SIRT3 promotes cellular metabolic reprogramming
Since SIRT3 activates enzymes involved in mitochondrial fuel catabolism (Verdin et al., 2010
), and SIRT3 loss increases glucose uptake (Kim et al., 2010
), we hypothesized that SIRT3 could serve as an important regulator of the balance between glycolytic and anabolic pathways and mitochondrial oxidative metabolism to regulate tumor cell growth. To test this idea, we first examined the influence of SIRT3 loss on metabolites from mouse embryonic fibroblasts (MEFs) using liquid chromatography-mass spectrometry (LC-MS). The metabolic profile of SIRT3 null (KO) MEFs demonstrated a clear shift towards glycolytic metabolism when compared with SIRT3 wild-type (WT) counterparts (), similar to the metabolic shift reported for transformed cells in culture (Lu et al., 2010
) and for cancer cells in vivo
(Denkert et al., 2008
; Hirayama et al., 2009
). In SIRT3 KO cells, intermediates of glycolysis were elevated, while TCA cycle metabolites were reduced (). Consistent with a pattern of increased glucose usage, SIRT3 KO cells had lower levels of intracellular glucose (), while levels of glucose-1-phosphate, a product of glycogenolysis, were increased (). Glucose-1-phosphate can be converted by phosphoglucomutase into glucose-6-phosphate (G6P) to provide substrates for glycolysis or the oxidative arm of the pentose phosphate pathway (PPP), generating NADPH and ribose. The nonoxidative arm of the PPP forms ribose-5-phosphate from fructose-6-phosphate or glyceraldehyde-3-phosphate (). Importantly, G6P, glycolytic intermediates and ribose-5-phosphate were all increased in SIRT3 KO cells (), suggesting that glucose metabolites were diverted into the PPP in order to provide the ribose necessary for nucleic acid synthesis. Notably, the pattern and the magnitude of metabolomic changes caused by SIRT3 loss were similar to those observed comparing tumors to nearby normal tissue (Hirayama et al., 2009
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 (more ...)
Increased metabolites involved in glycolysis and the PPP suggested that, like many cancer cells, SIRT3 KO MEFs might be using glucose to support increased proliferation by directing glucose away from the TCA cycle towards biosynthetic processes. Indeed, SIRT3 KO cells grew significantly faster than WT cells (). To test whether this increased growth rate required aerobic metabolism of glucose, we grew cells in media containing galactose instead of glucose, thereby reducing glycolytic flux and forcing the cell to rely on mitochondrial oxidative phosphorylation (Marroquin et al., 2007
). Under these conditions, WT and KO cells grew at the same rate, demonstrating that the increased proliferation of KO cells required enhanced glucose catabolism ().
To confirm that the metabolite patterns reflected an increase in glycolysis, we measured glucose uptake and lactate production. As expected, SIRT3 KO MEFs consumed more glucose and extruded more lactate into the media than did WT cells (). This effect was not specific to MEFs; HEK293T cells in which SIRT3 expression was stably reduced by lentiviral expression of shRNA against SIRT3 also showed an increase in glucose uptake and lactate production (Figure S1A and S1B
). Interestingly, the effect of SIRT3 loss on glucose uptake and lactate production was similar to the effect of pyruvate kinase M2 isoform overexpression or mTOR activation (Christofk et al., 2008
; Duvel et al., 2010
). These data suggest that loss of SIRT3 redirects cellular metabolism in favor of glycolysis, and as a result, cells with low levels of SIRT3 display features similar to the Warburg effect apparent in many cancer cells.
