Pathology & Oncology Research

, Volume 25, Issue 1, pp 137–148 | Cite as

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

  • Padmakrishnan CJ
  • Easwer HV
  • Vinod Vijayakurup
  • Girish R Menon
  • Suresh Nair
  • Srinivas Gopala
Original Article


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.


Glioma Autophagy LC3 Autophagic flux p62 Beclin 1 



low grade glioma


high grade glioma


progression free survival



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.


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).

Compliance with Ethical Standards

Conflict of Interest

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

Ethical Approval

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.

Informed Consent

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


  1. 1.
    Schwartzbaum JA, Fisher JL, Aldape KD, Wrensch M (2006) Epidemiology and molecular pathology of glioma. Nat Clin Pract Neurol 2(9):494–503 quiz 1 p following 16Google Scholar
  2. 2.
    Maher EA, Furnari FB, Bachoo RM, Rowitch DH, Louis DN, Cavenee WK et al (2001) Malignant glioma: genetics and biology of a grave matter. Genes Dev 15(11):1311–1333Google Scholar
  3. 3.
    Louis DN, Holland EC, Cairncross JG (2001) Glioma classification: a molecular reappraisal. Am J Pathol 159(3):779–786Google Scholar
  4. 4.
    Lefranc F, Kiss R (2006) Autophagy, the Trojan horse to combat glioblastomas. Neurosurg Focus 20(4):E7Google Scholar
  5. 5.
    Lefranc F, Brotchi J, Kiss R (2005) Possible future issues in the treatment of glioblastomas: special emphasis on cell migration and the resistance of migrating glioblastoma cells to apoptosis. J Clin Oncol Off J Am Soc Clin Oncol 23(10):2411–2422Google Scholar
  6. 6.
    Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S (2004) Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11(4):448–457Google Scholar
  7. 7.
    Ito H, Daido S, Kanzawa T, Kondo S, Kondo Y (2005) Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells. Int J Oncol 26(5):1401–1410Google Scholar
  8. 8.
    Lomonaco SL, Finniss S, Xiang C, Decarvalho A, Umansky F, Kalkanis SN et al (2009) The induction of autophagy by gamma-radiation contributes to the radioresistance of glioma stem cells. Int J Cancer 125(3):717–722Google Scholar
  9. 9.
    Jo GH, Bogler O, Chwae YJ, Yoo H, Lee SH, Park JB, et al (2014) Radiation-induced autophagy contributes to cell death and induces apoptosis partly in malignant glioma cells. Cancer Res Treat 47(2): 221-241Google Scholar
  10. 10.
    Ge P, Luo Y, Fu S, Ji X, Ling F (2009) Autophagy: a strategy for malignant gliomas’ resistance to therapy. Med Hypotheses 73(1):45–47Google Scholar
  11. 11.
    Zhuang W, Qin Z, Liang Z (2009) The role of autophagy in sensitizing malignant glioma cells to radiation therapy. Acta Biochim Biophys Sin 41(5):341–351Google Scholar
  12. 12.
    Cuervo AM (2004) Autophagy: in sickness and in health. Trends Cell Biol 14(2):70–77Google Scholar
  13. 13.
    Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R et al (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441(7095):885–889Google Scholar
  14. 14.
    Chen N, Debnath J (2010) Autophagy and tumorigenesis. FEBS Lett 584(7):1427–1435Google Scholar
  15. 15.
    Hippert MM, O'Toole PS, Thorburn A (2006) Autophagy in cancer: good, bad, or both? Cancer Res 66(19):9349–9351Google Scholar
  16. 16.
    He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93Google Scholar
  17. 17.
    Annovazzi L, Mellai M, Caldera V, Valente G, Tessitore L, Schiffer D (2009) mTOR, S6 and AKT expression in relation to proliferation and apoptosis/autophagy in glioma. Anticancer Res 29(8):3087–3094Google Scholar
  18. 18.
    Fan QW, Knight ZA, Goldenberg DD, Yu W, Mostov KE, Stokoe D et al (2006) A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell 9(5):341–349Google Scholar
  19. 19.
    Chang YY, Juhasz G, Goraksha-Hicks P, Arsham AM, Mallin DR, Muller LK et al (2009) Nutrient-dependent regulation of autophagy through the target of rapamycin pathway. Biochem Soc Trans 37(Pt 1):232–236Google Scholar
  20. 20.
    Kondo Y, Kanzawa T, Sawaya R, Kondo S (2005) The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 5(9):726–734Google Scholar
  21. 21.
    Eskelinen EL (2005) Doctor Jekyll and Mister Hyde: autophagy can promote both cell survival and cell death. Cell Death Differ 12(Suppl 2):1468–1472Google Scholar
  22. 22.
    Koukourakis MI, Mitrakas AG, Giatromanolaki A (2016) Therapeutic interactions of autophagy with radiation and temozolomide in glioblastoma: evidence and issues to resolve. Br J Cancer 114(5):485–496Google Scholar
  23. 23.
