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Expression of hypoxia inducible factor-1α in tumors of patients with glioblastoma multiforme and transitional meningioma
Available online at www.sciencedirect.com
Journal of Clinical Neuroscience 15 (2008) 1036–1042 www.elsevier.com/locate/jocn
Laboratory Study
Expression of hypoxia inducible factor-1a in tumors of patients with glioblastoma multiforme and transitional meningioma Mehmet Yasar Kaynar a, Galip Zihni Sanus a, Hakan Hnimoglu a, Tibet Kacira a, Rahsan Kemerdere a, Pinar Atukeren b, Koray Gumustas b, Bulent Canbaz a, Taner Tanriverdi a,* a b
Department of Neurosurgery, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey Received 5 April 2007; accepted 20 July 2007
Abstract Hypoxia-inducible factor-1 a (HIF-1a) is the major transcriptional factor involved in the adaptive response to hypoxia. The aim of this study was to assess HIF-1a in 22 patients with transitional meningioma (TM) and 26 patients with glioblastoma multiforme (GBM). HIF-1a was assessed using a commercially available enzyme-linked immunosorbent assay-based HIF-1 transcription factor assay. Levels of HIF-1a in TM and GBM were measured using optical density at 450 nm, and median values were found to be 0.35 for TM and 0.37 OD for GBM, respectively. There was no statistically significant difference between the two types of tumor (p = 0.264). These findings indicate that HIF-1a is elevated in both TM and GBM, suggesting that although hypoxia is one of the most important and powerful stimuli for HIF-1a elevation and consequently angiogenesis, other mechanisms may play roles in HIF-1a stimulation in benign brain tumors such as TM. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Brain tumors; Glioblastoma multiforme; HIF-1; Hypoxia; Meningioma
1. Introduction Hypoxia, a condition of low tissue oxygen (O2) concentration, is known to play an important role in normal physiological processes and tumor formation. Under hypoxic conditions, hypoxia-inducible factor-1 (HIF-1) binds to hypoxia-responsive elements (HRE) and induces transcription of various target genes involved in tumor angiogenesis, invasion, cell survival, and glucose metabolism.1–3 Since its discovery,4,5 HIF-1 has emerged as a key regulatory element in a variety of developmental, physiological and pathological responses to hypoxia. HIF-1 is composed of two subunits, hypoxia-inducible factor-1 al*
Corresponding author. Present address: P.K: 4, Cerrahpasa, 34301, Istanbul, Turkey. Tel./fax: +90 212 414 34 27. E-mail address: [email protected] (T. Tanriverdi). 0967-5868/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2007.07.080
pha (HIF-1a) and hypoxia-inducible factor-1 beta (HIF1b) (also known as aryl hydrocarbon receptor nuclear translocator) subunits, which belong to the basic helixloop-helix Per-Arnt-Sim (bHLH-PAS) family of transcription factors.1–6 These subunits, HIF-1 a and HIF-1b, must associate to form the active HIF heterodimer responsible for transcriptional activation.2–4 HIF-1 transcriptional activity mainly depends on the amount of HIF-1a protein in the cell, whereas HIF-1b is constitutively present regardless of O2 concentration; that is, HIF-1 a is the unique, O2regulated subunit that determines HIF-1 activity.2,3 In normoxic conditions, HIF-1a is rapidly degraded by the ubiquitin proteasome system. This degradation is dependent on O2 and iron-dependent hydroxylation at Pro-564 and Pro-402, followed by interaction with von Hippel-Lindau (VHL) E3 ubiquitin ligase.1 This degradation can be reduced by treating cells with agents that mimic hypoxic
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conditions, such as cobalt chloride, deferrioxamine, or dimethyloxalylglycine, regardless of the E3 ubiquitin ligase activity of VHL6. Under these conditions, stabilized HIF1a is transported into the nucleus where it dimerizes and creates an active complex with HIF-1b. HIF-1 is essential for angiogenesis, embryonic development, and is associated with tumor progression, erythropoiesis, vascular development/remodeling, vasodilation, and glucose/energy metabolism.1–8 Over-expression of HIF-1a has been demonstrated in many common human cancers and has been shown to correlate with tumor grade and tumor progression.9 Besides physiological hypoxia, genetic abnormalities frequently detected in human cancers, such as loss of function mutations (VHL, p53, and phosphatase and tensin homolog deleted on chromosome 10 [PTEN]), are associated with induction of HIF-1 activity and expression of HIF-1-inducible genes.3 HIF target genes include erythropoietin (EPO), heme oxidase 1 (HOX-1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS).1– 3,6,8 Cells with genetically deleted HIF-1a appear to be resistant to hypoxia-triggered apoptosis.8 A growing body of evidence from immunohistochemical analysis has shown that HIF-1a is present at higher levels in human tumors than in normal tissues.9,10 Most importantly, in gliomagenesis, HIF-1a has been shown to be a potent activator of angiogenesis and invasion through its up-regulation of target genes that are critical for these functions.11,12 Thus, since hypoxia is the main event in tumor progression and since HIF-1a expression and/or activity is regulating the cellular adaptation to hypoxia that appears central to tumor growth and progression, HIF-1 inhibition has become an anti-cancer target in recent years.13–20 Although HIF-1a expression in common human tumors including glioblastoma multiforme (grade-IV astrocytoma-GBM) is well established,9–12,21,22 there is little information about HIF-1a expression in meningioma.23 In this study, we examined the expression of HIF-1a in GBM and transitional meningioma (TM) and we compared the results for these two types of brain tumor. 2. Materials and methods 2.1. Study groups The present study involved two groups: patients with TM and patients with GBM. This study was conducted at the Department of Neurosurgery, Cerrahpasa Medical Faculty, Istanbul University, Turkey, in 2006. All patients or next of kin were fully informed and ethical approval for this study was obtained from the Human Investigations Committee at Istanbul University. We excluded patients who had any kind of chronic or acute infection, any immunological or metabolic disease, neoplastic disease of an other organ system, and any cardiovascular disease, and patients who had undergone a recent major surgical procedure.
