Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Effects of HIF-1

Effects of HIF-1 Hindawi Publishing Corporation Journal of Oncology Volume 2010, Article ID 757908, 14 pages doi:10.1155/2010/757908 Research Article Effects of HIF-1α and HIF2α on Growth and Metabolism of Clear-Cell Renal Cell Carcinoma 786-0 Xenografts 1, 2, 3 4, 5 2 4, 5 1 Swethajit Biswas, Helen Troy, Russell Leek, Yuen-Li Chung, Ji-liang Li, 1 2 2 2 4, 6 Raju R. Raval, Helen Turley, Kevin Gatter, Francesco Pezzella, John R. Griffiths, 4, 6 1 Marion Stubbs, and Adrian L. Harris Weatherall Institute of Molecular Medicine, University of Oxford, John RadcliffeHospital, Oxford OX39DU,UK CR UK Tumour Pathology Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John RadcliffeHospital, Oxford OX39DU,UK Northern Institute for Cancer Research (NICR), Newcastle University, Freeman Hospital, Newcastle-Upon-Tyne NE7 7DN, UK CR UK Biomedical Magnetic Resonance Research Group, Division of Basic Medical Sciences, St. George’s, University of London, London SW17 0RE, UK CRUK Clinical Magnetic Resonance Research Group, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK CRUK Cambridge Research Centre, Li Ka Shing Centre, Robinson Way, Cambridge CB2 ORE, UK Correspondence should be addressed to Marion Stubbs, [email protected] Received 16 September 2009; Revised 9 March 2010; Accepted 22 April 2010 Academic Editor: Dominic Fan Copyright © 2010 Swethajit Biswas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In cultured clear-cell renal carcinoma (CCRCC) 786-0 cells transfected with HIF1α (HIF-1+), HIF-2α (HIF-2+), or empty vector (EV), no significant differences were observed in the growth rates in vitro,but when grown in vivo as xenografts HIF- 2α significantly increased, and HIF-1α significantly decreased growth rates, compared to EV tumors. Factors associated with proliferation were increased and factors associated with cell death were decreased in HIF-2+ tumors. Metabolite profiles showed higher glucose and lower lactate and alanine levels in the HIF-2+ tumors whilst immunostaining demonstrated higher pyruvate dehydrogenase and lower pyruvate dehydrogenase kinase 1, compared to control tumors. Taken together, these results suggest that overexpression of HIF-2α in CCRCC 786-0 tumors regulated growth both by maintaining a low level of glycolysis and by allowing more mitochondrial metabolism and tolerance to ROS induced DNA damage. The growth profiles observed may be mediated by adaptive changes to a more oxidative phenotype. 1. Introduction (ETC) activity by altering the subunit composition of COX, minimising ROS generation [7]. In contrast, HIF-2α is The HIFα transcription factors, HIF-1α and HIF-2α, mediate uniquely involved in tumor growth and cell cycle progression adaptive responses to tumor hypoxia, as well as regulating through interaction with c-Myc [8, 9]. The most direct link between genetic events that pre- an extensive transcriptional response involving the induction of genes for angiogenesis, glucose metabolism/cellular ener- dispose to cancer and activation of the HIF pathway is getics, cell growth, metastasis, apoptosis, and extracellular observed in tumors associated with inactivation of the von matrix (ECM) remodelling [1]. HIF-1α and HIF-2α, despite Hippel-Lindau (VHL) tumor suppressor gene, particularly some overlapping effects, can uniquely regulate distinct VHL-associated clear-cell renal cell carcinoma (CCRCC) (for genes [2]. HIF-1α for example is primarily involved in review see [10]). The pseudohypoxic VHL-defective CCRCC glucose metabolism by upregulating glycolytic enzymes [3, cells show a bias toward HIF-2α, and overproduction of 4] whilst limiting pyruvate uptake by the mitochondria HIF2α (but not HIF1α), has been found to be sufficient to override the tumor suppressor function of VHL in xenograft [5, 6] and down regulating the electron transport chain 2 Journal of Oncology studies [11–13]. HIF-2α expression (in a mouse model of in pyruvate dehydrogenase (PDH) expression. This, together CCRCC) is necessary for the development of the typical with higher glucose and lower lactate and alanine levels clear-cell phenotype, demonstrating the important role of found in the HIF-2+ tumors (compared to both EV and HIF-2α in CCRCC [14]. HIF-1+ tumors), results in a more oxidative, DNA damage- Using retroviral transfection in 786-0 cells, Raval et tolerant phenotype that supports enhanced tumor growth, al. [12] confirmed that exogenous expression of HIF- similar to the Sporadic VHL-deficient clinical subtypes of 1α upregulated transcriptional products involved in pH CCRCC described by Gordan et al. [21]. regulation (CAIX) and cell fate (BNIP3), whereas expression of HIF-2α upregulated a different set of proteins which 2. Materials and Methods were involved in cell proliferation (cyclin D1), cell growth (TGF-α), and angiogenesis (VEGF). However, the in vitro 2.1. Human Clear-Cell Renal Cancer Cell Lines. Stable studies demonstrated no differences in the proliferation rate polyclonal pools of G418 selected 786-0 renal cancer cells between 786-0 cells that either exogenously expressed HIF1α expressing the relevant HIFα isoform (HIF-1+) were pro- (HIF-1+), overexpressed HIF-2α (HIF-2+) or were infected duced in vitro from the same pool as previously described with empty vector (EV) (control cells). But when these [12]. Polyclonal pools retrovirus-infected with the pBMN- manipulated 786-0 cells were grown in vivo as xenografts, a Z-IRES-Neo-based HIF-1α,HIF-2α (HIF-2+), or control different tumor growth profile emerged showing that HIF- (EV), were released by trypsinization and subsequently 2α caused significantly increased growth rates and HIF-1α resuspended in PBS. caused significantly decreased growth rates when compared to EV tumors. Similar findings (HIF-2α facilitating tumor 2.2. Xenografts. 786-0 cells (1 × 10 ) transfected with HIF- growth in vivo) have also been made in two nonepithelial 1or 2α or control in 100 μl PBS were injected subcutaneously tumors, teratoma [13] and neuroblastoma [15] in vivo. into the dorsal flanks of nu/nu mice. Three cohorts were However, apart from the growth curves, few studies have generated. Two consisting of 21 mice with 7 in each group been reported that investigate the role of HIF-2α in epithelial (HIF-1+, HIF 2+ and EV) for growth, immunohistochem- cancers in vivo (for review see [16]). Most epithelial cancer istry and histology; and one consisting of 15 mice with 5 cells rely on HIF-1α transcriptional products to mediate in each group (HIF-1+, HIF 2+ and EV) which were grown tumor metabolism including the effect first described by to at least 500 mm , the minimum size possible for in vivo Warburg [17] more than 80 years ago that leads to the repro- Magnetic Resonance Spectroscopy experiments. gramming of tumor cells from mitochondrial respiration Tumor size was measured bidirectionally in all experi- to aerobic glycolysis (see [18–20] for review). The human ments, twice per week using calipers, with the longest dimen- VHL -/- clear-cell renal cancer cell line, 786-0, provides a sion (l) and shortest dimension (s) measured postimplan- model for investigating the effects of both HIFα isoforms, tation. Tumor volume (mm ) was calculated by measuring particularly HIF-2α, on tumor growth and metabolism in length, width and depth using callipers and 1∗w∗d∗(π/6). vivo, since it constitutively expresses only HIF-2α. To further understand the role of HIF-2α in vivo,wehaveinvestigated 2.2.1. Tumor Processing. The mice from cohort (1 and 2) the effects of HIF-2α expression not only on CCRCC 786-0 were sacrificed by cervical dislocation at day 42, the tumors tumor growth, but also on metabolic adaptation to tumor bisected with one-half snap-frozen for storage at −80 C, progression by using Magnetic Resonance Spectroscopy and the other half-embedded in paraffin. The mice from (MRS) methods both noninvasively in vivo and ex vivo on cohort (3) were anesthetized with a single i.p. injection of tumor extracts. a Hypnovel/Hypnorm/water (1 : 1 : 2) mixture as previously The rationale for using 786-0 line in our experiments described [22] prior to the MR experiment (details below). is that CC-RCC comes in two HIF types that is HIF-2 only At the end of the experiment the tumors were freeze-clamped and HIF-2 + HIF-1. Therefore, using a CC-RCC, HIF-2 only and stored at −80 C. Subsequently the frozen tumors were expressing cell line is appropriate to investigate the role of extracted in 6% perchloric acid, as previously described HIF-1 in CC-RCC tumour growth/biology as well as the [23] and the neutralized extracts were freeze-dried and effects of HIF-2 overexpression on an endogenous HIF-1 reconstituted in 1 ml deuterium oxide for high resolution null background (see [21]). Expression of HIF-2α resulted MRS. Cyrostat sectioning of frozen tumors was performed in a significant increase in tumor growth rate similar to for mouse CD31 staining as previously described [24], and that observed previously [12] whilst HIF1+ tumors grew Fuhrman’s criteria were applied to histological grading for even more slowly than EV tumors. Immunohistochemistry characterization of nucleoli morphology [25]. (IHC) was chosen rather than Western Blotting because the necrosis and heterogeneity of in vivo tumors causes poor reproducibility. IHC allows heterogeneity to be scored 2.2.2. Immunohistochemistry. Tumors were prepared for and the extent of protein expression to be determined in a immunohistochemistry as follows. Briefly, endogenous tissue semiquantitative fashion. Using a combination of immunos- peroxidase activity was blocked using two drops of 0.3% 1 31 taining and/or Hor P Magnetic Resonance Spectroscopy hydrogen peroxide (1 : 100 dilution of 30% H O stock 2 2 (MRS), we also demonstrate that expression of HIF-2α (BDH Laboratory Supplies, Poole, UK) in distilled water), decreased the expression of HK-II, LDH5 and pyruvate to cover each section. After two rounds of immersion dehydrogenase kinase 1 (PDK1) with a concomitant increase in PBS, for 5 minutes each, 2.5% normal horse serum Journal of Oncology 3 (Normal horse serum concentrate—Vector Laboratories Inc, University of London, and tumors were positioned in the 1 31 California, USA, diluted in PBS) was applied to each section center of a 15-mm two-turn Hsurface or P MRS coil. for 30 minutes at room temperature to prevent nonspecific Voxels were selected from scout gradient echo images, primary antibody binding. and localized shimming yielded linewidths of the order Primary antibodies were directed against HIF-1α,HIF- of 20–30 Hz. The PRESS localization method with water 2α and CAIX; mouse monoclonals (ESEE122), (237/B5) and suppression with a repetition time of 2 seconds was used (M75), respectively, University of Oxford. Ki67, Cyclin D1: to detect choline [30]. For P MRS, image selected in mouse monoclonals (MIB-1) and (DSC-6), and GLUT-1: vivo spectroscopy (ISIS) [31] localised spectra of tumors rabbit polyclonal; DAKO, Ely, UK: Cleaved caspase-3; rabbit were acquired. MRUI software was used for all spectral monoclonal, R+D Systems, UK; BNIP3: mouse monoclonal processing programs, including preprocessing, fitting and (ANa40) SIGMA, UK; CD10: mouse monoclonal (56C6), quantification of peak areas of the observed metabolites. Abcam, UK; VEGF: SP28, rabbit monoclonal antibody, H MR spectra of the neutralised tumor extracts were Abcam,UK; Phospho-Ser Akt and PTEN: rabbit mon- obtained using a Bruker 600 MHz spectrometer (pulse angle oclonals 736E11 and 138G6 respectively, Cell Signalling, 45 ; repetition time, 3.5 seconds). The water resonance USA; Hexokinase-II: rabbit polyclonal, Chemicon, USA; was suppressed by gated irradiation centred on the water LDH5: sheep polyclonal, Abcam, UK; PDH E2 Complex: frequency. 