Regulation of cancer cell metabolism by hypoxia-inducible factor 1

Regulation of cancer cell metabolism by hypoxia-inducible factor 1

Seminars in Cancer Biology 19 (2009) 12–16 Contents lists available at ScienceDirect Seminars in Cancer Biology journal homepage: www.elsevier.com/l...

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Seminars in Cancer Biology 19 (2009) 12–16

Contents lists available at ScienceDirect

Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer

Review

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 [3]. HIF-1␣ levels increase dramatically as O2 concentration declines [4]. 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 [8], which is the substrate recognition subunit of an E3 ubiquitin-protein ligase [9]. 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 [13]. 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␣ [14]. 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

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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 [45], 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 [46]. 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 [22]. 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 [23]. 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 [25]. 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 [26]. These responses are lost in cells lacking cytochrome c [27]. 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 [15]. Many carcinomas also manifest an increased concentration of lactate, which is also associated with increased risk of metastasis [16]. The increased lactate production is associated with increased expression of LDH-A [17] and the monocarboxylate transporter MCT4, which transports lactate out of cancer cells [18]. 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 [19]. Expression of the LDHA, MCT4, NHE1, and CA9 genes is induced by hypoxia through the activity of HIF-1 [20]. Thus, along with its control of genes encoding glucose transporters and glycolytic enzymes [21], 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 [28]. 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 [29]. 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

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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 [30], 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 [32]. These observations may be of relevance to cancers in which cycles of hypoxia and reoxygenation occur as a result of dysfunctional tumor vasculature [33].

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 [34]. 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 [31]. 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 [31]. 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

Reference

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

[47] [48] [49] [50,51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66]

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 [39], 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 [42]. Inhibitors of CA9 have been developed and are under evaluation as anti-cancer agents [43]. 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 [44]. 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 [1] 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. [2] Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 1995;270:1230–7. [3] 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. [4] 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. [5] 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. [6] 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. [7] 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.

15

[8] Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, et al. The tumor suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 1999;399:271–5. [9] Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW. Activation of HIF-1␣ ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc Natl Acad Sci USA 2000;97:10430–5. [10] Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, et al. HIF␣ targeted for VHLmediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001;292:464–8. [11] Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-␣ to the von Hippel-Lindau ubiquitylation complex by O2 -regulated prolyl hydroxylation. Science 2001;292:468–72. [12] Yu F, White SB, Zhao Q, Lee FS. HIF-1␣ binding to VHL is regulated by stimulussensitive proline hydroxylation. Proc Natl Acad Sci USA 2001;98:9630–5. [13] Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke J, Mole DR, et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001;107:43–54. [14] Lando D, Peet DJ, Gorman JJ, Whelan DA, Whitelaw ML, Bruick RK. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev 2002;16:1466–71. [15] Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 2007;26:225–39. [16] Brizel DM, Schroeder T, Scher RL, Walenta S, Clough RW, Dewhirst MW, et al. Elevated tumor lactate concentrations predict for an increased risk of metastases in head-and-neck cancer. Int J Radiat Oncol Biol Phys 2001;51:349–53. [17] Koukourakis MI, Giatromanolaki A, Sivridis E, Gatter KC, Harris AL, Tumour Angiogenesis Research Group. Lactate dehydrogenase 5 expression in operable colorectal cancer: strong association with survival and activated vascular endothelial growth factor pathway—a report of the Tumour Angiogenesis Research Group. J Clin Oncol 2006;24:4301–8. [18] Koukourakis MI, Giatromanolaki A, Bougioukas G, Sivridis E. Lung cancer: a comparative study of metabolism related protein expression in cancer cells and tumor associated stroma. Cancer Biol Ther 2007;6:1476–9. [19] Fang JS, Gillies RD, Gatenby RA. Adaptation to hypoxia and acidosis in carcinogenesis and tumor progression. Semin Cancer Biol 2008;18:330–7. [20] Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer’s Achilles’ heel. Cancer Cell 2008;13:472–82. [21] Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, Wenger RH, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1␣. Genes Dev 1998;12:149–62. [22] Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 2006;3:177–85. [23] Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, Wesley JB, et al. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem 2008;283:10892–903. [24] Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab 2006;3:187–97. [25] Fukuda R, Zhang H, Kim JW, Shimoda L, Dang CV, Semenza GL. HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell 2007;129:111–22. [26] Guzy RD, Hoyos B, Robin E, Chen H, Liu L, Mansfield KD, et al. Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing. Cell Metab 2005;1:401–8. [27] Mansfield KD, Guzy RD, Pan Y, Young RM, Cash TP, Schumacker PT, et al. Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-␣ activation. Cell Metab 2005;1:393–9. [28] Warburg O. The metabolism of tumors. London: Arnold Constable; 1930. [29] Zhang H, Gao P, Fukuda R, Kumar G, Krishnamachary B, Zeller KI, et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHLdeficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell 2007;11:407–20. [30] Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721–32. [31] Gao P, Zhang H, Dinavahi R, Li F, Xiang Y, Raman V, et al. HIF-dependent antitumorigenic effect of antioxidants in vivo. Cancer Cell 2007;12:230–8. [32] Peng YJ, Yuan G, Ramakrishnan D, Sharma SD, Bosch-Marce M, Kumar GK, et al. Heterozygous HIF-1␣ deficiency impairs carotid body-mediated systemic responses and reactive oxygen species generation in mice exposed to intermittent hypoxia. J Physiol 2006;577:705–16. [33] Dewhirst MW, Cao Y, Moeller B. Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response. Nat Rev Cancer 2008;8:425–37. [34] Liu YV, Baek JH, Zhang H, Diez R, Cole RN, Semenza GL. RACK1 competes with HSP90 for binding to HIF-1␣ and is required for O2 -independent and HSP90 inhibitor-induced degradation of HIF-1␣. Mol Cell 2007;25:207–17. [35] Liao D, Johnson RS. Hypoxia: a key regulator of angiogenesis in cancer. Cancer Metastasis Rev 2007;26:281–90. [36] Semenza GL. Vasculogenesis, angiogenesis, and arteriogenesis: mechanisms of blood vessel formation and remodeling. J Cell Biochem 2007;102:840–7. [37] Melillo G. Targeting hypoxia cell signaling for cancer therapy. Cancer Metastasis Rev 2007;26:341–52. [38] Semenza GL. Evaluation of HIF-1 inhibitors as anticancer agents. Drug Discov Today 2007;12:853–9. [39] Ferrara N, Hillan KJ, Gerber HP, Novotny W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 2004;3:391–400.