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2.2. Patients with TM This study included 22 consecutive patients with histopathologically proven TM. Twelve and 10 lesions were located in the right and left hemispheres, respectively. The frontal lobe was the most commonly involved region (8 tumors), followed, in decreasing order of frequency, by the temporal (7 tumors), parietal (5) and occipital (1) lobes. The average age of the patients (13 women and 9 men) was 46 years. The postoperative period of all patients was uneventful and no recurrence was noted in the follow-up period. 2.3. Patients with GBM Twenty-six consecutive patients with histopathologically proven GBM were included. Sixteen lesions were located in the right hemisphere, whereas 10 were located in the left. The temporal lobe was the most affected region (10 GBM), followed by the occipital (5), frontal (4), parietal (3), fronto-parietal (2) and temporo-parietal (2) lobes. The average age of the patients (17 men and 9 women) was 51 years. The postoperative period was uneventful for all patients. All patients with GBM were referred to the Radiation Oncology Department for planning of further treatment, including chemotherapy and/or radiotherapy. All patients with either TM or GBM received corticosteroids and anticonvulsant medication (single agent) before, during and after surgery. 2.4. Specimen handling Twenty-two TMs and 26 GBMs were assayed for HIF-1a. For each patient, after exposure of the neoplasm, tumor tissue was obtained before thermal coagulation. All specimens were obtained from the core of the tumor. As soon as possible, each sample was frozen to –80°C until it was assayed. 2.5. Measurement of HIF-1a Prior to the Chemicon Transcription Factor Assay (catalog no. SGT 570), nuclear extracts were obtained using a nuclear extract kit (catalog no. 2900; Chemicon, Temecula, CA, USA). Cell pellets were washed in ice cold phosphatebuffered saline (PBS) (pH 7.5) and then warmed trypsin cell detachment buffer was added and the cells were washed again in PBS. Cell pellets were re-suspended in ice cold cytoplasmic lysis buffer containing dithiothreitol (DTT) and protease inhibitor cocktail. Using a syringe with a small (27 gauge) needle, the cell suspension was drawn into the syringe and then injected back into the sample tube to dissociate the cells. After centrifugation, the nuclear pellet was re-suspended in 2/3 of the original cell pellet volume of ice cold nuclear extraction buffer containing DTT and a protease inhibitor cocktail, thus, the nuclei were disrupted. After centrifugation, supernatants were stored at 80°C as nuclear extracts until the assay was performed.
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2.6. Statistical analysis We used a commercially available statistical software package (SPSS version 11.0; SPSS Inc., Chicago, IL, USA) for all statistical analyses. The medians, interquartile ranges and full ranges were calculated for HIF-1a. For all comparisons, the non-parametric Mann-Whitney U-test was used as a statistical method. Differences were considered statistically significant if the probability value was less than 0.05. 3. Results The median HIF-1a OD value at 450 nm in patients with TM was 0.35 (range: 0.29–0.39; interquartile range: 0.10), and in the GBM group it was 0.37 (range: 0.29– 0.42; interquartile range: 0.13) (Fig. 1). Although the median HIF-1a activity was higher in GBM than TM, the difference was not statistically significant (p = 0.264). 4. Discussion 4.1. General considerations Glioblastoma multiforme (GBM grade IV astrocytoma) is the most malignant brain tumor known, and accounts for 80% of all malignant astrocytomas.24 Despite great advances in both diagnostic and treatment modalities, including surgery, chemotherapy, radiotherapy and immunotherapy, patients with GBM have an extremely poor
P = 0.264
.44 .42 .40 .38 .36 .34 .32 .30 .28
GBM
.26 TM
HIF-1 activation was detected and quantified using a non-radioactive, enzyme-linked immunosorbent assay (ELISA)-based HIF-1 transcription factor assay (Chemicon), which combines the principle underlying the electrophoretic mobility shift assay (EMSA) with 96-well-based ELISA, enabling manual or high-throughput sample analysis with greater sensitivity than conventional EMSA assays. During the assay, the HIF-1 capture probe, a doublestranded biotinylated oligonucleotide containing the flanked HRE consensus sequence (50 - ACGTG -30 ) for HIF-1a binding, was mixed with cellular (nuclear) extract in the transcription factor assay buffer provided in the kit. When incubated together, the active form of HIF-1a contained in the nuclear extract binds to its consensus sequence. Nuclear cell extracts from Cos-7 cells treated with CoCl2 were used as positive controls. The extract/probe/buffer mixture was then directly transferred to a streptavidincoated plate. The active HIF-1a protein that was bound to the biotinylated double-stranded oligonucleotide was immobilized and any inactive, unbound material was washed away. The bound HIF-1a transcription factor was detected using a specific primary polyclonal anti-HIF-1a antibody. A horseradish peroxidase-conjugated secondary antibody was then used for detection in a spectrophotometric plate reader. Values are expressed as optical density (OD) at 450 nm.
HIF-1 alpha (OD 450)
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Fig. 1. Box plot comparing HIF-1a activity in patients with transitional meningioma and those with glioblastoma multiforme. The difference was not statistically significant. The horizontal lines within the boxes represent median values, and the boxes denote the interquartile range. Vertical lines denote the full ranges. GBM, glioblastoma multiforme; TM, transitional meningioma; HIF-1a, hypoxia inducible factor; OD, optical density.
