Adaptation to Hif1α Deletion in Hypoxic Cancer Cells by Upregulation of GLUT14 and Creatine Metabolism

Published OnlineFirst March 18, 2019; DOI: 10.1158/1541-7786.MCR-18-0315

Metabolism Molecular Cancer Research Adaptation to HIF1a Deletion in Hypoxic Cancer Cells by Upregulation of GLUT14 and Creatine Metabolism Alessandro Valli1,2, Matteo Morotti1, Christos E. Zois1, Patrick K. Albers3, Tomoyoshi Soga4, Katharina Feldinger1, Roman Fischer2, Martin Frejno2, Alan McIntyre1, Esther Bridges1, Syed Haider1, Francesca M. Buffa1, Dilair Baban3, Miguel Rodriguez5,6, Oscar Yanes5,6, Hannah J. Whittington7, Hannah A. Lake7, Sevasti Zervou7, Craig A. Lygate7, Benedikt M. Kessler2, and Adrian L. Harris1

Abstract

Hypoxia-inducible factor 1a is a key regulator of the than phosphofructokinase. Furthermore, glucose uptake hypoxia response in normal and cancer tissues. It is well could be maintained in hypoxia through upregulation of recognized to regulate glycolysis and is a target for therapy. GLUT14, not previously recognized in this role. Finally, However, how tumor cells adapt to grow in the absence of there was a marked adaptation and change of phospho- HIF1a is poorly understood and an important concept to creatine energy pathways, which made the cells susceptible understand for developing targeted therapies is the ﬂexi- to inhibition of creatine metabolism in hypoxic condi- bility of the metabolic response to hypoxia via alternative tions. Overall, our studies show a complex adaptation to pathways. We analyzed pathways that allow cells to survive hypoxia that can bypass HIF1a, but it is targetable and it hypoxic stress in the absence of HIF1a,usingtheHCT116 provides new insight into the key metabolic pathways colon cancer cell line with deleted HIF1a versus control. involved in cancer growth. Spheroids were used to provide a 3D model of metabolic gradients. We conducted a metabolomic, transcriptomic, Implications: Under hypoxia and HIF1 blockade, cancer cells and proteomic analysis and integrated the results. These adapt their energy metabolism via upregulation of the showed surprisingly that in three-dimensional growth, GLUT14 glucose transporter and creatine metabolism provid- a key regulatory step of glycolysis is Aldolase A rather ing new avenues for drug targeting.

Introduction conditions such as hypoxia, acidic pH, and a nutrient-poor microenvironment (1). Highly proliferating cancer cells must increase the import of Warburg first described the increase of oxidation of glucose nutrients and adopt a metabolic program that fulfils energetic, into lactate in cancer cells (2). Although yielding less ATP than redox, and biosynthetic requirements for increasing cell mass. OXPHOS, glycolysis is utilized predominantly by cancer cells and Metabolic flexibility is acquired through genetic and nonge- provides a more rapid response to the high energy demand of netic factors that allow tumors to grow in altered biochemical rapidly proliferating cancer cells (3). In hypoxia, this regulation represents the predominant way to produce ATP, even for normal cells (1). Although many solid tumors demonstrate the Warburg 1Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United 2 fi effect, some, such as prostate cancer, do not (4). Virus-infected Kingdom. Target Discovery Institute, Nuf eld Department of Medicine, Uni- þ versity of Oxford, Oxford, United Kingdom. 3The Wellcome Trust Centre for cells show increased glucose metabolism and CD4 T-cell growth Human Genetics, University of Oxford, Oxford, United Kingdom. 4Institute for is characterized by a high synthesis of ATP and biosynthetic Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan. 5Metabo- reactions (cell membrane and DNA biosynthesis). These observa- lomics Platform, IISPV, Department of Electronic Engineering, Universitat Rovira tions show that uncoupling glycolysis from OXPHOS can offer a 6 i Virgili, Tarragona, Spain. Spanish Biomedical Research Center in Diabetes and metabolic solution adequately beneficial for the high energy Associated Metabolic Disorders-CIBERDEM, Madrid, Spain. 7Division of Cardio- vascular Medicine, Radcliffe Department of Medicine, University of Oxford, demand while maintaining redox balance and biosynthetic Oxford, United Kingdom. requirements (5–7). Similarly, reprogramming of cancer metabolism activates distinct pathways in which oncogenic mutations Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). convey downstream signals to directed prometabolic traits (1). Adaptation to the hypoxic tumor microenvironment is driven B.M. Kessler and A.L. Harris are the co-senior authors of this article. by hypoxia-inducible factors HIF1a and HIF2a that induce a Corresponding Author: Alessandro Valli, University of Oxford, Oxford OX3 9DS, distinct transcriptional program (8). HIF1a regulates numerous United Kingdom. Phone: 4418-6522-2420; Fax: 4418-6522-2431; E-mail: metabolic genes, especially those involved in glycolysis and [email protected] and [email protected] related auxiliary processes (9). doi: 10.1158/1541-7786.MCR-18-0315 Because of the importance of HIF1 in tumor growth and the 2019 American Association for Cancer Research. potential of HIF1a as target, we investigated how a cell line

www.aacrjournals.org OF1

Valli et al.