25 μl of 10 mM Sodium 3-trimethylsilyl-2,2,3,3- mouse monoclonal (15D3), Invitrogen, USA; PDK-1: goat tetradeuterpropionate (TSP) was added to the samples for polyclonal, Santa Cruz, USA; TOM-20: mouse mono- chemical shift calibration and quantification. The pH was re- clonal (F10),Santa Cruz,USA;PGC-1 β:rabbitpoly- adjusted to pH 7 prior to HMRS. clonal, Santa Cruz, USA; γH2AX (phospho-Ser Histone H2A.X): Rabbit Monoclonal, 20E3, Cell Signaling, USA; 2.3.1. Statistics. For analysis of the immunohistochemical 8-Hydroxyguanosine (8-OH-G): goat polyclonal, Alexis expression of individual proteins between all tumor groups, Biochemicals, Nottingham, UK; OGG1 Rabbit polyclonal the nonparametric Kruskal-Wallis (ANOVA) test was used. (ab204) Abcam, UK. Results from one cohort were displayed as histograms with The Envision-HRP ChemMate polymer kit (DAKO, Ely, standard error of the mean (SEM) in the figures. Dunn’s UK) was used for detection of either mouse and rabbit post hoc test for all data sets was calculated if P< .05 monoclonal or rabbit polyclonal primary antibodies, as and displayed in the figures. Immunohistochemical protein per the manufacturer’s instructions. Where applicable, the expression between specific pairs of 786-0 tumor groups relevant secondary antibodies of antigoat (P0160, DAKO, Ely, was compared using the Mann-Whitney unpaired t-test, UK), antisheep (ab6747, Abcam, UK) and antirat (P0450, where mentioned in the text. The Spearman rank testing was DAKO,Ely,UK) were used. used to demonstrate correlations between non-parametric The majority of primary antibodies were detected with variables. For the MRS data a two-tailed t test was used a 3,3-diaminobenzidine (DAB+) chromogenic substrate sys- for significance levels. Significant results were designated if tem as part of the Envision kit. Nuclei were counterstained P< .05. with Haemotoxylin before mounting onto plastic coverslips with AquaMount (Gurr GmBH, Strasbourg, Germany). 3. Results For Ki67 (MIB-1 clone) detection, ChromogenSG (Vector Laboratories Inc., California, USA) was used and the nuclei 3.1. Effect of Transfection of Specific Retroviral HIFα Isoform in counterstained with Nuclear Fast Red (Sigma-Aldrich, St. CCRCC 786-0 Xenografts. We have previously demonstrated Louis, USA). Sections treated in both these ways were dehy- [12] that the appropriate HIFα isoform protein is expressed drated through methanol and xylene, before haemotoxylin after retrovirally-mediated infection of specific HIFα isoform counterstaining and mounting with DPX. constructs within a bicistronic IRES-neo cassette, in vitro. These in vitro findings were confirmed in vivo in tumors 2.2.3. Assessment of Tumor Immunostaining. Each tumor grown subcutaneously as xenografts in mice in all three section was assessed blindly and independently by two groups. HIF-1α expression was identified only in the HIF-1+ observers. Photomicrographs were taken at x100 hpf. Semi- tumors, and only within the nuclear compartment, whereas quantitative analysis of protein expression was performed the EV and HIF-2+ tumors showed no staining for HIF-1α using a modified “Histoscore” method, as previously (Figures 1(a)–1(d)). described [26]. For Ki67 and Cleaved caspase-3 scoring, positive and negatively stained cells within 5 individual However, HIF-2α expression was identified in both tumor areas, consisting of 100 cells each, were scored [27]. nuclear and cytoplasmic compartments in all 3 tumor Tumor necrosis was quantified as the % area of tumor groups (Figures 1(e)–1(h)), but showed a significant increase replaced by necrosis, as identified by light microscopy [28]. only within the nuclear compartment of HIF-2+ tumors CD31 Chalkley Vessel Count (CVC) was the average value (Figure 1(g)). No significant changes in HIF-2α expression from the three fields [29]. were seen in either compartment of the HIF1+ or the EV tumors (Figures 1(e)–1(f)). A further cohort (not shown in 2.3. Magnetic Resonance Spectroscopy (MRS). Anesthetized figures) of in vivo tumors confirmed the findings of HIF-1 mice were placed in the bore of a Varian 4.7 T nuclear and HIF-2α expression as well as a similar growth pattern in magnetic resonance (NMR) spectrometer at St. George’s the 3 tumor types. 4 Journal of Oncology EV HIF-1+ HIF-2+ Nuclear HIF-1α HIF-1α EV HIF-1+ HIF-2+ (a) (b) (c) (d) Nuclear HIF-2α HIF-2α EV HIF-1+ HIF-2+ (e) (f) (g) (h) (i) H+E (j) CD10 (k) PTEN (l) AKT (m) Phospho-Ser AKT Figure 1: Features of CCRCC 786-0 tumors. (a–d) (x100 hpf) HIF-1α nuclear staining only ( P< .0001). (e–h) Differential HIF-2α nuclear ∗ + staining ( P = .0004). (i) Clear-cell/sarcomatoid morphology (x40 hpf). (j) CD10 staining typical of clear-cell RCC lineage (x100 hpf). (k) PTEN staining; positive staining only identified in murine fibroblasts (x100 hpf). (l) Akt staining, (m) Phospho-Ser Akt staining (highest in HIF-2+ tumors). P values were calculated using an ANOVA test. 3.2. Effect of HIFα Isoform Expression on Grade and Phenotype demonstrating sarcomatoid dedifferentiation. There were no of CCRCC 786-0 Xenografts. 21 tumors were evaluated differences in Fuhrman’s grade between the three tumor after H+E staining, and each exhibited a high Fuhrman’s groups (a representative example is shown in Figure 1(i)). tumor grade of either 3 or 4, with the majority of tumors Only one tumor (an EV tumor) was morphologically wholly HIF-1α histoscore HIF-1α histoscore Journal of Oncology 5 clear-cell and one tumor (HIF-2+) was completely replaced with sarcomatoid de-differentiation which is a clinically recognised variant of high grade tumors. However despite their sarcomatoid de-differentiation the tumours retained expression of typical clear-cell renal cancer markers such as CD10 (Figure 1(j)), pancytokeratin and vimentin positive expression, on a CK-7 negative background (data not shown). 786-0 cells were PTEN negative (Figure 1(k)), but the surrounding murine fibroblasts demonstrated positive staining. This finding was confirmed by the high levels of phospho-Ser (activated) Akt expression in the 786-0 473 0 0 102030405060 70 cells of the EV tumors (Figure 1(l) and 1(m)). Expression Time (days) of activated Akt was increased in the HIF-2+ tumors compared to EV and HIF-1+ tumors. This may be because 7860-EV expression of TGFα was increased in the HIF-2+ tumors 7860-HIF-1 with a similar increase in activated EGFR (Tyr -EGFR) 7860-HIF-2 expression, compared with EV and HIF-1+ tumors (data not (a) shown). 3.3. Effect of HIFa Expression on Tumor Proliferation and Apoptosis in 786-0 Xenografts. In contrast to the growth patterns in vitro where the 3 cell types demonstrated similar proliferation rates [12], growth patterns in vivo showed that there were significant differences between HIF-1+, HIF-2+ and EV 786-0 tumors (Figure 2(a)). The differences in overall growth between the 3 tumor groups were dependent on the lag phase for each tumor group as well as the rate of 5 tumor growth. The HIF-2+ tumors had the shortest lag phase (21 days) followed by the EV tumors (27 days) with the HIF-1+ tumors taking the longest time (>32 days). Once HIF-2+ HIF-1+ EV the lag phase was over, the actual rates of growth were 45 ± 5.4mm /day for HIF-2+ (P = .09 compared to EV), (b) 35 ± 3.3mm /day for EV tumors (P = .0007 compared to Figure 2: (a) Growth curve kinetics of 786-0 CCRCC tumors in HIF-1) and 18±4.0mm /day for HIF-1+ tumors. There were ∗ ∗ vivo. (b) Areas of tumor necrosis (%) P >.1. P values were no significant differences in necrosis between the different calculated using an ANOVA test. tumor types (Figure 2(b)). The EV tumors demonstrated the highest Ki67 (MIB- 1%) proliferation rates in comparison to both the HIF- 1+ and HIF-2+ tumors (P = .0006) (Figures 3(a)–3(d)). the HIF-2+ tumors (Figures 4(a)–4(d)) in comparison to Cyclin D1 expression however was highest in the HIF- the other two tumor groups (P = .01), in contrast to 2+ tumors (P = .0010) (Figures 3(e)–3(h)). The overall the in vitro findings by Raval et al. [12]. Expression of rates of apoptosis measured by cleaved-caspase 3 were very two glycolytic enzyme proteins, HK-II (Figures 4(e)–4(h)) low (<0.5%) in all the tumor groups (Figure 3(i)–3(l)). and LDH5 (Figures 4(i)–4(l)), was significantly lower in the The HIF-1+ tumors had the highest rate of apoptosis (∼ HIF-2+ tumors in comparison to both the HIF-1+ and EV 0.4%) compared to controls (P = .0002), whereas the HIF- tumors, whereas there was no difference in the expression 2+ tumors had only 0.1% compared to the EV tumors of these glycolytic enzymes between HIF-1+ tumors and EV with ∼0.25%. Because the apoptotic rates were so low, tumors. we also considered potential regulators of alternative death pathways, such as BNIP3, which has been implicated in 1 31 3.5. Metabolites Measured by H MRS and P of 786-0 cancer cell autophagy [32, 33]. The intergroup expression of Xenografts and in Tumor Extracts. HMRS of in vivo tumors BNIP3 demonstrated that HIF-2+ tumors showed the lowest demonstrated higher levels of free choline (which resonates expression and HIF-1+ tumors the highest (P = .0002) at ∼3.2 ppm) in the HIF-2+ tumors (Figure 4(m))compared (Figures 3(m)–3(p)). However, as previously mentioned to HIF-1+ and EV tumors. After in vivo scanning the tumors there were no significant differences in the level of tumor were freeze-clamped and metabolites were measured at high necrosis between the different groups (Figure 2(b)). field in tumor extracts (which gives better resolution than in vivo)by HMRS (Figure 4(n) and Table 1). The MR spectra 3.4. Effect on Factors Related to Glucose Metabolism; Glut- shown in Figure 4(n) are representative samples of various 1, HKII, LDH. Expression of GLUT-1 was attenuated in spectral regions of the high resolution spectra obtained from Tumour size (mg) Necrosis (%) 6 Journal of Oncology EV HIF-1+ HIF-2+ Ki67 (MIB-1 %) proliferation Ki67 EV HIF-1+ HIF-2+ (a) (b) (c) (d) Cyclin D1 Cyclin D1 EV HIF-1+ HIF-2+ (e) (f) (g) (h) Cleaved caspase-3 % apoptosis 0.5 0.4 0.3 Cleaved 0.2 caspase-3 0.1 EV HIF-1+ HIF-2+ (i) (j) (k) (l) BNIP3 BNIP3 EV HIF-1+ HIF-2+ (m) (n) (o) (p) Figure 3: (x100 hpf). Growth and death markers in CCRCC 786-0 tumors. (a–d) Ki67 proliferation index ( P = .0006). (e–h) Cyclin D1 ∗ ∗ expression ( P = .001). (i–l) Apoptosis as measured by cleaved-caspase-3% index ( P = .0002). (m–p) BNIP3 expression (cytoplasm only) ∗ ∗ ( P = .0002). values were calculated using ANOVA test. the extracts of each tumor type. The significantly higher analysis [34], similar values for intracellular pH (pHi) were levels of choline/phosphocholine (PC) found in extracts of found in all 3 tumor groups. Similar to the in vivo results, HIF-2+ tumors reflected the raised choline found in the no differences were observed in the high energy phosphates 1 31 tumors in vivo by H MRS. In vivo PMRS of the tumors (ATP+ADP) between the different tumor types (Table 1). showed no significant differences between the parameters Signals from glucose, creatine (tCr), and taurine were ATP, PME, PDE, Pi (data not shown). Using Pi spectral shift also significantly higher in the HIF-2+ tumors, whereas MIB-1 (%) Cyclin D1 histoscore BNIP3 histoscore Cleaved caspase-3 (%) Journal of Oncology 7 Membranous GLUT-1 EV HIF-1+ HIF-2+ 4 ∗ GLUT-1 EV HIF-1+ HIF-2+ (a) (b) (c) (d) Hexokinase-II HK-II EV HIF-1+ HIF-2+ (e) (f) (g) (h) LDH5 LDH5 EV HIF-1+ HIF-2+ (i) (j) (k) (l) PC Lactate tCr Glucose Alanine HIF-2+ HIF-1+ HIF-2+ HIF-1+ EV EV 5.45.23.9 3.25 3.2 1.51.4 4 3 2 (ppm) (ppm) (ppm) (ppm) (ppm) (m) (n) Figure 4: Metabolism-related markers and metabolic profiles of CCRCC 786-0 tumors (x100 hpf). (a–d) GLUT-1 expression( P = .01). ∗ ∗ 1H (e–h) Hexokinase-II expression ( P = .0006). (i–l) LDH5 expression ( P = .004), (m) In vivo MRS of 786-0 tumors. (n) High-resolution 1H MR Spectra of tumor extracts. P values were calculated using an ANOVA test. HK-II histoscore LDH5 histoscore GLUT-1 histoscore 8 Journal of Oncology 4. Discussion Table 1: Metabolite levels measured by H MRS in 786-0 tumor extracts. The tumor grade of 786-0 tumors does not alter with Metabolite EV HIF-1+ HIF-2+ differential HIFα isoform expression on a HIF-2α-only expressing background, whether grown as cultured cells or Leucine 0.13 ± 0.01 0.17 ± 0.02 0.17 ± 0.01 as xenografts that demonstrate a high grade phenotype and Iso Leucine 0.06 ± 0.01 0.08 ± 0.005 0.08 ± 0.005 characteristic morphology. Although the patterns of HIFα a,b Lactate 5.13 ± 0.85 5.43 ± 0.51 2.54 ± 0.58 isoform expression in vivo were similar to those found in the a,b Alanine 0.84 ± 0.06 0.88 ± 0.05 0.63 ± 0.04 CCRCC 786-0 cells in vitro [12], there were some differences between the levels of specific transcription factors expressed Choline 0.17 ± 0.02 0.22 ± 0.04 0.29 ± 0.05 in vitro and in vivo. The expression of BNIP3, cyclin D1, a,b PC 0.73 ± 0.09 0.66 ± 0.14 1.14 ± 0.05 TGFα andVEGFinthe in vivo model were similar to HIFα Taurine 13.96 ± 1.55 13.02 ± 0.92 16.70 ± 0.66 isoform expression found in vitro. However the expression a,b of GLUT-1 was comparatively lower in the HIF-2+ tumors Cr 1.26 ± 0.14 1.46 ± 0.15 2.00 ± 0.17 a in vivo (see below for discussion) consistent with a more Glucose 0.63 ± 0.10 0.73 ± 0.12 1.19 ± 0.29 oxidative phenotype. ATP+ADP 0.91 ± 0.20 0.95 ± 0.13 1.14 ± 0.15 Metabolites expressed as μmol/g wet weight tissue (n = 3–5). denotes 4.1. Tumor Growth and Related Death Pathways. This in vivo statistically significant different from EV and denotes statistically signif- study showed that the growth of CCRCC 786-0 tumors was icant difference (P< .05) from HIF-1+. A two-tailed t test was used for biphasic, with an initial growth lag phase followed by growth significance levels. acceleration. The HIF-1+ tumors, which were overall the slowest growing of the three groups, had the longest lag phase whereas the EV tumors started to grow at day 27, and alanine and lactate were significantly lower compared to the the HIF-2+ tumors at day 21. The lag times and growth HIF-1+ and EV tumors. This is more clearly demonstrated in rates in vivo were similar to those observed previously [12]. the detailed analysis of the metabolites shown in Table 1 and These differences in early growth may reflect stress of a poor described below. These data imply a more oxidative and less blood supply which could have affected early establishment glycolytic phenotype for the HIF-2+ tumors. of the tumors, since the HIF-2+ tumors had the highest levels of CD31 angiogenesis and VEGF, but the shortest initial growth lag phase compared to EV and HIF-1+ tumors. 