Fumarate hydratase-deficient kidney cancer, a clear example of the Warburg effect in cancer, is an unusually aggressive form of type 2 papillary kidney cancer that metastasizes when the primary tumors are as small as 1 cm, and is nearly uniformly fatal once it has spread beyond the kidney. Here, we showed that HLRCC tumors and two different FH-deficient RCC cell lines depended on glycolysis and had low AMPK levels. In addition to promoting fatty acid and protein biosynthetic pathways (through the activation of ACC and S6), low AMPK levels reduced the expression of DMT1 and activated IRP1 and IRP2 (). Our studies demonstrated that p53 enhances expression of DMT1 (), and the AMPK-dependent reduction of p53 would thus be expected to cause cytosolic iron deficiency, activation of IRPs, and translational repression of the iron-storage protein ferritin. Previous studies have shown that synthesis of ferritin is decreased in cells that are stimulated by several oncogenes (Tsuji et al., 1995
; Wu et al., 1999
). Our studies demonstrated that an induction of p53 resulted in increased expression of DMT1 (), providing one mechanism for the previously reported p53-dependent decrease in IRP activities and increase in ferritin levels (Zhang et al., 2008
). In respiring cells, DMT1 expression is important for the uptake of iron for the biosynthesis of heme and Fe-S cluster cofactors in the mitochondrial respiratory complexes. The AMPK-dependent decrease of DMT1 levels may represent an integral part of the metabolic shift to glycolysis, as less iron may be needed to support energy metabolism in glycolytic cells compared to respiring cells. In addition, when cells are required to produce energy from glycolysis, low iron levels may consolidate the shift to glycolysis by stabilizing HIFs or other participants in the remodeled metabolism of FH-deficient cells.
Previous studies have shown that elevation of HIFα levels in FH-deficient cells resulted from fumarate accumulation which inhibits the PHDs that mark HIFα proteins for degradation (Isaacs et al., 2005
; Pollard et al., 2005
). However, many other factors may also contribute to FH-associated neoplasias. We suggest that HIF1α is increased in HLRCC in part because cytosolic iron deficiency also inhibits PHD activities and contributes to the stabilization of HIF-1α proteins. Furthermore, since the HIF-2α transcript contains an IRE in its 5’UTR (Sanchez et al., 2007
), whereas the HIF-1α transcript does not, activation of IRPs would be expected to selectively repress HIF-2α translation. We suggest that IRP activation explains why HIF-1α levels were markedly elevated whereas HIF-2α protein levels were very low in UOK262 and UOK268 cells (), even though HIF-2α transcript levels in UOK262 cells were comparable to control cells ().
The differential expression levels of HIFα proteins in 786-0 and UOK262 cells () have important implications for the development of targeted therapeutic treatment of VHL-deficient and FH-deficient RCC. Although HIF-1α has been shown to promote tumorigenesis in colon and pancreatic cancer cells (Chen et al., 2003
; Dang et al., 2006
), studies in RCC have shown that activation of HIF-2α, and not HIF-1α, is necessary for the tumorigenesis of VHL-deficient RCC (Kondo et al., 2002
; Maranchie et al., 2002
). HIF-1α and HIF-2α have both overlapping as well as distinct targets (Gordan and Simon, 2007
; Kaelin and Ratcliffe, 2008
; Raval et al., 2005
), and HIF-1α is uniquely involved in activating genes associated with glycolysis (Gordan and Simon, 2007
; Haase, 2010
). In this study, we showed that silencing of HIF-1α inhibited the invasion activities of UOK262 cells (), further suggesting that elevation of HIF-1α is critical for engineering the switch to glycolytic metabolism in UOK262 cells.
Notably, the coordination that we observed here between glucose and iron metabolisms in FH-deficient RCC has an evolutionary precedent in the metabolism of S. cerevisiae
. When grown in glucose-rich medium, S. cerevisiae
uses fermentation (a microbial equivalent of glycolysis) as its main metabolic pathway, and repression of the yeast AMPK homologue, SNF1, represses expression of iron uptake genes (Haurie et al., 2003
). As glucose concentrations decrease, activation of SNF1 induces expression of iron uptake genes and facilitates the shift to oxidative metabolism. Conversely, iron deficiency in S. cerevisiae
results in phosphorylation of SNF1, which increases glucose uptake and glycolysis, allowing the cells to switch from respiratory to fermentative metabolism (Puig et al., 2008
). Our results suggest that the high glycolytic flux in UOK262 cells reduces AMPK and DMT1 expression levels, whereas glucose depletion diminished glycolysis and increased AMPK signaling and DMT1 expression (). These studies suggest that AMPK/SNF1 signaling provides an evolutionarily conserved linkage between intermediary metabolism and intracellular iron homeostasis. Interestingly, while the activities of several Fe-dependent enzymes, including respiratory complex I and cytosolic aconitase, were low in UOK262 cells ( and ), mitochondrial aconitase activities were not diminished in UOK262 cells (), suggesting that these cells can preferentially direct iron to specific proteins or pathways.
