AMPK is a cellular energy sensor that coordinates metabolic activities in many tissues. Under conditions of energetic stress, AMPK activation suppresses cell growth and proliferation, leading to speculation that AMPK may function as part of a tumor suppressor pathway (Hardie, 2011
; Shackelford and Shaw, 2009
). Here we provide the first genetic evidence that AMPKα displays tumor suppressor activity in vivo
. Loss of AMPK signaling cooperates with oncogenic Myc to enhance tumorigenesis in a mouse model of lymphomagenesis, suggesting that AMPK may function as a tumor suppressor (). Moreover, we demonstrate that AMPK is a negative regulator of both aerobic glycolysis and cellular biosynthesis in cancer cells. Cells deficient for the catalytic alpha subunit(s) of AMPK display increased aerobic glycolysis marked by increased lactate production from glucose ( and ), and downregulation of AMPK activity is sufficient to induce the Warburg Effect in cancer cells (). We find that HIF-1α is a key mediator of AMPK-dependent effects on cellular metabolism. Reducing AMPKα levels in cells leads to increased HIF-1α protein levels under normoxia in both transformed and non-transformed cells (), and HIF-1α is required to drive both the Warburg effect and the growth of AMPKα1-deficient lymphomas in vivo
(–). The results presented here suggest that the downregulation of AMPK activity eliminates a key metabolic checkpoint that normally antagonizes anabolic pro-growth cellular metabolism. Thus, AMPK may act in cancer cells as a metabolic gatekeeper that functions to establish metabolic checkpoints that limit cell division, and its loss of function can enhance both tumorigenesis and tumor progression.
All cells must manage their energetic resources to survive. We and others have established that AMPK is the central mediator of a metabolic cell cycle checkpoint activated in response to nutrient limitation in mammalian cells (Gwinn et al., 2008
; Inoki et al., 2003
; Jones et al., 2005
). However, programs of ATP production and macromolecular synthesis must also be coordinated in proliferating cells to ensure proper cell division. The data presented here suggest that AMPK functions to regulate metabolic homeostasis in proliferating cells in the absence of acute energetic stress. Isogenic MEFs or cancer cells lacking AMPKα activity display a metabolic shift towards aerobic glycolysis, thus allowing cancer cells to engage aerobic glycolysis for ATP production and divert glucose-derived CAC intermediates towards lipid biosynthesis to support increased cell growth. AMPK may also influence lipid biosynthesis through regulation of ACC and other lipogenic enzymes, possibly through its effects on SREBP-1 (Li et al., 2011
). Thus, defective AMPKα signaling promotes the re-wiring of metabolic pathways to favor cell growth pathways.
Interestingly our data provide evidence that AMPKα-deficient tumors display increased activation of the TORC1 targets S6 and 4E-BP1, suggesting that AMPK, as opposed to other AMPK-related kinases, may be the key TORC1 regulator downstream of LKB1 in tumors. Consistent with past work (Inoki et al., 2003
; Liu et al., 2006
; Shaw et al., 2004a
), we find that AMPK functions to downregulate TORC1 activity specifically under conditions of energetic stress, when it is desirable to suppress ATP-consuming processes such as mRNA translation. This may provide a metabolic advantage to proliferating cells, where the loss of AMPK signaling promotes increased ATP production and resource accumulation without affecting the mitogenic properties of TORC1. By concurrently silencing AMPK while maintaining TORC1 signaling, cells may effectively bypass endogenous brakes on cellular metabolism, supporting increased tumor cell growth and proliferation.