16

G.L. Semenza / Seminars in Cancer Biology 19 (2009) 12–16

[40] Shim H, Dolde C, Lewis BC, Wu CS, Dang G, Jungmann RA, et al. c-Myc transactivation of LDH-A: implications for tumor metabolism and growth. Proc Natl Acad Sci USA 1997;94:6658–63. [41] Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 2006;9:425–34. [42] Pouysségur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 2006;441:437–43. [43] Thiry A, Dogné JM, Masereel B, Supuran CT. Targeting tumor-associated carbonic anhydrase IX in cancer therapy. Trends Pharmacol Sci 2006;27:566–73. [44] Cairns RA, Papandreou I, Sutphin PD, Denko NC. Metabolic targeting of hypoxia and HIF-1 in solid tumors can enhance cytotoxic chemotherapy. Proc Natl Acad Sci USA 2007;104:9445–50. [45] Baek JH, Mahon PC, Oh J, Kelly B, Krishnamachary B, Pearson M, et al. OS-9 interacts with hypoxia-inducible factor 1␣ and prolyl hydroxylases to promote oxygen-dependent degradation of HIF-1␣. Mol Cell 2005;17:503–12. [46] Baek JH, Liu YV, McDonald KR, Wesley JB, Hubbi ME, Byun H, et al. Spermidine/spermine-N1-acetyltransferase 2 is an essential component of the ubiquitin ligase complex that regulates hypoxia-inducible factor 1␣. J Biol Chem 2007;282:23572–80. [47] Korkolopoulou P, Patsouris E, Konstantinidou AE, Pavlopoulos PM, Kavantzas N, Boviatsis E, et al. Hypoxia-inducible factor 1␣/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis. Neuropathol Appl Neurobiol 2004;30:267–78. [48] Theodoropoulos VE, Lazaris AC, Kastriotis I, Spiliadi C, Theodoropoulos GE, Tsoukala V, et al. Evaluation of hypoxia-inducible factor 1␣ overexpression as a predictor of tumour recurrence and progression in superficial urothelial bladder carcinoma. BJU Int 2005;95:425–31. [49] Theodoropoulos VE, Lazaris ACh, Sofras F, Gerzelis I, Tsoukala V, Ghikonti I, et al. Hypoxia-inducible factor 1␣ expression correlates with angiogenesis and unfavorable prognosis in bladder cancer. Eur Urol 2004;46:200–8. [50] Vleugel MM, Greijer AE, Shvarts A, van der Groep P, van Berkel M, Aarbodem Y, et al. Differential prognostic impact of hypoxia induced and diffuse HIF-1␣ expression in invasive breast cancer. J Clin Pathol 2005;58:172–7. [51] Dales JP, Garcia S, Meunier-Carpentier S, Andrac-Meyer L, Haddad O, Lavaut MN, et al. Overexpression of hypoxia-inducible factor HIF-1␣ predicts early relapse in breast cancer: retrospective study in a series of 745 patients. Int J Cancer 2005;116:734–9. [52] Giatromanolaki A, Koukourakis MI, Simopoulos C, Sivridis E. Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original cerbB-2/HIF-1␣ phenotype. Cancer Biol Ther 2007;6:153–5. [53] Schindl M, Schoppmann SF, Samonigg H, Hausmaninger H, Kwasny W, Gnant M, et al. Overexpression of hypoxia-inducible factor 1␣ is associated with an unfavorable prognosis in lymph node-positive breast cancer. Clin Cancer Res 2002;8:1831–7.

