title: Methylation silencing of ULK2, an autophagy gene, is essential for astrocyte transformation and tumor growth.
abstract: Glioblastoma (GBM) is the most aggressive type of brain tumor and shows very poor prognosis. Here, using genome-wide methylation analysis, we show that G-CIMP+ and G-CIMP-subtypes enrich distinct classes of biological processes. One of the hypermethylated genes in GBM, ULK2, an upstream autophagy inducer, was found to be down-regulated in GBM. Promoter hypermethylation of ULK2 was confirmed by bisulfite sequencing. GBM and glioma cell lines had low levels of ULK2 transcripts, which could be reversed upon methylation inhibitor treatment. ULK2 promoter methylation and transcript levels showed significant negative correlation. Ectopic overexpression of ULK2-induced autophagy, which further enhanced upon nutrient starvation or temozolomide chemotherapy. ULK2 also inhibited the growth of glioma cells, which required autophagy induction as kinase mutant of ULK2 failed to induce autophagy and inhibit growth. Furthermore, ULK2 induced autophagy and inhibited growth in Ras-transformed immortalized Baby Mouse Kidney (iBMK) ATG5(+/+) but not in autophagy-deficient ATG5(-/-) cells. Growth inhibition due to ULK2 induced high levels of autophagy under starvation or chemotherapy utilized apoptotic cell death but not at low levels of autophagy. Growth inhibition by ULK2 also appears to involve catalase degradation and reactive oxygen species generation. ULK2 overexpression inhibited anchorage independent growth, inhibited astrocyte transformation in vitro and tumor growth in vivo. Of all autophagy genes, we found ULK2 and its homologue ULK1 were only down-regulated in all grades of glioma. Thus these results altogether suggest that inhibition of autophagy by ULK1/2 down-regulation is essential for glioma development.
authors.affiliation: From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Sudhanshu
authors.initials: S
authors.lastname: Shukla
authors.affiliation: From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Irene Rosita Pia
authors.initials: IR
authors.lastname: Patric
authors.affiliation: From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Vikas
authors.initials: V
authors.lastname: Patil
authors.affiliation: Neuropathology, National Institute of Mental Health and Neuro Sciences, Bangalore 560029, India.
authors.forename: Shivayogi D
authors.initials: SD
authors.lastname: Shwetha
authors.affiliation: the Sri Satya Sai Institute of Higher Medical Sciences, Bangalore 560066, India, and.
authors.forename: Alangar S
authors.initials: AS
authors.lastname: Hegde
authors.affiliation: the Departments of Neurosurgery and.
authors.forename: Bangalore A
authors.initials: BA
authors.lastname: Chandramouli
authors.affiliation: the Departments of Neurosurgery and.
authors.forename: Arimappamagan
authors.initials: A
authors.lastname: Arivazhagan
authors.affiliation: Neuropathology, National Institute of Mental Health and Neuro Sciences, Bangalore 560029, India.
authors.forename: Vani
authors.initials: V
authors.lastname: Santosh
authors.affiliation: From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India, skumar@mcbl.iisc.ernet.in.
authors.forename: Kumaravel
authors.initials: K
authors.lastname: Somasundaram
mesh_terms: D000818:Animals; D017209:Apoptosis; D001253:Astrocytes / Q000378:metabolism* / Q000473:pathology*; D001343:Autophagy / Q000235:genetics*; D000071189:Autophagy-Related Protein-1 Homolog; D001932:Brain Neoplasms / Q000235:genetics / Q000473:pathology; D016022:Case-Control Studies; D002374:Catalase / Q000378:metabolism; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation / Q000235:genetics*; D015536:Down-Regulation; D020868:Gene Silencing; D005909:Glioblastoma / Q000235:genetics / Q000473:pathology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D017382:Reactive Oxygen Species; D002374:Catalase; C401061:Ulk2 protein, mouse; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; C484754:ULK1 protein, human; C541703:Ulk2 protein, human
keywords: ATG; Apoptosis; Autophagy; DNA Methylation; Glioblastoma; Reactive Oxygen Species (ROS); ULK1; ULK2