Previous studies have found that SIRT3 loss increases glucose uptake (Kim et al., 2010
), yet the specific mechanism involved has not been elucidated. To test whether SIRT3 upregulates glycolysis as part of a compensatory response due to diminished oxidative capacity, we examined glucose uptake and lactic acid secretion in the presence of a mitochondrial respiratory inhibitor, rotenone, or an inhibitor of mitochondrial fatty acid oxidation, etomoxir. In WT cells, glycolysis is increased in the presence of both rotenone () and etomoxir (). Strikingly, glucose uptake and lactate production remain elevated in the SIRT3 KO cells even in the presence of oxidation inhibitors (). These data demonstrate that upregulated glycolysis in SIRT3 null cells does not result solely from nonspecific compensation for decreased mitochondrial oxidative functions. Instead, these data indicate that, surprisingly, SIRT3 may regulate glycolysis via activation of a specific signaling pathway.
We next asked whether SIRT3 KO mice exhibited signs of increased glucose usage. We injected mice with 18
F-FDG) and scanned animals using positron emission tomography-computed tomography (PET/CT) in order to monitor glucose uptake. We looked specifically in brown adipose tissue (BAT), which exhibits high glucose uptake (Cannon and Nedergaard, 2004
). In line with our cellular studies, we found that SIRT3 KO mice had a increased in 18
F-FDG uptake in brown adipose tissue (BAT) compared with WT mice (), even though the mass of BAT in SIRT3 KO mice was not larger than in WT mice (Figure S2A
). Glucose uptake in BAT is regulated by the β-adrenergic pathway and is thus dramatically increased by cold exposure (Shimizu et al., 1991
). We measured 18
F-FDG uptake in BAT of SIRT3 WT and KO after a 6 hour cold challenge and found that SIRT3 KO mice have higher 18
F-FDG uptake both at room temperature and at 4°C (Figure and S2B
), illustrating that SIRT3 WT and KO mice have a similar increase in BAT glucose uptake in response to β-adrenergic signaling. These differences in BAT glucose uptake occur independently of obvious changes in whole-body glucose homeostasis: we did not detect changes in blood glucose levels (Figure S2C
) as reported previously (Lombard et al., 2007
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). (more ...)
To examine mechanisms underlying increased glucose uptake in SIRT3 KO BAT, we performed genome-wide expression profiling on RNA isolated from BAT and performed gene set enrichment analysis (GSEA) using the ranked gene list from most up- to most down-regulated in SIRT3 KO mice in order to identify the biological pathways most significantly altered by SIRT3 loss. As SIRT3 is a mitochondrial deacetylase, we expected to see compensatory up-regulation of pathways involving mitochondrial function or energy production. To our surprise, SIRT3 loss up-regulated pathways important in tumorigenesis. Strikingly, of the nine gene sets most significantly overrepresented in SIRT3 KO BAT, three were independently defined as gene sets induced by exposure to hypoxia (Figure and S2B
). Hypoxia itself increases 18
F-FDG uptake (Clavo et al., 1995
) and is associated with many transcriptional changes that result in increased glucose uptake and utilization (Brahimi-Horn et al., 2007
). Thus, we hypothesized that the increase in glucose uptake in SIRT3 KO BAT could be explained by upregulation of the hypoxia response.
The similarity between gene signatures of SIRT3 KO mice and hypoxic cells was particularly notable because hypoxia induces a metabolic shift similar to that caused by loss of SIRT3, including a decrease in mitochondrial substrate oxidation and an increase in glycolysis (Semenza, 2010
). To test the role of SIRT3 in hypoxia-induced metabolic reprogramming, we analyzed metabolites isolated from MEFs cultured at 21% O2
(normoxia) or 1% O2
(hypoxia) for 12 hours. Strikingly, we observed that the increase in glycolytic intermediates caused by hypoxia was similar to the effects of SIRT3 deletion (). Furthermore, hypoxia and SIRT3 loss had additive effects: while intermediates of glycolysis, glycogenolysis and the PPP were elevated by hypoxia, levels of these metabolites were dramatically higher in SIRT3 KO MEFs under these conditions (Figure and S2E
). Consistent with the metabolite profiles, hypoxia increased glucose uptake in both cell lines, and SIRT3 KO or knock-down cells consumed even more glucose than control cells (Figure and S2F
). Taken together, these data illustrate that SIRT3 loss and hypoxia result in similar metabolic shifts and implicate dysregulated activation of the hypoxia pathway as a cause of the metabolic reprogramming of SIRT3 null cells.