deleted for HIF1a could survive in hypoxia. We studied the the increase in absorbance at 412 nm over 1 minute as a result of HCT116 colorectal cancer cell line wild-type (WT) and HIF1a 5-thio-2-nitrobenzoate (TNB2-) production. The assay was per- knockout (KO) under hypoxic conditions and analyzed transcrip- formed in duplicate and results were normalized by protein tomics, proteomics, metabolomics, and fluxomics. We integrated concentration. the multi-omics data to construct a map of hypoxic colorectal cancer cell energy metabolism. Our results show for the first time upregulation of glucose transporter type 14 (GTR14) to maximize Results nutrient uptake and an important role for creatine (Cr) energy HIF1a regulates hypoxic spheroid cancer growth pathways to sustain multidimensional growth under hypoxic We cultured WT and KO HCT116 cells as spheroids (WTs and conditions. KOs) for 72 hours, generating a hypoxic inner mass, and evaluated their morphology at three time points (72.5, 76, and 96 hours). Immunostaining of WTs and KOs spheroids at 96 hours showed Materials and Methods key differences in hypoxic adaptation. Pimonidazole (PIMO)- Cell culture positive staining, in both WTs and KOs, confirmed the presence of Cell lines were available from Clare Hall Laboratories hypoxic areas, and carbonic anhydrase 9 (CA9; HIF1a-target) (HCT116), or were a kind gift from Prof Walter Bodmer (Weath- positive stain in WTs demonstrated the presence of hypoxia and erall Institute of Molecular Medicine, Oxford, United Kingdom; HIF1a stabilization. In contrast, CA9 staining was absent in KOs, DLD-1, Ls174T). Cell line authentication was carried out by short demonstrating successful HIF1a knockout. Necrosis and cell tandem repeat analyses (LGC Standards) 6 months prior to the first death, as measured by H&E staining, was more pronounced in submission of the manuscript. Cells were cultured as described WTs than in KOs (Fig. 1A). previously, unless different conditions were specified (10). We evaluated differences in growth of WTs and KOs (Fig. 1B). HCT116 WTs and KOs proliferated over 24 hours, with a higher Enzyme activity assay rate in WTs (top). WTs volume was overall significantly bigger WT and KO HCT116 cells were cultivated at either 21% O2 or than that of KOs cells with no marked changes over time (middle). 1% O2 for 24 hours. Cells were collected, pelleted, and suspended Cell spheroid density (number of cells per spheroid/spheroid in 1 mL of ice-cold buffer (0.08 mmol/L K2HPO4, 1 mmol/L volume) showed a significant increased over time in both WTs EGTA, 0.02 mmol/L KH2PO4, and 1 mmol/L b-mercaptoethanol) and KOs and was more pronounced in KOs at 96 hours (bottom). while under experimental conditions. Samples were vortexed and 150 mL aliquots removed for determination of protein by the HIF1a metabolic effects in cancer spheroids Lowry method. A final concentration of 0.1% v/v Triton X-100 was Because WTs and KOs grow unequally, we optimized the added to permeabilize cells. Samples were stored on ice for at least formation of WTs and KOs with a comparable cell number, 30 minutes to allow precipitation and the supernatant was used morphology, volume, and diameter, which enabled the investi- for all enzyme activity measurements. gation of hypoxic gradients at the studied time points (72.5, 76, and 96 hours; data not shown). We measured 26 metabolites of Total creatine kinase activity six metabolic pathways: glycogen (GLG), glycolysis (GLY), TCA Twenty microliters of 1:5 (in ice buffer) diluted sample super- cycle, ATP, Cr, and redox metabolism (Fig. 1C). GLG metabolic natant was incubated with 1 mL of creatine kinase (CK)-NAC intermediates show a general decrease over time in both WTs and reagent (Thermo Fisher Scientific) at 30C. After 3-minute lag KOs. Glycolysis and Cr metabolic products accumulated mostly time, CK activity was quantified spectrophotometrically by mea- in WTs. TCA cycle metabolites did not show a defined trend, but suring the increase in absorbance at 340 nm over 2 minutes as a generally appeared to be more elevated in WTs at 96 hours and result of NADH production. The assay was performed in triplicate decreased in KOs over time. We observed higher amounts of ATP and results normalized to protein concentration. in WTs versus KOs, and this difference remained stable over time; þ ADP levels decreased at 96 hours in both in WTs and KOs. NAD Relative activity of creatine kinase isoenzymes levels were low overall, while ADP and AMP were lower in KOs Sample supernatant was diluted 1:200 in ice buffer and incu- versus WTs. HIF1a deletion correlated with higher AMP com- bated with 1% CK isoenzyme activator for 10 minutes prior to use. pared with ATP, but with less difference over time (Fig. 1D). Creatine kinase isoenzymes were separated according to their Supplementary Figure S1 shows statistical results for the meta- electrophoretic mobility on an agarose gel, followed by incuba- bolites detected in WTs and KOs. tion with CK isoenzyme chromogen, which allowed visualization of the bands. Relative activities of individual CK isoenzymes were Glycolytic and TCA flux in spheroids in hypoxia without HIF1a quantified by densitometry. All reagents were provided within the We next evaluated glycolysis and TCA cycle flux in spheroids in SAS-1 CK VIS-12 Isoenzyme Kit (Helena Biosciences). Absolute the presence and absence of HIF1a. Spheroids were grown for 72 13 activities for each isoenzyme were calculated by multiplying hours and we utilized C6 Glucose (Glu) for metabolic flux relative isoenzyme activity by total CK activity. analysis (MFA; Fig. 2A). We calculated the isotopolog ratio for each metabolite as [single fully labeled isotopolog value]/[sum of Citrate synthase activity the total isotopolog value] ratio (STIR). Next, we evaluated Fifty microliters of sample supernatant was incubated with glycolysis and TCA STIR distribution. We noted that the overall 850 mL of reaction mixture (0.35 mmol/L acetyl-CoA, 0.12 mmol/L STIR range was wider for metabolites of glycolysis compared with DTNB) at 25C. After 3 minutes, 100 mL of 1 mmol/L oxaloacetate the TCA cycle (STIR glycolysis 0.2–0.8; STIR TCA cycle: 0.3–0.45). was added into the reaction mixture. Citrate synthase (CS) activity In glycolytic MFA (Fig. 2B), Glu 6 phosphate (Glu-6P) STIR was immediately assessed spectrophotometrically by measuring increased linearly in WTs between 72.5 and 96 hours, increased

OF2 Mol Cancer Res; 2019 Molecular Cancer Research

Energy Proﬁling in Hypoxic Cancer Cells

Figure 1. Spheroid hypoxic phenotype and energy metabolism. A, Immunostaining of spheroids for protein expression of hypoxic markers. PIMO, CA9, and H&E expression in spheroids formed with HCT116 colorectal cancer cells HIF1a (WT) and HIF1a-deficient (KO). B, HIF1a WT and KO spheroid morphology. Spheroid- forming cell count (cell number, top), spheroid volume (cm3, middle), and spheroid cell density (cell number/spheroid volume ratio, bottom). C, Heatmap showing z-normalized intensities of energy-related metabolites in WT and KO spheroids at time points 72.5, 76, and 96 hours after formation. R.I., Relative intensity. D, Specific ATP, ADP, AMP, ATP/AMP ratio, and ATP/ADP ratio relative intensities (R.I. 0–1 normalized) in WT and KO spheroids at time points 72.5, 76, and 96 hours after formation. Metabolic pathways: glycogen (GLG), glycolysis (GLY), Krebs cycle (TCA), adenosine triphosphate (ATP), creatine (Cr), and oxidation–reduction (REDOX). Data are reported as average SD (N ¼ 3). Statistical comparisons were performed using two-way ANOVA, multiple post hoc group comparisons, or t test (P values corrected for multiple testing using the Benjamini–Hochberg procedure at a ¼ 1%), showing P value <0.05 (), <0.01 (), and <0.001 (). significantly in WTs versus KOs at 96 hours, but remained 96 hours, but remained stable in KOs. No effect was observed 13 13 stable in KOs. No effect was observed on C6 fructose 6 on C6 dihydroxyacetone phosphate (DHAP), 3-phosphoglyc- phosphate (Fru-6P) MFA. Fructose 1, 6 bis phosphate erate (3-PG), 2-phosphoglycerate (2-PG), and phosphoenol- (Fru-1,6BP) STIR decreased linearly in WTs between 72.5 and pyruvate (PEP) MFA in either spheroid type. Pyruvate (Pyr) and

www.aacrjournals.org Mol Cancer Res; 2019 OF3

Valli et al.

Figure 2. Metabolic flux in spheroids. A, Experimental design of metabolomics flux analysis in spheroids. B and C, Glycolytic and TCA metabolic flux analysis after 13 administration of C6 glucose. Isotopolog quantification is shown by STIR ratio [single fully labeled isotopolog value]/[sum of the total isotopolog value]. D, þ þ NAD / NADHþH ratio and CS activity and WTN, KON, WTH, and KOH cell proliferation after 24 hours in two O2 conditions (21% and 1%). Data are reported as average SD (N ¼ 3). Statistical comparisons were performed using two-way ANOVA, multiple post hoc group comparisons or t test (P values corrected using the Benjamini–Hochberg procedure at a ¼ 1%), showing P value <0.05 (), <0.01 (), and <0.001 ().

lactate STIR levels increased in WTs 96 hours versus earlier time In the TCA MFA (Fig. 2C), citrate STIR showed a linear increase points, whereas they accumulated earlier and to higher levels in KOs. It was signiﬁcantly lower in KOs than in WTs at 72.5 hours (76 hours) in KOs. and became signiﬁcantly higher at 96 hours, whereas citrate STIR