While previous studies on AMPK signaling have largely focused on the increase in AMPKα phosphorylation and its role in increasing catabolism during the acute response to metabolic stress (Hardie, 2007
), fewer studies have characterized the potential importance of low AMPK activity in promoting activities of anabolic pathways in tumorigenesis (Jones and Thompson, 2009
; Luo et al., 2005
; Shaw, 2006
). Here, we not only showed that levels of AMPKα-p were diminished in FH-deficient cells, but we also found that AMPK signaling was also regulated at the transcript and/or protein levels (). This metabolic remodeling was not unique to FH-deficient cells, since chemical inhibition of SDH in non-cancer cells also resulted in reduced levels of AMPK, p53, ACC-p, DMT1 and increased levels of S6-p (). Time-course studies indicated that, upon chemical inhibition of SDH, changes of AMPK activities inversely correlated with changes in intracellular ATP levels: ATP levels decreased initially upon SDH inhibition, and then gradually recovered over the following hours (), whereas after an initial rapid increase, AMPKα-p levels dropped below the levels of untreated cells (). After a 48 hr treatment with TTFA, there was also a marked decrease in AMPKα protein levels (), which further ensured that levels of AMPKα-p decreased regardless of the AMP:ATP ratio. These results are similar to the decreased levels of AMPKα-p, AMPKα and AMPKβ1 proteins and transcripts that were observed in FH-deficient cells (), and they show that the obligate shift to glycolysis in FH-deficient cells produces associated changes in AMPK activity that enhance anabolic reactions.
The high glycolytic rate, together with the decrease of AMPK signaling, can provide several potential growth advantages to FH-deficient RCC. Although the yield of ATP per glucose molecule is higher in OXPHOS than in glycolysis, aerobic glycolysis can provide sufficient energy for cell proliferation, provided that the glycolytic flux is high enough (DeBerardinis et al., 2008
). Aerobic glycolysis can also confer growth advantages by diverting some of the glucose into pathways that generate NADPH, acetyl-CoA, and ribose for fatty acid, protein and nucleotide synthesis (Vander Heiden et al., 2009
). Reduced AMPK activities would promote the efficient use of the NADPH, acetyl-CoA and ribose generated by aerobic glycolysis by activating anabolic factors involved in protein and fatty acid synthesis (Hardie, 2007
). Many cancers are characterized by upregulation of anabolic factors such as ACC, FAS, and mTOR, all of which are inhibited by AMPK activation (Jones and Thompson, 2009
; Luo et al., 2005
; Shaw, 2006
). Notably, reduced AMPK phosphorylation was recently reported in breast cancer specimens (Hadad et al., 2009
). Alterations in the LKB1/AMPK/TSC/mTOR pathway are associated with a wide variety of cancers and hamartomas, including Peutz-Jeghers syndrome and tuberous sclerosis complex (Inoki and Guan, 2009
; Jones and Thompson, 2009
; Shaw, 2006
). The tumor suppressor, p53, is a downstream effector of AMPK, and p53 mutations are found in 50% of all human cancers (Vousden and Ryan, 2009
). Here, we established that diminished p53 levels result from AMPK-dependent metabolic remodeling, adding to the potential mechanisms that impair p53 function in tumorigenesis. Thus, the marked increase in glycolysis and reduced AMPK levels in FH-deficient cancer cells together may promote anabolic metabolism (through the activation of ACC and S6) and repress senescence and apoptosis (as a result of the decrease of p53 levels) (Vousden and Ryan, 2009
) in a manner conducive to cancer cell proliferation ().
Our demonstration that metformin and AICAR reduced the invasion activities of UOK262 cells () is consistent with previous studies which showed that metformin and AICAR inhibited the proliferation of various cancer cell lines (Buzzai et al., 2007
; Hirsch et al., 2009
; Xiang et al., 2004
), and that diabetics treated with metformin exhibited decreases in cancer incidence (Evans et al., 2005
). Metformin can inhibit tumorigenesis by activating AMPK, and thereby inhibiting anabolic pathways (Shaw, 2006
) and inducing apoptosis (Buzzai et al., 2007
). However, our findings also suggest another potential mechanism for the anti-cancer effect of metformin: induction of DMT1 expression () may increase iron uptake, which may reduce HIF-1α levels and associated glycolysis, while promoting oxidative damage to DNA, proteins and lipids in cancer cells.
Interest in the Warburg effect (Vander Heiden et al., 2009
; Warburg, 1956
) has been rekindled by the mounting appreciation for the growth advantages that glycolysis can confer to tumor cells (Shaw, 2006
; Vander Heiden et al., 2009
; Warburg, 1956
). In neoplasia, oncogenic pathways may function to drive cell-autonomous nutrient uptake and anabolic metabolism via aerobic glycolysis (Vander Heiden et al., 2009
). We propose that the AMPK-dependent decrease in iron uptake in FH-deficient cells reflects a reversion to an evolutionarily conserved pathway that coordinates glucose and iron metabolisms. Our results suggest that a fundamental feature of HLRCC carcinogenesis involves reduced AMPK activities, which mediate a global remodeling of anabolic processes and iron metabolism to enhance cell proliferation and survival. The metabolic shift to aerobic glycolysis described in this aggressive form of cancer could provide insight into the most fundamental aspects of tumorigenesis and provide opportunities for the development of improved diagnostics and therapeutics in tumors characterized by aerobic glycolysis and impaired oxidative phosphorylation.