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. 2011 Mar 8;19(3):416-28.
doi: 10.1016/j.ccr.2011.02.014.

SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization

Lydia W S Finley  1 , Arkaitz Carracedo, Jaewon Lee, Amanda Souza, Ainara Egia, Jiangwen Zhang, Julie Teruya-Feldstein, Paula I Moreira, Sandra M Cardoso, Clary B Clish, Pier Paolo Pandolfi, Marcia C Haigis
Affiliations

SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization

Lydia W S Finley et al. Cancer Cell. .

Abstract

Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression.

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Figures

Figure 1
Figure 1
Metabolic profiles of SIRT3 KO MEFs reflect an increase in glycolytic pathways and a decrease in mitochondrial oxidative metabolism. (A) Schematic illustrating the metabolites that are increased (red) or decreased (blue) in SIRT3 KO MEFs compared to SIRT3 WT MEFs (n = 4), p < 0.1. Metabolites in parentheses were not measured. PPP, pentose phosphate pathway. The nonoxidative (red) and oxidative (black) arms of the PPP are shown. Levels of glycolytic intermediates (B), TCA cycle intermediates (C), glucose (D), glucose-1-phosphate (E) and ribose-5-phosphate (F). Growth curves of SIRT3 WT and KO MEFs (n = 3) cultured in media containing glucose (G) or galactose (H). Error bars, ±SD. (I-N) Glucose uptake and lactate production in SIRT3 WT and KO MEFs (n = 6). (I) Relative glucose uptake and (J) lactate production in SIRT3 WT and KO MEFs. (K) Relative glucose uptake and (L) relative lactate production in SIRT3 WT and KO MEFs incubated with or without 100 nM rotenone. (M) Glucose uptake and (N) lactate production in SIRT3 WT and KO MEFs cultured in the presence or absence of 50 μg/ml etomoxir. Cells were treated with drugs for 24 hours before measuring glucose uptake and lactate. All error bars (except growth curves), ±SEM. (*) p < 0.05; (**) p < 0.01, (***) p < 0.001. See also Figure S1.
Figure 2
Figure 2
SIRT3 KO mice have elevated glucose uptake and hypoxic signatures in vivo. 18F-fluorodeoxyglucose (18F-FDG) uptake in the brown adipose tissue (BAT) of SIRT3 WT and KO mice was measured using positron emission tomography-computed tomography (PET/CT). (A) Representative scans with color scale bar indicating relative levels of uptake from low (black) to high (white). (B) Quantification of BAT 18F-FDG uptake normalized to body weight (n = 6). (C) Gene set enrichment analysis of canonical pathways with the ranked genes list from most up- to most down-regulated in SIRT3 KO BAT. (D) Heat map comparing metabolite patterns of SIRT3 deletion and hypoxia. Red and blue indicate up- or down-regulation, respectively. SIRT3 WT and KO MEFs (n = 4) were cultured in 21% O2 (normoxia, N) or 1% O2 for 12 hours (hypoxia, H) and metabolites were analyzed by LC-MS. Relative levels of glycolytic intermediates (E) and ribose-5-phosphate (F). (G) Glucose uptake of MEFs cultured under hypoxia for 6 hours. Error bars, ±SEM. (*) p < 0.05; (**) p < 0.01. See also Figure S2.
Figure 3
Figure 3
SIRT3 regulates HIF1α stability. (A) Immunoblots of nuclear extracts from SIRT3 WT and KO MEFs cultured at 21% O2. Immunoblots of MEFs (B) or HEK293T cells expressing control shRNA (shNS) or shRNA targeted against SIRT3 (C) cultured at 1% O2 for the indicated times. (D) HIF1α target genes in SIRT3 WT and KO MEFs after 6 hours of hypoxia were measured by qRT-PCR and shown as a ratio of SIRT3 WT normoxia levels. (E) Immunoblots of HEK293T cells stably overexpressing empty vector or SIRT3 cultured at 1% O2 for the indicated times. (F) Expression of HIF1α target genes in HEK293T control and SIRT3-overexpressing cells after 6 hours of hypoxia. (G) SIRT3 WT and KO MEFs expressing shNS or shRNA against HIF1α (shHIF1#1,2) were cultured in normoxia or hypoxia (6 hours) and the fold change in Glut1 levels was measured by qRT-PCR. (H) Lactate produced by SIRT3 WT and KO MEFs expressing shNS or shHIF1α after 6 hours of hypoxia expressed as a ratio of SIRT3 WT shNS normoxic controls. Significance was assessed by two-way ANOVA. (I) Expression of Glut1 and Hk2 in the brown adipose tissue (left) and heart (right) of SIRT3 WT and KO mice (n =5-7) was measured by qRT-PCR. β-2-microglobulin or Rps16 were used as endogenous controls for qRT-PCR. Error bars, ±SEM (n = 4-6). (*) p <0.05; (**) p <0.01. See also Figure S3.
Figure 4
Figure 4
