Phosphomimetic Mutants of Pigment Epithelium-Derived Factor with Enhanced Antiangiogenic Activity as Potent Anticancer Agents

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 significantly 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 (glioblastoma) xenograft models. Importantly, the antitumor activity of the phosphomimetic mutants is comparable 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 intratumoral 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 apoptosis. 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 anticancer agents. Cancer Res; 70(15); 6247-57. ©2010 AACR.

Pigment Epithelium-derived Factor and Its Phosphomimetic Mutant Induce JNK-dependent Apoptosis and p38-mediated Migration Arrest

“…Apoptosis-According to our previous results (19), the antiproliferative effect of PEDF toward ECs was associated with an enhanced EC apoptosis. To verify this, we first compared the rate of WT-PEDF-and EEE-PEDF-induced apoptosis by TUNEL assay.…”

“…This effect was suggested to involve inhibition of activity and/or expression of VEGF (4, 31) as well as the proapoptotic activity of PEDF toward immature and migrating ECs (4, 31, 32). Yet, in our previous study, which employed three different xenograft models to test the anticancer effect of WT-PEDF and its mutants, these agents did not affect VEGF expression and activity in vivo, implying direct action on tumor ECs as a major determinant of the observed anticancer activity (19). Though still controversial, it has been reported that PEDF also exerts a direct antitumor effect, possibly by inducing either antiproliferative or prodifferentiation activities toward cancer cells (31,33).…”

“…In these studies, mutants mimicking either protein kinase CK2, or the accumulative protein kinase CK2 and protein kinase A phosphorylation (S24E114E227A, EEA-PEDF and S24E114E227E, EEE-PEDF) exhibited much stronger antiangiogenic activity than their WT counterpart (17,18). More recently, we have also shown that phosphomimetic mutants of PEDF, particularly EEE-PEDF, are significantly more efficient than WT-PEDF in inhibiting tumor growth and neovascularization in human breast, colon cancer, and glioblastoma xenograft models (19). Immunohistochemical analysis revealed that PEDF and its mutants affect mainly tumor-residing ECs and prevent the formation of intratumoral vascular network by facilitating EC apoptosis.…”

“…Cancer Cell Proliferation-We have previously shown that when administered to mice bearing MDA-MB-231, HCT116, or U87-MG xenografts, WT-PEDF and its phosphomimetic mutant EEE-PEDF predominantly affect ECs of the tumor vasculature, rather than cancer cells themselves (19). Thus, we undertook to further study the mechanisms of action of WT-PEDF and of EEE-PEDF, which caused the most profound inhibition of xenograft growth, in cultured endothelial and cancer cells.…”

“…2C). In contrast to BAEC, WT-PEDF as well as EEE-PEDF failed to induce apoptosis of cultured MDA-MB-231 cells as evaluated by TUNEL assay over a 48-h period (19). For further information on the apoptotic effect, we next used a FACS-based cell cycle analysis.…”