OF4 Mol Cancer Res; 2019 Molecular Cancer Research

Energy Proﬁling in Hypoxic Cancer Cells

remained stable in WTs. These data matched CS activity and its Lactate levels were significantly increased in WTH when compared þ þ allosteric enzymatic regulator NAD /NADHþH ratio both of with WTN and further increased in KOH when compared with which showed a significant increase only in HCT116 KO hypoxic WTH, matching to the 3D flux (Figs. 2B and 4A). TCA metabolites cells (KOH; Fig. 2D). were elevated in KOH demonstrated the predominant role of Cis-aconitate, isocitrate, and succinate STIR was essentially HIF1a in redirecting metabolism away from TCA cycle in hypoxia unchanged, but cis-aconitate accumulated in KOs at 96 hours. (Figs. 3C and 4A). Alpha-KG and fumarate STIR were not assessed as only the unlabeled isotopologs were detected. Malate STIR increased lin- Analysis of proteome. Many previously described enzymes were early in KOs between 72.5 and 96 hours and was significantly increased in WTH versus WTN (e.g., PGM1, GLGB, GTR1, HEXK1, higher than in WTs at 96 hours. Malate STIR showed a mild G6PI, and PFKAM). Comparing WTH versus KOH cells, enzymes increase between 72.5 and 96 hours in WTs (Fig. 2C). were similarly elevated in the WTH, demonstrating the predom- Thus, glucose uptake was maintained without HIF1a and a inant role of HIF1a (PGM1, GLGB, GTR1, G6PI, and PFKAM). clear block of conversion of Fru-1,6BP to DHAP occurred without In contrast, in KOH versus WTH, UGPAse, GYS1, GTR3/GTR14 HIF1a, at aldolase level, rather than the earlier step of conversion (proteomics did not differentiate between the two transporters), by phosphofructokinase of Fru-6P to Fru-1,6BP. There was, in PFKAL, PFKAP, PDP1, PDPR, ACLY, IDH3G, ADT2, ATAD2, addition, a surprising higher lactate and Pyr without HIF1a. KCRB, BLVRB, and ADK were upregulated. TIGAR was upregu- Because of this unexpected effect in the aldolase step on the lated (but decreased in WTH vs. WTN; Figs. 3D and E and 4B glycolytic flux, we evaluated the clinical relevance of aldolase in and C). colorectal cancer. We investigated whether tumor ALDOA mRNA expression levels correlated with patient prognosis using overall Analysis of transcriptome. We next evaluated mRNA features survival data derived from a cohort of 440 colon adenocarcinoma associated with the targeted proteomics signature. In WTH patients (The Cancer Genome Atlas). We found that, when the versus WTN, we observed an increase of GLGB, GSK3B, GTR1, patient group was divided in quartiles, the highest quartile had a GTR3/GTR14, HEXK1, PFKAL, ALDOA, PGK1, PGAM1, ACON, reduced 5-year survival (Kaplan–Meier analysis of overall surviv- and many others. PYGB, PDRP, IDH3A,andAAPK1 were al; log-rank P < 0.05; Supplementary Fig. S2A). downregulated. When we evaluated KOH versus WTH, we observed PYGB, HIF response in 2D growth UGPAse, GTR3 and/or GTR14, TIGAR, FUMH, ATPD, ATAD2, For further manipulations of the cells, we used 2D growth. We BLVRB, and AAPK1 upregulated. However, most of the genes were initially confirmed the biology for HCT116 WT and KO cells in lower (GTR1, PGM2, GLGB, GSK3B, GTR1, G6PI, PGK1, PGAM1, normoxia (N) and hypoxia (H; WTN, KON, WTH, and KOH; PKM, ACON, IDH3G, KCRU, and KAD4; Fig. 4D). ref. 10). Hypoxia reduced proliferation in WTH and KOH cells, and the lack of HIF1a reinforced this effect in both normoxia and HIF1a and hypoxia-dependent effects on transcription and hypoxia (Fig. 3A). We observed no difference in cell proliferation protein levels when experiments were conducted in 5 mmol/L or 25 mmol/L We correlated transcriptomics and proteomics by calculating Glu (data not shown). HIF1a expression was barely detectable in for each feature log2 fold changes (FC) for mRNA and proteins WTN, strongly accumulated in WTH, and was absent in both KON comparing the following models: KON-WTN (HIF1a effect in and KOH. CA9 accumulated only in WTH. HIF2a doubled in normoxia), WTH-WTN (HIF1a effect in hypoxia), KOH-KON expression in both WTH and KOH versus WTN and KON, show- (HIF1a-independent hypoxia effects), and KOH-WTH (HIF1a ing no notable compensatory effect in the absence of HIF1a effect in hypoxia, expected to be similar to WTH-WTN). (Fig. 3B). In the 2D experiments, we detected 27 metabolites We applied linear regression analysis to correlate mRNA log2 (Figs. 3C and 4A), 86 proteins (Figs. 3D and E and 4B and C), and FC and protein log2 FC for each model. The correlation of WTN- 76 mRNAs (Fig. 4D) involved in ATP metabolism and related WTH was similar to KOH-WTH. In contrast, KOH versus KON metabolic pathways that involve GLG, GLY, TCA cycle, ATP, Cr, showed no significant correlation. Interestingly, there was strong redox, and adenosyl metabolism. We observed the highest ATP evidence for a normoxic effect of HIF1a as the slope for KON level in WTN. Both hypoxia and lack of HIF1a lowered ATP in a versus WTN was similar to that for KOH versus WTH (Supple- cumulative fashion. Intracellular ADP did not show any signifi- mentary Fig. S3). cant differences. There was a significant increase in AMP levels in Significantly regulated genes at either RNA and protein level (63 hypoxia, with the effect reinforced by the lack of HIF1a in KOH for WTH-WTN and 56 for KOH-WTH) were compared to evaluate (Fig. 4E). transcription and/or translation patterns of regulation under hypoxia. Analysis of metabolome. More small molecules accumulated in They are shown (protein and mRNA) in a heatmap of log2 FC. WTH and in KOH than in WTN and KON. Metabolites increased For many previously well-characterized genes, there was a close in WTH versus WTN were GLG, Glu, Glu-6P, 2-PG, lactate, and correlation of mRNA and protein, but in some proteins went up þ NAD . Fumarate and malate were significantly increased in WTN. proportionally more (ACON, ATP5J, ATAD2, GTR14) suggesting Comparing WTH versus KOH cells, a set of metabolites increased a stronger posttranslational element. In others, mRNA increased in KOH (Glu-1P, UDP-Glu, Fru-1,6BP, lactate, succinate, fuma- without a change in protein (GSK3B and TIGAR). The most rate, malate, AMP, Cr). We assessed intracellular levels of PCr and regulated features were selected on the basis of residuals below Cr and calculated PCr/Cr ratio. We observed a decrease in ratio in and above 2s and 2s distribution for each experimental con- WTH versus KOH (P < 0.01); in addition, in both WTH and KOH, dition (Fig. 4D; Supplementary Fig. S3). the ratios were decreased versus their parental normoxic cells Protein kinase AMPK expression showed a significant increase WTN and KON (P < 0.001; Supplementary Fig. S6A and S6B). only in WTH versus WTN (50%, P < 0.01). Interestingly, p-AMPK

www.aacrjournals.org Mol Cancer Res; 2019 OF5

Valli et al.

Figure 3.

