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Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells.

ABSTRACT: Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h) dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia.

SUBMITTER: Lai MC

PROVIDER: S-EPMC4831676 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells.

Lai Ming-Chih MC Chang Chiao-May CM Sun H Sunny HS

PloS one 20160414 4

Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h) dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. ...[more]

PMID: 27078027

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Project description:Background Colon cancer is a common malignancy of the digestive tract. The search for effective drugs to treat colon cancer has become the focus of current researches. Tanshinone IIA (Tan IIA) is a fat-soluble component extracted from tanshinone, a traditional Chinese medicine. Tan IIA can modulate the occurrence and development of tumors, but its effect on autophagy in colon cancer cells has not been reported. Methods Two types of colon cancer cell lines were selected and different concentrations of Tan IIA were used to treat cells at different time points. Cell Counting Kit-8 assay (CCK-8) was used to detect the effect of Tan IIA on cell proliferation; transmission electron microscopy was used to observe the formation of autophagosomes; reverse transcription-polymerase chain reaction (RT-qPCR) and western blot were used to detect the expression of autophagy related genes and proteins. Cell transfection was used to interfere with MEK (mitogen-activated extracellular signal-regulated kinase) expression, and RT-qPCR and western blot were used to detect the expression of MEK/ERK/mTOR pathway-related proteins. Results Tan IIA resulted in a significant reduction in the viability of the two kinds of colon cancer cells. The number of autophagosomes increased significantly after the treatment of Tan IIA into these cells. Addition of autophagy inhibitor 3-MA (3-Methyladenine) improved the increase of autophagosomes in cells induced by Tan IIA. At the same time, Tan IIA induced the expression of autophagy-related proteins in the two colon cancer cell lines. When Tan IIA induced autophagy in colon cancer cells, the expression of MEK/ERK/mTOR pathway-related proteins increased significantly. After interfering with the expression of MEK, the expression of autophagy decreased significantly, indicating that Tan IIA promoted autophagy of colon cancer cells through MEK/ERK/mTOR pathway. Conclusions Tan IIA stimulates autophagy in colon cancer cells through MEK/ERK/mTOR pathway, hence inhibiting the growth of colon cancer cells.

Project description:Lysosomal Ca2+ contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif containing 6) is a Ca2+ channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca2+ levels. The Ca2+ efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca2+ level, in which ITPR-independent regulation of Ca2+ status was observed. Further, TMBIM6 regulated the local release of Ca2+ through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca2+ efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB (transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca2+ and the associated TFEB nuclear translocation were decreased. Furthermore, autophagy flux was significantly enhanced in the liver or kidney from starved Tmbim6+/+ mice compared with that in the counter tmbim6-/- mice. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca2+ release, leading to PPP3/calcineurin-mediated TFEB activation and subsequently enhanced autophagy. Thus, TMBIM6, an ER membrane protein, is suggested to be a lysosomal Ca2+ modulator that coordinates with autophagy to alleviate metabolism stress.Abbreviations: AVs: autophagic vacuoles; CEPIA: calcium-measuring organelle-entrapped protein indicator; ER: endoplasmic reticulum; GPN: glycyl-L-phenylalanine-beta-naphthylamide; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; LAMP1: lysosomal associated membrane protein 1; MCOLN/TRPML: mucolipin; MEF: mouse embryonic fibroblast; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TKO: triple knockout; TMBIM6/BI-1: transmembrane BAX inhibitor motif containing 6.

Dataset Information

Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells.

Publications

Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells.

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