SIRT3 regulates HIF1α stability through ROS. (A) Nuclear extracts from shNS and shSIRT3 HEK293T cells treated with or without 10 μM MG-132 for 1 hour or 1 mM DMOG for 4 hours as indicated were immunoblotted with antibodies specific to hydroxylated HIF1α (HIF-OH) or total HIF1α. (B) Fold induction of HIF1α target genes in response to hypoxia (n = 6) measured by qRT-PCR. The ratio of hypoxic to normoxic gene expression is shown. (C) Fold induction of Glut1 and Hk2 in response to DMOG treatment was measured by qRT-PCR and the ratio of untreated to DMOG-treated gene expression is shown (n = 6). (D) The increase in ROS production with hypoxia was calculated as the fold change in ROS in hypoxic cells relative to normoxic controls. (E) Immunoblots of SIRT3 WT and KO MEFs incubated with 10 mM N-acetylcysteine (NAC) and cultured under hypoxia. (F) Immunoblots of SIRT3 WT and KO MEFs cultured at 21% O2 with 10 mM NAC or 1 mM DMOG as indicated. (G) Glut1 expression was measured by qRT-PCR in SIRT3 WT and KO MEFs (n = 5) that were incubated with 10 mM NAC and cultured under hypoxia. Significance was assessed by one-way ANOVA. (G) Growth curves of SIRT3 WT and KO MEFs (n = 3) cultured in standard media or media supplemented with 10 mM NAC. Error bars, ±SD. Protein carbonyls (I) and lipid peroxidation (J) were measured in brown adipose tissue (BAT) of SIRT3 WT and KO mice (n = 7). (K) qRT-PCR analysis of Glut1 expression in BAT of SIRT3 WT and KO mice treated with 40 mM NAC. β-2-microglobulin or Rps16 were used as endogenous controls. Error bars (except for growth curves), ±SEM. (*) p < 0.05; (**) p < 0.01. See also Figure S4.
Figure 5
Figure 5
SIRT3 is significantly deleted in human breast cancer. (A) Soft agar assays using transformed SIRT3 WT and KO MEFs expressing shNS or shRNA against HIF1α (shHIF1) (n =4). (B) Quantitative RT-PCR on RNA isolated from xenograft tumors and normalized to expression of 36B4. (C) Hematoxylin and eosin (H&E) staining (left) and immunohistochemial analysis of GLUT1 expression (right) in xenograft tumors. One representative pair of contralateral tumors is shown. Scale bar, 50 μm. (D) Table summarizing SIRT3 deletion frequency across a panel of human tumors. (E) Schematic of copy number changes at the SIRT3-5 and TP53 loci. Amplifications are shown in red; deletions are shown in blue. (F) Expression levels of SIRT3 and several HIF1α target genes were determined using the Oncomine cancer microarray database (http://www.oncomine.org) in normal versus breast cancers. (G) Linear regression of SIRT3 and GLUT1 across the panel of normal and breast cancer samples described in (F). (H) Representative image of SIRT3 expression in normal breast epithelium and in breast tumor cells as assessed by immunohistochemistry. SIRT3 levels were classified as absent (0), weak scattered (1) or positive as strong (2) compared to normal epithelium and the percentage of patients classified in each category is depicted in histogram at right. Error bars, ±SEM (n = 4-6). (*) p <0.05; (**) p <0.01. See also Figure S5.
Figure 6
Figure 6
SIRT3 suppresses the Warburg effect in human breast cancer cells. (A) Lactate production and (B) glucose consumption of MCF7, T47D and CAMA1 cells stably expressing empty vector or SIRT3 and cultured under hypoxia expressed as a ratio of empty-vector normoxic controls. (C) Relative glucose uptake and (D) relative lactate production in CAMA1 control or SIRT3 overexpressing cells incubated with or without 100 nM rotenone. (E) Glucose uptake and (F) lactate production in CAMA1 cell lines cultured in the presence or absence of 50 μg/ml etomoxir. (G) Immunoblots of CAMA1 cells stably expressing control vector or SIRT3-FLAG cultured at 1% oxygen for the indicated. (H) qRT-PCR of HIF1α target genes in CAMA1 cells cultured at 1% oxygen. (I) Induction of HIF1α target genes in response to hypoxia measured by qRT-PCR in CAMA1 cells. The ratio of normoxic to hypoxic gene expression in each cell line is shown. (J) Induction of HIF1α target genes in response to 1 mM DMOG treatment measured by qRT-PCR in CAMA1 cells. The ratio of untreated to DMOG-treated gene expression in each cell line is shown. Growth curves of CAMA1 cells (n =3) cultured in glucose (K) or galactose (L). Error bars, ±SD. (M) Schematic of the regulation of HIF1α and the Warburg effect by SIRT3. β-2-microglobulin was used as an endogenous control for qRT-PCR. Error bars (except for growth curves), ±SEM. (*) p <0.05; (**) p <0.01. See also Figure S6.
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