Proteo-metabolomics profile of hypoxic HCT116. A, WTN, KON, WTH, and KOH cell proliferation after 24 hours in two O2 conditions (21% and 1%). B, Western blot analysis showing HIF1a stabilization in hypoxia and successful deletion in KO HCT116 cells. HIF1a, HIF2a, and CA9 protein expression in cells: HIF1a wild-type normoxia (WTN), HIF1a-deficient normoxia (KON), HIF1a wild-type hypoxia (WTH), and HIF1a-deficient hypoxia (KOH). C–E, Proteo-metabolomics heatmaps (C metabolites; D and E proteins) of the detected metabolites and proteins in WTN, KON, WTH, and KOH cells after 24-hour exposure to two O2 conditions (21% and 1%). Normalized levels refer to WTN. R.I., Relative intensity. Metabolic pathways: glycogen (GLG), glycolysis (GLY), Krebs cycle (TCA), adenosine triphosphate (ATP), creatine (Cr), oxidation–reduction (REDOX), and adenosyl metabolism. Data are reported as average SD (N ¼ 3). Statistical comparisons were performed using two-way ANOVA, multiple post hoc group comparisons, or t test, showing P value <0.05 (), <0.01 (), and <0.001 ().

was strongly expressed in the absence of HIF1a in both KON and hypoxia. PCR analysis showed GTR3 and GTR14 more highly KOH. AMPK/p-AMPK (levels normalized by b-actin) ratio was expressed in the absence of HIF1a in normoxia. Both genes were positive for KON and KOH, (13 and 9, respectively) while it was induced in WT cells in hypoxia, but more highly induced in the negative for WTN and WTH (Supplementary Fig. S6C). KO cells, KOH (Fig. 5A and B). Similar results were observed for more severe hypoxia O2 0.1% (Supplementary Figs. S4A and S5A) HIF1a/HIF2a hypoxic interaction regulates GTR14 expression and in two other colorectal cancer cell lines DLD1 (Supplemen- Flux analysis highlighted that Glu uptake was maintained in the tary Figs. S4B and S5B), and Ls174T (Supplementary Figs. S4C absence HIF1a; thus, we studied in more detail Glu transporters in and S5C).

OF6 Mol Cancer Res; 2019 Molecular Cancer Research

Energy Proﬁling in Hypoxic Cancer Cells

Figure 4. Analysis of the HCT116 hypoxic metabolic phenotype. A–C, Metabolomics- and proteomics-integrated circos-plots showing signiﬁcantly regulated features only in KON versus WTN, WTH versus WTN, KOH versus KON, and KOH versus WTH (upper side circos-plots). Adjusted P < 0.05 ()orP < 0.01 () are indicated for the different experimental comparisons (connecting lines inside circos-plots): KON/WTN (gray/white-gray connection) WTH/WTN (gold/white-brown connection), KOH/KON (blue/gray-violet connection), KOH/WTH (blue/gold-green connection). Colored boxes (lower side circos-plots linked to connecting lines) represent different metabolic pathways, while colored boxes (inside circos-plots linked to connecting lines) identify the biological condition where the feature is upregulated. D, Heatmap of integrated transomics (proteomics and transcriptomics); mRNA and protein log2 FC are calculated for WTH-WTN and KOH-WTH. Feature selection was based on departure from 95% conﬁdence intervals of the linear model distribution built for each comparison (WTH-WTN and KOH-WTH). Metabolic pathways: glycogen (GLG), glycolysis (GLY), Krebs cycle (TCA), adenosine triphosphate (ATP), creatine (Cr), oxidation–reduction (REDOX), and adenosyl metabolism (ADE). E, Targeted metabolomics showing distribution of ATP, ADP, AMP, ATP/AMP, and ATP/ADP ratios in WTN, KON,

WTH, and KOH cell after 24 hours in 21% and 1% O2. Raw intensities and ratios are shown as 0–1 normalized levels' relative intensity (R.I). Data are reported as average SD (N ¼ 3). Statistical comparisons were performed using two-way ANOVA, multiple post hoc group comparisons or t test (P values corrected using the Benjamini–Hochberg procedure at a ¼ 1%), showing P value <0.05 (), <0.01 (), and <0.001 ().

www.aacrjournals.org Mol Cancer Res; 2019 OF7

Valli et al.

Figure 5. Congruence between multi-omics metabolic reconstruction and hypoxia biochemistry. A, PCR analysis of CA9, GTR1, GTR3, GTR14, HEXK1, KCRU, KCRM, and

SLC6A8 mRNA levels in WTN, KON, WTH, and KOH cells after 24-hour exposure to two O2 conditions (21% and 1%). B, Statistical signiﬁcance for PCR analysis– measured features. C and D, Western blot analysis of GTR14, GTR3, KCRB, KCRU, and KCRM in WTN, KON, WTH, and KOH cells after 24-hour exposure to two O2 conditions (21% and 1%), and in WTs and KOs spheroids (96 hours). E, CK BB activity and WTN, KON, WTH, and KOH cell proliferation after 24-hour exposure to

two O2 conditions (21% and 1%). Data are reported as average SD (N ¼ 3). Statistical comparisons were performed using two-way ANOVA, multiple post hoc group comparisons, or t test (P values corrected using the Benjamini–Hochberg procedure at a ¼ 1%), showing P value <0.05 (), <0.01 (), and <0.001 ().

Western blot analysis showed GTR3 induced mostly in We studied the effects of HIF1a and HIF2a on GTR14 mod- KOH and slightly in WTH. GTR14 was suppressed by HIF1a ulation in hypoxia. We utilized HCT116 WT cells HIF1a and in both WTN and WTH versus KON and KOH. Hypoxia, in HIF2a single knockdown and HCT116 KO HIF2a knockdown absence of HIF1a, reinforced GTR14 expression protein exposed to 1% O2 (WT1KDH, WT2KDH, KO2KDH). CA9 sup- as shown by KOH versus KON (Fig. 5C; Supplementary pression in KON, WT1KDH, KOH, and KO2KDH versus WTN and Fig. S4E). WTH, conﬁrmed HIF1a absence. HIF2a suppression in WT2KDH

