Abstract
Autophagy is a lysosomal degradation and recycling process implicated in cancer progression and therapy resistance. We assessed the impact of basal autophagy in colon cancer (CC) in vitro and ex vivo. Functional autophagy was demonstrated in CC cell lines (LoVo; HT-29) showing a dose-dependent increase of the autophagy markers LC3B, p62 and autophagic vesciles upon increasing concentrations of the autophagy inhibitor chloroquine, which was demonstrated by immunoblotting, immunofluorescence and electron microscopy. Next, tissue microarrays with 292 primary resected CC, with cores from different tumor regions, and normal mucosa were analyzed by immunohistochemistry for LC3B and p62. CC tissue showed LC3B dot-like, p62 dot-like, cytoplasmic and nuclear staining in various levels without significant intratumoral heterogeneity. Tumoral LC3B and p62 expression was significantly higher than in normal tissue (p<0.001). No associations between staining patterns and pathological features (e.g. TNM categories; grading) were observed. Both low LC3B dot-like and low p62 dot-like-cytoplasmic staining were associated with worse overall survival (p=0.005 and p=0.002). The best prognostic discrimination, however, was seen for a combination of LC3B dot-like/p62 dot-like-cytoplasmic staining: high expression of both markers, indicative of impaired activated autophagy, was associated with the best overall survival. In contrast, high LC3B dot-like/low p62 dot-like-cytoplasmic expression, indicative of intact activated autophagy, was associated with the worst outcome (p<0.001 in univariate and HR=0.751; CI=0.607-0.928; p=0.008 in multivariate analysis). These specific expression patterns of LC3B and p62 pointing to different states of autophagy associated with diverging clinical outcomes highlighte the potential significance of basal autophagy in CC biology.
Highlights
Colon Cancer (CC) is a malignancy with one of the highest incidences and is a major cause of cancer-related death worldwide [1, 2]
Investigation of intact functional basal autophagy of CC cells in vitro The expression of the autophagy markers light chain 3 B (LC3B) and p62 were assessed via immunofluorescence and immunoblotting after pharmacological autophagy inhibition with chloroquine, which prevents the fusion of autophagosomes and lysosomes by increasing lysosomal pH, in HT-29 and LoVo CC cell lines
We investigated the impact of basal autophagy, focusing on two autophagy related proteins, in colon cancer
Summary
Colon Cancer (CC) is a malignancy with one of the highest incidences and is a major cause of cancer-related death worldwide [1, 2]. And functionally autophagy is characterized by the formation of double-membraned vesicles called autophagosomes. Autophagy is initiated via the formation of a phagophore or nucleation membrane to which cytoplasmic content is targeted. Physiological functions of autophagy include maintainance of energy homeostasis, elimination of defective organelles and proteins, prevention of reactive oxygen species and removal of intracellular pathogens. Autophagy maintains normal metabolism, prevents inflammation, oxidative stress and DNA damage by removing damaged organelles and proteins. It is theorized that in early stages of cancer, autophagy has tumor-suppressing properties. P62 targets ubiquitinated substrats to autophagosomes via its interacts with LC3B. During autophagic flux both proteins are subject to degradation in autolysosomes [7, 8]. We aimed to investigate the impact of autophagy under basal conditions in vitro in CC cell lines and ex vivo in CC tumor tissue
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