prognosis and median survival after initial diagnosis is still less than 1 year.25 Key histopathological features of GBM, such as necrosis and endothelial proliferation, distinguish these tumors from low-grade astrocytomas, which are associated with a better prognosis. GBM can occur as the result of progression from lower-grade astrocytomas or can arise de novo. Details of their pathogenesis remain to be elucidated. In contrast, meningiomas are generally considered to be benign tumors of the brain, and transitional or mixed types of meningiomas together with fibroblastic and meningothelial (syncytial) tumors are classed as type I meningiomas, which generally do not invade the brain tissue.26 They generally have a clear capsule, which enables the surgeon to easily remove them from the normal brain parenchyma; thus, total removal is achieved in most cases. The extent of resection depends largely on the location of the lesion and lesions can become problematic if located in certain positions, such as the medial sphenoid wing, posterior parasagittal region and posterior fossa, where total removal may damage important structures.26 Although several factors, including head trauma, radiation, hormones, chromosomal aberrations and viruses, have been identified as contributing to the development of meningiomas, the main etiological factor is unknown.26–29 Solid tumors, such as GBMs, have extensive areas of hypoxia and necrosis, which worsen a patient’s prognosis because necrosis and hypoxia promote a more malignant phenotype, and hypoxic cells are resistant to chemoand radiotherapy.9,10,12,16,19,29 It has been shown that hypoxia-induced angiogenesis is one of the most important steps in the survival of tumor cells.30 Elevated vascular cell
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endothelial growth factor (VEGF) has been shown to be associated with recurrence, neovascularization, peritumoral edema and malignancy.31–33 Transcription of VEGF is usually regulated by HIF-1a, a transcription factor that is critical in the adaptive response to reduced O2 concentration.34 Activation of HIF-1a leads to up-regulation of angiogenetic factors that are essential for the formation of new blood vessels and is one of the most important primary forces driving both physiological and pathological angiogenesis.35,36 4.2. Hypoxia inducible factor-1 HIF-1 is a heterodimer composed of a and b subunits, both of which belong to the basic helix-loop-helix (bHLH) Per Arnsim family of proteins.1–3,6–8 HIF-1b, an aryl hydrocarbon nuclear receptor translocator, heterodimerizes with aryl hydrocarbon receptor to form the functional dioxin receptor, and is a nuclear protein that is constitutively expressed, independent of O2 tension.37,38 HIF-1a is an 826-amino acid (120-kDa) protein that, in contrast to HIF-1b, is a cytoplasmic protein that is responsive to O2 levels.37,38 Under normoxic conditions, HIF-1a is continuously degraded by the ubiquitin-proteasome system.39 Under low O2 tension conditions, this degradation is inhibited, leading to increased HIF-1a, which translocates to the nucleus to activate gene transcription and heterodimerizes with HIF-1b subunits.39 The resultant product is an active HIF-1 protein that binds to specific HREs present in target genes, which encode erythropoietin, VEGF, various glycolytic enzymes, transferrin, and a variety of other proteins that are essential for systemic, local, and intracellular homeostasis.13,14,17–20 More importantly, it has been shown that the vast majority of these gene products are over-expressed in human tumor cells, suggesting that HIF-1 may play a role in tumor development and progression.9,21,40–44 Overall, these adaptive responses to low O2 levels serve as a compensatory mechanism for increasing delivery of O2 via new vessels (and nutrients) for any cells of the body or tumor cells with an inadequate O2 supply. 4.3. Hypoxia-inducible factor-1a and tumors Immunolabelling studies using monoclonal antibodies raised against the HIF-1a subunit, which determines HIF activity, have demonstrated increased HIF-1a expression in about 53% of malignant tumors, including brain, bladder, breast, colon, ovarian, pancreatic, renal and prostate tumors,9,21,23,41,42,44,45 whereas no expression has been detected in surrounding normal tissue, nor in benign tumors such as breast fibroadenoma and uterine leiomyoma.9,46 Clinically, increased HIF-1a levels are not only correlated with tumor grade and poor prognosis but also treatment failure in a number of cancers, including astrocytomas, oligodendrogliomas and meningiomas.21,23,45 In many cancers, over-expression of HIF-1a is seen at an early stage
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of tumor development, with expression correlating with the vascular density of the lesions. The same phenomenon is seen in brain tumors, suggesting that HIF-1 activity contributes to the angiogenic switch, most likely mediated by increased formation of pro-angiogenic factors such as VEGF.47 The pattern of HIF-1a protein expression in GBM is identical to that described for VEGF mRNA and meningiomas.21 GBM has significantly higher levels of VEGF expression and neovascularization compared with low-grade gliomas.48 Current evidence suggests that malignant gliomas take advantage of hypoxia-mediated HIF activation by up-regulation of at least two growth factors, platelet-derived growth factor (PDGF)-BB and VEGF.47,49 PDGF-BB, when expressed in glioma cells, sustains glioblastoma growth via autocrine and paracrine cell regulatory mechanisms.49 VEGF, as the major tumor angiogenesis and vascular permeability factor, supports glioma growth via a paracrine effect on endothelial cells.47 Increased HIF-1a levels have also been demonstrated in tumors, regardless of the presence of hypoxia. In such circumstances, it has been suggested that over-expression of HIF-1a is induced by nonphysiologic oncogenic stimuli rather than hypoxia, for example in hemangioblastoma, oligodendroglioma and low-grade epithelial ovarian tumors, despite their highly vascularized nature.44,45,50,51 HIF-1a is thought to be involved in tumoriogenesis through two main mechanisms: hypoxia-dependent mechanisms, which are observed close to the necrotic area, and hypoxia-independent mechanisms, such as oncogene activation and growth factor signaling pathways. The relevance of the HIF-1a system to tumor growth and progression is highlighted by the variety of mechanisms regulated by HIF-target genes, ranging from angiogenesis, to increased tissue oxygenation over glycolysis, and pH regulation, allowing for energy generation when oxygen is scarce, to cell proliferation and survival pathways.40 Intra-tumoral hypoxia correlates directly with the tumor’s biological aggressiveness, degree of malignancy and clinical recurrence, and inversely correlates with the post-operative survival of patients with gliomas.52,53 Since angiogenesis and cellular adaptation to hypoxia represent key steps in tumor progression, and since one of the key factors regulating cellular O2 homeostasis is HIF-1a, targeting HIF-1 may be beneficial in the treatment of aggressive brain tumors. Thus, in recent years HIF-1a has emerged as a potentially important therapeutic target for cancer treatment. Strategies suggested for HIF-1a targeting include disruption of the normal coactivational response to hypoxia, the use of decoy oligonucleotides, and a gene therapy approach based on HRE-regulated gene expression that exploits the presence of hypoxia/anoxia in tumors for the induction of therapeutic genes.13,17 4.4. Present study In the present study, we demonstrated that HIF-1a level is elevated in both TM and GBM. However, no significant