SIRT3 opposes the Warburg effect by destabilizing HIF1α
HIF1, comprised of the heterodimer HIF1α and HIF1β, is the primary driver of increased glycolysis and lactate production during hypoxia (Gordan and Simon, 2007
; Hu et al., 2003
; Seagroves et al., 2001
). Under conditions of low oxygen, HIF1α is stabilized and promotes transcription of many genes crucial for the cellular response to hypoxia (Kaelin and Ratcliffe, 2008
). Consequently, cells lacking HIF1α fail to upregulate glycolytic enzymes and lactic acid production in response to hypoxia (Seagroves et al., 2001
). Given the in vivo
hypoxic gene signature of SIRT3-null BAT, in addition to the striking similarity between the mitochondrial-independent glycolytic profiles of SIRT3 KO MEFs and hypoxic cells, we reasoned that the mechanism by which SIRT3 regulates glycolysis involves HIF1α. To test this idea, we first investigated whether SIRT3 directly modulates HIF1α stability under normoxic conditions. In the presence of high oxygen, HIF1α is rapidly degraded and difficult to measure from cell lysates, but HIF1α is detectable from isolated nuclei. Indeed, nuclei isolated from SIRT3 deficient cells during normoxia demonstrated elevated levels of HIF1α relative to WT cells (). Likewise, when MEFs were cultured under 1% O2
, HIF1α was stabilized earlier and to a higher degree in SIRT3 KO cells compared to WT cells in whole cell lysates (). We obtained comparable results in HEK293T cells in which SIRT3 expression was stably reduced by lentiviral expression of shRNA against SIRT3 (). Importantly, SIRT3 also regulates expression of HIF1α target genes. Both the glucose transporter Glut1
and hexokinase II (Hk2
)—HIF1α target genes that are critical for increased glucose uptake and catabolism via aerobic glycolysis or the PPP and are strongly implicated in tumorigenesis (O’Donnell et al., 2006
; Tennant et al., 2010
)—were elevated during hypoxia in SIRT3 KO MEFs and SIRT3 knock-down cells relative to control cells (Figure and S3A
). Furthermore, the HIF1α targets pyruvate dehydrogenase kinase 1 (Pdk1
), lactate dehydrogenase A (Ldha
), phosphoglycerate kinase (Pgk1
) and vascular endothelial growth factor A (Vegfa
) were significantly elevated in SIRT3 KO cells compared to WT cells during hypoxia (). Similar to the pattern we saw with metabolic intermediates of glycolysis, many of these genes were moderately elevated by SIRT3 loss under basal conditions (Figure S3B
), and SIRT3 deletion and hypoxia had additive effects on expression of HIF1α target genes ().
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 (more ...)
To test whether SIRT3 directly represses HIF1α, we examined the levels of HIF1α and its target genes in cells overexpressing SIRT3. SIRT3 overexpression clearly and reproducibly reduced the extent of HIF1α stabilization in hypoxic cells (). Importantly, the induction of GLUT1
during hypoxia was blunted by SIRT3 overexpression, demonstrating that SIRT3 directly inhibits HIF1α function (). SIRT3 catalytic activity was required for the full repression of HIF1α target genes: expression of a SIRT3 catalytic mutant did not significantly reduce hypoxic GLUT1
expression (Figure S3C
). Furthermore, using primary MEFs, we found that two SIRT3 KO lines exhibited increased Glut1
expression relative to two WT lines, suggesting that SIRT3 can regulate HIF1α activity in primary cell lines (Figure S3D
). Taken together, the data show that SIRT3 controls the stabilization of HIF1α and the induction of crucial HIF1α target genes that coordinate aerobic glucose consumption.
