The data presented in this report describe a more detailed model for metabolic regulation of HIF-1α stabilization in HLRCC tumors. Of key importance to ROS-mediated HIF-1α stabilization is the glycolytic switch that occurs rapidly in cells following loss of FH activity. While the mechanism underlying this phenomenon remains to be fully elucidated, initial fumarate accumulation that occurs in cells following loss of FH enzymatic activity may be a contributing factor. It is now well established that acute accumulation of intracellular fumarate following either pharmacologic inhibition of FH or its siRNA-mediated knockdown leads to HIF-1α stabilization mediated by competitive inhibition (fumarate competing with 2-OG) of HIF-1α proline hydroxylation. In turn, HIF-1α accumulation is associated with the rapid appearance of a glycolytic phenotype (16
). Our current findings suggest the possibility that an initial HIF-dependent increase in glucose uptake and metabolism occurring as an immediate and obligate response to reduced FH activity leads directly to heightened cellular ROS production, which further promotes HIF-1α stabilization by depleting cellular Fe2+
stores, thereby depriving HPH of an additional necessary cofactor. Elevated HIF-1α expression may drive glycolysis to establish a feed-forward signaling loop, and glucose-dependent ROS accumulation is, at least in part, mediated by NADPH oxidase and PKC-δ (Fig. ). Although there may be alternate mechanisms that permit the glycolytic switch independent of HIF, we believe it is likely that HIF-1α is integrally involved in this metabolic shift, given its established role in promoting glycolysis as opposed to oxidative phosphorylation.
Schema for ROS-mediated HIF-1α stabilization in HLRCC.
The biologic significance of these findings is severalfold. Since they are usually diagnosed with advanced disease, HLRCC patients have limited treatment options and suffer a poor prognosis. Thus, elucidation of the molecular pathogenesis of tumor formation will better inform the development of effective therapeutic strategies. Our current data highlight an important role for ROS in maintaining HIF-1α stabilization in these tumors and suggest various metabolic approaches to treatment. A rationale for the use of antioxidants in HIF-1α-driven tumors was recently provided by Gao et al., who examined the antitumorigenic effect of the antioxidant NAC (7
). They reported that NAC treatment resulted in reduced HIF-1α expression and in inhibition of in vivo tumor formation in a HIF-driven model of tumorigenesis. Their data support a role for ROS-mediated inhibition of HPH activity, as NAC was ineffective in a tumor model expressing a mutant HIF-1α allele that was resistant to HPH-dependent degradation.
In addition, our data add to the growing body of evidence linking derangements in mitochondrial metabolism to carcinogenesis. First described many years ago by Otto Warburg, the “Warburg effect” refers to the preference of cancer cells to obtain energy via glycolysis and not oxidative phosphorylation, even in normoxia (40
). HIF-1α has been suggested as a key regulator of this phenomenon, since it transcriptionally drives many components of glycolysis. In addition, HIF-1α shunts metabolic intermediates away from the TCA cycle by upregulating pyruvate dehydrogenase kinase 1 (PDK1) (20
). PDK1 phosphorylates and inhibits pyruvate dehydrogenase (PDH), thus limiting the ability of PDH to supply acetyl coenzyme A to the TCA cycle (by conversion from pyruvate). When the TCA cycle is genetically compromised, as is the case in HLRCC, glycolytic addiction of the tumor cells is ensured. This may prove to be an Achilles’ heel of HLRCC, even more so than in the case of other solid tumors. Because HLRCC tumors have become genetically obligated to glycolysis (as evidenced by their inability to substitute galactose for glucose), they must rapidly convert the pyruvate accumulating as a result of this metabolic process to lactic acid. Previous reports have suggested that failure to do so may slow glycolysis by-product feedback inhibition (4
). Others have suggested that rapid conversion of pyruvate to lactic acid is necessary to regenerate cellular NAD+
, which is required to support additional cycles of glycolysis (6
