Identification of a lung cancer cell line deficient in atg7-dependent autophagy

ABSTRACT

Autophagy is a major cellular process for bulk degradation of proteins and organelles in order to maintain metabolic homeostasis, and it represents an emerging target area for cancer. Initially proposed to be a cancer-restricting process for tumor initiation, recent studies suggest that autophagy can also promote cell survival in established tumors. ATG7 is an essential autophagy gene that encodes the E1 enzyme necessary for the lipidation of the LC3 family of ubiquitin-like proteins and autophagosome formation. In this study we identified a rare case of a cancer cell line, H1650 lung adenocarcinoma, which has lost ATG7 expression due to a focal biallelic deletion within the ATG7 locus. These cells displayed no evidence of ATG7 pathway activity; however, reconstituting the cells with wild-type ATG7 restored both LC3 lipidation and downstream autophagic consumption of autophagy substrates such as the SQSTM1/p62 protein. We characterized several phenotypes reported to be influenced by autophagy, and observed an ATG7-dependent increase in cell growth and clearance of proteasome-inhibitor induced protein aggregates. Cellular changes in mitochondrial metabolism or response to nutrient starvation were unaffected by ATG7 expression. In addition, parental H1650 cells that lacked ATG7 were still able to consume autophagy substrates SQSTM1, NBR1 and TAX1BP1 via a bafilomycin A₁-sensitive pathway, suggesting that these proteins were not exclusively degraded by autophagy. Overall, these findings highlight a unique outlier instance of complete loss of ATG7-dependent autophagy in a cancer cell line. The H1650 cell line may be a useful system for future studies to further understand the role of autophagy in tumorigenesis and potential redundant pathways that allow cells to circumvent the loss of ATG7-dependent autophagy in cancer.

KEYWORDS:

• Atg7
• lung cancer
• metabolism
• mitochondria
• proteasome
• ubiquitin

ABBREVIATIONS:

• ACTB, actin, beta
• ATG, autophagy related
• BAF, bafilomyin A₁
• ECAR, extracellular acidification rate
• GABARAP, GABA(A) receptor-associated protein
• HCQ, hydroxychloroquine
• LC3, microtubule-associated protein 1 light chain 3
• MTOR, mechanistic target of rapamycin (serine/threonine kinase)
• NBR1, neighbor of BRCA1 gene 1
• OCR, oxygen consumption rate
• PI, proteasome inhibitor
• SQSTM1, sequestosome 1
• TAX1BP1, Tax1 (human T-cell leukemia virus type I) binding protein 1
• Ubl, ubiquitin-like protein
• WT, wild-type
• UB, ubiquitin

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