Abstract 2510: Comparative proteomic analysis of melanoma cell secretomes reveals autophagy-related biomarkers.
Authors: Akl Adam , Amaravadi Ravi , Speicher David 
Published: August 2014

Abstract
Autophagy is a highly conserved mechanism of cellular self-digestion that serves as a major protein and organelle degrading pathway and a critical adaptive response to nutrient, endoplasmic reticulum, and oxidative stress. Autophagy has been implicated as a survival mechanism in melanoma tumorigenesis and has been recognized as a primary mechanism of chemotherapeutic resistance. Pre-treatment analyses of autophagy levels in melanoma tumor samples have shown a high degree of heterogeneity across patients, with high levels of autophagy correlating with significantly shorter survival. However, quantitative assays for autophagy remain elusive at the pre-clinical level, impeding further understanding as to its role in melanoma progression. Another challenge to developing effective markers of autophagy is the striking difference in autophagic response of cells grown in conventional monolayer culture versus cells growing in the tumor microenvironment. Recent studies have found that autophagy regulates ‘unconventional’ protein secretion pathways, resulting in secretion of a subset of cytosolic proteins. Therefore, the focus of this work was to investigate molecular changes in the melanoma cell secretome as a means of identifying key proteins diagnostic of autophagy levels. To approximate autophagy dynamics in the tumor microenvironment, human melanoma cells were grown as spheroids in Type I bovine collagen matrices. To enrich for autophagy-related secretions, secretomes of low-autophagy WM793 cells and the genetically closely related high-autophagy 1205Lu cells were directly compared at baseline. Conditioned media was collected and separated using 1-D gel electrophoresis, digested with trypsin, and analyzed using liquid chromatography-tandem mass spectrometry analysis on an LTQ-Orbitrap hybrid instrument. Mass spectra were analyzed using the Rosetta ELUCIDATOR proteomic pipeline, and significance analysis of microarrays (SAM) was used to identify differentially secreted proteins. From Gene Ontology (GO) analysis, candidate biomarkers were enriched for extracellular matrix components, inflammatory mediators, and metabolic enzymes implicated in autophagy and melanoma progression. A subset of these proteins was predicted to be secreted via a non-classical mechanism using SecretomeP and SignalP secretory protein prediction servers. Network analysis using the shortest paths algorithm identified densely connected sub-networks that illustrated interplay between inflammatory cytokines and metastasis-related proteins, providing insight into molecular pathways that may regulate constitutively elevated autophagy. Our findings indicate that differences in basal autophagy in human melanoma manifest as secretory changes that could be novel serological biomarkers of autophagy for cancer prognosis and therapy.
Citation Format: Adam Akl, Ravi Amaravadi, David Speicher. Comparative proteomic analysis of melanoma cell secretomes reveals autophagy-related biomarkers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2510. doi:10.1158/1538-7445.AM2013-2510