OF8 Mol Cancer Res; 2019 Molecular Cancer Research

Energy Proﬁling in Hypoxic Cancer Cells

and KO2KDH versus WTH, WTKD1H, and KOH, confirmed Interestingly, total KCR activity in HCT116 cells showed the HIF2a knockdown (Supplementary Figs. S4D and S5D). GTR1 same changes as KCRBB (Fig. 5E; Supplementary Fig. S7C). We RNA expression increased only in WTH and WT2KDH, that is, in evaluated total KCR activity also in MCF7 breast cancer cells WT response to HIF1a (Supplementary Figs. S4D and S5D). In or KO. In normoxia, total MCF7 KCR activity was increased in normoxia, GTR3 was higher in KO cells and further increased in KON versus WTN, showing a similar effect to HCT116 nor- the KOH. In hypoxia, the levels increased in WT cells to the same moxic experiments. Also, no difference was observed between level as KON. However, HIF2a KD had only a small effect on the KON and KOH. However, in contrast, in hypoxia, MCF7 KCR expression level in either cell line, suggesting that in the WT cells, it activity increased to the same level as KOH. This could reflect is mainly HIF1a regulated and the adaptation in KO cells is the much lower basal activity observed in this cell line (10 through a different pathway. Yet, in DLD1 cells, HIF2 is clearly times lower than HCT116; Supplementary Fig. S7B–S7D). the factor involved in GTR3 regulation in hypoxia (Supplemen- Finally, in 337 CODREAD patients with colorectal cancer tary Fig. S4B). KCRU (CKMT1A, and CKMT1B mitochondrial genes coding GTR14 was higher in KON than in WTN, and in WTH increased for KCRU) correlated positively with hypoxia signatures to a similar but lower level than KON. KOH increased further than (Supplementary Fig. S2B). KON. WTKD1H had higher levels than WTH. Knockdown of HIF2 in the KO cells substantially reduced the high GTR14 levels. These Creatine metabolism is essential for hypoxic tumor growth in data support the potential role of HIF2a competing with HIF1, so absence of HIF1a that in absence of HIF1, a great effect of HIF2a occurs in hypoxia. We tested the effect of Cr metabolism on growth by using Similar results were shown for DLD1 cells (Supplementary Fig. cyclocreatine (cc) to inhibit Cr kinase enzyme activity and methi- S4B), where HIF2a antagonized HIF1a and induction was greater onine (Met) deprivation, precursor in Cr biosynthesis through the with HIF1a KD, and ablated with HIF2a knockdown. In Western reaction mediated by S-adenosyl-L-methionine/Guanidinoace- blot analysis of GTR3 and GTR14 (Supplementary Fig. S4E), KOH tate N-methyltransferase. In HCT116, we observed that after cc had the highest levels of GTR3 and 14, reduced by HIF2a KD, but addition, there was an overall reduction in proliferation in WT not by HIF1a KD in WT cells, which had the lowest levels of both and KO in both normoxia and hypoxia (Fig. 6A). In normoxia, transporters. No effect on cell proliferation was observed when Met deprivation significantly reduced cell proliferation. In hyp- GTR3 was knocked down. However, GTR14 knockdown showed a oxia, the effect was less pronounced for KO cells and was not significant decrease of cell proliferation (about 40% in WTN and significant for WT HCT116 cells (Fig. 6B), showing compensation KON P < 0.001; 50% WTH and KOH P < 0.01 and P < 0.05) after by a partially HIF-dependent pathway in hypoxia. We confirmed 24-hour cultivation in any of the studied experimental condi- these results in the clonogenic capability of HCT116 cells with and tions, showing a high dependency of HCT116 on this glucose without cc. Interestingly, only KONcc and KOHcc showed a transporter (Supplementary Fig. S7A). FC in HCT116 cells' pro- significant reduction in clone formation with KOHcc substantial- liferation comparing normoxia and hypoxia upon GTR14 silenc- ly lower than KONcc (Fig. 6C and D). Finally, in spheroids, cc ing in HIF1a-deficient and control cells showed significant reduc- decreased the volume in KO-cc treated (day 10) as compared with tion in KOH/WTH (FC ¼ 0.3 0.06) as compared with KON/ KO controls (Fig. 6E and F). KCRB knocked down in HCT116 WTN (FC ¼ 0.6 0.08, P < 0.01), WTH/WTN (FC ¼ 0.5 0.06, P < showed a significant reduction of cell proliferation of more than 0.01), and KOH/KON (FC ¼ 0.6 0.1, P < 0.001). Finally, there 50% in all experimental conditions (WTN< KON< and WTH P < was a reduction in proliferation when HIF2a was knocked down, 0.00; KOH P < 0.01; Supplementary Fig. S7E). FC in HCT116 cells' which was greater in KOH HCT116 cells (about 50% in WTN, proliferation comparing normoxia and hypoxia upon KCRB KON, and WTH P < 0.001 to P < 0.01: and 60% in KOH P < 0.01; silencing in HIF1a-deficient and control showed significant Supplementary Fig. S7A). reduction in KOH/WTH (FC ¼ 0.2 0.05) compared with KON/WTN (FC ¼ 0.5 0.05, P < 0.001), WTH/WTN (FC ¼ HIF1a differentially regulates expression of CK isoenzymes 0.3 0.03, P < 0.05), and KOH/KON (FC ¼ 0.4 0.05, P < 0.001). Metabolic analysis highlighted Cr metabolism as a mechanism Altogether, these data confirm the results of cc experiments. that could compensate for loss of HIF1a activity by promoting the reversible reaction PCrþADP ¼ CrþATP. By proteomics analysis, KCRB was upregulated in the absence of HIF1a in both normoxia Discussion and hypoxia. KCRU was downregulated in the absence of HIF1a In this study, we show how hypoxic cancer cells can metabol- in both normoxia and hypoxia (Figs. 3E and 4C). The expression ically compensate for HIF1a loss in hypoxia. In particular, we also correlated with the transcriptomics data (Fig. 4D). These data identified two main adaptive metabolic processes allowing cancer were then validated by PCR (Fig. 5A and B) and by Western blot cells to support and meet their energetic and precursors needs for analysis (Fig. 5D). These data were confirmed in two additional proliferation in hypoxia in the absence of HIF1a (Fig. 7 summary colorectal cancer cell lines, DLD1, and Ls174T (Supplementary diagram). Figs. S4B, S4C, S5B, and S5C). The cytosolic enzyme KCRB was Hypoxic HIF1a-deficient cancer cells maintain a significant upregulated in the absence of HIF1a and the mitochondrial proliferation and metabolic capability. Several HIF1a-indepen- enzyme KCRU was upregulated in the presence of HIF1a dent hypoxic responses have been reported to sustain tumor (Fig. 5D). KCRB dimer isoenzyme (KCRBB) activity (measured growth via NFkB or PHD1/EglN2 (11–13) and support tumor in whole cell extract) matched protein and mRNA expression metabolism via fructose and lipid metabolic pathways (10, 14). (Fig. 5E). As expected, in hypoxia, HIF1a promotes GTR1 expression and a In addition, we found (by PCR analysis) that hypoxia upregu- dynamic induction of Glu-6P intracellular gradient increase. This lated cell membrane Cr transporter SLC6A8 equally in WTH and regulation, HIF1a/PI3K/Akt–mediated, ensures glucose supply to KOH versus normoxia (Fig. 5A and B). high glycolytic proliferating cancer cells (15).

www.aacrjournals.org Mol Cancer Res; 2019 OF9

Valli et al.

Figure 6. Creatine metabolism is essential for hypoxic tumor growth in absence of HIF1a. A, Effect of cc addition on WTN, KON, WTH, and KOH cells after 24-hour exposure to two O2 conditions (21% and 1%). B, Effect of methionine deprivation on WTN, KON, WTH, and KOH cells after 24-hour exposure to two O2 conditions (21% and 1%). C and D, Effect of cc addition on WTN, KON, WTH, and KOH cell clonogenic (number of clones counted) after 24-hour exposure to two O2 conditions (21% and 1%). E and F, Effect of cc addition on HCT116 WT and KO spheroid growth (volume) at days 5, 8, and 10 (cc added at day 3 and subsequent days after spheroid formation). Data are reported as average SD (N ¼ 3). Statistical comparisons were performed using two-way ANOVA, multiple post hoc group comparisons, or t test (P values corrected using the Benjamini–Hochberg procedure at a ¼ 1%), showing P value <0.05 (), <0.01 (), and <0.001 ().

However, in the absence of HIF1a, we found that low GTR1 share 95% nucleotide identity (16), and GTR14 was thought to be coexisted with a sustained intracellular Glu-6P level in hypoxia. a GTR3 variant (17). Indeed, we found that GTR14 antibody This metabolic phenotype was associated with an induction of detection yielded a signal for both GTR3 and GTR14 proteins, GTR3 and GTR14 at mRNA and protein level. GTR3 and GTR14 while mass spectrometry analysis did not result in a high enough

OF10 Mol Cancer Res; 2019 Molecular Cancer Research

Energy Proﬁling in Hypoxic Cancer Cells

Figure 7. The metabolic pathways and metabolites described in this study. A, Adaptation of hypoxic cancer cell metabolism by HIF1a maintains energetic, redox, and biosynthetic requirements. B, In this manuscript, Valli and colleagues reveal for the ﬁrst time that cancer cell growth and energy metabolism is maintained by HIF1a-independent metabolic pathways under hypoxia that involve glucose uptake by GLUT14 and creatine metabolism. Metabolic pathways: glycogen (GLG), glycolysis (GLY), Krebs cycle (TCA), adenosine triphosphate (ATP), creatine (Cr), oxidation–reduction (REDOX), and adenosyl metabolism. ("), Increase; (#), decrease, box size and lines/arrows' thickness describe the regulation in the different conditions.