3.6. Effects on Factors Related to Mitochondrial Regulation Tumor growth is a balance between cellular proliferation and Free Radical Damage; PDH, PDK-1, TOM-20, 8- and cell death. The increased levels of cyclin D1, an OH-Guanosine and OGG1. PDH (Figures 5(a)–5(d))was important regulator of cell cycle progression, were seen in upregulated and PDK-1 (Figures 5(e)–5(h)) down-regulated the faster growing HIF-2+ tumors, but surprisingly they in the faster growing HIF-2+ tumors. Higher expression had the lowest proliferation index (Ki67) and very low levels of the cellular mitochondrial load marker, TOM-20 levels (<0.5%) of apoptosis in vivo. This may be the result (Figures 5(i)–5(l)) was also seen in the HIF-2+ tumors of two independent background factors. Activated Akt is and in turn this was mirrored by an increase in expression constitutively expressed in the 786-0 xenografts, due to the of the mitochondrial biogenesis regulator, PGC-1β (data PTEN -/- status, facilitating tumor growth [35]and an not shown). Overall, this is consistent with an increase in antiapoptotic phenotype [36]. Since the levels of necrosis mitochondrial biosynthesis and activity. were similar between tumor groups, alternative cell death The HIF-2+ tumors were also under a comparatively mechanisms, such as autophagy, were considered to explain greater degree of oxidative stress, as manifest by higher the differences in growth between the tumor types. BNIP3 levels of 8-OH-guanosine staining compared to the other levels were significantly lower in HIF2+ tumors in vivo, two tumor groups (Figures 6(a)–6(d)). However immunos- and were consistent with the in vitro results of Raval et taining of γH2A.X (Figures 6(e)–6(h)) showed lower levels al. [12] showing that over-expression of HIF-2α attenuated indicating less DNA damage in HIF2+ than in HIF1+ or EV BNIP3 expression. Since both HIF-1+ and EV tumors had tumors. Expression of OGG1 (a DNA repair enzyme) was significantly higher levels of BNIP3, and since their levels higher in the HIF-2+ tumors compared to HIF-1+ and EV of apoptosis were very low, we hypothesize that BNIP3 tumors (Figures 6(i)–6(l)). induces autophagic cell death in this 786-0 model as a default death mechanism. In addition, phosphocholine and 3.7. Effects on Factors Related to Neoangiogenesis. VEGF, glycerophosphocholine were highest in the HIF2+ tumors identified only in the cytoplasm of tumor cells, was higher compared to HIF-1+ and EV tumors. Usually (although not in the HIF2+ tumors compared to HIF1+ and EV tumors always [37]) high levels of PC and GPC are associated with (Figures 7(a)–7(d)). The Chalkley Vessel Count (CVC) using increased proliferation and growth, but in the present study an anti-mouse CD31 antibody, was also higher in the HIF-2+ the HIF-2+ tumors had lower proliferation (Ki67) but higher tumors compared to both HIF-1+ and EV groups (Figures growth rates, compared to controls. The findings in the HIF- 7(e)–7(h)), which was consistent with the pattern of VEGF 2+ tumors combined with low apoptosis and autophagy are expression. in contrast to the tumor suppressor effects reported in both Journal of Oncology 9 PDH EV HIF-1+ HIF-2+ PDH EV HIF-1+ HIF-2+ (a) (b) (c) (d) PDK-1 PDK-1 EV HIF-1+ HIF-2+ (e) (f) (g) (h) TOM-20 TOM-20 EV HIF-1+ HIF-2+ (i) (j) (k) (l) Figure 5: Markers of oxidative phosphorylation and mitochondrial load in CCRCC 786-0 tumors. (a–d) PDH expression ( P = .003). (e–h) ∗ ∗ PDK-1 expression ( P = .006). (i–l) TOM-20 (mitochondrial marker) expression ( P = .004). P values were calculated using an ANOVA test. neuroblastoma [38]and coloncancer[39] xenograft models, rat GS9L orthotopic model, the tumor suppressive effect of as well as a rat GS9L orthotopic glioma model [40]. HIF-2α over-expression was caused by apoptosis [40]. This discrepancy in the growth profile between the 786- However in the CCRCC 786-0 model, we suggest that 0 CCRCC model and other non-CCRCC model systems over-expression of HIF-2α regulates growth both by main- may lie in the different HIFα backgrounds of the parental taining some glycolysis, albeit at a lower level, allowing cell lines which are different. The 786-0 CCRCC cell line more mitochondrial metabolism (higher PDH, lower PDK) only expresses HIF-2α, whereas both the N1E-115 neurob- and tolerance to DNA damage (γH2A.X) resulting from lastoma cell line [38] and the SW480 colon cancer line increased ROS (8-OH-guanosine) production. [39] endogenously expressed HIF-1α,aswellasHIF-2α.It A recent study by Gordan et al. [21] raises the possibility is the expression of HIF-1α in both of these other model that HIF1α acts as a tumor suppressor, and our data showing systems that is thought to facilitate tumor growth, and over- decreased growth rate of the HIF1+ compared to EV tumors expression of HIF-2α antagonises this effect. Similarly, in the seem to support this suggestion [10]. PDH histoscore PDK-1 histoscore TOM-20 histoscore 10 Journal of Oncology 8-OH-guanosine EV HIF-1+ HIF-2+ 8-OH-G EV HIF-1+ HIF-2+ (a) (b) (c) (d) γH2A.X 80 ∗ γH2A.X EV HIF-1+ HIF-2+ (e) (f) (g) (h) OGG1 OGG1 EV HIF-1+ HIF-2+ (i) (j) (k) (l) Figure 6: Oxidative stress and DNA damage/repair in CCRCC 786-0 tumors. (a–d) 8-OH-Guanosine staining (oxidative stress marker) ∗ ∗ ∗ ( P = .001). (e–h) γH2A.X staining (double-stranded DNA damage) ( P = .004). (i–l) OGG1 expression ( P = .006). P values were calculated using an ANOVA test. 4.2. Tumor Metabolism and Its Consequences. In non- HK-II and LDH5 and lower levels of lactate and alanine CCRCC cells in vitro, Akt signalling has also been demon- in the HIF-2+ tumors compared to both the EV and HIF- strated to positively regulate glycolysis in a HIF-1α indepen- 1+ tumors, suggested a decreased glycolytic flux in HIF2+ dent manner [41] mainly through mediating the localization tumors compared to HIF-1+ and EV tumors. However HIF- of GLUT-1. HIF-2+ tumors had lower expression of GLUT-1 1α (in an endogenous HIF-2α-only background) in vivo, in comparison to the EV tumors, despite supranormal levels appeared to have no effect on GLUT-1 expression since there of activated Akt. These findings are in contrast to the in vitro were no differences between glucose concentrations and findings of Raval et al. [12] who demonstrated that HIF- GLUT-1 expression in HIF-1+ and EV tumors. Interestingly, 2α was the principal regulator of GLUT-1 expression. An Cyclin D1 (which was higher in the HIF2+ tumors) has been explanation for this discrepancy between the in vitro and in shown in an in vivo mouse mammary cancer model to reduce vivo results could be that GLUT-1 expression is also sensitive the expression of both HK-II and LDH5 [42]. to changes in intracellular glucose concentration. Higher HIF-1 modulates multiple key metabolic pathways to concentrations of glucose were found in the HIF-2+ tumors, optimize use of O and glucose in response to changes in and could have attenuated GLUT-1 localization. This higher availability of these substrates, in order to most efficiently tumor glucose level along with decreased expression of generate ATP without excessive generation of ROS [7]. PDH Histoscore 786-0 nuclei (%) 786-0 nuclei (%) Journal of Oncology 11 EV HIF-1+ HIF-2+ VEGF VEGF EV HIF-1+ HIF-2+ (a) (b) (c) (d) CD31 Chalkley Vessel count Mouse CD31 EV HIF-1+ HIF-2+ (e) (f) (g) (h) ∗ + Figure 7: Markers of angiogenesis in CCRCC 786-0 tumors. (a–d) VEGF expression ( P = .003). (e–h) Murine CD31 vessel staining ∗ ∗ ( P = .001). values were calculated using an ANOVA test. is the key enzyme that determines whether pyruvate formed Since activated Akt is known to have the paradoxical during glycolysis from glucose will be metabolised to lactate effect of increasing mitochondrial O consumption and or oxidised in the TCA cycle. Its regulator, PDK, has been subsequently facilitating ROS generation [46], it could be shown to be expressed in a HIF-1α dependent manner [5, 6]. postulated that the supranormal levels of activated Akt status PDK negatively regulates PDH by phosphorylation, and in and the metabolic shift to greater oxidative metabolism in EV tumors the level of aerobic glycolysis was characterised the HIF-2+ tumors is mainly responsible for the higher levels by high PDK and low PDH indicating the basal level of of 8-OH-Guanosine immunostaining (high ROS stress) glycolysis in these tumors. A similarly high PDK, low PDH identified in these tumors. In spite of high ROS, γH2A.X was also found in HIF-1+ tumors, suggesting that the basal levels and OGG1 indicated resistance to DNA damage in level of aerobic glycolysis in EV 786-0 cells in vivo cannot be the HIF2+ tumors. These findings (summarised in Table 2) increased by exogenous expression of HIF-1α;alternatively are in agreement with Gordan et al. [8, 9] who have shown this may be due to mutually interacting effects of the pVHL that HIF-2α promotes cell cycle progression by enhancing -/-[43] and PTEN -/- status [44] of the parental 786-0 cell c-Myc mediated cyclin D2, leading to enhanced growth line. and resistance to DNA damage. This was not achieved In HIF-2+ tumors, in contrast, PDK-1 was decreased by modulating c-Myc levels, but by its interactions with and PDH was increased suggesting that the HIF2+ tumors partners. Although we did not stain for c-Myc in the rely on a less glycolytic, more oxidative metabolism. We 786-0 xenografts, it is highly likely that HIF-2α-mediated hypothesize that increased oxidation would supply more enhancement of c-Myc activity played a role in the xenografts reducing equivalents for the electron-transport chain (ETC), studied here. increase mitochondrial O consumption and thus increase the ATP supply to support the greater growth rate of 5. Conclusions the HIF2+ tumors. In support of this hypothesis were the findings of higher levels of TOM-20 (mitochondrial Tumor metabolism represents the end point of many signal load) and lower BNIP3 levels in HIF-2+ tumors, consistent cascades recruited by oncogenic activation. HIFα isoforms, with a higher mitochondrial mass, less mitophagy, and up- particularly HIF-1α, have been shown to be key regulators regulation of respiration, the converse of what was found of aerobic glycolysis in cancer cells. This is because HIF-1α with HIF-1α expression [45]. not only mediates the transcription of cytoplasmic glycolytic CVC score Histoscore 12 Journal of Oncology Table 2: Overview of molecular characteristics of HIF-1α and Abbreviations HIF-2α expression on CCRCC 786-0 xenografts compared to EV CCRCC: Clear-cell renal cell carcinoma xenografts. EV: CCRCC 786-0 tumors grown from cells Marker HIF-1+ HIF-2+ retrovirally infected with empty vector. Parameter HIF-1+: CCRCC 786-0 tumors grown from cells volume Growth retrovirally infected with expression of Ki67 ↓↓ HIF-1α. HIF-2+: CCRCC 786-0 tumors grown from cells Cyclin D1 — retrovirally infected with HIF-2α. Caspase-3 <0.5% <0.5% Apoptosis BNIP3 ↑ Autophagy Acknowledgments GLUT-1 — Glycolysis This paper was supported by Cancer Research UK, Li Ka HK II — Shing Foundation, and Hutchison Whampoa Ltd. LDH5 — Lactate — References Glucose — [1] G. L. Semenza, “Targeting HIF-1 for cancer therapy,” Nature Mitochondrial PDH/PDK ↓ Reviews Cancer, vol. 3, no. 10, pp. 721–732, 2003. Respiration [2] M. Zimmer, D. Doucette, N. Siddiqui, and O. Iliopoulos, TOM-20 — “Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL-/- tumors,” Molecular Cancer Research, 8-OH-guanosine ↓↑ ROS vol. 2, no. 2, pp. 89–95, 2004. γH2A.X ↑ DNA [3] V. Wang, D. A. Davis, M. Haque, L. E. Huang, and R. Yarchoan, “Differential gene up-regulation by hypoxia-inducible factor- OGG1 — damage/repair 1α and hypoxia-inducible factor-2α in HEK293T cells,” Cancer VEGF — Angiogenesis Research, vol. 65, no. 8, pp. 3299–3306, 2005. [4] C.