Targeting Hypoxia, HIF-1, and Tumor Glucose Metabolism to Improve Radiotherapy Efﬁcacy

CCR FOCUS

Tineke W.H. Meijer, Johannes H.A.M. Kaanders, Paul N. Span, and Johan Bussink

Abstract Radiotherapy, an important treatment modality in oncology, kills cells through induction of oxidative stress. However, malignant tumors vary in their response to irradiation as a consequence of resistance mechanisms taking place at the molecular level. It is important to understand these mechanisms of radioresistance, as counteracting them may improve the efﬁcacy of radiotherapy. In this review, we describe how the hypoxia-inducible factor 1 (HIF-1) pathway has a profound effect on the response to radiotherapy. The main focus will be on HIF-1–controlled protection of the vasculature postirradiation and on HIF-1 regulation of glycolysis and the pentose phosphate pathway. This aberrant cellular metabolism increases the antioxidant capacity of tumors, thereby countering the oxidative stress caused by irradiation. From the results of translational studies and the ﬁrst clinical phase I/II trials, it can be concluded that targeting HIF-1 and tumor glucose metabolism at several levels reduces the antioxidant capacity of tumors, affects the tumor microenvironment, and sensitizes various solid tumors to irradiation. Clin Cancer Res; 18(20); 5585–94. 2012 AACR.

Introduction The hypoxia-inducible factor 1 (HIF-1) pathway enables When ionizing radiation is absorbed in tissue, free radi- tumor cells to survive by changing glucose metabolism cals are produced as a result of ionizations either directly in toward a glycolytic phenotype, by inducing angiogenesis the DNA molecule itself or indirectly in other cellular and by regulating pH balance and proliferation rate. This review describes how the HIF-1 pathway impacts on molecules, primarily water (H2O). This indirect effect leads to the production of reactive oxygen species (ROS), which radioresistance of solid tumors, with special focus on are molecules that contain chemically active oxygen mole- HIF-1–induced changes in tumor glucose metabolism. Fur- cules including superoxide radical anions and hydroxyl thermore, inhibition of HIF-1 and glucose metabolism is radicals, thereby inducing oxidative stress. ROS can diffuse discussed as a new method to overcome radioresistance. to the DNA to produce similar free radicals. These free radicals, whether they are produced as a result of the direct HIF-1 Pathway or indirect mechanism, break chemical bonds and initiate Hypoxia-inducible factor 1 the chain of events that results in DNA damage. Oxygen HIF-1 is a heterodimeric protein consisting of an O2- molecules are able to react with the free radicals to yield a regulated HIF-1a and a constitutively expressed HIF-1b stable change in the chemical composition of the DNA subunit. Under normoxia, HIF-1a is rapidly degraded, damage. In this way, oxygen chemically "fixes" DNA dam- whereas hypoxia leads to stabilization and accumulation age and this is the basic mechanism for the therapeutic use of HIF-1a (Figs. 1 and 2A and B; refs. 7 and 8). However, of ionizing radiation in cancer (1). Hypoxia, a pathophys- under certain normoxic conditions, HIF-1a expression can iologic characteristic of solid malignancies (Fig. 1), inter- be increased; for example, mutations in the von Hippel- feres with the fixation of DNA damage and is therefore a Lindau protein stabilize HIF-1a protein and PI3K/AKT/ major cause of resistance to irradiation (2–5). A number of mTOR activity stimulates translation of HIF-1a mRNA therapeutic approaches have been designed to overcome (7–11). Moreover, reactive oxygen and nitrogen species tumor hypoxia (2, 4, 6). inhibit proteasomal degradation of HIF-1a (10, 12). After stabilization of HIF-1a, the heterodimeric protein activates transcription of numerous genes involved in angiogenesis, proliferation, glycolytic tumor metabolism, and pH regu- Authors' Affiliation: Department of Radiation Oncology, Radboud Uni- lation (refs. 7, 10, and 13; Fig. 2B). versity Nijmegen Medical Centre, Nijmegen, The Netherlands Corresponding Author: Johan Bussink, Department of Radiation Oncol- ogy, 874, Radboud University Nijmegen Medical Centre, P.O. Box 9101, Glycolytic cancer cell metabolism and aggressive tumor 6500 HB Nijmegen, The Netherlands. Phone: 31-243-614-515; Fax: 31- behavior 243-610-792; E-mail: [email protected] Under aerobic conditions, normal cells generate energy doi: 10.1158/1078-0432.CCR-12-0858 by processing glucose both via inefficient glycolysis and 2012 American Association for Cancer Research. via more efficient mitochondrial oxidation. As hypoxia

