Therapeutics, Targets, and Chemical Biology Phosphomimetic Mutants of Pigment Epithelium-Derived Factor with Enhanced Antiangiogenic Activity as Potent Anticancer Agents Alexander Konson, Sunila Pradeep, and Rony Seger Abstract Pigment epithelium-derived factor (PEDF) is an endogenous inhibitor of angiogenesis and a promising anticancer agent capable of suppressing solid tumor growth in animal cancer models. We have previously shown that PEDF can be phosphorylated and that distinct phosphorylation states of this factor differentially regulate its physiologic function. Here, we report that phosphomimetic mutants of PEDF, which possess sig- nificantly increased antiangiogenic activity, are much more efficient than wild-type (WT) PEDF in inhibiting growth and neovascularization in MDA-MB-231 (breast cancer), HCT116 (colon cancer), and U87-MG (glio- blastoma) xenograft models. Importantly, the antitumor activity of the phosphomimetic mutants is compa- rable with that of the established antiangiogenic agent bevacizumab. However, unlike bevacizumab, these compounds act in a vascular endothelial growth factor (VEGF)–independent manner, as they do not affect the levels of VEGF-A mRNA and VEGF receptor 2 phosphorylation. Further immunohistochemical analysis revealed that PEDF mutants affect mainly tumor-residing endothelial cells and prevent the formation of in- tratumoral vascular network by facilitating endothelial cell apoptosis. It was also found that PEDF mutants reduce survival of endothelial cells in culture much better than WT-PEDF, an effect that is apparent even in the presence of VEGF or basic fibroblast growth factor, and promote much stronger endothelial cell apopto- sis. On the other hand, PEDF and its mutants did not affect survival of cultured cancer cells, indicating that the antiangiogenic activity of these agents is the foremost element of the observed antitumor effect. These findings have specific implications on improving the properties of WT-PEDF, which is currently in preclinical development, and encourage the development of PEDF mutants as specific, neovascularization-targeting an- ticancer agents. Cancer Res; 70(15); 6247–57. ©2010 AACR. Introduction Neovascularization is a well-recognized target for anti- cancer therapy. Numerous angiogenesis-targeted anticancer agents have already been approved, while others are in clin- ical trials nowadays (1). As such, the pigment epithelium- derived factor (PEDF) was first isolated from the conditioned medium of primary human retinal pigment epithelial cells, where it was shown to act as a neurotrophic and antiangio- genic factor (2). In particular, PEDF plays a key role in the reduction of abnormal neovascularization in the eye, and its natural antiangiogenic activity is far greater than that of any other known endogenously produced factor (3, 4). Recently, it has become evident that PEDF is widely ex- pressed throughout the human body and is persistent in sys- temic circulation (5, 6), thereby suggesting its possible involvement in angiogenesis-associated processes, including development and growth of solid tumors. Indeed, downregu- lation of PEDF mRNA and/or protein has been detected in a wide range of human malignancies (7–11). PEDF expression was shown to be inversely correlated with cancer progression (7), intratumoral microvessel density (MVD; refs. 11, 12), me- tastatic potential (12, 13), and less favorable prognosis (11, 12). In addition, it has been shown that PEDF-overexpressing cancer cells exhibit reduced growth rate in vivo (14, 15) and that PEDF therapy results in a profound inhibition of tumor growth in animal cancer models (8, 16–19). Yet, the exact molecular mechanism by which PEDF causes tumor suppres- sion is not completely understood, and it has been proposed that PEDF may possess both indirect and direct antitumor effects (6). Indirectly, reduction in tumor growth is achieved through the antiangiogenic action of PEDF, in which the se- lective targeting of newly formed vasculature without harm- ing the existing blood vessels is of a particular importance (20). This effect was suggested to involve inhibition of activity and/or expression of vascular endothelial growth factor (VEGF; refs. 6, 21), as well as the proapoptotic activity of Authors' Affiliation: Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Rony Seger, Department of Biological Regula- tion, The Weizmann Institute of Science, POB 26, Herzel Str., 76100 Rehovot, Israel. Phone: 972-8-9343602; Fax: 972-8-9344116; E-mail: rony.seger@weizmann.ac.il. doi: 10.1158/0008-5472.CAN-10-0434 ©2010 American Association for Cancer Research. Cancer Research www.aacrjournals.org 6247 Research. on October 3, 2021. © 2010 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst July 7, 2010; DOI: 10.1158/0008-5472.CAN-10-0434