sequence coverage to discriminate between the two glucose the variation that can occur in this regulation. Because of the high transporters. afﬁnity for Glu, GTR3 overexpression could provide growth Adaptation of cancer cells to local glucose and Cr concentra- advantages in tumors already at low Glu levels (18). Interestingly, tions occurs by speciﬁc regulation of Glu transporters expression, a link between hypoxia, GTR3, and Glu uptake was suggested to be glycolytic enzymes, or ATP levels (18, 19). In our data, GTR3 important for lipid biosynthesis in macrophages (21). In line with regulation did not display typical HIF2a dependency although this, HIF2a was reported to play a major role in lipid metabo- expressed at higher basal levels in the KO cells, it was induced in lism (22). GTR14 has been associated with low survival in human WT by hypoxia to the same level, suggesting, in this case, another testis and in patients with gastric adenocarcinoma (23). Alterna- mechanism, for example, NfkB, has replaced HIF1a (20) while tive mechanisms by which GTR3 and GTR1 can be regulated GTR14 upregulation was driven by HIF2a in the absence of include HIF2a via Ptpmt1 and HIF1a via IGF-1, respectively HIF1a. However, in DLD1, GTR3 is clearly a HIF2 target, showing (24, 25). The reason that GTR14KD, but not GT3KD, affects

www.aacrjournals.org Mol Cancer Res; 2019 OF11

Valli et al.

HCT116 cell proliferation may be related to relative flux through In HCT116 spheroids and 2D hypoxic cells, we found a strong the pathways if GTR14 was the major transporter, or because increase of AMP levels in absence of HIF1a. This was accompa- GTR14 is also a dehydroascorbate transporter (26), but these nied by a strong expression of p-AMPK in HCT116 KO cells only. require further analysis. Previous data report AMPK being activated in low-oxygen con- HIF1a in hypoxia enhances tumor glycolytic enzymes expres- ditions by a HIF1a-independent mechanism (38), potentially sion and glycolytic flux (27). In hypoxia, in the absence of HIF1a, responding to cellular energy status through AMP binding to we found an accumulation of most of the glycolytic intermediates, AMPK g subunit, so promoting phosphorylation by upstream which was accompanied by low glycolysis enzymatic levels and kinases and finally inhibiting AMPK dephosphorylation (39). In glycolytic flux. addition, in our results intracellular ATP levels were higher in the Our results match previous data in HCT116 HIF1b KD and presence of HIF1a, although ATP is maintained in its absence. hepatoma cancer cells where Glu uptake was maintained in either This was found to be consistent with a generally reduced protein normoxia or hypoxia despite the downregulation of many gly- synthesis (as assessed by mRNA/protein ratios) as this process is a colytic genes (28) with a mechanism that involved autophagy. An primary energy-consuming process that can account for up to increase of Glu-6P and Fru-1,6BP metabolic flux not only con- 70% of cellular ATP utilization (40). Under hypoxia, a rapid tributes to glycolytic ATP production, but can also provide pre- inhibition of global mRNA translation represents a major pro- cursors for auxiliary pathways such as, glycogen, PPP, and tective strategy to maintain energy (41). lipids (10, 14, 29, 30). However, ATP maintenance is a process linked to ATP re/ In hypoxia, in the absence of HIF1a,wefoundablockage generating metabolic systems that relies on biochemical regula- at the metabolic step mediated by fructose-bisphosphate tory systems at subcellular level (42). For example, in skeletal aldolase (Aldolase A, ALDOA), catabolizing the cleavage of muscle, the high availability of PCr and Cr allows ATP mainte- Fru-1,6BP to glyceraldehyde 3-phosphate and dihydroxyace- nance by promoting the reversible reaction PCrþADP ¼ CrþATP. tone phosphate. Previously, it has been shown that PFKB3 The Cr kinase system, highly expressed in human tissues with was key in regulating glycolysis one step earlier, and has been high-energy demand, can enhance neuromuscular performance the focus of several approaches to develop inhibitors (31). on an anaerobic short extent and during anaerobic intermittent Our data highlight that ALDOA should be considered as a exercise (43). Indeed, active skeletal muscles capable of ATP target for therapy now. ALDOA was shown to stabilize HIF1a anaerobic biosynthesis keep global ATP reasonably constant via PDH in lung cancer and to promote cancer progression in during exercise. vivo (32, 33). ALDO is activated via PI3K, which recruits This is accomplished by the Cr kinase metabolic system that ALDO from the cytoskeleton and redistributes it in the replenishes the rate of ATP use at a fast pace (44). HIF2a- cytosol where it can increase glycolysis (34). Finally, ALDOA modulated hypoxic response elements have been reported to be was shown to be induced by hypoxia in colorectal cancer- involved in Cr metabolism (45). Interestingly, our study shows derived cell lines and to be an independent prognostic factor that both in normoxia and hypoxia, HIF1a maintains levels of the for colorectal cancer (35). mitochondrial CK isoenzyme, KCRU, which was associated with a In hypoxia, in the presence of HIF1a, we found that Pyr and reduced TCA cycle. In contrast, the cytoplasmic CK isoenzyme, lactate levels were lower compared with HIF1a KO This could be KCRB, is lower in WT cells. Strikingly, in cells without HIF1a, this due to a faster metabolism of Pyr into lactate by LDHA and its distribution was reversed, irrespective of hypoxia. This shows the subsequent removal through transporters (15). As LDHA and importance of low levels of HIF1a expression in regulating lactate transporters are HIF1a targets, this flux can be reduced in metabolic pathways. The complex effects of altering these hypoxic HIF1a-deficient cells, thus explaining the observed high enzymes on cell survival is shown by KCRU overexpression, which intracellular levels found for Pyr and lactate. enhanced the survival of breast cancer cells, while KCRB KD We also found that in hypoxia, citrate levels are elevated and Pyr induced cell death in hypoxic ovarian cancer cells (43). Cr oxidation into the TCA is overall maintained in the absence of metabolism, which translocates ATP from sites of production to HIF1a. Under hypoxic conditions, HIF1a drives phosphorylation locations of ATP consumption, acts as a spatial/temporal energy of PDK that inhibits PDH and shunts Pyr away from mitochon- buffer utilizing Cr, ATP, and ADP at distinct subcellular compart- dria to lactate, reducing the rate of oxidative decarboxylation and ments (46). Consistent with this, colon cancer cells were shown to diminishing acetyl-CoA formation (36). Consequently, replen- secrete KCRB extracellularly to generate and import PCr to ishment of TCA cycle in mitochondria is altered. In such condi- enhance energy (19). KCRB and KCRM genes emerged as tions, citrate biosynthesis is prevented, reductive carboxylation is HIF2a-selective targets, and regulation of Cr kinase by HIF was enhanced, and a HIF1a-dependent mechanism allows hypoxic shown in the gut (46), suggesting a role for HIF2a in energy cells to metabolize glutamine into fatty acids trough citrate maintenance. In the absence of HIF1a, we found that cc reduces formation (37). Consistent with this, the high fully labeled malate HCT116 cells' clonogenic capability and spheroid volume. KCRB content could be due to malate formation associated with a depletion/inhibition in combination with chemotherapeutic decreased malate transport outside the mitochondria (M2OM) agents showed synergistic effects in cancer therapy (47). Cr kinase and a reduced metabolism that is MDHM/MDHC-dependent metabolic inhibition by cc was shown to decrease cell viability, (Fig. 4). Finally, STIR reduction in TCA versus glycolysis (more promote cell-cycle arrest, and apoptosis in EVI1 (48). Inhibition evident in the presence of HIF1a) shows the metabolic uncou- of the colon cancer cell line KCRB by preincubation with cc pling that is HIF1a-dependent in hypoxic cancer cells. Our finding reduced liver metastasis formation and depleted PCr (49) and that, in hypoxia, fully labeled citrate formation is favored and recently, mice inoculated with colorectal cancer cells and treated malate is accumulated in the absence of HIF1a, suggests dimin- with cc showed a significant reduction in metastatic coloniza- ished reductive carboxylation as the predominant metabolic tion (19). Our novel experimental work has shown that silencing regulation. of GTR14 or KCRB reduces the proliferation rate of HIF1a-KO