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decreases the rate of mitochondrial oxidation, tumor cells switch to glycolysis for energy production under hypoxic N circumstances (14). This process in which pyruvate, lactate, and hydrogen ions are produced is called anaerobic glycolysis or Pasteur effect (9, 15). However, a hallmark of cancer cells is a high rate of glucose consumption and lactate generation even in the presence of oxygen (aerobic glycolysis or Warburg effect; refs. 9, and 16–18). HIF-1 initiates transcription of genes that encode transporters and enzymes regulating glycolysis and the pentose phosphate pathway (Fig. 3; refs. 8 and 19). The glycolytic products pyruvate and lactate induce HIF-1a accumulation (20, 21), suggesting a feed-forward mechanism in N which HIF-1 causes a glycolytic metabolism with elevated pyruvate/lactate concentrations, which in turn increase © 2012 American Association for Cancer Research HIF-1 activity (Fig. 2B; refs. 19 and 22). Besides HIF-1, activated oncogenes (e.g., RAS and MYC)andthePI3K/ AKT/mTOR pathway are involved in the regulation of the metabolic shift to (aerobic) glycolysis (9, 16, 18). Figure 1. Immunofluorescent image of a human head and neck squamous cell carcinoma xenograft tumor (SCCNij172) showing HIF-1a expression The glycolytic switch may seem counterintuitive, given in hypoxic regions as assessed by pimonidazole staining. Red, HIF-1a; the less efficient energy production by glycolysis com- green, pimonidazole; white, vessels; N, necrosis. Magnification 100. pared with mitochondrial oxidation. However, the m Scale bar represents 100 m. glycolytic intermediate glucose-6-phosphate is also used

A HIF-1α degradation under B Stabilization and activation of HIF-1 normoxia

Growth factor Mutations: HIF-1α Physiological stress: stimulation: VHL - Hypoxia → Hydroxylation EGFR - Oxygen and PI3K/AKT/mTOR by nitrogen species prolyl hydroxylase activity HIF-1α OH α Binding of HIF-1 VHL protein accumulation α HIF-1 OH HIF-1α β VHL HIF-1 Cytoplasm

Nucleus HIF-1α Degradation HIF-1β HRE DNA

pH Proliferation regulation Angiogenesis Glycolysis producing pyruvate and lactate

Figure 2. Schematic representation of the HIF-1 pathway. A, under normoxic conditions, HIF-1a is rapidly degraded because of hydroxylation of HIF-1a by prolyl hydroxylase and subsequent binding of the von Hippel-Lindau protein. B, physiologic stress (hypoxia and reactive oxygen and nitrogen species), mutations in the von Hippel-Lindau protein and PI3K/AKT/mTOR activity contribute to HIF-1a stabilization and accumulation. Upon stabilization of HIF-1a,itbindstoHIF-1b. This HIF-1 complex binds to the hypoxia responsive element (HRE) of the DNA, thereby initiating the transcription of its target genes. This results in a glycolytic tumor metabolism among others. The glycolytic end products pyruvate and lactate induce HIF-1a accumulation, which in turn increases HIF-1 activity. AKT, protein kinase B; EGFR, epidermal growth factor receptor; HIF-1, hypoxia-inducible factor 1; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol-3-kinase;VHL,vonHippel-Lindauprotein.

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Glucose Glucose + H HCO3- GLUT3 GLUT1 + CAIX H Fibroblast

Metalloproteinase NHE1 H2O CO2 Glycolysis Hyaluronidase Glucose H+ Degradation of HK H O + NADP+ NADPH 2 H extracellular H+ matrix and Glucose-6-P 6-P-gluconolactone 6-P-gluconate tumor-cell G6PD NADP+ migration Pentose Fructose-6-P phosphate shunt NADPH H+ Fructose-1,6-P 2 CO2 CO2 Erythrose-4-phosphate Glyceraldehyde-3-P TKL/TAL Ribose-5-phosphate Nucleotide and amino acid synthesis TCA Pyruvate required for proliferation Immune PDH LDHA escape ROS and free PDK1 Lactate radical Radioresistance scavenging Immune cell Metalloproteinase MCT4 Hyaluronidase

