Seminars in Cancer Biology 19 (2009) 12–16
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Regulation of cancer cell metabolism by hypoxia-inducible factor 1 Gregg L. Semenza ∗ Vascular Program, Institute for Cell Engineering, Departments of Pediatrics, Medicine, Oncology, and Radiation Oncology, and McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, United States
a r t i c l e
i n f o
Keywords: BNIP3 Glucose transporter Glycolysis HIF-1 Lactate dehydrogenase MCT4 Mitochondrial autophagy NHE1 Oxygen PDK1 Warburg effect
a b s t r a c t The induction of hypoxia-inducible factor 1 (HIF-1) activity, either as a result of intratumoral hypoxia or loss-of-function mutations in the VHL gene, leads to a dramatic reprogramming of cancer cell metabolism involving increased glucose transport into the cell, increased conversion of glucose to pyruvate, and a concomitant decrease in mitochondrial metabolism and mitochondrial mass. Blocking these adaptive metabolic responses to hypoxia leads to cell death due to toxic levels of reactive oxygen species. Targeting HIF-1 or metabolic enzymes encoded by HIF-1 target genes may represent a novel therapeutic approach to cancer. © 2008 Published by Elsevier Ltd.
1. Introduction For decades, students of biochemistry have learned that in the presence of O2 cells generate ATP by completely oxidizing glucose to carbon dioxide and water through the activity of glycolytic enzymes, pyruvate dehydrogenase (PDH), the tricarboxylic acid (TCA) cycle enzymes, and the electron transport chain. In contrast, under hypoxic conditions, glucose is converted to lactate, through the activity of glycolytic enzymes and lactate dehydrogenase A (LDHA), which is a much less efﬁcient means of generating ATP. Lactate production has been viewed as a default pathway that is followed when O2 is not available for respiration. However, lactate production increases several-fold when cells are exposed to 1% O2 (corresponding to a partial pressure [PO2 ] of ∼7 mmHg at sea level), which is well above the critical O2 concentration required for electron transport chain activity in isolated mitochondria (∼0.1 M; PO2 = 0.05 mmHg). Recent studies have demonstrated that the switch from oxidative to glycolytic metabolism is an active response to hypoxia that is mediated by hypoxia-inducible factor 1 (HIF1). 2. HIF-1 HIF-1 is a heterodimeric protein, composed of HIF-1␣ and HIF1␤ subunits [1,2], which modulates the regulation of hundreds of
genes according to the cellular O2 concentration . HIF-1␣ levels increase dramatically as O2 concentration declines . Under normoxic conditions, HIF-1␣ is subjected to ubiquitination and proteasomal degradation [5-7] due to the binding of the von Hippel-Lindau tumor suppressor protein , which is the substrate recognition subunit of an E3 ubiquitin-protein ligase . VHL binds to HIF-1␣ only when the latter is hydroxylated on proline residue 402 and/or 564 [10–12]. The hydroxylation reaction is performed by prolyl hydroxylases (PHDs) that utilize O2 and ␣-ketoglutarate as substrates and generate carbon dioxide and succinate as byproducts . Under hypoxic conditions, hydroxylation, ubiquitination and degradation are inhibited, leading to the accumulation of HIF1␣ (Fig. 1). Under normoxic conditions, asparagine residue 803 is also hydroxylated. This reaction, which is mediated by factor inhibiting HIF-1 (FIH-1), prevents the binding of the co-activators CBP and p300 to HIF-1␣ . Thus, O2 -dependent hydroxylation regulates both the stability and transcriptional activity of HIF-1. Once activated, HIF-1 mediates a variety of adaptive responses to hypoxia. Two general classes of responses are, ﬁrst, those that serve to increase O2 delivery (for example, by stimulating angiogenesis by activation of the gene encoding vascular endothelial growth factor [VEGF]); and, second, those that serve to regulate O2 utilization. Recent discoveries with regard to the latter responses are described below. 3. Intratumoral PO2 , lactate, and pH
∗ Correspondence address: Broadway Research Building, Suite 671, 733 North Broadway, Baltimore, MD 21205, United States. Fax: +1 443 287 5618. E-mail address: [email protected]
1044-579X/$ – see front matter © 2008 Published by Elsevier Ltd. doi:10.1016/j.semcancer.2008.11.009
The mean PO2 in human tumors is signiﬁcantly reduced compared to surrounding normal tissue and tumors with the greatest reduction in PO2 are most likely to invade, metastasize, and kill
G.L. Semenza / Seminars in Cancer Biology 19 (2009) 12–16
4. Effects of HIF-1␣ deﬁciency on cell metabolism
Fig. 1. Regulation of HIF-1. Under normoxic conditions, HIF-1␣ is hydroxylated by PHD2. The protein OS-9 binds to both HIF-1␣ and PHD2, thereby promoting the hydroxylation reaction , in which O2 and ␣-ketoglutarate are consumed, with CO2 and succinate generated as byproducts. Hydroxylated HIF-1␣ is bound by VHL, which recruits an ubiquitin-protein ligase complex consisting of Elongin C, Elongin B, Cullin 2 (CUL2), Ring Box Protein 1 (RBX1), and an E2 ubiquitin conjugating enzyme (E2). SSAT2 promotes ubiquitination by binding to HIF-1␣, VHL, and Elongin C . Ubiquitinated HIF-1␣ is subjected to degradation by the 26S proteasome.
