-κB stimulation by TNFα, and showed reduced expression of many NF-κB target genes (3-5). A selective apoptotic effect on transformed cells, but not on endothelial cells, of the anti-oxidants may be related to the reduced expression of the NF-κB dependent survival factors Bcl2 and Birc5/surviving, that are selectively over-expressed in transformed cells, by these factors. The repression of the NF-κB pathway suggests an overall anti-inflammatory activity of the anti-oxidant compounds that may represent an additional mechanism of angiogenesis inhibition. For example, the green tea flavonoid EGCG targets inflammatory cells, including neutrophils, and inhibits inflammation-associated angiogenesis. The link between angiogenesis and immunity is further underscored by our recent data that demonstrate a key role for the immune system in generating anti-angiogenic responses induced by anti-angiogenesis inhibitors. Angiostatin, an anti-angiogenic fragment of plasminogen originally isolated through functional assays, still lacks a definitive mode of action. Although most studies on angiostatin action have focused on endothelial cells in culture, in microarray analyses we noted that exposure of endothelial cells in vitro to angiostatin had very limited effects on gene expression profiles as compared to that generated by anti-angiogenic cytokines. Since angiostatin exerts effects on leukocytes such as neutrophils and monocytes that are primary sources of cytokines, we examined their role in AST induced angiogenesis inhibition in vivo. Using function blocking antibodies and gene targeted animals, we discovered that IL-12 is required for transduction of the angiostatin signal for angiogenesis inhibition. However, microarray analyses demonstrated that endothelial cells neither produced IL-12 upon angiostatin stimulation nor respond to IL-12 in vitro. These data clearly show that the immune system is a key component in tumor angiogenesis and a critical target in cancer prevention. We would extend our observations beyond the concept of angioprevention, and suggest that prevention of the tumor microenvironment, even before transformation, represents the future for cancer chemoprevention. References 1. Albini, A., Tosetti, F., Benelli, R. and Noonan, D.M. Tumor inflammatory angiogenesis and its chemoprevention. Cancer Res 65, 10637-41 (2005). 2. Tosetti, F., Ferrari, N., De Flora, S. and Albini, A. Angioprevention': angiogenesis is a common and key target for cancer chemopreventive agents. Faseb J 16, 2-14. (2002). 3. Pfeffer, U. et al. Molecular mechanisms of action of angiopreventive anti-oxidants on endothelial cells: Microarray gene expression analyses. Mutat Res 591, 198-211 (2005). 4. Albini, A. et al. Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kappaB and Akt as targets. Faseb J 20, 527-9 (2006). 5. Dell'Eva, R. et al. The Akt inhibitor deguelin, is an angiopreventive agent also acting on the NF-{kappa}B pathway. Carcinogenesis Sep 4 (2006). 6. Ferrari, N. et al. The transforming growth factor-beta family members bone morphogenetic protein-2 and macrophage inhibitory cytokine-1 as mediators of the antiangiogenic activity of N-(4-hydroxyphenyl)retinamide. Clin Cancer Res 11, 4610-9 (2005).