Abstract
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 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 toward the highly aggressive mesenchymal/CSC stage; low expression of TCN2 has an effect similar to hypoxia, whereas high expression of TCN2 can reverse it. We conclude that hypoxia induces sequential metabolic responses of one-carbon metabolism in tumor cells. Our mass spectrometry data are available via ProteomeXchange with identifiers PXD005487 (TMT-labeling) and PXD007280 (label-free).
Highlights
From the ‡Department of Pharmacology, University of Texas Medical Branch, Galveston (UTMB), Texas, 77555; §Institute for Translational Sciences, UTMB, Galveston, Texas, 77555; ¶Sealy Center for Molecular Medicine, UTMB, Galveston, Texas, 77555; ʈDepartment of Neuroscience and Cell Biology, UTMB, Galveston, Texas, 77555
Of the 14 glucose transporter (GLUT) isoforms, our proteomic data indicate that GLUT1 is the most rapidly and highly upregulated protein under hypoxia before glycolysis (Figs. 3, 6, and 4S)
The GLUT1 protein is one of the low-oxygen stressinduced or HIF-1␣ transcriptionally regulated proteins, which include the HIF-1␣ stabilizer protein Thrombopoietin (TPO) [29, 51]; these two proteins belong to the HIF-1␣ signaling pathway and were upregulated in hypoxic U87 cells as shown by tandem mass tag (TMT) proteomics (Fig. 2)
Summary
From the ‡Department of Pharmacology, University of Texas Medical Branch, Galveston (UTMB), Texas, 77555; §Institute for Translational Sciences, UTMB, Galveston, Texas, 77555; ¶Sealy Center for Molecular Medicine, UTMB, Galveston, Texas, 77555; ʈDepartment of Neuroscience and Cell Biology, UTMB, Galveston, Texas, 77555. Hypoxia induces deficient mitochondria redox-oxidation cycles normally needed for energy production [14, 15]. To compensate for this deficiency, anaerobic cells metabolize citric acid cycle intermediates and glucose, an adaptation historically known as the Warburg effect [16, 17]. We analyzed the hypoxic proteome by tandem mass tag (TMT) and label-free LC-MS/MS in U87. We have innovatively adapted the SILAC technique to TMT and label-free proteomics and the quantification of proteins in replicating cells in which light arginine (LR) was incorporated into the proteins, so that the effects from preexisting proteome of old cells, in which the arginines in proteins were originally labeled with heavy isotopes (13C615N4-arginine, denoted HR), can be minimized. An additional benefit of quantification starting with HR-labeled cells is that the cell proliferation rates can be readily obtained by calculation of the LR/HR ratios when proteins/ peptides are analyzed by LC-MS
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