-J. Hu,L.-Y. Wang,L.A.Chodosh,B.Keith,and M. CD31 — C. Simon, “Differential roles of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α in hypoxic gene regulation,” Molecular and Cellular Biology, vol. 23, no. 24, pp. 9361–9374, 2003. [5] J.-W. Kim, I. Tchernyshyov, G. L. Semenza, and C. V. enzymes and PDK-1, which phosphorylates and inactivates Dang, “HIF-1-mediated expression of pyruvate dehydroge- PDH, but also attenuates mitochondrial function by down nase kinase: a metabolic switch required for cellular adapta- regulating ETC activity, leading to a consequent reduc- tion to hypoxia,” Cell Metabolism, vol. 3, no. 3, pp. 177–185, tion of oxidative phosphorylation [7]. However, it could [6] I. Papandreou, R. A. Cairns, L. Fontana, A. L. Lim, and be speculated that expression of HIF-2α, in manipulated N. C. Denko, “HIF-1 mediates adaptation to hypoxia by CCRCC 786-0 tumors, overcomes the HIF-1α effects, which actively downregulating mitochondrial oxygen consumption,” results in a more oxidative tumor phenotype that supports Cell Metabolism, vol. 3, no. 3, pp. 187–197, 2006. a more aggressive phenotype. These results (see Table 2)are [7] G. L. Semenza, “Oxygen-dependent regulation of mitochon- in general agreement with the recent findings of Gordan drial respiration by hypoxia-inducible factor 1,” Biochemical et al. [8] who showed that HIF-2α expression in pVHL- Journal, vol. 405, no. 1, pp. 1–9, 2007. deficient CCRCC tumor lines potentiated c-Myc activity, [8] J. D. Gordan, J. A. Bertout, C.-J. Hu, J. A. Diehl, and M. resulting in enhanced growth and resistance to replication C. Simon, “HIF-2α promotes hypoxic cell proliferation by stress. We propose that the growth profiles observed in the enhancing c-Myc transcriptional activity,” Cancer Cell, vol. 11, HIF-1+ and HIF-2+ tumors that we have studied may be no. 4, pp. 335–347, 2007. mediated by HIF-1α inhibition of the c-Myc oncoprotein [9] J. D. Gordan, C. B. Thompson, and M. C. Simon, “HIF and c-Myc: sibling rivals for control of cancer cell metabolism and (slowing HIF-1+ growth) whilst HIF-2α potentiates c- proliferation,” Cancer Cell, vol. 12, no. 2, pp. 108–113, 2007. Myc transcriptional activity (HIF-2+) and promotes tumor [10] W. G. Kaelin Jr., “The von Hippel-Lindau tumour suppressor growth by an adaptive change to a more oxidative phenotype. protein: O2 sensing and cancer,” Nature Reviews Cancer, vol. In addition, the overall results from our study are consistent 8, no. 11, pp. 865–873, 2008. with the findings of Gordan et al. [21] who showed that [11] J. K. Maranchie, J. R. Vasselli, J. Riss, J. S. Bonifacino, W. clinical CCRCC tumors expressing only HIF-2α were bigger M. Linehan, and R. D. Klausner, “The contribution of VHL in size and more resistant to replicative stress compared to substrate binding and HIF1-α to the phenotype of VHL loss in those that expressed both HIF-1α and HIF-2 α.Thismay renal cell carcinoma,” Cancer Cell, vol. 1, no. 3, pp. 247–255, define a critical role for HIF-2α in the biology of VHL -/- CCRCC enabling greater growth; this demonstrates that in [12] R. R. Raval, K. W. Lau, M. G. B. Tran et al., “Contrasting certain contexts HIF-1α can act as a tumor suppressor (see properties of hypoxia-inducible factor 1 (HIF-1) and HIF- also [10, 47]). 2 in von Hippel-Lindau-associated renal cell carcinoma,” Journal of Oncology 13 Molecular and Cellular Biology, vol. 25, no. 13, pp. 5675–5686, of the breast; correlation of BNip3 levels with necrosis and 2005. grade,” Journal of Pathology, vol. 201, no. 4, pp. 573–580, 2003. [13] K. L. Covello, M. C. Simon, and B. Keith, “Targeted replace- [29] S. B. Fox, R. D. Leek, M. P. Weekes, R. M. Whitehouse, K. C. ment of hypoxia-inducible factor-1α by a hypoxia-inducible Gatter, and A. L. Harris, “Quantitation and prognostic value of factor-2α knock-in allele promotes tumor growth,” Cancer breast cancer angiogenesis: comparison of microvessel density, Research, vol. 65, no. 6, pp. 2277–2286, 2005. Chalkley count, and computer image analysis,” Journal of [14] K. Kondo, J. Klco, E. Nakamura, M. Lechpammer, and W. Pathology, vol. 177, no. 3, pp. 275–283, 1995. G. Kaelin Jr., “Inhibition of HIF is necessary for tumor [30] B. Madhu, S. P. Robinson, F. A. Howe, and J. R. Griffiths, suppression by the von Hippel-Lindau protein,” Cancer Cell, “Effect of Gd-DTPA-BMA on choline signals of HT29 tumors vol. 1, no. 3, pp. 237–246, 2002. detected by in vivo H MRS,” Journal of Magnetic Resonance Imaging, vol. 28, no. 5, pp. 1201–1208, 2008. [15] L. Holmquist-Mengelbier, E. Fredlund, T. Lofst ¨ edt et al., “Recruitment of HIF-1α and HIF-2α to common target genes [31] R. J. Ordidge, A. Connelly, and J. A. B. Lohman, “Image- is differentially regulated in neuroblastoma: HIF-2α promotes selected in vivo spectroscopy (ISIS). A new technique for an aggressive phenotype,” Cancer Cell, vol. 10, no. 5, pp. 413– spatially selective NMR spectroscopy,” Journal of Magnetic 423, 2006. Resonance, vol. 66, no. 2, pp. 283–294, 1986. [16] G. Qing and M. C. Simon, “Hypoxia inducible factor-2α:a [32] M. B. Azad, Y. Chen, E. S. Henson et al., “Hypoxia induces critical mediator of aggressive tumor phenotypes,” Current autophagic cell death in apoptosis-competent cells through a Opinion in Genetics & Development, vol. 19, no. 1, pp. 60–66, mechanism involving BNIP3,” Autophagy,vol. 4, no.2,pp. 2009. 195–204, 2008. [17] O. Warburg, The Metabolism of Tumors, Constable, London, [33] H. R. Mellor and A. L. Harris, “The role of the hypoxia- UK, 1930. inducible BH3-only proteins BNIP3 and BNIP3L in cancer,” Cancer and Metastasis Reviews, vol. 26, no. 3-4, pp. 553–566, [18] P. Vizan, ´ S. Mazurek, and M. Cascante, “Robust metabolic adaptation underlying tumor progression,” Metabolomics, vol. 2007. 4, no. 1, pp. 1–12, 2008. [34] A. S. E. Ojugo, P. M. J. McSheehy, D. J. O. McIntyre et al., “Measurement of the extracellular pH of solid tumours in [19] N. C. Denko, “Hypoxia, HIF1 and glucose metabolism in the solid tumour,” Nature Reviews Cancer, vol. 8, no. 9, pp. 705– mice by magnetic resonance spectroscopy: a comparison of 19 31 exogenous Fand Pprobes,” NMR in Biomedicine, vol. 12, 713, 2008. no. 8, pp. 495–504, 1999. [20] G. L. Semenza, “Regulation of cancer cell metabolism by hypoxia-inducible factor 1,” Seminars in Cancer Biology, vol. [35] B. Govindarajan, J. E. Sligh, B. J. Vincent et al., “Overex- pression of Akt converts radial growth melanoma to vertical 19, no. 1, pp. 12–16, 2009. growth melanoma,” Journal of Clinical Investigation, vol. 117, [21] J. D. Gordan, P. Lal, V. R. Dondeti et al., “HIF-α effects on c- no. 3, pp. 719–729, 2007. Myc distinguish two subtypes of sporadic VHL-deficient clear [36] R. B. Robey and N. Hay, “Mitochondrial hexokinases, novel cell renal carcinoma,” Cancer Cell, vol. 14, no. 6, pp. 435–446, 2008. mediators of the antiapoptotic effects of growth factors and Akt,” Oncogene, vol. 25, no. 34, pp. 4683–4696, 2006. [22] P. M. J. McSheehy,M.T.Seymour,A.S.E.Ojugo et al., [37] E. Ackerstaff, K. Glunde, and Z. M. Bhujwalla, “Choline “A pharmacokinetic and pharmacodynamic study in vivo of 19 31 human HT29 tumours using Fand P magnetic resonance phospholipid metabolism: a target in cancer cells?” Journal of Cellular Biochemistry, vol. 90, no. 3, pp. 525–533, 2003. spectroscopy,” European Journal of Cancer, vol. 33, no. 14, pp. 2418–2427, 1997. [38] J. Favier, S. Lapointe, R. Maliba, and M. G. Sirois, “HIF2α [23] H. U. Bergmeyer, Methods of Enzymatic Analysis,Verlag reduces growth rate but promotes angiogenesis in a mouse model of neuroblastoma,” BMC Cancer, vol. 7, article 139, Chemie, Weinheim, Germany, 2nd edition, 1974. [24] J.-L. Li, R. C. A. Sainson, W. Shi et al., “Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular [39] T. Imamura, H. Kikuchi, M.-T. Herraiz et al., “HIF-1α and HIF-2α have divergent roles in colon cancer,” International function, and promotes tumor growth in vivo,” Cancer Research, vol. 67, no. 23, pp. 11244–11253, 2007. Journal of Cancer, vol. 124, no. 4, pp. 763–771, 2009. [25] S. A. Fuhrman, L. C. Lasky, and C. Limas, “Prognostic sig- [40] T. Acker, A. Diez-Juan, J. Aragones et al., “Genetic evidence for a tumor suppressor role of HIF-2α,” Cancer Cell,vol. 8, no.2, nificance of morphologic parameters in renal cell carcinoma,” American Journal of Surgical Pathology, vol. 6, no. 7, pp. 655– pp. 131–141, 2005. 663, 1982. [41] D. R. Plas and C. B. Thompson, “Akt-dependent transforma- tion: there is more to growth than just surviving,” Oncogene, [26] H. M. E. Scheres, A. F. P. M. De Goeij, M. J. M. Rousch et al., “Quantification of oestrogen receptors in breast cancer: vol. 24, no. 50, pp. 7435–7442, 2005. radiochemical assay on cytosols and cryostat sections com- [42] T. Sakamaki, M. C. Casimiro, X. Ju et al., “Cyclin D1 pared with semiquantitative immunocytochemical analysis,” determines mitochondrial function in vivo,” Molecular and Journal of Clinical Pathology, vol. 41, no. 6, pp. 623–632, 1988. Cellular Biology, vol. 26, no. 14, pp. 5449–5469, 2006. [27] W. R. Duan, D. S. Gamer, S. D. Williams, C. L. Funckes- [43] E. Hervouet, J. Demont, P. Pecina et al., “A new role for the Shippy, I. S. Spath, and E. A. G. Blomme, “Comparison of von Hippel-Lindau tumor suppressor protein: stimulation of immunohistochemistry for activated caspase-3 and cleaved mitochondrial oxidative phosphorylation complex biogene- cytokeratin 18 with the TUNEL method for quantification sis,” Carcinogenesis, vol. 26, no. 3, pp. 531–539, 2005. of apoptosis in histological sections of PC-3 subcutaneous [44] R. L. Elstrom, D. E. Bauer, M. Buzzai et al., “Akt stimulates xenografts,” Journal of Pathology, vol. 199, no. 2, pp. 221–228, aerobic glycolysis in cancer cells,” Cancer Research, vol. 64, no. 11, pp. 3892–3899, 2004. [28] H. M. Sowter, M. Ferguson, C. Pym et al., “Expression of the [45] G. L. Semenza, “Mitochondrial autophagy: life and breath of cell death genes BNip3 and NIX in ductal carcinoma in situ the cell,” Autophagy, vol. 4, no. 4, pp. 534–536, 2008. 14 Journal of Oncology [46] V. Nogueira, Y. Park, C.-C. Chen et al., “Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis,” Cancer Cell, vol. 14, no. 6, pp. 458–470, 2008. [47] W. G. Kaelin Jr. and P. J. Ratcliffe, “Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway,” Molecular Cell, vol. 30, no. 4, pp. 393–402, 2008. MEDIATORS of INFLAMMATION The Scientific Gastroenterology Journal of World Journal Research and Practice Diabetes Research Disease Markers Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Journal of Immunology Research Endocrinology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com BioMed PPAR Research Research International Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Obesity Evidence-Based Journal of Journal of Stem Cells Complementary and Ophthalmology International Alternative Medicine Oncology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Parkinson’s Disease Computational and Behavioural Mathematical Methods AIDS Oxidative Medicine and in Medicine Research and Treatment Cellular Longevity Neurology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Oncology Wiley

Effects of HIF-1

Download PDF
15 pages

Loading next page...