    Yan Y, Xu Z, Dai S, Qian L, Sun L, Gong Z (2016) Targeting autophagy to sensitive glioma to temozolomide treatment. J Exp Clin Cancer Res 35:23Google Scholar
  24. 24.
    Lefranc F, Facchini V, Kiss R (2007) Proautophagic drugs: a novel means to combat apoptosis-resistant cancers, with a special emphasis on glioblastomas. Oncologist 12(12):1395–1403Google Scholar
  25. 25.
    Ballman KV, Buckner JC, Brown PD, Giannini C, Flynn PJ, LaPlant BR et al (2007) The relationship between six-month progression-free survival and 12-month overall survival end points for phase II trials in patients with glioblastoma multiforme. Neuro-Oncology 9(1):29–38Google Scholar
  26. 26.
    Tang JY, Hsi E, Huang YC, Hsu NC, Chu PY, Chai CY (2013) High LC3 expression correlates with poor survival in patients with oral squamous cell carcinoma. Hum Pathol 44(11):2558–2562Google Scholar
  27. 27.
    Lee YJ, Hah YJ, Kang YN, Kang KJ, Hwang JS, Chung WJ et al (2013) The autophagy-related marker LC3 can predict prognosis in human hepatocellular carcinoma. PLoS One 8(11):e81540Google Scholar
  28. 28.
    Liu JL, Chen FF, Lung J, Lo CH, Lee FH, Lu YC et al (2014) Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma. Br J Cancer 111(5):944–954Google Scholar
  29. 29.
    Wang XJ, Zhou SL, Fu XD, Zhang YY, Liang B, Shou JX et al (2015) Clinical and prognostic significance of high-mobility group box-1 in human gliomas. Exp Ther Med 9(2):513–518Google Scholar
  30. 30.
    Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4(2):151–175Google Scholar
  31. 31.
    Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I (2003) Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res 63(9):2103–2108Google Scholar
  32. 32.
    Carew JS, Espitia CM, Esquivel JA 2nd, Mahalingam D, Kelly KR, Reddy G et al (2011) Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis. J Biol Chem 286(8):6602–6613Google Scholar
  33. 33.
    Klionsky DJ (2005) The correct way to monitor autophagy in higher eukaryotes. Autophagy 1(2):65Google Scholar
  34. 34.
    Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8(4):445–544Google Scholar
  35. 35.
    Rubinsztein DC, Cuervo AM, Ravikumar B, Sarkar S, Korolchuk V, Kaushik S et al (2009) In search of an “autophagomometer”. Autophagy 5(5):585–589Google Scholar
  36. 36.
    Sadasivan S, Waghray A, Larner SF, Dunn WA Jr, Hayes RL, Wang KK (2006) Amino acid starvation induced autophagic cell death in PC-12 cells: evidence for activation of caspase-3 but not calpain-1. Apoptosis: Int J Programmed Cell Death 11(9):1573–1582Google Scholar
  37. 37.
    Ni HM, Bockus A, Wozniak AL, Jones K, Weinman S, Yin XM et al (2011) Dissecting the dynamic turnover of GFP-LC3 in the autolysosome. Autophagy 7(2):188–204Google Scholar
  38. 38.
    Kathryn J. Huber-Keener and Jin-Ming Yang (2011). Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy In: Prof. Clark Chen (Ed.), Advances in the Biology, Imaging and Therapies for Glioblastoma, InTech, CroatiaGoogle Scholar
  39. 39.
    Kang C, Avery L (2008) To be or not to be, the level of autophagy is the question: dual roles of autophagy in the survival response to starvation. Autophagy 4(1):82–84Google Scholar
  40. 40.
    Huang X, Bai HM, Chen L, Li B, Lu YC (2010) Reduced expression of LC3B-II and Beclin 1 in glioblastoma multiforme indicates a down-regulated autophagic capacity that relates to the progression of astrocytic tumors. J Clin Neurosci: Off J Neurosurg Soc Australasia 17(12):1515–1519Google Scholar
  41. 41.
    Ge PF, Zhang JZ, Wang XF, Meng FK, Li WC, Luan YX et al (2009) Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells. Acta Pharmacol Sin 30(7):1046–1052Google Scholar
  42. 42.
    Pirtoli L, Cevenini G, Tini P, Vannini M, Oliveri G, Marsili S et al (2009) The prognostic role of Beclin 1 protein expression in high-grade gliomas. Autophagy 5(7):930–936Google Scholar
  43. 43.
    Giatromanolaki A, Sivridis E, Mitrakas A, Kalamida D, Zois CE, Haider S et al (2014) Autophagy and lysosomal related protein expression patterns in human glioblastoma. Cancer Biol Ther 15(11):1468–1478Google Scholar
  44. 44.
    Klionsky DJ, Elazar Z, Seglen PO, Rubinsztein DC (2008) Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy 4(7):849–850Google Scholar
  45. 45.
    Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T et al (2003) MAP-LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis. FASEB J: Off Publ Fed Am Soc Exp Biol 17(9):1165–1167Google Scholar
  46. 46.
    Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1(2):84–91Google Scholar
  47. 47.
    Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y et al (2009) Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell 20(7):1981–1991Google Scholar
  48. 48.
    Kamada Y, Yoshino K, Kondo C, Kawamata T, Oshiro N, Yonezawa K et al (2010) Tor directly controls the Atg1 kinase complex to regulate autophagy. Mol Cell Biol 30(4):1049–1058Google Scholar
  49. 49.
    Hau AM, Greenwood JA, Lohr CV, Serrill JD, Proteau PJ, Ganley IG et al (2013) Coibamide a induces mTOR-independent autophagy and cell death in human glioblastoma cells. PLoS One 8(6):e65250Google Scholar
  50. 50.
    Rodriguez FJ, Raabe EH (2014) mTOR: a new therapeutic target for pediatric low-grade glioma? CNS Oncol 3(2):89–91Google Scholar
  51. 51.
    Franz DN, Leonard J, Tudor C, Chuck G, Care M, Sethuraman G et al (2006) Rapamycin causes regression of astrocytomas in tuberous sclerosis complex. Ann Neurol 59(3):490–498Google Scholar
  52. 52.
    McBride SM, Perez DA, Polley MY, Vandenberg SR, Smith JS, Zheng S et al (2010) Activation of PI3K/mTOR pathway occurs in most adult low-grade gliomas and predicts patient survival. J Neuro-Oncol 97(1):33–40Google Scholar
  53. 53.
    Hao CL, Li Y, Yang HX, Luo RZ, Zhang Y, Zhang MF et al (2014) High level of microtubule-associated protein light chain 3 predicts poor prognosis in resectable esophageal squamous cell carcinoma. Int J Clin Exp Pathol 7(7):4213–4221Google Scholar
  54. 54.
    Karpathiou G, Sivridis E, Koukourakis MI, Mikroulis D, Bouros D, Froudarakis ME et al (2011) Light-chain 3A autophagic activity and prognostic significance in non-small cell lung carcinomas. Chest 140(1):127–134Google Scholar
  55. 55.
    Wan XB, Fan XJ, Chen MY, Xiang J, Huang PY, Guo L et al (2010) Elevated Beclin 1 expression is correlated with HIF-1alpha in predicting poor prognosis of nasopharyngeal carcinoma. Autophagy 6(3):395–404Google Scholar
  56. 56.
    Fujii S, Mitsunaga S, Yamazaki M, Hasebe T, Ishii G, Kojima M et al (2008) Autophagy is activated in pancreatic cancer cells and correlates with poor patient outcome. Cancer Sci 99(9):1813–1819Google Scholar
  57. 57.
    Han C, Sun B, Wang W, Cai W, Lou D, Sun Y et al (2011) Overexpression of microtubule-associated protein-1 light chain 3 is associated with melanoma metastasis and vasculogenic mimicry. Tohoku J Exp Med 223(4):243–251Google Scholar
  58. 58.
    Guo JY, Karsli-Uzunbas G, Mathew R, Aisner SC, Kamphorst JJ, Strohecker AM et al (2013) Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. Genes Dev 27(13):1447–1461Google Scholar
  59. 59.
    Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H et al (1999) Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402(6762):672–676Google Scholar
  60. 60.
    Hu YL, DeLay M, Jahangiri A, Molinaro AM, Rose SD, Carbonell WS et al (2012) Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma. Cancer Res 72(7):1773–1783Google Scholar
  61. 61.
    Kimmelman AC (2011) The dynamic nature of autophagy in cancer. Genes Dev 25(19):1999–2010Google Scholar
  62. 62.
    Knizhnik AV, Roos WP, Nikolova T, Quiros S, Tomaszowski KH, Christmann M et al (2013) Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage. PLoS One 8(1):e55665Google Scholar
  63. 63.
    Winardi D, Tsai HP, Chai CY, Chung CL, Loh JK, Chen YH et al (2014) Correlation of altered expression of the autophagy marker LC3B with poor prognosis in astrocytoma. Biomed Res Int 2014:723176Google Scholar
  64. 64.
    Karagounis IV, Kalamida D, Mitrakas A, Pouliliou S, Liousia MV, Giatromanolaki A et al (2016) Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy. Br J Cancer 115(3):312–321Google Scholar
  65. 65.
    Koukourakis MI, Kalamida D, Mitrakas A, Pouliliou S, Kalamida S, Sivridis E et al (2015) Intensified autophagy compromises the efficacy of radiotherapy against prostate cancer. Biochem Biophys Res Commun 461(2):268–274Google Scholar
  66. 66.
    Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M et al (2013) Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis 4:e838Google Scholar
  67. 67.
    Fimia GM, Kroemer G, Piacentini M (2013) Molecular mechanisms of selective autophagy. Cell Death Differ 20(1):1–2Google Scholar

Copyright information

© Arányi Lajos Foundation 2017

Authors and Affiliations

  • Padmakrishnan CJ
    • 1
  • Easwer HV
    • 2
  • Vinod Vijayakurup
    • 1
  • Girish R Menon
    • 2
    • 3
  • Suresh Nair
    • 2
  • Srinivas Gopala
    • 1
  1. 1.Department of BiochemistrySree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
  2. 2.Department of NeurosurgerySree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuramIndia
  3. 3.Department of NeurosurgeryKasturba HospitalManipalIndia

Personalised recommendations