Lactate Fibroblast Angiogenesis

VEGF Endothelial cell

Figure 3. Tumor glucose metabolism and aggressive tumor behavior. HIF-1 activates transcription of transporters and enzymes regulating glycolysis and the pentose phosphate shunt. These transporters and enzymes are involved in glucose delivery into the cell (GLUT1 and GLUT3), phosphorylation of glucose to form glucose-6-phosphate (HK), conversion of glucose-6-phosphate into 6-phosphogluconolactone to support the pentose phosphate shunt (G6PD), conversion of pyruvate into lactate (LDHA), lactate transport (MCT4), and pH regulation (CAIX, NHE1). Furthermore, activation of PDK1 by HIF-1 inhibits the conversion of pyruvate to acetyl CoA, which results in a decreased flux through oxidative phosphorylation. Glycolysis is involved in aggressive tumor behavior because it causes radioresistance, creates a tumor microenvironment favorable for tumor cell migration, induces angiogenesis, and contributes to the immunologic escape of tumors. HIF-1–regulated transporters and enzymes are shown in orange; blocked lines indicate an inhibitory effect. CAIX, carbonic anhydrase IX; GLUT1, glucose transporter 1; GLUT3, glucose transporter 3; G6PD, glucose-6-phosphate dehydrogenase; NHE1, sodium-hydrogen exchanger 1; P, phosphate; P2, biphosphate; PDK1, pyruvate dehydrogenase kinase 1; PDH, pyruvate dehydrogenase; ROS, reactive oxygen species; TCA, tricarboxylic acid cycle; TKL/TAL, transketolase/transaldolase; HK, hexokinase; LDHA, lactate dehydrogenase A; MCT4, monocarboxylate transporter 4; VEGF, vascular endothelial growth factor. in the pentose phosphate pathway. This pathway synthe- Moreover, glycolytic metabolism in malignancies corre- sizes precursors of nucleotides and amino acids, which lates with radioresistance and will be described in detail are macromolecules required for tumor cell growth later (24, 25), and lactate accumulation predicts for metas- and proliferation. When cells do not require these macro- tases formation during follow-up in different tumor entities molecules for metabolism, intermediates of the pentose (26–28). The higher incidence of metastases could be phosphate pathway (fructose-6-phosphate and glyceral- explained by the fact that the glycolytic products lactate dehyde-3-phosphate) can be recycled back into glycolysis and acid induce secretion of matrix-degrading hyaluroni- to produce pyruvate and lactate (Fig. 3). During the dase and metalloproteinases by tumor-associated fibro- pentose phosphate pathway, CO2 is released, leading to blasts, creating a tumor microenvironment favorable for extracellular matrix acidification by carbonic anhydrase tumor cell migration (9, 15, 29, 30). Furthermore, lactate IX (CAIX; Fig. 3; refs. 18 and 23). inhibits the activity of dendritic cells and T cells, which