 
/lp/wiley/effects-of-hif-1-NO28fHn0n0

References (52)

Publisher
Wiley
Copyright
Copyright © 2010 Swethajit Biswas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN
1687-8450
eISSN
1687-8469
DOI
10.1155/2010/757908
Publisher site
See Article on Publisher Site

Abstract

Hindawi Publishing Corporation Journal of Oncology Volume 2010, Article ID 757908, 14 pages doi:10.1155/2010/757908 Research Article Effects of HIF-1α and HIF2α on Growth and Metabolism of Clear-Cell Renal Cell Carcinoma 786-0 Xenografts 1, 2, 3 4, 5 2 4, 5 1 Swethajit Biswas, Helen Troy, Russell Leek, Yuen-Li Chung, Ji-liang Li, 1 2 2 2 4, 6 Raju R. Raval, Helen Turley, Kevin Gatter, Francesco Pezzella, John R. Griffiths, 4, 6 1 Marion Stubbs, and Adrian L. Harris Weatherall Institute of Molecular Medicine, University of Oxford, John RadcliffeHospital, Oxford OX39DU,UK CR UK Tumour Pathology Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John RadcliffeHospital, Oxford OX39DU,UK Northern Institute for Cancer Research (NICR), Newcastle University, Freeman Hospital, Newcastle-Upon-Tyne NE7 7DN, UK CR UK Biomedical Magnetic Resonance Research Group, Division of Basic Medical Sciences, St. George’s, University of London, London SW17 0RE, UK CRUK Clinical Magnetic Resonance Research Group, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK CRUK Cambridge Research Centre, Li Ka Shing Centre, Robinson Way, Cambridge CB2 ORE, UK Correspondence should be addressed to Marion Stubbs, [email protected] Received 16 September 2009; Revised 9 March 2010; Accepted 22 April 2010 Academic Editor: Dominic Fan Copyright © 2010 Swethajit Biswas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In cultured clear-cell renal carcinoma (CCRCC) 786-0 cells transfected with HIF1α (HIF-1+), HIF-2α (HIF-2+), or empty vector (EV), no significant differences were observed in the growth rates in vitro,but when grown in vivo as xenografts HIF- 2α significantly increased, and HIF-1α significantly decreased growth rates, compared to EV tumors. Factors associated with proliferation were increased and factors associated with cell death were decreased in HIF-2+ tumors. Metabolite profiles showed higher glucose and lower lactate and alanine levels in the HIF-2+ tumors whilst immunostaining demonstrated higher pyruvate dehydrogenase and lower pyruvate dehydrogenase kinase 1, compared to control tumors. Taken together, these results suggest that overexpression of HIF-2α in CCRCC 786-0 tumors regulated growth both by maintaining a low level of glycolysis and by allowing more mitochondrial metabolism and tolerance to ROS induced DNA damage. The growth profiles observed may be mediated by adaptive changes to a more oxidative phenotype. 1. Introduction (ETC) activity by altering the subunit composition of COX, minimising ROS generation [7]. In contrast, HIF-2α is The HIFα transcription factors, HIF-1α and HIF-2α, mediate uniquely involved in tumor growth and cell cycle progression adaptive responses to tumor hypoxia, as well as regulating through interaction with c-Myc [8, 9]. The most direct link between genetic events that pre- an extensive transcriptional response involving the induction of genes for angiogenesis, glucose metabolism/cellular ener- dispose to cancer and activation of the HIF pathway is getics, cell growth, metastasis, apoptosis, and extracellular observed in tumors associated with inactivation of the von matrix (ECM) remodelling [1]. HIF-1α and HIF-2α, despite Hippel-Lindau (VHL) tumor suppressor gene, particularly some overlapping effects, can uniquely regulate distinct VHL-associated clear-cell renal cell carcinoma (CCRCC) (for genes [2]. HIF-1α for example is primarily involved in review see [10]). The pseudohypoxic VHL-defective CCRCC glucose metabolism by upregulating glycolytic enzymes [3, cells show a bias toward HIF-2α, and overproduction of 4] whilst limiting pyruvate uptake by the mitochondria HIF2α (but not HIF1α), has been found to be sufficient to override the tumor suppressor function of VHL in xenograft [5, 6] and down regulating the electron transport chain 2 Journal of Oncology studies [11–13]. HIF-2α expression (in a mouse model of in pyruvate dehydrogenase (PDH) expression. This, together CCRCC) is necessary for the development of the typical with higher glucose and lower lactate and alanine levels clear-cell phenotype, demonstrating the important role of found in the HIF-2+ tumors (compared to both EV and HIF-2α in CCRCC [14]. HIF-1+ tumors), results in a more oxidative, DNA damage- Using retroviral transfection in 786-0 cells, Raval et tolerant phenotype that supports enhanced tumor growth, al. [12] confirmed that exogenous expression of HIF- similar to the Sporadic VHL-deficient clinical subtypes of 1α upregulated transcriptional products involved in pH CCRCC described by Gordan et al. [21]. regulation (CAIX) and cell fate (BNIP3), whereas expression of HIF-2α upregulated a different set of proteins which 2. Materials and Methods were involved in cell proliferation (cyclin D1), cell growth (TGF-α), and angiogenesis (VEGF). However, the in vitro 2.1. Human Clear-Cell Renal Cancer Cell Lines. Stable studies demonstrated no differences in the proliferation rate polyclonal pools of G418 selected 786-0 renal cancer cells between 786-0 cells that either exogenously expressed HIF1α expressing the relevant HIFα isoform (HIF-1+) were pro- (HIF-1+), overexpressed HIF-2α (HIF-2+) or were infected duced in vitro from the same pool as previously described with empty vector (EV) (control cells). But when these [12]. Polyclonal pools retrovirus-infected with the pBMN- manipulated 786-0 cells were grown in vivo as xenografts, a Z-IRES-Neo-based HIF-1α,HIF-2α (HIF-2+), or control different tumor growth profile emerged showing that HIF- (EV), were released by trypsinization and subsequently 2α caused significantly increased growth rates and HIF-1α resuspended in PBS. caused significantly decreased growth rates when compared to EV tumors. Similar findings (HIF-2α facilitating tumor 2.2. Xenografts. 786-0 cells (1 × 10 ) transfected with HIF- growth in vivo) have also been made in two nonepithelial 1or 2α or control in 100 μl PBS were injected subcutaneously tumors, teratoma [13] and neuroblastoma [15] in vivo. into the dorsal flanks of nu/nu mice. Three cohorts were However, apart from the growth curves, few studies have generated. Two consisting of 21 mice with 7 in each group been reported that investigate the role of HIF-2α in epithelial (HIF-1+, HIF 2+ and EV) for growth, immunohistochem- cancers in vivo (for review see [16]). Most epithelial cancer istry and histology; and one consisting of 15 mice with 5 cells rely on HIF-1α transcriptional products to mediate in each group (HIF-1+, HIF 2+ and EV) which were grown tumor metabolism including the effect first described by to at least 500 mm , the minimum size possible for in vivo Warburg [17] more than 80 years ago that leads to the repro- Magnetic Resonance Spectroscopy experiments. gramming of tumor cells from mitochondrial respiration Tumor size was measured bidirectionally in all experi- to aerobic glycolysis (see [18–20] for review). The human ments, twice per week using calipers, with the longest dimen- VHL -/- clear-cell renal cancer cell line, 786-0, provides a sion (l) and shortest dimension (s) measured postimplan- model for investigating the effects of both HIFα isoforms, tation. Tumor volume (mm ) was calculated by measuring particularly HIF-2α, on tumor growth and metabolism in length, width and depth using callipers and 1∗w∗d∗(π/6). vivo, since it constitutively expresses only HIF-2α. To further understand the role of HIF-2α in vivo,wehaveinvestigated 2.2.1. Tumor Processing. The mice from cohort (1 and 2) the effects of HIF-2α expression not only on CCRCC 786-0 were sacrificed by cervical dislocation at day 42, the tumors tumor growth, but also on metabolic adaptation to tumor bisected with one-half snap-frozen for storage at −80 C, progression by using Magnetic Resonance Spectroscopy and the other half-embedded in paraffin. The mice from (MRS) methods both noninvasively in vivo and ex vivo on cohort (3) were anesthetized with a single i.p. injection of tumor extracts. a Hypnovel/Hypnorm/water (1 : 1 : 2) mixture as previously The rationale for using 786-0 line in our experiments described [22] prior to the MR experiment (details below). is that CC-RCC comes in two HIF types that is HIF-2 only At the end of the experiment the tumors were freeze-clamped and HIF-2 + HIF-1. Therefore, using a CC-RCC, HIF-2 only and stored at −80 C. Subsequently the frozen tumors were expressing cell line is appropriate to investigate the role of extracted in 6% perchloric acid, as previously described HIF-1 in CC-RCC tumour growth/biology as well as the [23] and the neutralized extracts were freeze-dried and effects of HIF-2 overexpression on an endogenous HIF-1 reconstituted in 1 ml deuterium oxide for high resolution null background (see [21]). Expression of HIF-2α resulted MRS. Cyrostat sectioning of frozen tumors was performed in a significant increase in tumor growth rate similar to for mouse CD31 staining as previously described [24], and that observed previously [12] whilst HIF1+ tumors grew Fuhrman’s criteria were applied to histological grading for even more slowly than EV tumors. Immunohistochemistry characterization of nucleoli morphology [25]. (IHC) was chosen rather than Western Blotting because the necrosis and heterogeneity of in vivo tumors causes poor reproducibility. IHC allows heterogeneity to be scored 2.2.2. Immunohistochemistry. Tumors were prepared for and the extent of protein expression to be determined in a immunohistochemistry as follows. Briefly, endogenous tissue semiquantitative fashion. Using a combination of immunos- peroxidase activity was blocked using two drops of 0.3% 1 31 taining and/or Hor P Magnetic Resonance Spectroscopy hydrogen peroxide (1 : 100 dilution of 30% H O stock 2 2 (MRS), we also demonstrate that expression of HIF-2α (BDH Laboratory Supplies, Poole, UK) in distilled water), decreased the expression of HK-II, LDH5 and pyruvate to cover each section. After two rounds of immersion dehydrogenase kinase 1 (PDK1) with a concomitant increase in PBS, for 5 minutes each, 2.5% normal horse serum Journal of Oncology 3 (Normal horse serum concentrate—Vector Laboratories Inc, University of London, and tumors were positioned in the 1 31 California, USA, diluted in PBS) was applied to each section center of a 15-mm two-turn Hsurface or P MRS coil. for 30 minutes at room temperature to prevent nonspecific Voxels were selected from scout gradient echo images, primary antibody binding. and localized shimming yielded linewidths of the order Primary antibodies were directed against HIF-1α,HIF- of 20–30 Hz. The PRESS localization method with water 2α and CAIX; mouse monoclonals (ESEE122), (237/B5) and suppression with a repetition time of 2 seconds was used (M75), respectively, University of Oxford. Ki67, Cyclin D1: to detect choline [30]. For P MRS, image selected in mouse monoclonals (MIB-1) and (DSC-6), and GLUT-1: vivo spectroscopy (ISIS) [31] localised spectra of tumors rabbit polyclonal; DAKO, Ely, UK: Cleaved caspase-3; rabbit were acquired. MRUI software was used for all spectral monoclonal, R+D Systems, UK; BNIP3: mouse monoclonal processing programs, including preprocessing, fitting and (ANa40) SIGMA, UK; CD10: mouse monoclonal (56C6), quantification of peak areas of the observed metabolites. Abcam, UK; VEGF: SP28, rabbit monoclonal antibody, H MR spectra of the neutralised tumor extracts were Abcam,UK; Phospho-Ser Akt and PTEN: rabbit mon- obtained using a Bruker 600 MHz spectrometer (pulse angle oclonals 736E11 and 138G6 respectively, Cell Signalling, 45 ; repetition time, 3.5 seconds). The water resonance USA; Hexokinase-II: rabbit polyclonal, Chemicon, USA; was suppressed by gated irradiation centred on the water LDH5: sheep polyclonal, Abcam, UK; PDH E2 Complex: frequency. 