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Proteome analysis of hypoxic glioblastoma cells reveals sequential metabolic adaptation of one-carbon metabolic pathways

ABSTRACT: Rapidly proliferating tumors are exposed to a hypoxic microenvironment due to their density, high metabolic consumption, and interruptions in blood flow due to immature angiogenesis. Cellular responses to hypoxia promote highly malignant and metastatic behavior, as well as a chemotherapy-resistant state. In order to better understand the complex relationships between hypoxic adaptations and cancer progression, we studied the dynamic proteome responses of glioblastoma cells exposed to hypoxia via an innovative approach: quantification of newly synthesized proteins using heavy stable-isotope arginine labeling combined with accurate assessment of cell replication by quantification of the light/heavy arginine ratio of peptides in histone H4. We found that hypoxia affects cancer cells in multiple intertwined ways: inflammation, typically with over-expressed glucose transporter (GLUT1), DUSP4/ MKP2, and RelA proteins; a metabolic adaptation with overexpression of all glycolytic pathway enzymes for pyruvate/lactate synthesis; and the EMT (epithelial-mesenchymal transition) and cancer stem cell (CSC) renewal with characteristic morphological changes and mesenchymal/CSC protein expression profiles. For the first time, we identified the vitamin B12 transporter protein TCN2, which is essential for one-carbon metabolism, as being significantly downregulated. Further, we found, by knockdown and overexpression experiments, that TCN2 plays an important role in controlling cancer cell transformation towards the highly aggressive mesenchymal/CSC stage; low expression of TCN2 has an effect similar to hypoxia, while high expression of TCN2 can reverse it. We conclude that hypoxia induces sequential metabolic responses of one-carbon metabolism in tumor cells.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Brain Glioblastoma Multiforme,Brain Cancer

SUBMITTER: Kangling Zhang

LAB HEAD: Kangling Zhang

PROVIDER: PXD007280 | Pride | 2017-09-07

REPOSITORIES: Pride

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Proteome Analysis of Hypoxic Glioblastoma Cells Reveals Sequential Metabolic Adaptation of One-Carbon Metabolic Pathways.

Zhang Kangling K Xu Pei P Sowers James L JL Machuca Daniel F DF Mirfattah Barsam B Herring Jason J Tang Hui H Chen Yan Y Tian Bing B Brasier Allan R AR Sowers Lawrence C LC

Molecular & cellular proteomics : MCP 20170905 11

Rapidly proliferating tumors are exposed to a hypoxic microenvironment because of their density, high metabolic consumption, and interruptions in blood flow because of immature angiogenesis. Cellular responses to hypoxia promote highly malignant and metastatic behavior, as well as a chemotherapy-resistant state. To better understand the complex relationships between hypoxic adaptations and cancer progression, we studied the dynamic proteome responses of glioblastoma cells exposed to hypoxia via ...[more]

PMID: 28874504

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Project description:The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic ATP production, an event with consequences for both cell bioenergetics and cell fate. This response is regulated at the transcriptional level by HIF-1-dependent transcriptional upregulation of all ten glycolytic enzymes. However, this response alone does not account for the levels of ATP produced in hypoxia. Here, we investigated additional mechanisms of regulating glycolysis in hypoxia. We found that both intestinal epithelial cells treated with inhibitors of transcription and translation and human platelets (which lack nuclei) maintained the capacity for hypoxia-induced glycolysis, suggesting the involvement of a non-transcriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. Mass spectrometric analysis of the interactome of immunoprecipitated rate-limiting glycolytic enzymes identified hypoxia-sensitive complexes comprising multiple glycolytic enzymes and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of glycolytic complexes, though not dependent upon transcription, occurs via a HIF-1?-dependent mechanism, suggesting that HIF-1? may play a moonlighting role in the formation / maintenance of glycolytic complexes. Furthermore, we provide evidence for the presence of HIF-1? in cytosolic fractions of hypoxic cells which physically associated with the glucose transporter GLUT1 and the glycolytic enzyme PFKP in a hypoxia-sensitive manner. In conclusion, we hypothesize that HIF-1? plays a role in initiation and/or maintenance of glycolytic complexes in intestinal epithelial cells under hypoxic conditions in a manner which optimizes catalytic efficiency of the pathway by facilitating substrate channeling of glycolytic intermediates between sequential pathway enzymes. In hypoxia, cells undergo a metabolic switch to increased glycolysis. This has important implications for cell behavior, phenotype, and fate in both healthy and cancerous cells. Here we describe a mechanism by which HIF-1, in addition to increasing glycolytic enzyme expression, promotes glycolysis via the formation of a metabolic complex.

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