Next, to examine the requirement for HIF1α in the glycolytic shift observed in SIRT3 null cells, we used two separate shRNA constructs against HIF1α to generate SIRT3 WT and KO MEFs with HIF1α levels stably reduced (Figure S3E
). We measured normoxic and hypoxic Glut1
expression in these cell lines and found, as predicted, that control (shNS) SIRT3 KO MEFs demonstrated an exaggerated response to hypoxia, measured as the fold change in Glut1
expression, compared to control WT MEFs (). In contrast, WT and SIRT3 KO MEFs expressing either shRNA against HIF1α had comparable responses to hypoxia (Figure and S3F
). Importantly, the increase in lactate production caused by SIRT3 deletion required HIF1α both in normoxia and hypoxia (Figure and S3G
). Together, these data demonstrate that SIRT3 regulates aerobic glycolysis through HIF1α.
To probe for evidence of increased HIF1α activation in vivo
, we measured levels of HIF1α and HIF1α target genes from tissues of SIRT3 WT and KO mice. Levels of HIF1α protein and many HIF1α target genes involved in glycolysis were significantly elevated in the BAT of SIRT3 KO mice (Figures and S3H-J
), consistent with our studies demonstrating increased glucose uptake in SIRT3 KO BAT. Similarly, several HIF1α target genes showed a trend of increased expression in SIRT3 KO heart (Figure and S3K
The regulation of HIF1α is complex and not completely understood (Kaelin and Ratcliffe, 2008
). During normoxia, HIF1α is hydroxlylated at two proline residues by a family of oxygen-dependent prolyl hydroxylases (PHD1-3), enabling the tumor suppressor von Hippel-Lindau (VHL) to bind and target HIF1α for ubiquitination and proteasomal degradation (Kaelin and Ratcliffe, 2008
). As we did not detect changes in HIF1α mRNA levels (), we tested whether SIRT3 exerted a post-translational effect on HIF1α stability. SIRT1 binds HIF1α and regulates its activity through direct deacetylation (Lim et al., 2010
). To test whether SIRT3 might act through a similar mechanism, we immunoprecipitated SIRT1 or SIRT3 and probed for interactions with HIF1α. SIRT1, but not SIRT3, pulled down HIF1α (Figure S4A
), suggesting that SIRT3 does not interact with HIF1α directly.
We next hypothesized that SIRT3 regulates HIF1α stability by modulating PHD activity by measuring the extent of HIF1α hydroxylation. We assessed PHD activity in control and SIRT3 knock-down HEK293T cells by treating cells with the proteasomal inhibitor MG-132 (to prevent hydroxylated HIF1α from being degraded) or with DMOG (dimethyloxaloylglycine, to inhibit PHDs). Although SIRT3 knock-down cells accumulated more HIF1α during MG-132 treatment, they had significantly less hydroxylated HIF1α, indicating that PHD activity is lower in SIRT3 knock-down cells (). Similarly, SIRT3 WT MEFs demonstrated higher levels of HIF1α hydroxylation than KO MEFs (Figure S4B
). As a potent PHD inhibitor, we predicted that DMOG would overcome any differences between HIF1α levels SIRT3 WT and KO MEFs if SIRT3 acts at the level of the PHDs. Indeed, we observed that at every time point examined, SIRT3 WT and KO MEFs have equal levels of HIF1α stabilized in response to DMOG treatment (Figure S4C
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 (more ...)
To confirm that SIRT3 influences HIF1α through the PHDs, we performed a series of experiments comparing the effects of hypoxia and DMOG treatment on SIRT3 WT and KO MEFs. We observed that both hypoxia and DMOG stabilize HIF1α and induce expression of HIF1α target genes (). The relative responses of SIRT3 WT and KO MEFs to hypoxia and DMOG underscore the PHDs as the point of regulation by SIRT3. During hypoxia, HIF1α target genes are induced more strongly in SIRT3 KO cells, illustrating the physiological importance of SIRT3 in regulating the metabolic response to hypoxia (). In contrast, SIRT3 deletion represses the induction of HIF1α target genes in response to DMOG (). These data support a model whereby PHD activity is already reduced in SIRT3 KO cells. Consequently, when PHD activity is potently blocked by DMOG, SIRT3 KO cells have a smaller change in PHD activity and thus a smaller induction of HIF1α target genes. Together, these results point to reduced PHD activity as the mechanism of increased HIF1α expression in SIRT3 deficient cells.
