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

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Pathology & Oncology Research

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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Abbreviations

LGG:

low grade glioma

HGG:

high grade glioma

PFS:

progression free survival

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Acknowledgements

We thank all the patients involved in the study. We are grateful to Department of Neurosurgery and Department of Pathology for providing tissue samples and sections. We thank Dr. Neelima Radhakrishnan for reviewing the IHC slides.

Funding

This work was supported by the Department of Biotechnology Research Grant, Government of India (BT/PR11624/med/30/154/2008) (GS) and research fellowship from Sree Chitra Tirunal Institute for Medical Sciences & Technology (PCJ) and Council of Scientific and Industrial Research (VV).

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  1. Girish R Menon

    Present address: Department of Neurosurgery, Kasturba Hospital, Manipal, 576104, India

Authors and Affiliations

  1. Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, 695011, India

    Padmakrishnan CJ, Vinod Vijayakurup & Srinivas Gopala

  2. Department of Neurosurgery, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, 695011, India

    Easwer HV, Girish R Menon & Suresh Nair

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  1. Padmakrishnan CJ
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  2. Easwer HV
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Correspondence to Srinivas Gopala.

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All authors (PCJ, EHV, VV, GMR, SN, SG) declare no conflict of interest.

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All procedures performed in the above study were in accordance with the ethical standards of the Institutional Human Ethical Committee and with the 1964 Helsinki declaration and its later amendments or comparable standards.

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Informed consent was obtained from all individual participants included in the study.

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Highlights

• Increased expression of LC3/Beclin 1 was more prominent in high grade glioma as evidenced by immunohistochemistry and Western Blot.

• For the first time, autophagic flux assay done in in vitro cultured primary glioma cells revealed autophagy was non-defective in both low and high-grade gliomas.

• Patients with high expression of LC3/beclin 1 had worse PFS than patients with low expression of LC3/beclin 1 in their tumors.

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CJ, P., HV, E., Vijayakurup, V. et al. High LC3/Beclin Expression Correlates with Poor Survival in Glioma: a Definitive Role for Autophagy as Evidenced by In Vitro Autophagic Flux. Pathol. Oncol. Res. 25, 137–148 (2019). https://doi.org/10.1007/s12253-017-0310-7

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