). Our data suggest a third, not mutually exclusive, possibility—that accumulation of pyruvate causes a reduction in cellular ROS level sufficient to negatively impact HIF-1α stabilization. Since lactate dehydrogenase is itself transcriptionally regulated by HIF-1α, as are glucose uptake, glycolysis, and downregulation of oxidative phosphorylation, interference in this process (e.g., LDH inhibition, ROS scavenging) provides the potential to convert a feed-forward stimulatory signaling loop into a reverse inhibitory loop and offers several potential treatment strategies. An initial report describing the impact of LDH knockdown on the viability of cells experimentally knocked down for FH supports this hypothesis (43
The initial link between loss of FH activity and ROS generation could be HIF-1α itself, but this hypothesis does not explain the elevated ROS levels that we observed in fumarase-deficient yeast (which do not express HIF proteins). However, like UOK262 cells, yeast lacking fumarase activity cannot use mitochondrial respiration to grow on nonfermentable carbon sources (22
), suggesting that addiction to glycolysis in the absence of FH activity is a highly conserved property of eukaryotic cells. Indeed, the enforced glycolysis and resultant rapid generation and secretion of lactic acid characteristic of FH-deficient cells may underlie the high metastatic potential of HLRCC, as lactic acid is reported to stimulate endothelial cell migration, which is a crucial component of a tumor's metastatic phenotype (1
). In patients with cervical cancer, high lactate levels have been directly correlated with likelihood of tumor metastasis and reduced patient survival (38
Exposure to high glucose levels stimulates NADPH-mediated ROS production in vascular smooth muscle and endothelial cells (15
), and this has been associated with increased activity of PKC isozymes, including PKC-δ (14
). Furthermore, ROS production is elevated in adipocytes obtained from high-fat-diet-induced obese and insulin-deficient mice, and both increased PKC-δ activity and NADPH oxidase have been proposed to mediate high glucose-dependent ROS production in these settings (36
). Our data identify both NADPH oxidase and PKC-δ as contributors to the elevated HIF-1α level found in UOK262 cells, as inhibition of both proteins led to decreased HIF-1α expression. A similar effect on HIF-1α was noted following RNA interference knockdown of the NADPH oxidase subunit p47phox
and following molecular knockdown of PKC-δ.
Inhibition of HPH activity by either excess fumarate or elevated ROS causes the phenotype we have described in this study. While ROS levels are clearly elevated following experimental FH knockdown, it is unclear whether fumarate levels are elevated sufficiently in HLRCC renal tumor cells to inhibit HPH. These results parallel findings with SDH
) where both succinate and ROS have been proposed to lead to HIF-1α stabilization (12
). However, more recent evidence suggests that elevation of the succinate level alone, in the absence of ROS, is not sufficient to support HIF-1α stabilization (12
Additional mechanisms also may account for the pseudohypoxic expression profile of HLRCC. As FH is a mitochondrial enzyme, another potential source for ROS may be the electron transport chain, which is the source of ROS generation in the context of SDH mutations. Alternatively, the mTOR pathway has been implicated in HIF-1α synthesis. Although neither the inhibition of the electron transport chain nor the inhibition of mTOR impacts HIF-1α expression over the short time frame of our experiments (data not shown), our results do not exclude the possibility that HIF-1α in UOK262 cells may be impacted by these or other inputs over a longer time scale.
In addition, the role of pseudohypoxia in HLRCC tumorigenesis, while implied, has not been proven. The HPH enzymes belong to a family of dioxygenases that uniformly require 2-OG and Fe2+ as cosubstrate and cofactor, respectively. These enzymes include collagen hydroxylases and certain histone demethylases. Thus, elevated levels of fumarate and/or ROS may impact additional cellular processes unrelated to HIF-1α. It is certainly possible that inhibition of these or other dioxygenases, whether in concert with or independent of HIF-1α stabilization, may contribute to HLRCC tumor formation.
In summary, our findings link loss of FH activity to glycolytic addiction, chronic ROS production and HIF-1α stabilization, and we show that these phenomena are interdependent. The UOK262 cell line represents a unique model system by which to further evaluate the impact on HLRCC tumorigenesis of various strategies aimed at interdicting this signaling axis, with the potential to identify novel therapeutic approaches to target this disease.