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contributes to the immunologic escape of tumors (31, 32). contribute to tumor cell death (45–48). Radiation-induced Finally, lactate is able to induce angiogenesis (Fig 3; ref. 29). endothelial apoptosis starts at exposure to single-dose radi- These findings indicate that a glycolytic tumor cell metab- ation of 8 to 10 Gy, being maximal at 20 to 25 Gy (45). olism contributes to aggressive tumor behavior. Tumor blood flow decreases after a single dose of 10 to 15 Gy with subsequently indirect killing of tumor cells. Doses higher than 15 to 20 Gy cause a marked and lasting HIF-1 and Radioresistance deterioration of the vasculature (49). In the clinical situa- Activation of the HIF-1 pathway after radiotherapy tion, radiation-induced vascular damage may thus contrib- Radiation-induced reoxygenation is the process by which ute to tumor response in case of high-dose hypofractionated the surviving hypoxic tumor cells become better oxygenated treatment regimens (45, 49). The effect of standard frac- after irradiation due to the aerobic population being killed. tionation using 1.5 to 2 Gy per fraction on endothelial cells As a result, the fraction of hypoxic tumor cells falls. This is a may be minor (49), albeit that this will also depend on the highly variable process within tumors, however, with the total dose delivered. final level of oxygenation depending on the degree of During fractionated radiotherapy, the tumor microvas- reoxygenation (1, 33). culature is protected through action of HIF-1. HIF-1 upre- This process elicits a cascade of HIF-1–related events. gulation stimulates tumor cells to produce VEGF and other Within hours postirradiation, intratumoral HIF-1 activity proangiogenic factors, which induce angiogenesis and pro- decreases due to von Hippel-Lindau–dependent HIF-1a tect the microvasculature from radiation-induced endothe- degradation under these reoxygenated conditions (34). lial apoptosis (36, 45, 50–52). Moreover, vasculogenesis is However, due to reoxygenation, ROS arise that induce responsible for the recovery of tumor blood flow and the HIF-1a stabilization (12). As a result, HIF-1 expression recurrence of glioblastomas after ionizing radiation in an in increases in a hypoxia-independent manner 18 to 24 hours vivo tumor model (43). Irradiation induces influx of bone after radiotherapy in in vivo mice studies (34–38). This marrow–derived cells. HIF-1–dependent upregulation of upregulation endures up to 1 week (38). In an in vivo tumor stromal-derived factor 1 (SDF1) and VEGF plays a crucial model, it was shown that some HIF-1 target genes are also role in the recruitment of these bone marrow–derived cells upregulated after irradiation, including vascular endothe- (43, 53). These cells promote neovascularization, thereby lial growth factor (VEGF) and CAIX (36–38). In these restoring the radiation-damaged vasculature and stimulat- preclinical studies, tumors were irradiated with 3 fraction ing regrowth of surviving tumor cells. Furthermore, VEGF of 5 Gy or single fractions of 5 to 8 Gy. However, during and SDF1 activate resident endothelial cells (10). In short, fractionated radiotherapy in clinical practice, these process- HIF-1 is responsible for vascular protection, recovery of es are likely to be much more complex with schedules of 25 tumor blood and nutrient supply, and tumor recurrence to 40 fractions of 1.8 to 2 Gy, delivered over several weeks. postirradiation by stimulating angiogenesis and/or vascu- Initially, the responses in the tumor microenvironment logenesis (Fig. 4). regarding reoxygenation, ROS, and HIF-1 upregulation may High HIF-1a expression correlates with poor locoregio- to some extent follow the patterns described earlier. How- nal control and an increased risk of tumor-related death in ever, with increasing cumulative dose toward the end of the patients with head and neck, cervical, and prostate cancer treatment, it is conceivable that there will be a progressive treated with radiotherapy (54–59). Preclinical research disregulation of the response pathways and disruptions of strongly suggests that HIF-1–mediated vascular protection the microenvironmental structure. It remains to be eluci- is also responsible for the tumor resistance to irradiation in dated whether HIF-1 upregulation by cycles of reoxygena- patients (36, 37, 60). tion-induced ROS can be sustained for several weeks. Several other reoxygenation-dependent mechanisms are HIF-1 inhibition, tumor microenvironment, and also able to upregulate HIF-1 and its downstream targets response to radiotherapy postirradiation, including depolymerization of cytoplasmic Several existing classes of drugs such as anthracyclines stress granules containing HIF-1–regulated mRNA tran- (doxorubicin), epidermal growth factor receptor (EGFR) scripts (36, 39, 40) and AKT/mTOR signaling (35). Further- inhibitors (cetuximab), and topoisomerase I inhibitors more, nitrogen monoxide (NO), which is a free radical (topotecan) inhibit HIF-1 activity and tumor xenograft produced by tumor-infiltrating macrophages, prevents HIF- growth (10). 1a from degradation after radiotherapy (41, 42). Without Topotecan inhibits HIF-1a mRNA translation (61) and continued treatment and due to renewed tumor progres- recently, the effect of this compound on the tumor micro- sion, after 1 or 2 weeks postirradiation, hypoxia will environment was studied in 16 patients with advanced solid increase again resulting in an increase in tumor HIF-1 levels tumors expressing nuclear HIF-1a (62). After treatment, (33, 43). nuclear HIF-1a expression decreased, becoming undetect- able in 4 of 7 available patients. Furthermore, the HIF-1 HIF-1 and vascular radiosensitivity downstream targets glucose transporter 1 (GLUT1) and Apart from the radiosensitivity of tumor cells as an VEGF decreased after HIF-1 inhibition. Decreased tumor important determinant of tumor response (44), the degree blood flow on dynamic contrast-enhanced magnetic reso- of radiation-induced microvascular destruction might nance imaging was seen in 7 of 10 patients after 1 cycle of