25 μl of 10 mM Sodium 3-trimethylsilyl-2,2,3,3- mouse monoclonal (15D3), Invitrogen, USA; PDK-1: goat tetradeuterpropionate (TSP) was added to the samples for polyclonal, Santa Cruz, USA; TOM-20: mouse mono- chemical shift calibration and quantification. The pH was re- clonal (F10),Santa Cruz,USA;PGC-1 β:rabbitpoly- adjusted to pH 7 prior to HMRS. clonal, Santa Cruz, USA; γH2AX (phospho-Ser Histone H2A.X): Rabbit Monoclonal, 20E3, Cell Signaling, USA; 2.3.1. Statistics. For analysis of the immunohistochemical 8-Hydroxyguanosine (8-OH-G): goat polyclonal, Alexis expression of individual proteins between all tumor groups, Biochemicals, Nottingham, UK; OGG1 Rabbit polyclonal the nonparametric Kruskal-Wallis (ANOVA) test was used. (ab204) Abcam, UK. Results from one cohort were displayed as histograms with The Envision-HRP ChemMate polymer kit (DAKO, Ely, standard error of the mean (SEM) in the figures. Dunn’s UK) was used for detection of either mouse and rabbit post hoc test for all data sets was calculated if P< .05 monoclonal or rabbit polyclonal primary antibodies, as and displayed in the figures. Immunohistochemical protein per the manufacturer’s instructions. Where applicable, the expression between specific pairs of 786-0 tumor groups relevant secondary antibodies of antigoat (P0160, DAKO, Ely, was compared using the Mann-Whitney unpaired t-test, UK), antisheep (ab6747, Abcam, UK) and antirat (P0450, where mentioned in the text. The Spearman rank testing was DAKO,Ely,UK) were used. used to demonstrate correlations between non-parametric The majority of primary antibodies were detected with variables. For the MRS data a two-tailed t test was used a 3,3-diaminobenzidine (DAB+) chromogenic substrate sys- for significance levels. Significant results were designated if tem as part of the Envision kit. Nuclei were counterstained P< .05. with Haemotoxylin before mounting onto plastic coverslips with AquaMount (Gurr GmBH, Strasbourg, Germany). 3. Results For Ki67 (MIB-1 clone) detection, ChromogenSG (Vector Laboratories Inc., California, USA) was used and the nuclei 3.1. Effect of Transfection of Specific Retroviral HIFα Isoform in counterstained with Nuclear Fast Red (Sigma-Aldrich, St. CCRCC 786-0 Xenografts. We have previously demonstrated Louis, USA). Sections treated in both these ways were dehy- [12] that the appropriate HIFα isoform protein is expressed drated through methanol and xylene, before haemotoxylin after retrovirally-mediated infection of specific HIFα isoform counterstaining and mounting with DPX. constructs within a bicistronic IRES-neo cassette, in vitro. These in vitro findings were confirmed in vivo in tumors 2.2.3. Assessment of Tumor Immunostaining. Each tumor grown subcutaneously as xenografts in mice in all three section was assessed blindly and independently by two groups. HIF-1α expression was identified only in the HIF-1+ observers. Photomicrographs were taken at x100 hpf. Semi- tumors, and only within the nuclear compartment, whereas quantitative analysis of protein expression was performed the EV and HIF-2+ tumors showed no staining for HIF-1α using a modified “Histoscore” method, as previously (Figures 1(a)–1(d)). described [26]. For Ki67 and Cleaved caspase-3 scoring, positive and negatively stained cells within 5 individual However, HIF-2α expression was identified in both tumor areas, consisting of 100 cells each, were scored [27]. nuclear and cytoplasmic compartments in all 3 tumor Tumor necrosis was quantified as the % area of tumor groups (Figures 1(e)–1(h)), but showed a significant increase replaced by necrosis, as identified by light microscopy [28]. only within the nuclear compartment of HIF-2+ tumors CD31 Chalkley Vessel Count (CVC) was the average value (Figure 1(g)). No significant changes in HIF-2α expression from the three fields [29]. were seen in either compartment of the HIF1+ or the EV tumors (Figures 1(e)–1(f)). A further cohort (not shown in 2.3. Magnetic Resonance Spectroscopy (MRS). Anesthetized figures) of in vivo tumors confirmed the findings of HIF-1 mice were placed in the bore of a Varian 4.7 T nuclear and HIF-2α expression as well as a similar growth pattern in magnetic resonance (NMR) spectrometer at St. George’s the 3 tumor types. 4 Journal of Oncology EV HIF-1+ HIF-2+ Nuclear HIF-1α HIF-1α EV HIF-1+ HIF-2+ (a) (b) (c) (d) Nuclear HIF-2α HIF-2α EV HIF-1+ HIF-2+ (e) (f) (g) (h) (i) H+E (j) CD10 (k) PTEN (l) AKT (m) Phospho-Ser AKT Figure 1: Features of CCRCC 786-0 tumors. (a–d) (x100 hpf) HIF-1α nuclear staining only ( P< .0001). (e–h) Differential HIF-2α nuclear ∗ + staining ( P = .0004). (i) Clear-cell/sarcomatoid morphology (x40 hpf). (j) CD10 staining typical of clear-cell RCC lineage (x100 hpf). (k) PTEN staining; positive staining only identified in murine fibroblasts (x100 hpf). (l) Akt staining, (m) Phospho-Ser Akt staining (highest in HIF-2+ tumors). P values were calculated using an ANOVA test. 3.2. Effect of HIFα Isoform Expression on Grade and Phenotype demonstrating sarcomatoid dedifferentiation. There were no of CCRCC 786-0 Xenografts. 21 tumors were evaluated differences in Fuhrman’s grade between the three tumor after H+E staining, and each exhibited a high Fuhrman’s groups (a representative example is shown in Figure 1(i)). tumor grade of either 3 or 4, with the majority of tumors Only one tumor (an EV tumor) was morphologically wholly HIF-1α histoscore HIF-1α histoscore Journal of Oncology 5 clear-cell and one tumor (HIF-2+) was completely replaced with sarcomatoid de-differentiation which is a clinically recognised variant of high grade tumors. However despite their sarcomatoid de-differentiation the tumours retained expression of typical clear-cell renal cancer markers such as CD10 (Figure 1(j)), pancytokeratin and vimentin positive expression, on a CK-7 negative background (data not shown). 786-0 cells were PTEN negative (Figure 1(k)), but the surrounding murine fibroblasts demonstrated positive staining. This finding was confirmed by the high levels of phospho-Ser (activated) Akt expression in the 786-0 473 0 0 102030405060 70 cells of the EV tumors (Figure 1(l) and 1(m)). Expression Time (days) of activated Akt was increased in the HIF-2+ tumors compared to EV and HIF-1+ tumors. This may be because 7860-EV expression of TGFα was increased in the HIF-2+ tumors 7860-HIF-1 with a similar increase in activated EGFR (Tyr -EGFR) 7860-HIF-2 expression, compared with EV and HIF-1+ tumors (data not (a) shown). 3.3. Effect of HIFa Expression on Tumor Proliferation and Apoptosis in 786-0 Xenografts. In contrast to the growth patterns in vitro where the 3 cell types demonstrated similar proliferation rates [12], growth patterns in vivo showed that there were significant differences between HIF-1+, HIF-2+ and EV 786-0 tumors (Figure 2(a)). The differences in overall growth between the 3 tumor groups were dependent on the lag phase for each tumor group as well as the rate of 5 tumor growth. The HIF-2+ tumors had the shortest lag phase (21 days) followed by the EV tumors (27 days) with the HIF-1+ tumors taking the longest time (>32 days). Once HIF-2+ HIF-1+ EV the lag phase was over, the actual rates of growth were 45 ± 5.4mm /day for HIF-2+ (P = .09 compared to EV), (b) 35 ± 3.3mm /day for EV tumors (P = .0007 compared to Figure 2: (a) Growth curve kinetics of 786-0 CCRCC tumors in HIF-1) and 18±4.0mm /day for HIF-1+ tumors. There were ∗ ∗ vivo. (b) Areas of tumor necrosis (%) P >.1. P values were no significant differences in necrosis between the different calculated using an ANOVA test. tumor types (Figure 2(b)). The EV tumors demonstrated the highest Ki67 (MIB- 1%) proliferation rates in comparison to both the HIF- 1+ and HIF-2+ tumors (P = .0006) (Figures 3(a)–3(d)). the HIF-2+ tumors (Figures 4(a)–4(d)) in comparison to Cyclin D1 expression however was highest in the HIF- the other two tumor groups (P = .01), in contrast to 2+ tumors (P = .0010) (Figures 3(e)–3(h)). The overall the in vitro findings by Raval et al. [12]. Expression of rates of apoptosis measured by cleaved-caspase 3 were very two glycolytic enzyme proteins, HK-II (Figures 4(e)–4(h)) low (<0.5%) in all the tumor groups (Figure 3(i)–3(l)). and LDH5 (Figures 4(i)–4(l)), was significantly lower in the The HIF-1+ tumors had the highest rate of apoptosis (∼ HIF-2+ tumors in comparison to both the HIF-1+ and EV 0.4%) compared to controls (P = .0002), whereas the HIF- tumors, whereas there was no difference in the expression 2+ tumors had only 0.1% compared to the EV tumors of these glycolytic enzymes between HIF-1+ tumors and EV with ∼0.25%. Because the apoptotic rates were so low, tumors. we also considered potential regulators of alternative death pathways, such as BNIP3, which has been implicated in 1 31 3.5. Metabolites Measured by H MRS and P of 786-0 cancer cell autophagy [32, 33]. The intergroup expression of Xenografts and in Tumor Extracts. HMRS of in vivo tumors BNIP3 demonstrated that HIF-2+ tumors showed the lowest demonstrated higher levels of free choline (which resonates expression and HIF-1+ tumors the highest (P = .0002) at ∼3.2 ppm) in the HIF-2+ tumors (Figure 4(m))compared (Figures 3(m)–3(p)). However, as previously mentioned to HIF-1+ and EV tumors. After in vivo scanning the tumors there were no significant differences in the level of tumor were freeze-clamped and metabolites were measured at high necrosis between the different groups (Figure 2(b)). field in tumor extracts (which gives better resolution than in vivo)by HMRS (Figure 4(n) and Table 1). The MR spectra 3.4. Effect on Factors Related to Glucose Metabolism; Glut- shown in Figure 4(n) are representative samples of various 1, HKII, LDH. Expression of GLUT-1 was attenuated in spectral regions of the high resolution spectra obtained from Tumour size (mg) Necrosis (%) 6 Journal of Oncology EV HIF-1+ HIF-2+ Ki67 (MIB-1 %) proliferation Ki67 EV HIF-1+ HIF-2+ (a) (b) (c) (d) Cyclin D1 Cyclin D1 EV HIF-1+ HIF-2+ (e) (f) (g) (h) Cleaved caspase-3 % apoptosis 0.5 0.4 0.3 Cleaved 0.2 caspase-3 0.1 EV HIF-1+ HIF-2+ (i) (j) (k) (l) BNIP3 BNIP3 EV HIF-1+ HIF-2+ (m) (n) (o) (p) Figure 3: (x100 hpf). Growth and death markers in CCRCC 786-0 tumors. (a–d) Ki67 proliferation index ( P = .0006). (e–h) Cyclin D1 ∗ ∗ expression ( P = .001). (i–l) Apoptosis as measured by cleaved-caspase-3% index ( P = .0002). (m–p) BNIP3 expression (cytoplasm only) ∗ ∗ ( P = .0002). values were calculated using ANOVA test. the extracts of each tumor type. The significantly higher analysis [34], similar values for intracellular pH (pHi) were levels of choline/phosphocholine (PC) found in extracts of found in all 3 tumor groups. Similar to the in vivo results, HIF-2+ tumors reflected the raised choline found in the no differences were observed in the high energy phosphates 1 31 tumors in vivo by H MRS. In vivo PMRS of the tumors (ATP+ADP) between the different tumor types (Table 1). showed no significant differences between the parameters Signals from glucose, creatine (tCr), and taurine were ATP, PME, PDE, Pi (data not shown). Using Pi spectral shift also significantly higher in the HIF-2+ tumors, whereas MIB-1 (%) Cyclin D1 histoscore BNIP3 histoscore Cleaved caspase-3 (%) Journal of Oncology 7 Membranous GLUT-1 EV HIF-1+ HIF-2+ 4 ∗ GLUT-1 EV HIF-1+ HIF-2+ (a) (b) (c) (d) Hexokinase-II HK-II EV HIF-1+ HIF-2+ (e) (f) (g) (h) LDH5 LDH5 EV HIF-1+ HIF-2+ (i) (j) (k) (l) PC Lactate tCr Glucose Alanine HIF-2+ HIF-1+ HIF-2+ HIF-1+ EV EV 5.45.23.9 3.25 3.2 1.51.4 4 3 2 (ppm) (ppm) (ppm) (ppm) (ppm) (m) (n) Figure 4: Metabolism-related markers and metabolic profiles of CCRCC 786-0 tumors (x100 hpf). (a–d) GLUT-1 expression( P = .01). ∗ ∗ 1H (e–h) Hexokinase-II expression ( P = .0006). (i–l) LDH5 expression ( P = .004), (m) In vivo MRS of 786-0 tumors. (n) High-resolution 1H MR Spectra of tumor extracts. P values were calculated using an ANOVA test. HK-II histoscore LDH5 histoscore GLUT-1 histoscore 8 Journal of Oncology 4. Discussion Table 1: Metabolite levels measured by H MRS in 786-0 tumor extracts. The tumor grade of 786-0 tumors does not alter with Metabolite EV HIF-1+ HIF-2+ differential HIFα isoform expression on a HIF-2α-only expressing background, whether grown as cultured cells or Leucine 0.13 ± 0.01 0.17 ± 0.02 0.17 ± 0.01 as xenografts that demonstrate a high grade phenotype and Iso Leucine 0.06 ± 0.01 0.08 ± 0.005 0.08 ± 0.005 characteristic morphology. Although the patterns of HIFα a,b Lactate 5.13 ± 0.85 5.43 ± 0.51 2.54 ± 0.58 isoform expression in vivo were similar to those found in the a,b Alanine 0.84 ± 0.06 0.88 ± 0.05 0.63 ± 0.04 CCRCC 786-0 cells in vitro [12], there were some differences between the levels of specific transcription factors expressed Choline 0.17 ± 0.02 0.22 ± 0.04 0.29 ± 0.05 in vitro and in vivo. The expression of BNIP3, cyclin D1, a,b PC 0.73 ± 0.09 0.66 ± 0.14 1.14 ± 0.05 TGFα andVEGFinthe in vivo model were similar to HIFα Taurine 13.96 ± 1.55 13.02 ± 0.92 16.70 ± 0.66 isoform expression found in vitro. However the expression a,b of GLUT-1 was comparatively lower in the HIF-2+ tumors Cr 1.26 ± 0.14 1.46 ± 0.15 2.00 ± 0.17 a in vivo (see below for discussion) consistent with a more Glucose 0.63 ± 0.10 0.73 ± 0.12 1.19 ± 0.29 oxidative phenotype. ATP+ADP 0.91 ± 0.20 0.95 ± 0.13 1.14 ± 0.15 Metabolites expressed as μmol/g wet weight tissue (n = 3–5). denotes 4.1. Tumor Growth and Related Death Pathways. This in vivo statistically significant different from EV and denotes statistically signif- study showed that the growth of CCRCC 786-0 tumors was icant difference (P< .05) from HIF-1+. A two-tailed t test was used for biphasic, with an initial growth lag phase followed by growth significance levels. acceleration. The HIF-1+ tumors, which were overall the slowest growing of the three groups, had the longest lag phase whereas the EV tumors started to grow at day 27, and alanine and lactate were significantly lower compared to the the HIF-2+ tumors at day 21. The lag times and growth HIF-1+ and EV tumors. This is more clearly demonstrated in rates in vivo were similar to those observed previously [12]. the detailed analysis of the metabolites shown in Table 1 and These differences in early growth may reflect stress of a poor described below. These data imply a more oxidative and less blood supply which could have affected early establishment glycolytic phenotype for the HIF-2+ tumors. of the tumors, since the HIF-2+ tumors had the highest levels of CD31 angiogenesis and VEGF, but the shortest initial growth lag phase compared to EV and HIF-1+ tumors. 