Several intracellular signals, in addition to changes in oxygen concentration, are known to regulate PHD activity. Notably, reactive oxygen species (ROS) have been shown to inhibit the PHDs and stabilize HIF1α (Gerald et al., 2004
; Kaelin and Ratcliffe, 2008
). Moreover, hypoxia triggers an increase in ROS production that is required for the hypoxic activation of HIF1α (Chandel et al., 1998
; Hamanaka and Chandel, 2009
). Because SIRT3 is a well-known inhibitor of ROS (Kawamura et al., 2010
; Kim et al., 2010
; Kong et al., 2010
; Sundaresan et al., 2009
), we hypothesized that increased ROS in SIRT3-deficient cells would contribute to the inhibition of the PHDs. Thus, we tested whether SIRT3 loss would magnify the increase in ROS associated with hypoxia. We found that the hypoxia-triggered increase in ROS was significantly higher in SIRT3 KO MEFs (), providing a mechanistic explanation for why SIRT3 null cells have an exaggerated response to hypoxia.
Next, we treated cells with the anti-oxidant N-acetylcysteine (NAC) in order to probe the model that suppressing ROS could block the effects of SIRT3 deletion. Indeed, we observed that while SIRT3 KO MEFs had higher levels of HIF1α during hypoxia, NAC treatment reduced HIF1α to comparable levels in SIRT3 WT and KO MEFs (). In contrast, SIRT3 WT and KO MEFs have comparable levels of HIF1α induced by DMOG (), and NAC could no longer destabilize HIF1α in the presence of DMOG (Figure and S4D
). As predicted by the decrease in HIF1α observed in NAC-treated KO MEFs, NAC treatment restored Glut1
expression in KO MEFs to WT levels (). Finally, to test whether increased ROS could underlie the proliferative phenotype of SIRT3 KO MEFs, we cultured cells with NAC and measured growth rates. Strikingly, we found that NAC rescued the increased proliferation of SIRT3 KO MEFs, restoring their growth to WT levels (). Thus, regulation of ROS by SIRT3 plays an important role in stabilization of HIF1α and activation of glycolytic metabolism in SIRT3 null cells.
To examine the contribution of increased ROS to altered BAT metabolism in vivo
, we first looked for evidence of increased ROS in SIRT3 KO tissues. We found that two measures of oxidative damage, protein carbonyls and lipid peroxidation, were significantly elevated in SIRT3 KO BAT (). Because antioxidant treatment rescued the HIF1α-driven gene expression in cultured cells, we hypothesized that NAC treatment would reverse the glycolytic signature in SIRT3 KO tissues. To test this idea, we treated mice with NAC for one month and measured expression of HIF1α target genes in BAT. Strikingly, we found that NAC repressed expression of HIF1α target genes in SIRT3 KO mice, but not in SIRT3 WT mice (Figures , S4E,F
). These data demonstrate that increased ROS production in vivo
contribute to enhanced glycolytic gene expression in SIRT3 deficient mice.
