Pigment epithelial-derived factor (PEDF), an angiogenesis inhibitor with neurotrophic properties, balances angiogenesis in the eye and blocks tumor progression. Its neurotrophic function and the ability to block vascular leakage is replicated by the PEDF 44-mer peptide (residues 58-101). We analyzed PEDFs' three-dimensional structure and identified a potential receptor-binding surface. Seeking PEDF-based antiangiogenic agents we generated and tested peptides representing the middle and lower regions of this surface. We identified previously unknown antiangiogenic epitopes consisting of the 34-mer (residues 24-57) and a shorter proximal peptide (TGA, residues 16-26) with the critical stretch L 19 VEEED 24 and a fragment within the 44-mer (ERT, residues 78-94), which retained neurotrophic activity. The 34-mer and TGA, but not the 44-mer reproduced PEDF angioinhibitory signals hinged on c-jun-NH 2 -kinase-dependent nuclear factor of activated T cell deactivation and caused apoptosis. Conversely, the ERT, but not the 34-mer/TGA induced neuronal differentiation. For the 44-mer/ERT, we showed a novel ability to cause neuroendocrine differentiation in prostate cancer cells. PEDF and the peptides bound endothelial and PC-3 prostate cancer cells. Bound peptides were displaced by PEDF, but not by each other, suggesting multiple receptors. PEDF and its active fragments blocked tumor formation when conditionally expressed by PC-3 cells. The 34-and 44-mer used distinct mechanisms: the 34-mer acted on endothelial cells, blocked angiogenesis, and induced apoptosis whereas 44-mer prompted neuroendocrine differentiation in cancer cells. Our results map active regions for the two PEDF functions, signaling via distinct receptors, identify candidate peptides, and provide their mechanism of action for future development of PEDFbased tumor therapies. (Cancer Res 2005; 65(12): 5144-52)

Pigment epithelium-derived factor (PEDF) is a 50 kDa secreted glycoprotein that belongs to the non-inhibitory serpin family group. PEDF has been described as a natural angiogenesis inhibitor with neurotrophic and immune-modulation properties; it balances angiogenesis in the eye and blocks tumor progression. The mechanisms underlying most of these events are not completely clear; however, it appears that PEDF acts via multiple high affinity ligands and cell receptors. In this review article, we will summarize the current knowledge on the biochemical properties of PEDF and its receptors, the multimodal activities of PEDF and finally address the therapeutic potential of PEDF in treating angiogenesis-, neurodegeneration- and inflammation-related diseases.

Id proteins are helix-loop-helix transcription factors that regulate tumor angiogenesis. In order to identify downstream effectors of Id1 involved in the regulation of angiogenesis, we performed PCR-select subtractive hybridization on wild-type and Id1 knockout mouse embryo fibroblasts (MEFs). Here we demonstrate that thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis, is a target of transcriptional repression by Id1. We also show that Id1-null MEFs secrete an inhibitor of endothelial cell migration, which is completely inactivated by depletion of TSP-1. Furthermore, in vivo studies revealed decreased neovascularization in matrigel assays in Id1-null mice compared to their wild-type littermates. This decrease was completely reversed by a TSP-1 neutralizing antibody. We conclude that TSP-1 is a major target for Id1 effects on angiogenesis.

It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial–derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis.We identified c-Jun NH2-terminal kinases (JNKs) as essential upstream regulators of NFAT activity in angiogenesis. We distinguished JNK-2 as responsible for NFATc2 cytoplasmic retention by PEDF and JNK-1 and JNK-2 as mediators of PEDF-driven NFAT nuclear export.We identified a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain–like interleukin 1β–converting enzyme inhibitory protein (c-FLIP), whose expression was coregulated by VEGF and PEDF. Chromatin immunoprecipitation showed VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo, which was attenuated by PEDF. We propose that one possible mechanism of concerted angiogenesis regulation by activators and inhibitors may be modulation of the endothelial cell apoptosis via c-FLIP controlled by NFAT and its upstream regulator JNK.

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NFjB, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NFjB suppression, since it was restricted to the cells lacking nuclear (active) NFjB. Thus we for the first time identified combined decrease of NFjB and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. ' 2007 Wiley-Liss, Inc.

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Antiangiogenic thrombospondin-1 (TSP1) induces endothelial cell death via a CD95-mediated cascade. We used this signaling pathway, where CD95/Fas is a rate-limiting intermediate, as a target to optimize the efficacy of TSP1 active peptide, DI-TSP. Like TSP1, DI-TSP upregulated endothelial CD95L in vivo. To modulate CD95 levels, we chose chemotherapy agent doxorubicin (DXR). DXR caused sustained upregulation of CD95 in the activated endothelium at 1/ 100 of the maximal tolerated dose. DI-TSP and DXR synergistically induced endothelial apoptosis in vitro, and in vivo, in developing murine vessels. Fas decoy, TSP1 receptor antibody and Pifithrin, a p53 inhibitor, severely decreased apoptosis and restored angiogenesis by DXR-DI-TSP combination, evidencing critical roles of CD95 and TSP1. Combined therapy synergistically blocked neovascularization and progression of the bladder and prostate carcinoma. Such informed design of a complex antiangiogenic therapy based on the rate-limiting molecular targets is a novel concept, which may yield new approaches to cancer treatment.

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