3.6. Effects on Factors Related to Mitochondrial Regulation Tumor growth is a balance between cellular proliferation and Free Radical Damage; PDH, PDK-1, TOM-20, 8- and cell death. The increased levels of cyclin D1, an OH-Guanosine and OGG1. PDH (Figures 5(a)–5(d))was important regulator of cell cycle progression, were seen in upregulated and PDK-1 (Figures 5(e)–5(h)) down-regulated the faster growing HIF-2+ tumors, but surprisingly they in the faster growing HIF-2+ tumors. Higher expression had the lowest proliferation index (Ki67) and very low levels of the cellular mitochondrial load marker, TOM-20 levels (<0.5%) of apoptosis in vivo. This may be the result (Figures 5(i)–5(l)) was also seen in the HIF-2+ tumors of two independent background factors. Activated Akt is and in turn this was mirrored by an increase in expression constitutively expressed in the 786-0 xenografts, due to the of the mitochondrial biogenesis regulator, PGC-1β (data PTEN -/- status, facilitating tumor growth [35]and an not shown). Overall, this is consistent with an increase in antiapoptotic phenotype [36]. Since the levels of necrosis mitochondrial biosynthesis and activity. were similar between tumor groups, alternative cell death The HIF-2+ tumors were also under a comparatively mechanisms, such as autophagy, were considered to explain greater degree of oxidative stress, as manifest by higher the differences in growth between the tumor types. BNIP3 levels of 8-OH-guanosine staining compared to the other levels were significantly lower in HIF2+ tumors in vivo, two tumor groups (Figures 6(a)–6(d)). However immunos- and were consistent with the in vitro results of Raval et taining of γH2A.X (Figures 6(e)–6(h)) showed lower levels al. [12] showing that over-expression of HIF-2α attenuated indicating less DNA damage in HIF2+ than in HIF1+ or EV BNIP3 expression. Since both HIF-1+ and EV tumors had tumors. Expression of OGG1 (a DNA repair enzyme) was significantly higher levels of BNIP3, and since their levels higher in the HIF-2+ tumors compared to HIF-1+ and EV of apoptosis were very low, we hypothesize that BNIP3 tumors (Figures 6(i)–6(l)). induces autophagic cell death in this 786-0 model as a default death mechanism. In addition, phosphocholine and 3.7. Effects on Factors Related to Neoangiogenesis. VEGF, glycerophosphocholine were highest in the HIF2+ tumors identified only in the cytoplasm of tumor cells, was higher compared to HIF-1+ and EV tumors. Usually (although not in the HIF2+ tumors compared to HIF1+ and EV tumors always [37]) high levels of PC and GPC are associated with (Figures 7(a)–7(d)). The Chalkley Vessel Count (CVC) using increased proliferation and growth, but in the present study an anti-mouse CD31 antibody, was also higher in the HIF-2+ the HIF-2+ tumors had lower proliferation (Ki67) but higher tumors compared to both HIF-1+ and EV groups (Figures growth rates, compared to controls. The findings in the HIF- 7(e)–7(h)), which was consistent with the pattern of VEGF 2+ tumors combined with low apoptosis and autophagy are expression. in contrast to the tumor suppressor effects reported in both Journal of Oncology 9 PDH EV HIF-1+ HIF-2+ PDH EV HIF-1+ HIF-2+ (a) (b) (c) (d) PDK-1 PDK-1 EV HIF-1+ HIF-2+ (e) (f) (g) (h) TOM-20 TOM-20 EV HIF-1+ HIF-2+ (i) (j) (k) (l) Figure 5: Markers of oxidative phosphorylation and mitochondrial load in CCRCC 786-0 tumors. (a–d) PDH expression ( P = .003). (e–h) ∗ ∗ PDK-1 expression ( P = .006). (i–l) TOM-20 (mitochondrial marker) expression ( P = .004). P values were calculated using an ANOVA test. neuroblastoma [38]and coloncancer[39] xenograft models, rat GS9L orthotopic model, the tumor suppressive effect of as well as a rat GS9L orthotopic glioma model [40]. HIF-2α over-expression was caused by apoptosis [40]. This discrepancy in the growth profile between the 786- However in the CCRCC 786-0 model, we suggest that 0 CCRCC model and other non-CCRCC model systems over-expression of HIF-2α regulates growth both by main- may lie in the different HIFα backgrounds of the parental taining some glycolysis, albeit at a lower level, allowing cell lines which are different. The 786-0 CCRCC cell line more mitochondrial metabolism (higher PDH, lower PDK) only expresses HIF-2α, whereas both the N1E-115 neurob- and tolerance to DNA damage (γH2A.X) resulting from lastoma cell line [38] and the SW480 colon cancer line increased ROS (8-OH-guanosine) production. [39] endogenously expressed HIF-1α,aswellasHIF-2α.It A recent study by Gordan et al. [21] raises the possibility is the expression of HIF-1α in both of these other model that HIF1α acts as a tumor suppressor, and our data showing systems that is thought to facilitate tumor growth, and over- decreased growth rate of the HIF1+ compared to EV tumors expression of HIF-2α antagonises this effect. Similarly, in the seem to support this suggestion [10]. PDH histoscore PDK-1 histoscore TOM-20 histoscore 10 Journal of Oncology 8-OH-guanosine EV HIF-1+ HIF-2+ 8-OH-G EV HIF-1+ HIF-2+ (a) (b) (c) (d) γH2A.X 80 ∗ γH2A.X EV HIF-1+ HIF-2+ (e) (f) (g) (h) OGG1 OGG1 EV HIF-1+ HIF-2+ (i) (j) (k) (l) Figure 6: Oxidative stress and DNA damage/repair in CCRCC 786-0 tumors. (a–d) 8-OH-Guanosine staining (oxidative stress marker) ∗ ∗ ∗ ( P = .001). (e–h) γH2A.X staining (double-stranded DNA damage) ( P = .004). (i–l) OGG1 expression ( P = .006). P values were calculated using an ANOVA test. 4.2. Tumor Metabolism and Its Consequences. In non- HK-II and LDH5 and lower levels of lactate and alanine CCRCC cells in vitro, Akt signalling has also been demon- in the HIF-2+ tumors compared to both the EV and HIF- strated to positively regulate glycolysis in a HIF-1α indepen- 1+ tumors, suggested a decreased glycolytic flux in HIF2+ dent manner [41] mainly through mediating the localization tumors compared to HIF-1+ and EV tumors. However HIF- of GLUT-1. HIF-2+ tumors had lower expression of GLUT-1 1α (in an endogenous HIF-2α-only background) in vivo, in comparison to the EV tumors, despite supranormal levels appeared to have no effect on GLUT-1 expression since there of activated Akt. These findings are in contrast to the in vitro were no differences between glucose concentrations and findings of Raval et al. [12] who demonstrated that HIF- GLUT-1 expression in HIF-1+ and EV tumors. Interestingly, 2α was the principal regulator of GLUT-1 expression. An Cyclin D1 (which was higher in the HIF2+ tumors) has been explanation for this discrepancy between the in vitro and in shown in an in vivo mouse mammary cancer model to reduce vivo results could be that GLUT-1 expression is also sensitive the expression of both HK-II and LDH5 [42]. to changes in intracellular glucose concentration. Higher HIF-1 modulates multiple key metabolic pathways to concentrations of glucose were found in the HIF-2+ tumors, optimize use of O and glucose in response to changes in and could have attenuated GLUT-1 localization. This higher availability of these substrates, in order to most efficiently tumor glucose level along with decreased expression of generate ATP without excessive generation of ROS [7]. PDH Histoscore 786-0 nuclei (%) 786-0 nuclei (%) Journal of Oncology 11 EV HIF-1+ HIF-2+ VEGF VEGF EV HIF-1+ HIF-2+ (a) (b) (c) (d) CD31 Chalkley Vessel count Mouse CD31 EV HIF-1+ HIF-2+ (e) (f) (g) (h) ∗ + Figure 7: Markers of angiogenesis in CCRCC 786-0 tumors. (a–d) VEGF expression ( P = .003). (e–h) Murine CD31 vessel staining ∗ ∗ ( P = .001). values were calculated using an ANOVA test. is the key enzyme that determines whether pyruvate formed Since activated Akt is known to have the paradoxical during glycolysis from glucose will be metabolised to lactate effect of increasing mitochondrial O consumption and or oxidised in the TCA cycle. Its regulator, PDK, has been subsequently facilitating ROS generation [46], it could be shown to be expressed in a HIF-1α dependent manner [5, 6]. postulated that the supranormal levels of activated Akt status PDK negatively regulates PDH by phosphorylation, and in and the metabolic shift to greater oxidative metabolism in EV tumors the level of aerobic glycolysis was characterised the HIF-2+ tumors is mainly responsible for the higher levels by high PDK and low PDH indicating the basal level of of 8-OH-Guanosine immunostaining (high ROS stress) glycolysis in these tumors. A similarly high PDK, low PDH identified in these tumors. In spite of high ROS, γH2A.X was also found in HIF-1+ tumors, suggesting that the basal levels and OGG1 indicated resistance to DNA damage in level of aerobic glycolysis in EV 786-0 cells in vivo cannot be the HIF2+ tumors. These findings (summarised in Table 2) increased by exogenous expression of HIF-1α;alternatively are in agreement with Gordan et al. [8, 9] who have shown this may be due to mutually interacting effects of the pVHL that HIF-2α promotes cell cycle progression by enhancing -/-[43] and PTEN -/- status [44] of the parental 786-0 cell c-Myc mediated cyclin D2, leading to enhanced growth line. and resistance to DNA damage. This was not achieved In HIF-2+ tumors, in contrast, PDK-1 was decreased by modulating c-Myc levels, but by its interactions with and PDH was increased suggesting that the HIF2+ tumors partners. Although we did not stain for c-Myc in the rely on a less glycolytic, more oxidative metabolism. We 786-0 xenografts, it is highly likely that HIF-2α-mediated hypothesize that increased oxidation would supply more enhancement of c-Myc activity played a role in the xenografts reducing equivalents for the electron-transport chain (ETC), studied here. increase mitochondrial O consumption and thus increase the ATP supply to support the greater growth rate of 5. Conclusions the HIF2+ tumors. In support of this hypothesis were the findings of higher levels of TOM-20 (mitochondrial Tumor metabolism represents the end point of many signal load) and lower BNIP3 levels in HIF-2+ tumors, consistent cascades recruited by oncogenic activation. HIFα isoforms, with a higher mitochondrial mass, less mitophagy, and up- particularly HIF-1α, have been shown to be key regulators regulation of respiration, the converse of what was found of aerobic glycolysis in cancer cells. This is because HIF-1α with HIF-1α expression [45]. not only mediates the transcription of cytoplasmic glycolytic CVC score Histoscore 12 Journal of Oncology Table 2: Overview of molecular characteristics of HIF-1α and Abbreviations HIF-2α expression on CCRCC 786-0 xenografts compared to EV CCRCC: Clear-cell renal cell carcinoma xenografts. EV: CCRCC 786-0 tumors grown from cells Marker HIF-1+ HIF-2+ retrovirally infected with empty vector. Parameter HIF-1+: CCRCC 786-0 tumors grown from cells volume Growth retrovirally infected with expression of Ki67 ↓↓ HIF-1α. HIF-2+: CCRCC 786-0 tumors grown from cells Cyclin D1 — retrovirally infected with HIF-2α. Caspase-3 <0.5% <0.5% Apoptosis BNIP3 ↑ Autophagy Acknowledgments GLUT-1 — Glycolysis This paper was supported by Cancer Research UK, Li Ka HK II — Shing Foundation, and Hutchison Whampoa Ltd. LDH5 — Lactate — References Glucose — [1] G. L. Semenza, “Targeting HIF-1 for cancer therapy,” Nature Mitochondrial PDH/PDK ↓ Reviews Cancer, vol. 3, no. 10, pp. 721–732, 2003. Respiration [2] M. Zimmer, D. Doucette, N. Siddiqui, and O. Iliopoulos, TOM-20 — “Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL-/- tumors,” Molecular Cancer Research, 8-OH-guanosine ↓↑ ROS vol. 2, no. 2, pp. 89–95, 2004. γH2A.X ↑ DNA [3] V. Wang, D. A. Davis, M. Haque, L. E. Huang, and R. Yarchoan, “Differential gene up-regulation by hypoxia-inducible factor- OGG1 — damage/repair 1α and hypoxia-inducible factor-2α in HEK293T cells,” Cancer VEGF — Angiogenesis Research, vol. 65, no. 8, pp. 3299–3306, 2005. [4] C.