When HIF-1␣-null mouse embryo ﬁbroblasts (MEFs) are subjected to hypoxia for 3 days, the cells die due to increased production of reactive oxygen species (ROS) [22,23]. In contrast, ROS levels decrease in wild type (WT) cells in response to chronic hypoxia. This adaptive response is mediated by HIF-1 through the transactivation of genes encoding pyruvate dehydrogenase kinase 1 (PDK1) and BNIP3. In hypoxic cells, PDK1 phosphorylates and inactivates PDH, thereby blocking the conversion of pyruvate to acetyl coenzyme A (CoA), which is required for entry into the TCA cycle [22,24]. In concert with LDH-A, PDK1 leads to the preferential conversion of pyruvate to lactate rather than acetyl CoA. Overexpression of PDK1 in HIF-1␣-null MEFs is sufﬁcient to reduce ROS levels and prevent cell death . A second key response to hypoxia that is mediated by HIF-1 is the induction of BNIP3, which is a member of the BCL2 family of mitochondrial proteins. Expression of BNIP3 triggers selective mitochondrial autophagy in WT MEFs, but not in HIF-1␣-null MEFs, which results in a two- to fourfold decrease in mitochondrial mass and O2 consumption within 48 h . Experimental knockdown of BNIP3 or a key component of the autophagy machinery, such as Atg5 or Beclin1, phenocopies the effect of HIF-1␣ deﬁciency, whereas forced overexpression of BNIP3 in HIF-1␣-null MEFs reduces ROS levels and cell death, similar to the effect of PDK1 overexpression. These data suggest that there is an optimal PO2 for mitochondrial respiration, that increased or decreased PO2 is associated with increased ROS production, and that a major role of HIF-1 is in balancing energy and redox homeostasis. This hypothesis is supported by the ﬁnding that HIF-1 also regulates the composition of cytochrome c oxidase (COX; electron transport complex IV) by activating transcription of the COX4I2 gene, which encodes COX4-2, a regulatory subunit that allows the enzyme to function optimally under hypoxic conditions, and of the LON gene, which encodes a mitochondrial protease that is required for degradation of the COX4-1 subunit under hypoxic conditions . Thus, in response to modest reduction of PO2 , this subunit switch may provide a mechanism that allows continued ATP production via oxidative phosphorylation without increased ROS production. Previous studies have demonstrated that acute hypoxia results in increased ROS levels, which are required for the inhibition of PHD activity and stabilization of HIF-1␣ in hypoxic cells . These responses are lost in cells lacking cytochrome c . Thus, hypoxia leads to increased mitochondrial ROS, which induce HIF-1, which then mediates adaptive responses to reduce ROS levels through the modulation of mitochondrial oxidative metabolism. 5. The molecular basis of the Warburg effect in renal clear-cell carcinoma
the patient . Many carcinomas also manifest an increased concentration of lactate, which is also associated with increased risk of metastasis . The increased lactate production is associated with increased expression of LDH-A  and the monocarboxylate transporter MCT4, which transports lactate out of cancer cells . Cancer cells also overexpress the sodium–hydrogen exchanger NHE1 and carbonic anhydrase 9, which function to maintain an alkaline intracellular pH and an acidic extracellular pH . Expression of the LDHA, MCT4, NHE1, and CA9 genes is induced by hypoxia through the activity of HIF-1 . Thus, along with its control of genes encoding glucose transporters and glycolytic enzymes , HIF-1 coordinately regulates all of the proteins required for glucose uptake and its conversion to lactate (Fig. 2). The induction of CA9, MCT4, and NHE1 allows cancer cells to maintain an alkaline intracellular pH and an acidic extracellular pH, which are critical for cell proliferation and invasion, respectively.
