CCR FOCUS Targeting Hypoxia, HIF-1, and Tumor Glucose Metabolism to Improve Radiotherapy Efficacy 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. Ó2012 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 Ó2012 American Association for Cancer Research. via more efficient mitochondrial oxidation. As hypoxia www.aacrjournals.org 5585 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2012 American Association for Cancer Research. CCR FOCUS 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 glycol- ysis 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 glycol- ysis or Warburg effect; refs. 9, and 16–18). HIF-1 initiates transcription of genes that encode trans- porters and enzymes regulating glycolysis and the pentose phosphate pathway (Fig. 3; refs. 8 and 19). The glycolytic products pyruvate and lactate induce HIF-1a accumula- tion (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 100Â. 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 © 2012 American Association for Cancer Research CCR Focus 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. 5586 Clin Cancer Res; 18(20) October 15, 2012 Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2012 American Association for Cancer Research. HIF-1, Glucose Metabolism, and Resistance to Radiotherapy 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 © 2012 American Association for Cancer Research CCR Focus 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