OF12 Mol Cancer Res; 2019 Molecular Cancer Research

Energy Proﬁling in Hypoxic Cancer Cells

HCT116 cells in normoxic and hypoxic conditions. Because Acquisition of data (provided animals, acquired and managed patients, GRT14 and KCRB are more highly expressed in HIF1a-KO hyp- provided facilities, etc.): A. Valli, M. Morotti, C.E. Zois, T. Soga, oxic HCT116 cells, silencing these genes alters in a larger extent K. Feldinger, R. Fischer, A. McIntyre, E. Bridges, D. Baban, M. Rodriguez, O. Yanes, H.J. Whittington, H.A. Lake, C.A. Lygate HCT116 HIF1a-KO cells' proliferation in hypoxia than control. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, In summary, our work shows for the first time that hypoxic computational analysis): A. Valli, P.K. Albers, K. Feldinger, R. Fischer, cancer cells harbor the capability to compensate for HIF1a- M. Frejno, S. Haider, F.M. Buffa, D. Baban, O. Yanes, H.A. Lake, S. Zervou, deficient metabolic impasses by inducing GTR14-mediated Glu C.A. Lygate, B.M. Kessler, A.L. Harris internalization in a HIF1a/HIF2a antagonistic fashion. We also Writing, review, and/or revision of the manuscript: A. Valli, M. Morotti, report Cr metabolism is tuned to function as a fast ATP buffering R. Fischer, A. McIntyre, O. Yanes, C.A. Lygate, B.M. Kessler, A.L. Harris Administrative, technical, or material support (i.e., reporting or organizing biochemical system. Aldolase A is shown to be a control point in data, constructing databases): A. Valli, E. Bridges, D. Baban, A.L. Harris glycolysis by HIF1a. As these alternate metabolic pathways were Study supervision: A. Valli, F.M. Buffa, B.M. Kessler, A.L. Harris shown for the first time in 3D cancer cell cultures, they provide a Other (conducted experiments): C.E. Zois model relevant to micrometastases and drug targeting. Under hypoxia and HIF1 blockade, cancer cells adapt their energy Acknowledgments metabolism via upregulation of the GLUT14 glucose transporter This work was supported by a Cancer Research UK (CRUK) program grant and creatine metabolism providing new avenues for drug (C602/A18974, to A.L. Harris). B.M. Kessler is supported by the Biomedical Research Centre (NIHR) Oxford, United Kingdom. C.A. Lygate is supported by targeting. British Heart Foundation Programme Grant (RG/13/8/30266). fl Disclosure of Potential Con icts of Interest The costs of publication of this article were defrayed in part by the payment of fl No potential con icts of interest were disclosed. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: A. Valli, A. McIntyre, B.M. Kessler, A.L. Harris Received March 30, 2018; revised January 28, 2019; accepted March 13, 2019; Development of methodology: A. Valli, D. Baban, M. Rodriguez, S. Zervou published first March 18, 2019.

References 1. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the War- 15. Schulze A, Harris AL. How cancer metabolism is tuned for proliferation and burg effect: the metabolic requirements of cell proliferation. Science 2009; vulnerable to disruption. Nature 2012;491:364–73. 324:1029–33. 16. Augustin R. The protein family of glucose transport facilitators: it's not only 2. Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. about glucose after all. IUBMB Life 2010;62:315–33. J Gen Physiol 1927;8:519–30. 17. Schlosser HA, Drebber U, Urbanski A, Haase S, Baltin C, Berlth F, et al. 3. Pfeiffer T, Schuster S, Bonhoeffer S. Cooperation and competition in the Glucose transporters 1, 3, 6, and 10 are expressed in gastric cancer and evolution of ATP-producing pathways. Science 2001;292:504–7. glucose transporter 3 is associated with UICC stage and survival. 4. Eidelman E, Twum-Ampofo J, Ansari J, Siddiqui MM. The metabolic Gastric Cancer 2015;20:83–91. phenotype of prostate cancer. Front Oncol 2017;7:131. 18. Birsoy K, Possemato R, Lorbeer FK, Bayraktar EC, Thiru P, Yucel B, et al. 5. Palmer CS, Cherry CL, Sada-Ovalle I, Singh A, Crowe SM. Glucose metab- Metabolic determinants of cancer cell sensitivity to glucose limitation and olism in T cells and monocytes: new perspectives in HIV pathogenesis. biguanides. Nature 2014;508:108–12. EBioMedicine 2016;6:31–41. 19. Loo JM, Scherl A, Nguyen A, Man FY, Weinberg E, Zeng Z, et al. Extracellular 6. Pavlova NN, Thompson CB. The emerging hallmarks of cancer metabo- metabolic energetics can promote cancer progression. Cell 2015;160: lism. Cell Metab 2016;23:27–47. 393–406. 7. Liberti MV, Locasale JW. The Warburg effect: how does it benefit cancer 20. Christensen DR, Calder PC, Houghton FD. GLUT3 and PKM2 regulate cells? Trends Biochem Sci 2016;41:211–8. OCT4 expression and support the hypoxic culture of human embryonic 8. Xie H, Simon MC. Oxygen availability and metabolic reprogramming in stem cells. Sci Rep 2015;5:17500. cancer. J Biol Chem 2017;292:16825–32. 21. Li L, Liu B, Haversen L, Lu E, Magnusson LU, Stahlman M, et al. The 9. Mole DR, Blancher C, Copley RR, Pollard PJ, Gleadle JM, Ragoussis J, et al. importance of GLUT3 for de novo lipogenesis in hypoxia-induced lipid Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and loading of human macrophages. PLoS One 2012;7:e42360. HIF-2alpha DNA binding with expression profiling of hypoxia-inducible 22. Rankin EB, Rha J, Selak MA, Unger TL, Keith B, Liu Q, et al. Hypoxia- transcripts. J Biol Chem 2009;284:16767–75. inducible factor 2 regulates hepatic lipid metabolism. Mol Cell Biol 2009; 10. Valli A, Rodriguez M, Moutsianas L, Fischer R, Fedele V, Huang HL, et al. 29:4527–38. Hypoxia induces a lipogenic cancer cell phenotype via HIF1alpha- 23. Berlth F, Monig S, Pinther B, Grimminger P, Maus M, Schlosser H, et al. dependent and -independent pathways. Oncotarget 2015;6:1920–41. Both GLUT-1 and GLUT-14 are independent prognostic factors in gastric 11. Klein O, Rohwer N, de Molina KF, Mergler S, Wessendorf P, Herrmann M, adenocarcinoma. Ann Surg Oncol 2015;22Suppl 3:S822–31. et al. Application of two-dimensional gel-based mass spectrometry to 24. Xu QQ, Xiao FJ, Sun HY, Shi XF, Wang H, Yang YF, et al. Ptpmt1 induced by functionally dissect resistance to targeted cancer therapy. Proteomics Clin HIF-2alpha regulates the proliferation and glucose metabolism in erythro- Appl 2013;7:813–24. leukemia cells. Biochem Biophys Res Commun 2016;471:459–65. 12. Rohwer N, Bindel F, Grimm C, Lin SJ, Wappler J, Klinger B, et al. Annexin A1 25. Yu J, Li J, Zhang S, Xu X, Zheng M, Jiang G, et al. IGF-1 induces hypoxia- sustains tumor metabolism and cellular proliferation upon stable loss of inducible factor 1alpha-mediated GLUT3 expression through PI3K/ HIF1A. Oncotarget 2016;7:6693–710. Akt/mTOR dependent pathways in PC12 cells. Brain Res 2012;1430: 13. Li Y, Padmanabha D, Gentile LB, Dumur CI, Beckstead RB, Baker KD. HIF- 18–24. and non-HIF-regulated hypoxic responses require the estrogen-related 26. Amir Shaghaghi M, Zhouyao H, Tu H, El-Gabalawy H, Crow GH, Levine M, receptor in Drosophila melanogaster. PLoS Genet 2013;9:e1003230. et al. The SLC2A14 gene, encoding the novel glucose/dehydroascorbate 14. Armitage EG, Kotze HL, Allwood JW, Dunn WB, Goodacre R, Williams KJ. transporter GLUT14, is associated with inflammatory bowel disease. Am J Metabolic profiling reveals potential metabolic markers associated with Clin Nutr 2017;106:1508–13. Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells. 27. Marin-Hernandez A, Gallardo-Perez JC, Ralph SJ, Rodriguez-Enriquez S, Sci Rep 2015;5:15649. Moreno-Sanchez R. HIF-1alpha modulates energy metabolism in cancer