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difference was found in terms of HIF-1a expression between TM and GBM, which are benign and malignant tumors of the brain, respectively. These findings may be explained by both hypoxic and non-hypoxic mechanisms. In GBM, there is almost always a hypoxic region, thus the elevated HIF-1a level is due to hypoxia, which up-regulates the angiogenic factors in order to compensate for the demands of rapidly growing tumors. In contrast, overexpression of HIF-1a in TM in our study could not be attributed to hypoxia alone because such tumors grow slowly enough that they rarely outgrow their blood supply. Therefore, we suggest that elevated expression of HIF-1a is induced by non-physiologic oncogenic stimuli in TM rather than hypoxia, as in GBM, which is analogous to the situation in hemangioblastomas51 (51) and oligodendrogliomas.45 Although only a small number of patients with TM were included in our study, we speculate that three mechanisms, loss of p53, PTEN and/or VHL function, can explain over-expression of HIF-1a in TMs. p53 is a tumor suppressor gene and loss of function is a frequent event in tumorigenesis. Although the function of p53 is still not well understood, many lines of evidence indicate that meningiomas have low levels of p53, leading to speculations that p53 mutations may be involved in the etiology of these tumors.54–56 It has been demonstrated that wildtype p53 promotes degradation of HIF-1a57 so it may be expected that inactivation of p53 either results from somatic mutations or from functional inactivation, which would lead to increased HIF-1a expression in meningiomas. If functional p53 is lost, the proapoptotic effect of HIF-1a is lost and tumor progression is stimulated by HIF-1a via induction of angiogenesis. Furthermore, loss of p53 activity in meningioma cells may be associated with increased HIF-1a expression and HIF-1 DNA binding activity, as a consequence promoting tumor angiogenesis by enhancing levels of the angiogenic factor VEGF.35 Immunohistochemically detectable accumulation of p53 and HIF-1a has been found in glioma,22 oligodendroglioma,45,58 and highly vascularized ovarian carcinoma.59 Similar to p53, PTEN is a recently identified tumor suppressor gene that has been found to be inactive in various human cancers, including GBM.60 Although PTEN mutations are rarely seen in low-grade meningiomas,54,61,62 it has been suggested that PTEN is a progression-associated tumor suppressor gene in meningiomas.61,62 Furthermore, mice that are heterozygous for PTEN are predisposed to various forms of cancer, including meningiomas.63 In addition, germline mutations in PTEN have been detected in patients with Cowden disease,64 which is an autosomal dominant disorder characterized by hamartomas of multiple organs, macrocephaly and carcinoma of the breast and thyroid. Patients with Cowden disease have an increased risk of developing meningiomas.65 It has been shown that wild-type PTEN downregulates HIF-1-mediated gene expression.53,66 Induced expression of PTEN in glioma cells has been shown to decrease accumulation of HIF-1a in gliomas, reduce angiogenesis66 under hypoxic conditions
and lead to attenuation of the HIF transcriptional response, possibly by inhibiting the P13K (kinase)-AKT pathway.67 Another mechanism that may explain the overexpression of HIF-1a in TMs is loss of VHL function. The VHL gene product (pVHL) is required for regulation of the HIF-1 transcriptional control system.68 It has recently been demonstrated that VHL inactivation is frequently detected in sporadic hemangioblastoma and renal cell carcinoma,69,70 both of which are highly vascular tumors, like meningiomas. The common finding of lesions associated with VHL loss of function is an angiogenic phenotype that suggests that angiogenic factors such as VEGF are constitutively activated. More importantly, HIF-1a degradation involves a physical association between pVHL and HIF-a subunits.69,70 Additionally, nonfunctional VHL also results in the accumulation of HIF-1a in normoxic conditions.68 Given these data, we speculate that HIF-1 is involved in tumorigenesis through a number of different mechanisms, as suggested by the results of our study. On the one hand, hypoxia, in the case of GBM, induces the activation of HIF-1, which increases the expression of several glycolytic enzymes and hence the glycolysis rate for cells’ adaptive modifications to hypoxia. On the other hand, HIF-1 is involved in the establishment of vascular supply, and neoangiogenesis can be triggered by tumor cells becoming hypoxic while enlarging, as in cases of meningioma. 5. Conclusion We conclude that HIF-1a is elevated in both TMs and GBMs, suggesting that although hypoxia is one of the most important and powerful stimuli for HIF-1a elevation and consequent angiogenesis, other mechanisms may play roles in HIF-1a stimulation in benign brain tumors such as TM. References 1. Chan DA, Sutphin PD, Denko NC, et al. Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1a. J Biol Chem 2002;277:40112–7. 2. Chun YS, Choi E, Kim TY, et al. A dominant-negative isoform lacking exons 11 and 12 of the human hypoxiainducible factor-1a gene. Biochem J 2002;362:71–9. 3. Rapisarda A, Uranchimeg B, Scudiero DA, et al. Identification of small molecule inhibitors of hypoxia-inducible factor-1 transcriptional activation pathway. Cancer Res 2002;62:4316–24. 4. Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995;92:5510–4. 5. Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 1995;270:1230–7. 6. Chun YS, Choi E, Kim GT, et al. Cadmium blocks hypoxia-inducible factor (HIF)-1-mediated response to hypoxia by stimulating the proteasome-dependent degradation of HIF-1a. Eur J Biochem 2000;267:4198–204. 7. Narravula S, Colgan SP. Hypoxia-Inducible Factor-1-mediated inhibition of peroxisome proliferator-activated receptor a expression during hypoxia. J Immunol 2001;166:7543–8.