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Nutrient supply Glucose Recruitment of BMDC Vasculogenesis

GLUT1 VEGF SDF-1 Angiogenesis Glucose

TKTL1 TKTL1 G6P R5P E4P F6P G3P NADP+ NADPH

Glycolysis Pentose phospate pathway Nutrient supply

GSH GSSG Pyruvate ROS LDHA R. H2O Indirect . Lactate R action Ionizing DNA irradiation Direct Angiogenesis action MCT4

Endothelial Oxygen fixes DNA damage VEGF Lactate cell R˙ + O2 RO2˙ ROOH

Figure 4. HIF-1 pathway and tumor glucose metabolism as cellular defense mechanisms against radiotherapy. Ionizing irradiation induces production of DNA- damaging ROS and free radicals through a direct and indirect effect. Oxygen molecules are able to react with free radicals to yield a stable change in the chemical composition of the DNA damage. So, oxygen chemically "fixes" DNA damage and hypoxia interferes with the fixation of DNA damage. Another cellular defense mechanism against ionizing irradiation is the activation of the HIF-1 pathway. In response to HIF-1 upregulation after radiotherapy, tumor cells secrete VEGF and SDF1, which stimulate angiogenesis and vasculogenesis. This results in recovery of tumor blood and nutrient supply and tumor recurrence. An active HIF-1 pathway also leads to changes in tumor glucose metabolism with accumulation of lactate, pyruvate, gluthathione, and NADPH. These molecules effectively scavenge free radicals and ROS, thereby protecting the tumor cell from free radical–mediated DNA damage. Furthermore, lactate stimulates endothelial cells to produce VEGF, thereby contributing to angiogenesis. HIF-1–regulated growth factors, transporters, and enzymes are shown in orange. Blocked lines show the inhibitory effect of pyruvate, lactate, NADPH, and glutathione on free radicals and ROS. BMDC, bone marrow–derived cells; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; GLUT1, glucose transporter 1; G3P, glyceraldehyde-3-phosphate; GSH, glutathione (reduced form); GSSG, glutathione disulfide (oxidized form); MCT4; monocarboxylate transporter 4; R*, free radicals; R5P, ribose-5-phosphate; SDF1, stromal-derived factor 1; TKTL1, transketolase-like protein-1; G6P, glucose-6-phosphate; HK, hexokinase; LDHA, lactate dehydrogenase A; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor. topotecan. These results indicate that HIF-1a inhibition mRNA via suppression of the PI3K/AKT/mTOR pathway lowers the level of HIF-1a and the downstream targets and and Akt/NF-kB signaling (11). Furthermore, YC-1 regu- affects tumor vascularization in patients. Only one patient lates HIF-1a functional activity at the posttranslational in this study had a partial response lasting for 6 cycles of level (65). topotecan, which was accompanied by both a reduction in In in vivo mice studies, YC1-treated stomach, renal, and HIF-1a level and a decreased tumor blood flow (62). cervical carcinomas are smaller and less vascularized. These Newly developed drugs with HIF-1 inhibitory capacity tumors express lower levels of HIF-1a and VEGF with higher are YC-1 (3-[50-hydroxymethyl-20furyl]-1-benzyl inda- hypoxic fractions compared with control tumors (34, 66). zole) and PX-478 (S-2-amino-3-[40-N,N,-bis(chlor- Apparently, YC-1 halts tumor growth by blocking HIF-1 oethyl)amino]phenyl propionic acid N-oxide dihy- activity and subsequent vascularization (66). Treatment of drochloride). PX-478 is an agent that inhibits HIF-1a by tumor-bearing mice with radiotherapy followed by YC1 reducing HIF-1a mRNA levels, by inhibiting HIF-1a suppresses upregulation of HIF-1 activity, dramatically translation, and, to a lesser extent, by inhibiting HIF- increases radiation-induced vascular destruction, and 1a deubiquitination (63). YC-1 induces HIF-1a protein delays tumor growth (34, 36). Adversely, administration degradation (64), and it inhibits the translation of HIF-1a of YC-1 before radiotherapy increases tumor hypoxia and