SIRT3 loss increases glycolytic signatures in tumors
HIF1α activity and aerobic glycolysis are strongly implicated in the Warburg effect (Semenza, 2010
), and so we reasoned that SIRT3 may exert its tumor suppressive activity by opposing the HIF1α-mediated activation of the Warburg effect. Previously, SIRT3 deletion was shown to increase colony formation in a soft agar colony growth assay (Kim et al., 2010
). To investigate the contribution of HIF1α to this tumorigenic phenotype, we transformed primary MEFs by expressing the Ras and E1a oncogenes and then stably knocked down HIF1α. As previously shown (Kim et al., 2010
), we found that SIRT3 loss increased colony formation (). Importantly, knock down of HIF1α rescued the increased colony formation of SIRT3 KO cells (). Furthermore, SIRT3 WT and KO MEFs formed colonies at equivalent rates when cultured in media containing galactose instead of glucose (Figure S5A
), suggesting that colony formation required glucose metabolism. Taken together, these data suggest that the metabolic reprogramming mediated by SIRT3 via HIF1α could be an important contributor of the tumor-suppressive role of SIRT3.
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 (more ...)
Next, we performed xenograft assays with the transformed MEFs in order to probe the metabolic status of SIRT3 null tumors. As has previously been shown (Kim et al., 2010
), we found that tumors lacking SIRT3 had a growth advantage: tumors formed from 64% of KO injections but only 27% of WT injections and tumors lacking SIRT3 grew faster and were bigger than WT tumors (Figures S5B-F
). As tumors are subject to intermittent hypoxia (Gatenby and Gillies, 2004
), we examined expression of rate-limiting glycolytic genes in the xenograft tumors. Strikingly, HIF1α target genes were elevated in SIRT3 KO tumors (); SIRT3 KO tumors also showed higher levels of GLUT1 protein (). Taken together, these data suggest that increased levels of glycolytic enzymes, perhaps as part of a heightened response to hypoxia, provides a growth advantage for tumor cells lacking SIRT3 in vivo
SIRT3 is deleted in many human cancers
Our data indicate that SIRT3 may regulate tumor cell metabolism and anabolic growth pathways. In order to determine the relevance of SIRT3 in human cancers, we first examined the copy-number variations of SIRT3 that are associated with the progression of multiple types of human cancer (Beroukhim et al., 2010
). Strikingly, at least one copy of the SIRT3
gene is deleted in 20% of all human cancers and 40% of breast and ovarian cancers present in the dataset (). SIRT3
is significantly focally deleted (deletions of less than a chromosome arm) across all cancers, and focal deletions of SIRT3
were especially frequent in breast and ovarian tumors (). In contrast, SIRT4
were not significantly focally deleted in any of the 14 subtypes analyzed ( and data not shown). TP53, a tumor suppressor known to be frequently deleted in many human cancers, is included as a control (Fisher, 2001
) (Figure and S4G,H
). Our analysis of copy-number changes at the SIRT3
locus revealed no evidence of focal amplifications across 14 types of cancer. Most of the genomic SIRT3
deletions are heterozygous, and SIRT3
deletion frequencies are similar to the well-known breast cancer tumor suppressors, BRCA1 and BRCA2, which are heterozygously deleted in 43% and 40% of human breast cancers, respectively (data not shown). Intriguingly, the peak region of deletion that includes SIRT3
(11p15.5) does not contain any known tumor suppressor (Beroukhim et al., 2010
Because breast cancers exhibited exceptionally high frequency of SIRT3 deletions compared to other tumor types () (Kim et al., 2010
), we further examined SIRT3 in human breast cancers. Elevated HIF1α expression in breast carcinomas is associated with tumor aggressiveness and poor prognosis (Chaudary and Hill, 2006
). Many breast cancer cells exhibit increased glycolysis, and expression of GLUT1 is a characteristic feature of may breast cancer biopsies (Rivenzon-Segal et al., 2003
). In xenograft models, SIRT3 loss increases expression of HIF1α target genes and results in strong GLUT1 expression (). Thus, we looked for a relationship between SIRT3 loss and HIF1α targets in human breast cancer. Gene expression profiling of 7 normal breast samples and 40 ductal breast carcinomas revealed that SIRT3
expression is significantly reduced (p = 3.53e−8
) in breast carcinomas (Richardson et al., 2006
) (). Moreover, several HIF1α target genes—most notably GLUT1
—were significantly increased in the same dataset (). We further analyzed the correlation between SIRT3
expression in individual samples from this data set and found that SIRT3
is significantly inversely correlated with GLUT1
(p = 0.0008) (). Our results demonstrate that SIRT3 loss is associated with increased expression of HIF1α target genes in vivo
and in human breast cancer and provide a metabolic link between SIRT3 deletion and breast cancer tumorigenesis.