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34. Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1a. Genes Dev 1998;12:149–62. 35. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor I. Mol Cell Biol 1996;16:4604–13. 36. Gleadle JM, Ratcliffe PJ. Induction of hypoxia-inducible factor-1, erythropoietin, vascular endothelial growth factor, and glucose transporter-1 by hypoxia: evidence against a regulatory role for Src kinase. Blood 1997;89:503–9. 37. Jiang BH, Semenza GL, Bauer C, et al. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am J Physiol 1996;271:C1172–80. 38. Semenza GL. Regulation of mammalian O2 homeostasis by hypoxiainducible factor 1. Annu Rev Cell Dev Biol 1999;15:551–78. 39. Semenza GL, Nejfelt MK, Chi SM, et al. Hypoxia-inducible nuclear factors bind to enhancer element located 3’ to the human erythropoietin gene. Proc Natl Acad Sci 1991;88:5680–4. 40. Brat DJ, Mapstone TB. Malignant glioma physiology: Cellular response to hypoxia and its role in tumor progression. Ann Intern Med 2003;138:659–68. 41. Compostella A, Tosoni A, Blatt V, et al. Prognostic factors for anaplastic astrocytomas. J Neurooncol 2006;26:1–9. 42. Flamme I, Krieg M, Plate KH. Up-regulation of vascular endothelial growth factor in stromal cells of hemangioblastomas is correlated with up-regulation of the transcription factor HRF/HIF-2a. Am J Pathol 1998;153:25–9. 43. Jensen RL, Ragel BT, Whang K, et al. Inhibition of hypoxia-inducible factor-1a (HIF-1a) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas. J NeuroOncol 2006;78:233–47. 44. Preusser M, Wolfsberger S, Haberler C, et al. Vascularization and expression of hypoxia-related tissue factors in intracranial ependymoma and their impact on patient survival. Acta Neuropathol 2005;109:211–6. 45. Birner P, Gatterbauer B, Oberhuber G, et al. Expression of hypoxiainducible factor-1 a in oligodendrogliomas. Its impact on prognosis and on neoangiogenesis. Cancer 2001;92:165–71. 46. Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1a and HIF-2a in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol 2000;157:411–21. 47. Plate KH, Risau W. Angiogenesis in malignant gliomas. Glia 1995;15:339–47. 48. Pietsch T, Valter MM, Wolf HK, et al. Expression and distribution of vascular endothelial growth factor protein in human brain tumors. Acta Neuropathol 1997;93:109–17. 49. Smits A, Funa K. Platelet-derived growth factor (PDGF) in primary brain tumors of neuroglial origin. Histol Histopathol 1998;13:511– 20. 50. Birner P, Schindl M, Obermair A, et al. Expression of hypoxiainducible factor (HIF) 1a in epithelial ovarian tumors: its impact on prognosis and on response to chemotherapy. Clin Cancer Res 2001;7:1661–8. 51. Zagzag D, Zhong H, Scalzetti J, et al. Expression of hypoxia-inducible factor-1a in brain tumors: association with angiogenesis, invasion and progression. Cancer 2000;88:2606–18. 52. Acker T, Plate KH. Hypoxia and hypoxia inducible factors (HIF) as important regulators of tumor physiology. Cancer Treat Res 2004;117:219–48. 53. Fischer I, Gagner JP, Law M, et al. Angiogenesis in gliomas: Biology and molecular pathophysiology. Brain Pathol 2005;15:297–310. 54. Al-Khalaf HH, Lach B, Allam A, et al. The p53/p21 DNA damagesignaling pathway is defective in most meningioma cells. J Neurooncol 2007;83:9–15. 55. Wang JL, Zhang ZJ, Hartman M, et al. Detection of TP53 gene mutation in human meningiomas: a study using immunohistochemistry, polymerase chain reaction/single-strand conformation polymor-
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63. Newton HB. Molecular neuro-oncology and development of targeted therapeutic strategies for brain tumors. Part 2: PI3K/Akt/PTEN, mTOR, SHH/PTCH and angiogenesis. Expert Rev Anticancer Ther 2004;4:105–28. 64. Liaw D, Marsh DJ, Li J, et al. Germ line mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nature Genet 1997;16:64–7. 65. Lyons CJ, Wilson CB, Horton JC. Association between meningioma and Cowden’s disease. Neurology 1993;43:1436–7. 66. Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev 2000;14:391–6. 67. Zhong H, Chiles K, Feldser D, et al. Modulation of hypoxia-inducible factor 1 alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 2000;60:1541–5. 68. Maxwell PH, Wiesener MS, Chang GW, et al. The tumor suppressor protein VHL targets hypoxia-inducible factor for oxygen-dependent proteolysis. Nature 1999;399:271–5. 69. Maxwell PH, Pugh CW, Ratcliffe PJ. Insights into the role of the von Hippel-Lindau gene product. A key player in hypoxic regulation. Exp Nephrol 2001;9:235–40. 70. Yang H, Kaelin Jr WG. Moleculer pathogenesis of the von HippelLindau hereditary cancer syndrome: implications for oxygen sensing. Cell Growth Differ 2001;12:447–55.
Journal of Clinical Neuroscience 15 (2008) 1036–1042 www.elsevier.com/locate/jocn
Laboratory Study
Expression of hypoxia inducible factor-1a in tumors of patients with glioblastoma multiforme and transitional meningioma Mehmet Yasar Kaynar a, Galip Zihni Sanus a, Hakan Hnimoglu a, Tibet Kacira a, Rahsan Kemerdere a, Pinar Atukeren b, Koray Gumustas b, Bulent Canbaz a, Taner Tanriverdi a,* a b
Department of Neurosurgery, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey Received 5 April 2007; accepted 20 July 2007
Abstract Hypoxia-inducible factor-1 a (HIF-1a) is the major transcriptional factor involved in the adaptive response to hypoxia. The aim of this study was to assess HIF-1a in 22 patients with transitional meningioma (TM) and 26 patients with glioblastoma multiforme (GBM). HIF-1a was assessed using a commercially available enzyme-linked immunosorbent assay-based HIF-1 transcription factor assay. Levels of HIF-1a in TM and GBM were measured using optical density at 450 nm, and median values were found to be 0.35 for TM and 0.37 OD for GBM, respectively. There was no statistically significant difference between the two types of tumor (p = 0.264). These findings indicate that HIF-1a is elevated in both TM and GBM, suggesting that although hypoxia is one of the most important and powerful stimuli for HIF-1a elevation and consequently angiogenesis, other mechanisms may play roles in HIF-1a stimulation in benign brain tumors such as TM. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Brain tumors; Glioblastoma multiforme; HIF-1; Hypoxia; Meningioma
1. Introduction Hypoxia, a condition of low tissue oxygen (O2) concentration, is known to play an important role in normal physiological processes and tumor formation. Under hypoxic conditions, hypoxia-inducible factor-1 (HIF-1) binds to hypoxia-responsive elements (HRE) and induces transcription of various target genes involved in tumor angiogenesis, invasion, cell survival, and glucose metabolism.1–3 Since its discovery,4,5 HIF-1 has emerged as a key regulatory element in a variety of developmental, physiological and pathological responses to hypoxia. HIF-1 is composed of two subunits, hypoxia-inducible factor-1 al*