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suppresses the therapeutic effect of irradiation (34). Also, In this way, tumor glucose metabolism is involved in the PX-478 radiosensitizes glioma tumor xenografts through production of reducing species, which protect the DNA inhibition of HIF-1–dependent proangiogenic signaling from free radical–mediated damage (Fig. 4; ref. 68). (38). Lactate accumulation has been related to radioresis- PX-478 was tested in 40 patients with advanced solid tance in tumor xenografts derived from human head and tumors in a phase I dose-escalation trial (NCT00522652; neck squamous cell carcinomas (24, 25). This lactate- ref. 67). Only a limited number of severe events were associated radioresistance was hypoxia independent, that reported, including severe thrombocytopenia without is, well-oxygenated high-lactate tumors were radioresis- bleeding (n ¼ 1), anemia (n ¼ 1), acute renal failure tant as well (24). Lactate is able to scavenge superoxide (n ¼ 1), hypotension (n ¼ 1), and elevated liver enzymes and hydroxyl radicals in vitro (74). Monocarboxylate (n ¼ 1). A relatively high proportion of patients (39%) transporters (MCT) are involved in lactate transport. achieved stable disease. This single-agent study excluded Therefore, MCT activity could influence the capacity of patients who received radiation therapy within 4 weeks tumors to counter oxidative or radical stress. The effect of before entry into the study. Therefore, no conclusions can MCT inhibition on the intracellular redox status has been be drawn about potential interactions of PX-478 treatment studied in human glioma cells (75). Gliomas are highly with radiotherapy. glycolytic and produce large amounts of lactate. Surpris- As HIF-1a is not expressed in all cancer cells in a tumor, ingly, when lactate efflux in glioma cells is blocked by inhibition of HIF-1 activity is probably not effective as a-cyano-4-hydroxycinnamicacid(ACCA),thelevelof monotherapy. Therefore, clinical trials should combine intracellular lactate and glutathione decreases, which is HIF-1 inhibition with radiotherapy and should focus on accompaniedbyenhancedradiosensitivity. Colen and optimal patient selection. For example, tumors showing colleagues suggested that pyruvate is redirected into mito- little or no hypoxia cannot undergo radiation-induced chondrial respiration as a consequence of lactate efflux reoxgenation and HIF-1 activation (37). Furthermore, inhibition (75). Because ROS are synthesized during establishing optimal timing of HIF-1 inhibitory treatment mitochondrial respiration, an increased rate of mitochon- relative to fractionated radiotherapy is a prerequisite for drial oxidation might exacerbate the ROS-induced DNA positive results. damage caused by radiotherapy (69, 75). Pyruvate can be reduced to acetate, thereby scavenging hydrogen peroxide, which is a source of hydroxyl radicals Tumor Glucose Metabolism and Radioresistance (68, 73). Despite the fact that pyruvate is theoretically an Tumor glucose metabolism, the cellular redox status, efficient radical scavenger, it does not predict the radiosen- and radioresistance sitivity of tumors in an in vivo tumor model (25). However, Hypoxic tumor cells are more resistant to radiotherapy the ratio lactate:pyruvate reaches 200:1 in malignancies as a consequence of the interference of hypoxia with the (68), possibly minimizing the antioxidant contribution of fixation of free radical–induced DNA damage (Fig. 4). pyruvate, and explaining the lack of correlation between Besides this protective mechanism, tumor cells counter pyruvate and radioresistance. the direct and indirect action of radiotherapy, that is In conclusion, redox adaptation is an important radiation-induced radical and oxidative stress, by upre- mechanistic concept that explains why cancer cells gulation of their endogenous antioxidant capacity become resistant to radiotherapy (69). Several preclin- through accumulation of pyruvate, lactate, and the redox ical studies have shown that tumor glucose metabolism couples glutathione/glutathione disulfide and NAD(P)H/ is likely to be involved in alterations of the cellular redox þ NAD(P) (68, 69). These molecules constitute an intra- status and radioresistance. Interfering with glucose cellular redox buffer network that effectively scavenges metabolism of tumor cells to reduce the levels of anti- free radicals and ROS, and are products of glucose metab- oxidant metabolites could therefore improve response olism (68, 70). to radiotherapy (29). The major pathways of glucose metabolism after the first step of glycolysis, that is, the conversion of glucose into Modulating tumor glucose metabolism and the cellular glucose-6-phosphate via action of hexokinase, are glycolysis redox status to overcome radioresistance and the pentose phosphate pathway (Fig. 4). Tumor cells, Tumor glucose metabolism can be targeted at several predominantly using glycolysis, produce pyruvate and lac- levels, directly via inhibiting enzymes and transporters þ tate and show an increase in the cytosolic NADH/NAD involved in glucose metabolism and indirectly by anti- ratio due to decreased NADH oxidation by oxidative phos- HIF-1 therapy (71). HIF-1 inhibition results in metabolic phorylation in the mitochondria (71). The pentose phos- changes with a decreased rate of glucose uptake and þ phate pathway regenerates NADPH from NADP . Quanti- lactate production in vitro (76) and an increase in oxygen tatively, glutathione is a very important cellular redox consumption, reflecting enhanced mitochondrial oxida- buffer. NADPH obtained from the pentose phosphate tion in in vivo mice studies (76, 77). As ROS are pro- pathway is required to keep glutathione in the reduced duced during mitochondrial oxidation, these metabolic state (68, 72). Furthermore, pyruvate contributes to the alterations could enhance the therapeutic efficacy of recovery of the glutathione pool after reoxygenation (73). radiotherapy (69).

