Phosphomimetic Mutants of Pigment Epithelium-Derived Factor with Enhanced Anti-Choroidal Melanoma Cell Activity In Vitro and In Vivo

Abstract

Currently, choroidal melanoma is chemoresistant and there is no routine adjuvant chemotherapy for it. We investigated whether pigment epithelium-derived factor (PEDF) and its triple phosphomimetic mutants could more efficiently suppress melanoma tumor growth and metastasis, as well as how the triple phosphomimetic mutants act as antitumor agents. Phosphomimetic mutants of PEDF were constructed by site mutagenesis. Lentiviruses carrying wild type (WT) PEDF, S24E114E227A (EEA)-PEDF, and S24E114E227E (EEE)-PEDF were produced in 293 fast-growing, highly transfectable (FT) cells and used to infect human choroidal melanoma cell line (OCM-1). The growth, migration, invasion and metastasis abilities of OCM-1 cells expressing WT-PEDF, EEA-PEDF or EEE-PEDF were investigated in vitro and in vivo, while the underlying mechanism of PEDF phosphomimetic mutants were investigated via Western blotting. OCM-1 cells infected with lentiviruses carrying WT-PEDF, EEA-PEDF, and EEE-PEDF displayed reduced proliferation, migration and invasion abilities, and were more prone to apoptosis. Cell media containing WT-PEDF, EEA-PEDF, or EEE-PEDF protein inhibited the tube forming capacity of human umbilical vein endothelial cells (HUVEC) in vitro. OCM-1 cells expressing WT-PEDF, EEA-PEDF, or EEE-PEDF displayed significantly reduced tumor growth and metastasis in the melanoma xenograft of nude mice models, with the PEDF mutants displaying much stronger effects than the wild type. The antitumor effects of PEDF are associated with the inhibition of VEGF and nuclear factor kappa-B (NF-κB) expression, as well as further inhibition of Akt phosphorylation. The phosphomimetic mutants of PEDF showed enhanced anti-melanoma activity by directly affecting tumor cells and indirectly affecting angiogenesis. These findings encourage the development of PEDF mutants as innovative anticancer agents.