Corresponding author. Present address: P.K: 4, Cerrahpasa, 34301, Istanbul, Turkey. Tel./fax: +90 212 414 34 27. E-mail address: [email protected] (T. Tanriverdi). 0967-5868/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2007.07.080
pha (HIF-1a) and hypoxia-inducible factor-1 beta (HIF1b) (also known as aryl hydrocarbon receptor nuclear translocator) subunits, which belong to the basic helixloop-helix Per-Arnt-Sim (bHLH-PAS) family of transcription factors.1–6 These subunits, HIF-1 a and HIF-1b, must associate to form the active HIF heterodimer responsible for transcriptional activation.2–4 HIF-1 transcriptional activity mainly depends on the amount of HIF-1a protein in the cell, whereas HIF-1b is constitutively present regardless of O2 concentration; that is, HIF-1 a is the unique, O2regulated subunit that determines HIF-1 activity.2,3 In normoxic conditions, HIF-1a is rapidly degraded by the ubiquitin proteasome system. This degradation is dependent on O2 and iron-dependent hydroxylation at Pro-564 and Pro-402, followed by interaction with von Hippel-Lindau (VHL) E3 ubiquitin ligase.1 This degradation can be reduced by treating cells with agents that mimic hypoxic
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conditions, such as cobalt chloride, deferrioxamine, or dimethyloxalylglycine, regardless of the E3 ubiquitin ligase activity of VHL6. Under these conditions, stabilized HIF1a is transported into the nucleus where it dimerizes and creates an active complex with HIF-1b. HIF-1 is essential for angiogenesis, embryonic development, and is associated with tumor progression, erythropoiesis, vascular development/remodeling, vasodilation, and glucose/energy metabolism.1–8 Over-expression of HIF-1a has been demonstrated in many common human cancers and has been shown to correlate with tumor grade and tumor progression.9 Besides physiological hypoxia, genetic abnormalities frequently detected in human cancers, such as loss of function mutations (VHL, p53, and phosphatase and tensin homolog deleted on chromosome 10 [PTEN]), are associated with induction of HIF-1 activity and expression of HIF-1-inducible genes.3 HIF target genes include erythropoietin (EPO), heme oxidase 1 (HOX-1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS).1– 3,6,8 Cells with genetically deleted HIF-1a appear to be resistant to hypoxia-triggered apoptosis.8 A growing body of evidence from immunohistochemical analysis has shown that HIF-1a is present at higher levels in human tumors than in normal tissues.9,10 Most importantly, in gliomagenesis, HIF-1a has been shown to be a potent activator of angiogenesis and invasion through its up-regulation of target genes that are critical for these functions.11,12 Thus, since hypoxia is the main event in tumor progression and since HIF-1a expression and/or activity is regulating the cellular adaptation to hypoxia that appears central to tumor growth and progression, HIF-1 inhibition has become an anti-cancer target in recent years.13–20 Although HIF-1a expression in common human tumors including glioblastoma multiforme (grade-IV astrocytoma-GBM) is well established,9–12,21,22 there is little information about HIF-1a expression in meningioma.23 In this study, we examined the expression of HIF-1a in GBM and transitional meningioma (TM) and we compared the results for these two types of brain tumor. 2. Materials and methods 2.1. Study groups The present study involved two groups: patients with TM and patients with GBM. This study was conducted at the Department of Neurosurgery, Cerrahpasa Medical Faculty, Istanbul University, Turkey, in 2006. All patients or next of kin were fully informed and ethical approval for this study was obtained from the Human Investigations Committee at Istanbul University. We excluded patients who had any kind of chronic or acute infection, any immunological or metabolic disease, neoplastic disease of an other organ system, and any cardiovascular disease, and patients who had undergone a recent major surgical procedure.
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2.2. Patients with TM This study included 22 consecutive patients with histopathologically proven TM. Twelve and 10 lesions were located in the right and left hemispheres, respectively. The frontal lobe was the most commonly involved region (8 tumors), followed, in decreasing order of frequency, by the temporal (7 tumors), parietal (5) and occipital (1) lobes. The average age of the patients (13 women and 9 men) was 46 years. The postoperative period of all patients was uneventful and no recurrence was noted in the follow-up period. 2.3. Patients with GBM Twenty-six consecutive patients with histopathologically proven GBM were included. Sixteen lesions were located in the right hemisphere, whereas 10 were located in the left. The temporal lobe was the most affected region (10 GBM), followed by the occipital (5), frontal (4), parietal (3), fronto-parietal (2) and temporo-parietal (2) lobes. The average age of the patients (17 men and 9 women) was 51 years. The postoperative period was uneventful for all patients. All patients with GBM were referred to the Radiation Oncology Department for planning of further treatment, including chemotherapy and/or radiotherapy. All patients with either TM or GBM received corticosteroids and anticonvulsant medication (single agent) before, during and after surgery. 2.4. Specimen handling Twenty-two TMs and 26 GBMs were assayed for HIF-1a. For each patient, after exposure of the neoplasm, tumor tissue was obtained before thermal coagulation. All specimens were obtained from the core of the tumor. As soon as possible, each sample was frozen to –80°C until it was assayed. 2.5. Measurement of HIF-1a Prior to the Chemicon Transcription Factor Assay (catalog no. SGT 570), nuclear extracts were obtained using a nuclear extract kit (catalog no. 2900; Chemicon, Temecula, CA, USA). Cell pellets were washed in ice cold phosphatebuffered saline (PBS) (pH 7.5) and then warmed trypsin cell detachment buffer was added and the cells were washed again in PBS. Cell pellets were re-suspended in ice cold cytoplasmic lysis buffer containing dithiothreitol (DTT) and protease inhibitor cocktail. Using a syringe with a small (27 gauge) needle, the cell suspension was drawn into the syringe and then injected back into the sample tube to dissociate the cells. After centrifugation, the nuclear pellet was re-suspended in 2/3 of the original cell pellet volume of ice cold nuclear extraction buffer containing DTT and a protease inhibitor cocktail, thus, the nuclei were disrupted. After centrifugation, supernatants were stored at 80°C as nuclear extracts until the assay was performed.
