Hypoxia Negatively Regulates Antimetastatic PEDF in Melanoma Cells by a Hypoxia Inducible Factor-Independent, Autophagy Dependent Mechanism

Asunción Fernández-Barral,Valentí Gomez,Luis Del Peso,José Luis Orgaz,Benilde Jiménez,María José Calzada

doi:10.1371/journal.pone.0032989

Abstract

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent antiangiogenic and antimetastatic activity in a broad range of tumor types. Melanocytes and low aggressive melanoma cells secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas. PEDF efficiently abrogates a number of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization, proliferation, migration, invasiveness and extravasation. In this study, we identify hypoxia as a relevant negative regulator of PEDF in melanocytes and low aggressive melanoma cells. PEDF was regulated at the protein level. Importantly, although downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia. Decreased PEDF protein was not mediated by inhibition of translation through untranslated regions (UTRs) in melanoma cells. Degradation by metalloproteinases, implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory mechanism in melanoma cells. Instead, we found that degradation by autophagy was critical for PEDF downregulation under hypoxia in human melanoma cells. Our findings show that hypoxic conditions encountered during primary melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of aggressive melanoma cells.

Highlights

Serine protease inhibitor (SERPIN) is a large superfamily of genes that codes for serine protease inhibitors in mammals [1]
Seeking for regulators of Pigment epithelium-derived factor (PEDF) relevant in the context of melanoma progression we explored whether hypoxia could be a candidate mechanism
In primary cultures of human skin melanocytes we found that extracellular levels of PEDF protein (PEDFe) detected by western blot analysis of conditioned medium gradually decreased under hypoxic (1% O2) (Fig. 1A) and anoxic (0% O2) conditions (Fig. S1)

Summary

Introduction

Serine protease inhibitor (SERPIN) is a large superfamily of genes that codes for serine protease inhibitors in mammals [1]. There is a small number of SERPIN family members with non-inhibitory protease activity, among which is included pigment epithelium-derived factor (PEDF, gene symbol SERPINF1) [1,2,3,4,5]. PEDF was originally described as the most potent angiostatic factor in the eye [6]. PEDF is produced at high levels by retinal pigment epithelial (RPE) cells, and counteracts a number of potent angiogenic growth factors in the retina; ensuring the right balance of angiogenic regulators that leads to an optimum physiological pattern of blood vessels for a correct retinal function. Levels of angiostatic PEDF decrease during the progression of a number of cancers, such as hepatocellular carcinoma [11], prostate carcinoma [12,13], breast adenocarcinoma [14], glioblastoma [15] and Wilm’s tumors [16]

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