www.aacrjournals.org Mol Cancer Res; 2019 OF13

Valli et al.

cells by inducing over-expression of specific glycolytic isoforms. Mini Rev glucose deprivation conditions found in solid-tumor microenvironments. Med Chem 2009;9:1084–101. Mol Cell Biol 2006;26:5336–47. 28. Frezza C, Zheng L, Tennant DA, Papkovsky DB, Hedley BA, Kalna G, et al. 39. Li YH, Luo J, Mosley YY, Hedrick VE, Paul LN, Chang J, et al. AMP-Activated Metabolic profiling of hypoxic cells revealed a catabolic signature required protein kinase directly phosphorylates and destabilizes Hedgehog pathway for cell survival. PLoS One 2011;6:e24411. transcription factor GLI1 in medulloblastoma. Cell Rep 2015;12:599–609. 29. Favaro E, Bensaad K, Chong MG, Tennant DA, Ferguson DJ, Snell C, 40. Koritzinsky M, Wouters BG. Hypoxia and regulation of messenger RNA et al. Glucose utilization via glycogen phosphorylase sustains prolifer- translation. Methods Enzymol 2007;435:247–73. ation and prevents premature senescence in cancer cells. Cell Metab 41. Staudacher JJ, Naarmann-de Vries IS, Ujvari SJ, Klinger B, Kasim M, Benko 2012;16:751–64. E, et al. Hypoxia-induced gene expression results from selective mRNA 30. Bensaad K, Favaro E, Lewis CA, Peck B, Lord S, Collins JM, et al. Fatty acid partitioning to the endoplasmic reticulum. Nucleic Acids Res 2015;43: uptake and lipid storage induced by HIF-1alpha contribute to cell growth 3219–36. and survival after hypoxia-reoxygenation. Cell Rep 2014;9:349–65. 42. Kazak L, Chouchani ET, Jedrychowski MP, Erickson BK, Shinoda K, Cohen 31. Lu L, Chen Y, Zhu Y. The molecular basis of targeting PFKFB3 as a P, et al. A creatine-driven substrate cycle enhances energy expenditure and therapeutic strategy against cancer. Oncotarget 2017;8:62793–802. thermogenesis in beige fat. Cell 2015;163:643–55. 32. Chang YC, Chan YC, Chang WM, Lin YF, Yang CJ, Su CY, et al. Feedback 43. Yan YB. Creatine kinase in cell cycle regulation and cancer. Amino Acids regulation of ALDOA activates the HIF-1alpha/MMP9 axis to promote lung 2016;48:1775–84. cancer progression. Cancer Lett 2017;403:28–36. 44. Khan JR, Iftikar FI, Herbert NA, Gnaiger E, Hickey AJ. Thermal plasticity of 33. Sanzey M, Abdul Rahim SA, Oudin A, Dirkse A, Kaoma T, Vallar L, et al. skeletal muscle mitochondrial activity and whole animal respiration in a Comprehensive analysis of glycolytic enzymes as therapeutic targets in the common intertidal triplefin fish, Forsterygion lapillum (Family: Tripter- treatment of glioblastoma. PLoS One 2015;10:e0123544. ygiidae). J Comp Physiol B 2014;184:991–1001. 34. Hu H, Juvekar A, Lyssiotis CA, Lien EC, Albeck JG, Oh D, et al. Phosphoi- 45. Zheng L, Kelly CJ, Colgan SP. Physiologic hypoxia and oxygen homeostasis nositide 3-kinase regulates glycolysis through mobilization of aldolase in the healthy intestine. A review in the theme: cellular responses to from the actin cytoskeleton. Cell 2016;164:433–46. hypoxia. Am J Physiol Cell Physiol 2015;309:C350–60. 35. Kawai K, Uemura M, Munakata K, Takahashi H, Haraguchi N, Nishimura J, 46. Kitzenberg D, Colgan SP, Glover LE. Creatine kinase in ischemic and et al. Fructose-bisphosphate aldolase A is a key regulator of hypoxic inflammatory disorders. Clin Transl Med 2016;5:31. adaptation in colorectal cancer cells and involved in treatment resistance 47. Li XH, Chen XJ, Ou WB, Zhang Q, Lv ZR, Zhan Y, et al. Knockdown of and poor prognosis. Int J Oncol 2017;50:525–34. creatine kinase B inhibits ovarian cancer progression by decreasing gly- 36. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expres- colysis. Int J Biochem Cell Biol 2013;45:979–86. sion of pyruvate dehydrogenase kinase: a metabolic switch required for 48. Fenouille N, Bassil CF, Ben-Sahra I, Benajiba L, Alexe G, Ramos A, et al. The cellular adaptation to hypoxia. Cell Metab 2006;3:177–85. creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute 37. Metallo CM, Gameiro PA, Bell EL, Mattaini KR, Yang J, Hiller K, et al. myeloid leukemia. Nat Med 2017;23:301–13. Reductive glutamine metabolism by IDH1 mediates lipogenesis under 49. Lillie JW, O'Keefe M, Valinski H, Hamlin HA Jr, Varban ML, Kaddurah- hypoxia. Nature 2011;481:380–4. Daouk R. Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) inhi- 38. Laderoute KR, Amin K, Calaoagan JM, Knapp M, Le T, Orduna J, et al. 50- bits growth of a broad spectrum of cancer cells derived from solid tumors. AMP-activated protein kinase (AMPK) is induced by low-oxygen and Cancer Res 1993;53:3172–8.

OF14 Mol Cancer Res; 2019 Molecular Cancer Research

Adaptation to HIF1α Deletion in Hypoxic Cancer Cells by Upregulation of GLUT14 and Creatine Metabolism

Alessandro Valli, Matteo Morotti, Christos E. Zois, et al.

Mol Cancer Res Published OnlineFirst March 18, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-18-0315

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2019/03/16/1541-7786.MCR-18-0315.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/early/2019/04/16/1541-7786.MCR-18-0315. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.