High LC3/Beclin Expression Correlates with Poor Survival in Glioma: a Definitive Role for Autophagy as Evidenced by In Vitro Autophagic Flux

Abstract

Recent studies suggest the role of autophagy, an evolutionarily conserved catabolic process, in determining the response of gliomas to treatment either positively or negatively. The study attempts to characterize autophagy in low and high-grade glioma by investigating the autophagic flux and clinical significance of autophagy proteins (LC3 and beclin 1) in a group of glioma patients. We evaluated the expression of autophagic markers in resected specimens of low-grade glioma (LGG) and high-grade glioma (HGG) tissues, by immunohistochemistry and Western blotting. Our results show that expression of autophagy proteins were more prominent in HGG than in LGG. Increased level of autophagic proteins in HGG can be due to an increased rate of autophagy or can be because of blockage in the final degradation step of autophagy (defective autophagy). To distinguish these possibilities, the autophagic flux assay which helps to determine the rate of degradation/synthesis of autophagic proteins (LC3-II and p62) over a period of time by blocking the final degradation step of autophagy using bafilomycin A1 was used . The assessment of autophagic flux in ex vivo culture of primary glioma cells revealed for the first time increased turnover of autophagy in high grade compared to low grade-glioma. Though autophagic markers were reduced in LGG, functionally autophagy was non defective in both grades of glioma. We then investigated whether autophagy in gliomas is regulated by nutrient sensing pathways including mTOR and promote cell survival by providing an alternate energy source in response to metabolic stress. The results depicted that the role of autophagy during stress varies with tissue and has a negative correlation with mTOR substrate phosphorylation. We also evaluated the expression of LC3 and beclin 1 with progression free survival (PFS) using Kaplan-Meier survival analysis and have found that patients with low LC3/beclin 1 expression had better PFS than those with high expression of LC3/beclin 1 in their tumors. Together, we provide evidence that autophagy is non-defective in glioma and also show that high LC3/beclin 1 expression correlates with poor PFS in both LGG and HGG.

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