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þ 2-Deoxyglucose competes with glucose for transmem- production, glutathione content, and NADPH/NADP brane transport. Hexokinase converts 2-deoxyglucose into ratio, as well as an increase in ROS-induced apoptosis. 2-deoxyglucose-6-phosphate, which cannot be further Lower amounts of these antioxidants may alter the ability metabolized within the cancer cell. Treatment with 2-deoxy- of cells to detoxify ROS (86). The cellular redox status can glucose is cytotoxic and radiosensitizes human glioma, also be modulated by directly targeting glutathione. Glu- cervical carcinoma, and pancreatic carcinoma cells tathione depletion followedbyradiotherapyresultsin (72, 78, 79). The combination of radiotherapy with 2- large areas of apoptosis and significantly enhances the deoxyglucose shows a trend toward increased growth delay sensitivity of xenografted head and neck squamous cell of pancreatic carcinomas in an in vivo tumor model, relative carcinomas and non–small cell lung carcinomas to irra- to either agent alone (72). This cytotoxic and radiosensiti- diation (87, 88). zing effect of 2-deoxyglucose is mediated by disruptions in Besides the alteration of the cellular redox status as glutathione metabolism and decreased NADPH content described earlier (75), Sonveaux and colleagues identified (72, 79), which reflects the close link between tumor another mechanism explaining the radiosensitizing effect glucose metabolism and an altered cellular redox status. of MCT inhibition (89). Lactate produced by glycolytic However, glycolysis inhibitors may improve the therapeutic hypoxic tumor cells can be taken up through MCT1 and efficacy of radiotherapy not only through disruptions in the used as a fuel for mitochondrial oxidation by aerobic cellular redox status, but also by depletion of ATP levels tumor cells (89–92). The use of lactate instead of glucose (71). Inhibition of GLUT1 and hexokinase decreases ATP by oxygenated cells saves glucose for the hypoxic cells to levels resulting in reduced cancer cell viability in vitro, which support glycolysis. MCT1 inhibition blocks lactate uptake inhibits tumor growth in in vivo tumor models (80, 81). and forces aerobic cells to use glucose for their energy Addition of ATP rescues GLUT1-inhibited cancer cells (81), metabolism. As a consequence, hypoxic cells located at suggesting that glycolysis inhibition has an anticancer effect largedistancefromthevasculaturearedeprivedofglu- partially through ATP depletion. cose and die. MCT1 inhibition induces extensive necrosis, Phase I/II clinical trials examined the effect of the com- decreases tumor hypoxia, and delays tumor growth in in bination of 2-deoxyglucose with hypofractionated radio- vivo tumor models. Combining MCT1 inhibition with therapy in patients with cerebral glioma (82, 83). 2-Deox- irradiation further delays tumor growth. Radiotherapy yglucose was given 30 minutes before every fraction of may complement MCT1 inhibition by eliminating the radiotherapy in a dose-escalation regimen (83). This treat- remaining oxygenated tumor cells in the vicinity of blood ment strategy was well tolerated without severe acute tox- vessels (89). icity and did not lead to any apparent increase in the late CAIX inhibition with indanesulfonamide (11c) also radiation damage to normal brain tissue (83). This regimen results in a significantly slowertumorgrowthinacolo- resulted in a moderate increase in survival with a signifi- rectal carcinoma tumor model. Tumor growth is further cantly improved quality of life (82). Daily administration of delayed by combining 11c with single-dose radiotherapy. 2-deoxyglucose alone and in combination with docetaxel However, in vitro incubation of colorectal carcinoma cells has very recently been tested in lung, breast, gastric, with 11c before irradiation does not enhance the effect of pancreatic, and head and neck cancer in a phase I dose- radiotherapy. Targeting CAIX under hypoxia reduces the escalation trial (NCT00096707), but results are not yet rate of extracellular acidification and proliferation and available. One other 2-deoxyglucose study has been termi- induces apoptosis of colorectal carcinoma cells (93). nated due to slow accrual (NCT00633087) and a third trial Therefore, CAIX inhibition might kill hypoxic cells, has been cancelled before enrollment of patients because of whereas radiotherapy attacks normoxic cells, resulting in logistic reasons (NCT00247403). the therapeutic enhancement of the combined treatment Another target of tumor glucose metabolism is the pen- strategy in the colorectal carcinoma tumor model. How- tose phosphate pathway, which is closely connected to ever, the effect of intracellular acidification on tumor glycolysis via the glycolytic intermediate glucose-6-phos- metabolism and the cellular redox status has not yet been phate. Lowering the amount of glucose-6-phosphate determined. through inhibition of hexokinase may reduce flux into the In conclusion, blocking tumor glucose metabolism at pentose phosphate pathway. The enzyme transketolase several levels has been shown to decrease the amount of (TKT) has a key role in the regulation of the pentose antioxidant molecules and to radiosensitize different solid phosphate pathway (84). Transketolase-like protein-1 tumors in preclinical studies. The administration of the (TKTL1) is significantly overexpressed in different human glycolysis inhibitor 2-deoxyglucose combined with hypo- cancers and is responsible for 60% to 70% of transketolase fractionated radiotherapy shows promising results in the activity in human hepatoma and colon cancer cells (84, 85). first phase I/II clinical trials without severe toxicity. How- TKTL1 increases the generation of fructose-6-phosphate and ever, daily use of antiglucose metabolism treatment for glyceraldehyde-3-phosphate, accompanied by significantly several weeks, which might be useful as an additive treat- enhanced levels of pyruvate and lactate in vitro. These data ment in combination with daily-fractionated radiotherapy, indicate that TKTL1 overexpression increases glycolysis (Fig. could cause more severe side effects. For example, the brain 4; ref. 22). Human colon carcinoma cells with suppressed is highly dependent on glucose for its energy metabolism. TKTL1 activity display reduced cell proliferation, lactate Therefore, more clinical studies examining the efficacy and