[54] Bos R, van der Groep P, Greijer AE, Shvarts A, Meijer S, Pinedo HM, et al. Levels of hypoxia-inducible factor-1␣ independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer 2003;97:1573–81. [55] Birner P, Schindl M, Obermair A, Plank C, Breitenecker G, Oberhuber G. Overexpression of hypoxia-inducible factor 1␣ is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res 2000;60:4693–6. [56] Burri P, Djonov V, Aebersold DM, Lindel K, Studer U, Altermatt HJ, et al. Significant correlation of hypoxia-inducible factor-1␣ with treatment outcome in cervical cancer treated with radical radiotherapy. Int J Radiat Oncol Biol Phys 2003;56:494–501. [57] Bachtiary B, Schindl M, Pötter R, Dreier B, Knocke TH, Hainfellner JA, et al. Overexpression of hypoxia-inducible factor 1␣ indicates diminished response to radiotherapy and unfavorable prognosis in patients receiving radical radiotherapy for cervical cancer. Clin Cancer Res 2003;9:2234–40. [58] Sivridis E, Giatromanolaki A, Gatter KC, Harris AL, Koukourakis MI. Association of hypoxia-inducible factors 1␣ and 2␣ with activated angiogenic pathways and prognosis in patients with endometrial carcinoma. Cancer 2002;95:1055– 63. [59] Griffiths EA, Pritchard SA, Valentine HR, Whitchelo N, Bishop PW, Ebert MP, et al. Hypoxia-inducible factor-1␣ expression in the gastric carcinogenesis sequence and its prognostic role in gastric and gastro-oesophageal adenocarcinomas. Br J Cancer 2007;96:95–103. [60] Takahashi R, Tanaka S, Hiyama T, Ito M, Kitadai Y, Sumii M, et al. Hypoxiainducible factor-1␣ expression and angiogenesis in gastrointestinal stromal tumor of the stomach. Oncol Rep 2003;10:797–802. [61] Swinson DE, Jones JL, Cox G, Richardson D, Harris AL, O’Byrne KJ. Hypoxiainducible factor-1␣ in non small cell lung cancer: relation to growth factor, protease and apoptosis pathways. Int J Cancer 2004;111:43–50. [62] Giatromanolaki A, Sivridis E, Kouskoukis C, Gatter KC, Harris AL, Koukourakis MI. Hypoxia-inducible factors 1␣ and 2␣ are related to vascular endothelial growth factor expression and a poorer prognosis in nodular malignant melanomas of the skin. Melanoma Res 2003;13:493–501. [63] Birner P, Gatterbauer B, Oberhuber G, Schindl M, Rössler K, Prodinger A, et al. Expression of hypoxia-inducible factor-1␣ in oligodendrogliomas: its impact on prognosis and on neoangiogenesis. Cancer 2001;92:165–71. [64] Aebersold DM, Burri P, Beer KT, Laissue J, Djonov V, Greiner RH, et al. Expression of hypoxia-inducible factor-1␣: a novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer. Cancer Res 2001;61:2911–6. [65] Birner P, Schindl M, Obermair A, Breitenecker G, Oberhuber G. Expression of hypoxia-inducible factor 1␣ in epithelial ovarian tumors: its impact on prognosis and on response to chemotherapy. Clin Cancer Res 2001;7:1661– 8. [66] Sun HC, Qiu ZJ, Liu J, Sun J, Jiang T, Huang KJ, et al. Expression of hypoxia-inducible factor-1␣ and associated proteins in pancreatic ductal adenocarcinoma and their impact on prognosis. Int J Oncol 2007;30:1359–67.