Although many cancer cells utilize the physiological responses to hypoxia described above, in some cancers, genetic alterations can result in a ﬁxed and O2 -independent reprogramming of metabolism. Warburg noted increased production of lactate in the tissue culture media of liver tumor explants as compared to normal liver explants cultured under aerobic conditions . In renal cell carcinoma lines in which VHL is inactivated by mutation, HIF-1␣ and HIF-2␣ are constitutively expressed and mediate glycolytic metabolism. Reintroduction of WT VHL into the cell results in loss of HIF-1␣ and HIF-2␣ expression under aerobic conditions and a dramatic increase in mitochondrial mass and O2 consumption . In VHL-deﬁcient renal carcinoma cells HIF-1 blocks the biogenesis of mitochondria through inhibition of MYC, which would otherwise activate transcription of the gene encoding PGC-1␤, a transcription factor that controls mitochondrial biogenesis. Loss of
G.L. Semenza / Seminars in Cancer Biology 19 (2009) 12–16
Fig. 2. Regulation of glucose and energy metabolism by HIF-1. Metabolic substrates and products are shown in blue and HIF-1-regulated gene products are shown in purple. Red arrows, mitochondrial biogenesis mediated by C-MYC (in renal carcinoma cells). Green blocked arrow, mitochondrial autophagy mediated by BNIP3 (in mouse embryo ﬁbroblasts). AcCoA, acetyl coenzyme A; ALD, aldolase; CA9, carbonic anhydrase; COX, cytochrome c oxidase; ENO, enolase; G-6-P, glucose-6-phosphate; GLUT, glucose transporter; HK, hexokinase; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; NHE, sodium–hydrogen exchanger; PDH, pyruvate dehydrogenase; PDK, PDH kinase; PFK, phosphofructokinase; PGC, PPAR-␥ co-activator; PGK, phosphoglycerate kinase.
VHL activity and the subsequent dysregulation of HIF-1 represents the most well understood mechanism through which cancer cells can be reprogrammed from oxidative to glycolytic metabolism (the Warburg effect) as a result of a single genetic alteration. Many other mutations that either activate oncoproteins or inactivate tumor suppressors also lead to increased HIF-1 activity , suggesting that upregulation of HIF-1 represents a general mechanism underlying the Warburg effect in human cancer.
elevated despite the induction of HIF-1. A feedforward mechanism involving ROS and HIF-1 has been shown to play a key role in the pathogenic cardiovascular responses to intermittent hypoxia that occur in patients with sleep apnea . These observations may be of relevance to cancers in which cycles of hypoxia and reoxygenation occur as a result of dysfunctional tumor vasculature .