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2.6. Statistical analysis We used a commercially available statistical software package (SPSS version 11.0; SPSS Inc., Chicago, IL, USA) for all statistical analyses. The medians, interquartile ranges and full ranges were calculated for HIF-1a. For all comparisons, the non-parametric Mann-Whitney U-test was used as a statistical method. Differences were considered statistically significant if the probability value was less than 0.05. 3. Results The median HIF-1a OD value at 450 nm in patients with TM was 0.35 (range: 0.29–0.39; interquartile range: 0.10), and in the GBM group it was 0.37 (range: 0.29– 0.42; interquartile range: 0.13) (Fig. 1). Although the median HIF-1a activity was higher in GBM than TM, the difference was not statistically significant (p = 0.264). 4. Discussion 4.1. General considerations Glioblastoma multiforme (GBM grade IV astrocytoma) is the most malignant brain tumor known, and accounts for 80% of all malignant astrocytomas.24 Despite great advances in both diagnostic and treatment modalities, including surgery, chemotherapy, radiotherapy and immunotherapy, patients with GBM have an extremely poor
P = 0.264
.44 .42 .40 .38 .36 .34 .32 .30 .28
GBM
.26 TM
HIF-1 activation was detected and quantified using a non-radioactive, enzyme-linked immunosorbent assay (ELISA)-based HIF-1 transcription factor assay (Chemicon), which combines the principle underlying the electrophoretic mobility shift assay (EMSA) with 96-well-based ELISA, enabling manual or high-throughput sample analysis with greater sensitivity than conventional EMSA assays. During the assay, the HIF-1 capture probe, a doublestranded biotinylated oligonucleotide containing the flanked HRE consensus sequence (50 - ACGTG -30 ) for HIF-1a binding, was mixed with cellular (nuclear) extract in the transcription factor assay buffer provided in the kit. When incubated together, the active form of HIF-1a contained in the nuclear extract binds to its consensus sequence. Nuclear cell extracts from Cos-7 cells treated with CoCl2 were used as positive controls. The extract/probe/buffer mixture was then directly transferred to a streptavidincoated plate. The active HIF-1a protein that was bound to the biotinylated double-stranded oligonucleotide was immobilized and any inactive, unbound material was washed away. The bound HIF-1a transcription factor was detected using a specific primary polyclonal anti-HIF-1a antibody. A horseradish peroxidase-conjugated secondary antibody was then used for detection in a spectrophotometric plate reader. Values are expressed as optical density (OD) at 450 nm.
HIF-1 alpha (OD 450)
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Fig. 1. Box plot comparing HIF-1a activity in patients with transitional meningioma and those with glioblastoma multiforme. The difference was not statistically significant. The horizontal lines within the boxes represent median values, and the boxes denote the interquartile range. Vertical lines denote the full ranges. GBM, glioblastoma multiforme; TM, transitional meningioma; HIF-1a, hypoxia inducible factor; OD, optical density.
prognosis and median survival after initial diagnosis is still less than 1 year.25 Key histopathological features of GBM, such as necrosis and endothelial proliferation, distinguish these tumors from low-grade astrocytomas, which are associated with a better prognosis. GBM can occur as the result of progression from lower-grade astrocytomas or can arise de novo. Details of their pathogenesis remain to be elucidated. In contrast, meningiomas are generally considered to be benign tumors of the brain, and transitional or mixed types of meningiomas together with fibroblastic and meningothelial (syncytial) tumors are classed as type I meningiomas, which generally do not invade the brain tissue.26 They generally have a clear capsule, which enables the surgeon to easily remove them from the normal brain parenchyma; thus, total removal is achieved in most cases. The extent of resection depends largely on the location of the lesion and lesions can become problematic if located in certain positions, such as the medial sphenoid wing, posterior parasagittal region and posterior fossa, where total removal may damage important structures.26 Although several factors, including head trauma, radiation, hormones, chromosomal aberrations and viruses, have been identified as contributing to the development of meningiomas, the main etiological factor is unknown.26–29 Solid tumors, such as GBMs, have extensive areas of hypoxia and necrosis, which worsen a patient’s prognosis because necrosis and hypoxia promote a more malignant phenotype, and hypoxic cells are resistant to chemoand radiotherapy.9,10,12,16,19,29 It has been shown that hypoxia-induced angiogenesis is one of the most important steps in the survival of tumor cells.30 Elevated vascular cell
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endothelial growth factor (VEGF) has been shown to be associated with recurrence, neovascularization, peritumoral edema and malignancy.31–33 Transcription of VEGF is usually regulated by HIF-1a, a transcription factor that is critical in the adaptive response to reduced O2 concentration.34 Activation of HIF-1a leads to up-regulation of angiogenetic factors that are essential for the formation of new blood vessels and is one of the most important primary forces driving both physiological and pathological angiogenesis.35,36 4.2. Hypoxia inducible factor-1 HIF-1 is a heterodimer composed of a and b subunits, both of which belong to the basic helix-loop-helix (bHLH) Per Arnsim family of proteins.1–3,6–8 HIF-1b, an aryl hydrocarbon nuclear receptor translocator, heterodimerizes with aryl hydrocarbon receptor to form the functional dioxin receptor, and is a nuclear protein that is constitutively expressed, independent of O2 tension.37,38 HIF-1a is an 826-amino acid (120-kDa) protein that, in contrast to HIF-1b, is a cytoplasmic protein that is responsive to O2 levels.37,38 Under normoxic conditions, HIF-1a is continuously degraded by the ubiquitin-proteasome system.39 Under low O2 tension conditions, this degradation is inhibited, leading to increased HIF-1a, which translocates to the nucleus to activate gene transcription and heterodimerizes with HIF-1b subunits.39 The resultant product is an active HIF-1 protein that binds to specific HREs present in target genes, which encode erythropoietin, VEGF, various glycolytic enzymes, transferrin, and a variety of other proteins that are essential for systemic, local, and intracellular homeostasis.13,14,17–20 More importantly, it has been shown that the vast majority of these gene products are over-expressed in human tumor cells, suggesting that HIF-1 may play a role in tumor development and progression.9,21,40–44 Overall, these adaptive responses to low O2 levels serve as a compensatory mechanism for increasing delivery of O2 via new vessels (and nutrients) for any cells of the body or tumor cells with an inadequate O2 supply. 4.3. Hypoxia-inducible factor-1a and tumors Immunolabelling studies using monoclonal antibodies raised against the HIF-1a subunit, which determines HIF activity, have demonstrated increased HIF-1a expression in about 53% of malignant tumors, including brain, bladder, breast, colon, ovarian, pancreatic, renal and prostate tumors,9,21,23,41,42,44,45 whereas no expression has been detected in surrounding normal tissue, nor in benign tumors such as breast fibroadenoma and uterine leiomyoma.9,46 Clinically, increased HIF-1a levels are not only correlated with tumor grade and poor prognosis but also treatment failure in a number of cancers, including astrocytomas, oligodendrogliomas and meningiomas.21,23,45 In many cancers, over-expression of HIF-1a is seen at an early stage
