Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas.

Olivia Adams,Anna M Schläfli,Rupert Langer,Sabina Berezowska,Christian A Seiler,Bastian Dislich,Dino Kroell,Mario P Tschan

doi:10.18632/oncotarget.9649

Abstract

Esophageal adenocarcinomas (EAC) are aggressive tumors with considerable rates of chemoresistance. Autophagy is a lysosome-dependent degradation process, characterized by the formation of vesicles called autophagosomes, and has been implicated in cancer. Protein light chain 3 B (LC3B) and p62 are associated with autophagosomal membranes and degraded. We aimed to assess the impact of basal autophagy on EAC. In EAC cell lines, an increase in LC3B and p62 was observed with increasing concentrations of the autophagy inhibitor chloroquine, which indicates functional basal autophagy. LC3B and p62 immunohistochemistry was performed on primary resected EAC. High LC3B and p62 expression was associated with earlier tumor stages (p < 0.05). High nuclear and cytoplasmic p62 staining were associated with a better prognosis (p = 0.006; p = 0.028). Various combinations of p62 expression with or without LC3B expression identified different prognostic groups. Tumors with low total p62 (p = 0.007) or low LC3B/low p62 expression had the worst outcome (p = 0.007; p = 0.005). A combination score of dot-like/cytoplasmic p62 and nuclear p62 staining was an independent prognostic parameter (p = 0.033; HR = 0.6). This study highlights the potential significance of basal autophagy in EAC biology. Tumors with low LC3B and p62 expression show the most aggressive behavior and may be candidates for autophagy regulating therapeutics.

Highlights

Esophageal Adenocarcinomas (EAC), usually arising from metaplastic Barrett’s esophagus (BE), are highly aggressive tumors and often locally and systemically advanced upon diagnosis
To access functional basal autophagy in EAC in vitro the autophagy markers light chain 3 B (LC3B) and p62 were assessed via immunoblotting and immunofluorescence upon pharmacological autophagy inhibition with chloroquine
EAC cell lines OE19 and OE33 were treated with chloroquine (CQ) for 48 hours followed by immunoblotting for LC3B and p62, with total protein was visualized as loading control

Summary

Introduction

Esophageal Adenocarcinomas (EAC), usually arising from metaplastic Barrett’s esophagus (BE), are highly aggressive tumors and often locally and systemically advanced upon diagnosis. Functional basal autophagy can be defined as the continual autophagic flux under steady state culture conditions in the absence of chemo-, radio or targeted therapy. This is of relevance given the recent characterization of cancer cell lines lacking core autophagy machinery components and proving unable to undergo functional basal autophagic flux [7, 8]. As LC3B-II is membranebound and subsequently degraded, the accumulation of this isoform upon pharmacological autophagy inhibition is indicative of intact basal autophagy. The accumulation of p62, which is associated with the autophagosomal membrane and subject to degradation, upon pharmacological autophagy inhibition is an indicator of functional basal autophagy [9]. Using a previously established staining and scoring protocol [10] LC3B and p62 immunohistochemistry was performed on generation tissue microarrays (ngTMAs) of formalin fixed paraffin embedded (FFPE) samples of primary resected EAC, Barrett’s mucosa, adjacent nonneoplastic esophageal mucosa, adjacent non-neoplastic gastric mucosa, EAC lymph node metastasis and EAC distant metastasis and the results were correlated with clinicopathological parameters

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