Our work here establishes HIF-1α as an key mediator of the metabolic transformation triggered by reduced AMPKα activity in cancer cells. We show that downregulation of AMPK signaling is sufficient to induce normoxic HIF-1α stabilization and enhance the Warburg effect. TORC1 activity appears to contribute in part to this process, as silencing the mTORC1 binding partner Raptor reduces levels of hif1a
mRNA in AMPKα-deficient cells. However, silencing Raptor moderately reduces HIF-1α protein levels and has a minimal effect on the glycolytic phenotype of AMPKα-deficient cells, suggesting that AMPK may regulate HIF-1α-dependent Warburg metabolism through additional mechanisms. Interestingly, TORC1 inhibition reduces hif1a
mRNA and reduces glycolysis in cell regardless of AMPK expression, suggesting that TORC1 may function on a more global level as a positive regulator of glycolysis beyond specific effects on HIF-1α expression (Duvel et al., 2010
). Given that TORC1 signaling is elevated in AMPKα1-deficient lymphomas, this may have implications for tumor metabolism in vivo
. HIF-1α mRNA levels are unaffected by AMPK expression; thus, AMPK may affect normoxic HIF-1α protein expression either through decreased protein turnover or differential translation of HIF-1α mRNA (Choo et al., 2008
). Overall we propose that AMPK functions to coordinate glycolytic and oxidative metabolism in proliferating cells by restricting HIF-1α function.
One consequence of AMPK loss in cells is enhanced flux of glucose-derived carbon to citrate for lipid biosynthesis, promoting biomass accumulation and increased cell size. This may appear counterintuitive, as HIF-1α-dependent upregulation of PDK1 under hypoxia is proposed to direct glucose-derived carbon away from the CAC (Kim et al., 2006
; Papandreou et al., 2006
). However, glucose-to-citrate flux is not blocked in AMPKα-null cells despite elevated PDK1 levels. Rather, the reduced levels of citrate(m+4) in AMPKα-null cells may result from increased use of glucose-derived citrate (m+2) for lipid biosynthesis. Reducing AMPK levels significantly decreases ACC1 inhibition in both tumor cells and tumor tissue, which would permit maximal activity of ACC1 for lipid biosynthesis. Thus, AMPK may regulate lipid biosynthesis and biomass accumulation on multiple levels: substrate availability (HIF-1α-dependent glucose-derived citrate) and ACC activity.
We propose that AMPK may function as a metabolic tumor suppressor, limiting the growth of cancer cells by regulating key bioenergetic and biosynthetic pathways required to support unchecked proliferation. Thus, selection against AMPK activity may represent an important regulatory step for tumor initiation and progression, allowing tumor cells to gain a metabolic growth advantage. Reduced AMPK activity has been detected in primary human breast cancer (Hadad et al., 2009
), and reduced expression of prkaa2
, the gene that encodes for AMPKα2, has been linked to human breast, ovarian, and gastric cancer (Hallstrom et al., 2008
; Kim et al., 2012
). It is also well documented that LKB1-deficiency (Shackelford and Shaw, 2009
) or genetic events that target LKB1 activity (Godlewski et al., 2010
; Zheng et al., 2009
) lead to reduced AMPK signaling in tumor cells. Thus, there may be several routes by which AMPK function is suppressed in tumors to provide a selective metabolic growth advantage.
While selection for loss of AMPK function may favor the Warburg effect in tumor cells, it may also eliminate metabolic checkpoints essential for cellular adaptation to stress. AMPK normally plays a protective role to block cell growth in response to poor nutrient conditions, and as such its loss or suppression during tumorigenesis may sensitize tumor cells to apoptosis under hypoxic or nutrient depleted environments (Svensson and Shaw, 2012
). Consistent with this, silencing AMPKα1 in Eμ-Myc lymphomas conferred sensitivity to apoptosis induced by the glycolytic inhibitor 2-DG. The increased levels of ACC phosphorylation observed in Eμ-Myc tumors () infer that lymphomas experience metabolic stress and AMPK activation in vivo
. Thus, while ablation of AMPK signaling may enhance tumorigenesis, inhibition of this central energy-sensing pathway may offer unique a therapeutic window for the treatment of tumors with metabolic inhibitors. Our data provide a mechanistic rationale in support of the use of AMPK agonists such as metformin for cancer therapy (Buzzai et al., 2007
; Evans et al., 2005
), as the efficacy of these agents against tumor growth may lie in their ability to engage AMPK-dependent metabolic checkpoints to restrict anabolic growth. Understanding the reprogramming of cellular metabolic networks by AMPK in cancer may aid in the development of novel approaches for cancer therapy.