6. Increased ROS levels and HIF-1␣-dependent growth of tumor xenografts
Clinical studies are warranted to determine whether inhibition of HIF-1 by antioxidants may improve outcome in patients with cancers in which HIF-1␣ overexpression is associated with poor prognosis (Table 1). However, this approach would not be efﬁcacious in renal clear-cell carcinoma in which PHD2 activity is irrelevant due to VHL loss-of-function. Fortunately, other HIF-1 inhibitors have a mechanism of action that is independent of the PHD2-VHL pathway. For example, HSP90 inhibitors, such as 17-allylaminogeldanamycin, promote HIF-1␣ degradation by inducing the binding of RACK1, which recruits the same Elongin C-containing ubiquitin ligase that is recruited by VHL, but it does so in an O2 - and PHD2-independent manner . Because HIF-1 mediates adaptive angiogenic responses that serve to increase O2 availability [35,36], as well as those metabolic adaptations that serve to regulate O2 utilization, blocking HIF-1 activity has profound effects on tumor growth and progression . Inhibition
The induction of HIF-1 activity by ROS appears to play an important role in cancer biology. Treatment of mice bearing tumor xenografts with N-acetyl cysteine or ascorbic acid results in a marked inhibition of tumor growth that is dependent upon the ability of these antioxidants to induce degradation of HIF-1␣ via the PHD2-VHL pathway . Forced expression in cancer cells of a mutant form of HIF-1␣ that is resistant to PHD2-VHL-dependent degradation renders the cells resistant to the anti-tumor effect of N-acetyl cysteine or ascorbic acid in vivo. It is important to note that whereas the ROS-dependent induction of HIF-1 activity in normal (non-transformed) cells, such as mouse embryo ﬁbroblasts, leads to adaptive responses that serve to reduce ROS levels under conditions of chronic hypoxic [22,23], in cancer cells ROS levels remain
7. Therapeutic implications
G.L. Semenza / Seminars in Cancer Biology 19 (2009) 12–16 Table 1 Association of HIF-1␣ overexpression with patient mortality. Cancer type
Astrocytoma, diffuse Bladder, superﬁcial urotheliala Bladder, transitional cell Breast Breast, c-erbB-2-positive Breast, LN-positive Breast, LN-negative Cervix, early stage Cervix, RTX Cervix, IB-IIIB, RTX Endometrial Gastric GIST, stomach Lung, NSCLC Malignant melanomab Oligodendroglioma Oropharynx-SCC Ovariana Pancreas
   [50,51]               
LN, lymph node; RTX, radiation therapy; GIST, gastrointestinal stromal tumor; NSCLC, non-small cell lung carcinoma; SCC, squamous cell carcinoma. a Combination of HIF-1␣ overexpression and mutant p53 is associated with mortality in this cancer. b HIF-2␣ overexpression is associated with mortality in this cancer.
of HIF-1 activity may contribute to the efﬁcacy of a large number of novel and established anti-cancer drugs [37,38]. Another strategy is to inhibit the proteins regulated by HIF-1 target genes. Just as VEGF is now targeted for cancer therapy , inhibition of key metabolic enzymes regulated by HIF-1 may be of therapeutic beneﬁt. The inhibition of LDHA has been shown to inhibit cancer cell growth under hypoxic conditions ex vivo and tumor xenograft growth in vivo [40,41]. NHE1 loss-of-function in Ras transformed cells is associated with reduced tumorigenesis . Inhibitors of CA9 have been developed and are under evaluation as anti-cancer agents . Inhibition of HIF-1 or PDK-1 was shown to increase oxygen consumption and thereby increase the anti-cancer efﬁcacy of the hypoxic cytotoxin tirapazamine . Combining anti-HIF-1 and anti-angiogenic therapy to block both O2 delivery and adaptation to hypoxia may represent another effective strategy for killing cancer cells. Thus, the recent advances in understanding tumor metabolism may lead to major advances in the treatment of cancer patients. Conﬂict of interest None declared. References  Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basichelix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995;92:5510–4.  Wang GL, Semenza GL. Puriﬁcation and characterization of hypoxia-inducible factor 1. J Biol Chem 1995;270:1230–7.  Manalo DJ, Rowan A, Lavoie T, Natarajan L, Kelly BD, Ye SQ, et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood 2005;105:659–69.  Jiang BH, Semenza GL, Bauer C, Marti HH. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am J Physiol 1996;271:C1172–80.  Salceda S, Caro J. Hypoxia-inducible factor 1␣ (HIF-1 ␣) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 1997;272:22642–7.  Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1␣ is mediated by an O2 -dependent degradation domain via the ubiquitinproteasome pathway. Proc Natl Acad Sci USA 1998;95:7987–92.  Kallio PJ, Wilson WJ, O’Brien S, Makino Y, Poellinger L. Regulation of the hypoxiainducible transcription factor 1␣ by the ubiquitin-proteasome pathway. J Biol Chem 1990;274:6519–25.
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