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side effects of antitumor glucose metabolism treatment are metabolism and predict response to radiation, is required required. for optimal patient selection.

Conclusions and Future Perspectives Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed. This review describes that the HIF-1 pathway is involved in the tumor-protective response to radiotherapy, both via Authors' Contributions vascular protection postirradiation and via enhancing the Conception and design: T.W.H. Meijer, J.H.A.M. Kaanders, P.N. Span, J. tumor antioxidant capacity through initiating a glycolytic Bussink Analysis and interpretation of data (e.g., statistical analysis, biosta- tumor metabolism. Targeting HIF-1 and tumor glucose tistics, computational analysis): T.W.H. Meijer, J.H.A.M. Kaanders, P.N. metabolism affects the tumor microenvironment, induces Span, J. Bussink metabolic alterations, and sensitizes various solid tumors to Writing, review, and/or revision of the manuscript: T. W. H. Meijer, J.H. A.M. Kaanders, P. N. Span, J. Bussink irradiation. Study supervision: J.H.A.M. Kaanders, J. Bussink Future studies should examine whether HIF-1 and glucose metabolism inhibitors are useful in clinical practice. Grant Support Special attention should be paid to the effect of HIF-1 This work was supported by METOXIA (Metastatic Tumors Facilitated by Hypoxic Tumor Micro-Environment; EU 7th Research Framework Pro- inhibition on the glycolytic and redox status of tumors, gramme - Theme HEALTH; Grant No. 222741). and optimal timing of these inhibitory treatments in rela- tion to radiotherapy. Furthermore, validation of imaging Received May 20, 2012; revised July 11, 2012; accepted August 28, 2012; techniques, which establish HIF-1 expression and glucose published online October 15, 2012.

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