-J. Hu,L.-Y. Wang,L.A.Chodosh,B.Keith,and M. CD31 — C. Simon, “Differential roles of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α in hypoxic gene regulation,” Molecular and Cellular Biology, vol. 23, no. 24, pp. 9361–9374, 2003. [5] J.-W. Kim, I. Tchernyshyov, G. L. Semenza, and C. V. enzymes and PDK-1, which phosphorylates and inactivates Dang, “HIF-1-mediated expression of pyruvate dehydroge- PDH, but also attenuates mitochondrial function by down nase kinase: a metabolic switch required for cellular adapta- regulating ETC activity, leading to a consequent reduc- tion to hypoxia,” Cell Metabolism, vol. 3, no. 3, pp. 177–185, tion of oxidative phosphorylation [7]. However, it could [6] I. Papandreou, R. A. Cairns, L. Fontana, A. L. Lim, and be speculated that expression of HIF-2α, in manipulated N. C. Denko, “HIF-1 mediates adaptation to hypoxia by CCRCC 786-0 tumors, overcomes the HIF-1α effects, which actively downregulating mitochondrial oxygen consumption,” results in a more oxidative tumor phenotype that supports Cell Metabolism, vol. 3, no. 3, pp. 187–197, 2006. a more aggressive phenotype. These results (see Table 2)are [7] G. L. Semenza, “Oxygen-dependent regulation of mitochon- in general agreement with the recent findings of Gordan drial respiration by hypoxia-inducible factor 1,” Biochemical et al. [8] who showed that HIF-2α expression in pVHL- Journal, vol. 405, no. 1, pp. 1–9, 2007. deficient CCRCC tumor lines potentiated c-Myc activity, [8] J. D. Gordan, J. A. Bertout, C.-J. Hu, J. A. Diehl, and M. resulting in enhanced growth and resistance to replication C. Simon, “HIF-2α promotes hypoxic cell proliferation by stress. We propose that the growth profiles observed in the enhancing c-Myc transcriptional activity,” Cancer Cell, vol. 11, HIF-1+ and HIF-2+ tumors that we have studied may be no. 4, pp. 335–347, 2007. mediated by HIF-1α inhibition of the c-Myc oncoprotein [9] J. D. Gordan, C. B. Thompson, and M. C. Simon, “HIF and c-Myc: sibling rivals for control of cancer cell metabolism and (slowing HIF-1+ growth) whilst HIF-2α potentiates c- proliferation,” Cancer Cell, vol. 12, no. 2, pp. 108–113, 2007. Myc transcriptional activity (HIF-2+) and promotes tumor [10] W. G. Kaelin Jr., “The von Hippel-Lindau tumour suppressor growth by an adaptive change to a more oxidative phenotype. protein: O2 sensing and cancer,” Nature Reviews Cancer, vol. In addition, the overall results from our study are consistent 8, no. 11, pp. 865–873, 2008. with the findings of Gordan et al. [21] who showed that [11] J. K. Maranchie, J. R. Vasselli, J. Riss, J. S. Bonifacino, W. clinical CCRCC tumors expressing only HIF-2α were bigger M. Linehan, and R. D. Klausner, “The contribution of VHL in size and more resistant to replicative stress compared to substrate binding and HIF1-α to the phenotype of VHL loss in those that expressed both HIF-1α and HIF-2 α.Thismay renal cell carcinoma,” Cancer Cell, vol. 1, no. 3, pp. 247–255, define a critical role for HIF-2α in the biology of VHL -/- CCRCC enabling greater growth; this demonstrates that in [12] R. R. Raval, K. W. Lau, M. G. B. Tran et al., “Contrasting certain contexts HIF-1α can act as a tumor suppressor (see properties of hypoxia-inducible factor 1 (HIF-1) and HIF- also [10, 47]). 2 in von Hippel-Lindau-associated renal cell carcinoma,” Journal of Oncology 13 Molecular and Cellular Biology, vol. 25, no. 13, pp. 5675–5686, of the breast; correlation of BNip3 levels with necrosis and 2005. grade,” Journal of Pathology, vol. 201, no. 4, pp. 573–580, 2003. [13] K. L. Covello, M. C. Simon, and B. Keith, “Targeted replace- [29] S. B. Fox, R. D. Leek, M. P. Weekes, R. M. Whitehouse, K. C. ment of hypoxia-inducible factor-1α by a hypoxia-inducible Gatter, and A. L. Harris, “Quantitation and prognostic value of factor-2α knock-in allele promotes tumor growth,” Cancer breast cancer angiogenesis: comparison of microvessel density, Research, vol. 65, no. 6, pp. 2277–2286, 2005. Chalkley count, and computer image analysis,” Journal of [14] K. Kondo, J. Klco, E. Nakamura, M. Lechpammer, and W. Pathology, vol. 177, no. 3, pp. 275–283, 1995. G. Kaelin Jr., “Inhibition of HIF is necessary for tumor [30] B. Madhu, S. P. Robinson, F. A. Howe, and J. R. Griffiths, suppression by the von Hippel-Lindau protein,” Cancer Cell, “Effect of Gd-DTPA-BMA on choline signals of HT29 tumors vol. 1, no. 3, pp. 237–246, 2002. detected by in vivo H MRS,” Journal of Magnetic Resonance Imaging, vol. 28, no. 5, pp. 1201–1208, 2008. [15] L. Holmquist-Mengelbier, E. Fredlund, T. Lofst ¨ edt et al., “Recruitment of HIF-1α and HIF-2α to common target genes [31] R. J. Ordidge, A. Connelly, and J. A. B. Lohman, “Image- is differentially regulated in neuroblastoma: HIF-2α promotes selected in vivo spectroscopy (ISIS). A new technique for an aggressive phenotype,” Cancer Cell, vol. 10, no. 5, pp. 413– spatially selective NMR spectroscopy,” Journal of Magnetic 423, 2006. Resonance, vol. 66, no. 2, pp. 283–294, 1986. [16] G. Qing and M. C. Simon, “Hypoxia inducible factor-2α:a [32] M. B. Azad, Y. Chen, E. S. Henson et al., “Hypoxia induces critical mediator of aggressive tumor phenotypes,” Current autophagic cell death in apoptosis-competent cells through a Opinion in Genetics & Development, vol. 19, no. 1, pp. 60–66, mechanism involving BNIP3,” Autophagy,vol. 4, no.2,pp. 2009. 195–204, 2008. [17] O. Warburg, The Metabolism of Tumors, Constable, London, [33] H. R. Mellor and A. L. Harris, “The role of the hypoxia- UK, 1930. inducible BH3-only proteins BNIP3 and BNIP3L in cancer,” Cancer and Metastasis Reviews, vol. 26, no. 3-4, pp. 553–566, [18] P. Vizan, ´ S. Mazurek, and M. Cascante, “Robust metabolic adaptation underlying tumor progression,” Metabolomics, vol. 2007. 4, no. 1, pp. 1–12, 2008. [34] A. S. E. Ojugo, P. M. J. McSheehy, D. J. O. McIntyre et al., “Measurement of the extracellular pH of solid tumours in [19] N. C. Denko, “Hypoxia, HIF1 and glucose metabolism in the solid tumour,” Nature Reviews Cancer, vol. 8, no. 9, pp. 705– mice by magnetic resonance spectroscopy: a comparison of 19 31 exogenous Fand Pprobes,” NMR in Biomedicine, vol. 12, 713, 2008. no. 8, pp. 495–504, 1999. [20] G. L. Semenza, “Regulation of cancer cell metabolism by hypoxia-inducible factor 1,” Seminars in Cancer Biology, vol. [35] B. Govindarajan, J. E. Sligh, B. J. Vincent et al., “Overex- pression of Akt converts radial growth melanoma to vertical 19, no. 1, pp. 12–16, 2009. growth melanoma,” Journal of Clinical Investigation, vol. 117, [21] J. D. Gordan, P. Lal, V. R. Dondeti et al., “HIF-α effects on c- no. 3, pp. 719–729, 2007. Myc distinguish two subtypes of sporadic VHL-deficient clear [36] R. B. Robey and N. Hay, “Mitochondrial hexokinases, novel cell renal carcinoma,” Cancer Cell, vol. 14, no. 6, pp. 435–446, 2008. mediators of the antiapoptotic effects of growth factors and Akt,” Oncogene, vol. 25, no. 34, pp. 4683–4696, 2006. [22] P. M. J. McSheehy,M.T.Seymour,A.S.E.Ojugo et al., [37] E. Ackerstaff, K. Glunde, and Z. M. Bhujwalla, “Choline “A pharmacokinetic and pharmacodynamic study in vivo of 19 31 human HT29 tumours using Fand P magnetic resonance phospholipid metabolism: a target in cancer cells?” Journal of Cellular Biochemistry, vol. 90, no. 3, pp. 525–533, 2003. spectroscopy,” European Journal of Cancer, vol. 33, no. 14, pp. 2418–2427, 1997. [38] J. Favier, S. Lapointe, R. Maliba, and M. G. Sirois, “HIF2α [23] H. U. Bergmeyer, Methods of Enzymatic Analysis,Verlag reduces growth rate but promotes angiogenesis in a mouse model of neuroblastoma,” BMC Cancer, vol. 7, article 139, Chemie, Weinheim, Germany, 2nd edition, 1974. [24] J.-L. Li, R. C. A. Sainson, W. Shi et al., “Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular [39] T. Imamura, H. Kikuchi, M.-T. Herraiz et al., “HIF-1α and HIF-2α have divergent roles in colon cancer,” International function, and promotes tumor growth in vivo,” Cancer Research, vol. 67, no. 23, pp. 11244–11253, 2007. Journal of Cancer, vol. 124, no. 4, pp. 763–771, 2009. [25] S. A. Fuhrman, L. C. Lasky, and C. Limas, “Prognostic sig- [40] T. Acker, A. Diez-Juan, J. Aragones et al., “Genetic evidence for a tumor suppressor role of HIF-2α,” Cancer Cell,vol. 8, no.2, nificance of morphologic parameters in renal cell carcinoma,” American Journal of Surgical Pathology, vol. 6, no. 7, pp. 655– pp. 131–141, 2005. 663, 1982. [41] D. R. Plas and C. B. Thompson, “Akt-dependent transforma- tion: there is more to growth than just surviving,” Oncogene, [26] H. M. E. Scheres, A. F. P. M. De Goeij, M. J. M. Rousch et al., “Quantification of oestrogen receptors in breast cancer: vol. 24, no. 50, pp. 7435–7442, 2005. radiochemical assay on cytosols and cryostat sections com- [42] T. Sakamaki, M. C. Casimiro, X. Ju et al., “Cyclin D1 pared with semiquantitative immunocytochemical analysis,” determines mitochondrial function in vivo,” Molecular and Journal of Clinical Pathology, vol. 41, no. 6, pp. 623–632, 1988. Cellular Biology, vol. 26, no. 14, pp. 5449–5469, 2006. [27] W. R. Duan, D. S. Gamer, S. D. Williams, C. L. Funckes- [43] E. Hervouet, J. Demont, P. Pecina et al., “A new role for the Shippy, I. S. Spath, and E. A. G. Blomme, “Comparison of von Hippel-Lindau tumor suppressor protein: stimulation of immunohistochemistry for activated caspase-3 and cleaved mitochondrial oxidative phosphorylation complex biogene- cytokeratin 18 with the TUNEL method for quantification sis,” Carcinogenesis, vol. 26, no. 3, pp. 531–539, 2005. of apoptosis in histological sections of PC-3 subcutaneous [44] R. L. Elstrom, D. E. Bauer, M. Buzzai et al., “Akt stimulates xenografts,” Journal of Pathology, vol. 199, no. 2, pp. 221–228, aerobic glycolysis in cancer cells,” Cancer Research, vol. 64, no. 11, pp. 3892–3899, 2004. [28] H. M. Sowter, M. Ferguson, C. Pym et al., “Expression of the [45] G. L. Semenza, “Mitochondrial autophagy: life and breath of cell death genes BNip3 and NIX in ductal carcinoma in situ the cell,” Autophagy, vol. 4, no. 4, pp. 534–536, 2008. 14 Journal of Oncology [46] V. Nogueira, Y. Park, C.-C. Chen et al., “Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis,” Cancer Cell, vol. 14, no. 6, pp. 458–470, 2008. [47] W. G. Kaelin Jr. and P. J. Ratcliffe, “Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway,” Molecular Cell, vol. 30, no. 4, pp. 393–402, 2008. MEDIATORS of INFLAMMATION The Scientific Gastroenterology Journal of World Journal Research and Practice Diabetes Research Disease Markers Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Journal of Immunology Research Endocrinology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com BioMed PPAR Research Research International Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Obesity Evidence-Based Journal of Journal of Stem Cells Complementary and Ophthalmology International Alternative Medicine Oncology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Parkinson’s Disease Computational and Behavioural Mathematical Methods AIDS Oxidative Medicine and in Medicine Research and Treatment Cellular Longevity Neurology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Journal

Journal of Oncology – Wiley

Published: Jun 27, 2010

There are no references for this article.