To confirm that SIRT3 expression is reduced in human breast cancers, we analyzed SIRT3 protein levels by immunohistochemistry in normal breast epithelium in addition to a large panel of human breast cancer tissue. Out of 46 patient samples, only 6 demonstrated SIRT3 staining that was positive or as strong as SIRT3 staining in normal epithelium (). Strikingly, 87% of patients showed decreased or undetectable SIRT3 staining in adjacent cancer tissue and 20% of patients showed no detectable SIRT3 (). Similarly, gene expression profiling of an independent set of human breast cancer samples (Richardson et al., 2006
) revealed that 25% of breast cancers exhibited at least a six-fold reduction in the mRNA of SIRT3
compared to normal breast epithelium (Figure S5I
). This independent dataset provides additional validation for the observation that SIRT3
is deleted in human tumors () (Beroukhim et al., 2010
). Furthermore, an earlier high-resolution analysis of copy number variation in 171 human breast tumors similarly found significant reduction in SIRT3
copy number (Chin et al., 2007
). These findings also support those of Kim et al. who first reported that SIRT3 KO mice develop mammary tumors and that SIRT3 levels were decreased in human breast cancer (Kim et al., 2010
The studies of SIRT3 expression in human cancers suggest that SIRT3 may function as a tumor suppressor in part by preventing the metabolic shift that facilitates tumor growth. In order to examine whether SIRT3 can actively repress the Warburg effect in tumor cells, we stably overexpressed SIRT3 in three independent breast cancer cell lines: MCF7, T47D and CAMA1 (Figure S6A
). We analyzed the glucose uptake and lactate secretion in cells during hypoxia in order to simulate the tumor microenvironment. We found that SIRT3 repressed both lactate production and glucose uptake in every cell line tested (). These data clearly demonstrate that overexpression of SIRT3 in tumor cells is sufficient to reverse the metabolic shift associated with the Warburg effect.
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 (more ...)
Because SIRT3 robustly suppressed glucose uptake and lactate production in the CAMA1 cells, we chose to further analyze these cell lines. To examine the contribution of complex I activity or fatty acid oxidation on these phenotypes, we measured glucose uptake and lactate production in the presence of rotenone and etomoxir. Both rotenone and etomoxir increased glucose uptake and lactate production to a similar degree in both control and SIRT3 overexpressing cell lines, indicating that the repression of glycolysis by SIRT3 is independent of the influence of SIRT3 on fatty acid oxidation or complex I activity ().
We next examined whether SIRT3 repressed HIF1α in CAMA1 cells. SIRT3 overexpression strongly reduced HIF1α protein levels and expression of HIF1α target genes in hypoxic cells (). Moreover, when we examined the fold change of HIF1α targets in response to hypoxia or DMOG treatment, we found the inverse of the results using SIRT3 KO MEFs. SIRT3 overexpression blunted the response to hypoxia () while increasing the response to DMOG (). This is consistent with a model of elevated PHD activity in SIRT3 overexpressing cells and illustrates the importance of SIRT3 in regulating the physiological response to hypoxia at the level of the PHDs.
Next, we tested the hypothesis that SIRT3-mediated control of glucose metabolism could influence cancer cell proliferation. SIRT3 overexpression significantly repressed proliferation of CAMA1 cells cultured in high glucose (). Remarkably, control and SIRT3-expressing cells proliferated at similar rates when cultured in media containing galactose instead of glucose (). These data illustrate that SIRT3 regulates cancer cell growth by influencing the use of glucose for anabolic processes.