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of tumor development, with expression correlating with the vascular density of the lesions. The same phenomenon is seen in brain tumors, suggesting that HIF-1 activity contributes to the angiogenic switch, most likely mediated by increased formation of pro-angiogenic factors such as VEGF.47 The pattern of HIF-1a protein expression in GBM is identical to that described for VEGF mRNA and meningiomas.21 GBM has significantly higher levels of VEGF expression and neovascularization compared with low-grade gliomas.48 Current evidence suggests that malignant gliomas take advantage of hypoxia-mediated HIF activation by up-regulation of at least two growth factors, platelet-derived growth factor (PDGF)-BB and VEGF.47,49 PDGF-BB, when expressed in glioma cells, sustains glioblastoma growth via autocrine and paracrine cell regulatory mechanisms.49 VEGF, as the major tumor angiogenesis and vascular permeability factor, supports glioma growth via a paracrine effect on endothelial cells.47 Increased HIF-1a levels have also been demonstrated in tumors, regardless of the presence of hypoxia. In such circumstances, it has been suggested that over-expression of HIF-1a is induced by nonphysiologic oncogenic stimuli rather than hypoxia, for example in hemangioblastoma, oligodendroglioma and low-grade epithelial ovarian tumors, despite their highly vascularized nature.44,45,50,51 HIF-1a is thought to be involved in tumoriogenesis through two main mechanisms: hypoxia-dependent mechanisms, which are observed close to the necrotic area, and hypoxia-independent mechanisms, such as oncogene activation and growth factor signaling pathways. The relevance of the HIF-1a system to tumor growth and progression is highlighted by the variety of mechanisms regulated by HIF-target genes, ranging from angiogenesis, to increased tissue oxygenation over glycolysis, and pH regulation, allowing for energy generation when oxygen is scarce, to cell proliferation and survival pathways.40 Intra-tumoral hypoxia correlates directly with the tumor’s biological aggressiveness, degree of malignancy and clinical recurrence, and inversely correlates with the post-operative survival of patients with gliomas.52,53 Since angiogenesis and cellular adaptation to hypoxia represent key steps in tumor progression, and since one of the key factors regulating cellular O2 homeostasis is HIF-1a, targeting HIF-1 may be beneficial in the treatment of aggressive brain tumors. Thus, in recent years HIF-1a has emerged as a potentially important therapeutic target for cancer treatment. Strategies suggested for HIF-1a targeting include disruption of the normal coactivational response to hypoxia, the use of decoy oligonucleotides, and a gene therapy approach based on HRE-regulated gene expression that exploits the presence of hypoxia/anoxia in tumors for the induction of therapeutic genes.13,17 4.4. Present study In the present study, we demonstrated that HIF-1a level is elevated in both TM and GBM. However, no significant
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difference was found in terms of HIF-1a expression between TM and GBM, which are benign and malignant tumors of the brain, respectively. These findings may be explained by both hypoxic and non-hypoxic mechanisms. In GBM, there is almost always a hypoxic region, thus the elevated HIF-1a level is due to hypoxia, which up-regulates the angiogenic factors in order to compensate for the demands of rapidly growing tumors. In contrast, overexpression of HIF-1a in TM in our study could not be attributed to hypoxia alone because such tumors grow slowly enough that they rarely outgrow their blood supply. Therefore, we suggest that elevated expression of HIF-1a is induced by non-physiologic oncogenic stimuli in TM rather than hypoxia, as in GBM, which is analogous to the situation in hemangioblastomas51 (51) and oligodendrogliomas.45 Although only a small number of patients with TM were included in our study, we speculate that three mechanisms, loss of p53, PTEN and/or VHL function, can explain over-expression of HIF-1a in TMs. p53 is a tumor suppressor gene and loss of function is a frequent event in tumorigenesis. Although the function of p53 is still not well understood, many lines of evidence indicate that meningiomas have low levels of p53, leading to speculations that p53 mutations may be involved in the etiology of these tumors.54–56 It has been demonstrated that wildtype p53 promotes degradation of HIF-1a57 so it may be expected that inactivation of p53 either results from somatic mutations or from functional inactivation, which would lead to increased HIF-1a expression in meningiomas. If functional p53 is lost, the proapoptotic effect of HIF-1a is lost and tumor progression is stimulated by HIF-1a via induction of angiogenesis. Furthermore, loss of p53 activity in meningioma cells may be associated with increased HIF-1a expression and HIF-1 DNA binding activity, as a consequence promoting tumor angiogenesis by enhancing levels of the angiogenic factor VEGF.35 Immunohistochemically detectable accumulation of p53 and HIF-1a has been found in glioma,22 oligodendroglioma,45,58 and highly vascularized ovarian carcinoma.59 Similar to p53, PTEN is a recently identified tumor suppressor gene that has been found to be inactive in various human cancers, including GBM.60 Although PTEN mutations are rarely seen in low-grade meningiomas,54,61,62 it has been suggested that PTEN is a progression-associated tumor suppressor gene in meningiomas.61,62 Furthermore, mice that are heterozygous for PTEN are predisposed to various forms of cancer, including meningiomas.63 In addition, germline mutations in PTEN have been detected in patients with Cowden disease,64 which is an autosomal dominant disorder characterized by hamartomas of multiple organs, macrocephaly and carcinoma of the breast and thyroid. Patients with Cowden disease have an increased risk of developing meningiomas.65 It has been shown that wild-type PTEN downregulates HIF-1-mediated gene expression.53,66 Induced expression of PTEN in glioma cells has been shown to decrease accumulation of HIF-1a in gliomas, reduce angiogenesis66 under hypoxic conditions
and lead to attenuation of the HIF transcriptional response, possibly by inhibiting the P13K (kinase)-AKT pathway.67 Another mechanism that may explain the overexpression of HIF-1a in TMs is loss of VHL function. The VHL gene product (pVHL) is required for regulation of the HIF-1 transcriptional control system.68 It has recently been demonstrated that VHL inactivation is frequently detected in sporadic hemangioblastoma and renal cell carcinoma,69,70 both of which are highly vascular tumors, like meningiomas. The common finding of lesions associated with VHL loss of function is an angiogenic phenotype that suggests that angiogenic factors such as VEGF are constitutively activated. More importantly, HIF-1a degradation involves a physical association between pVHL and HIF-a subunits.69,70 Additionally, nonfunctional VHL also results in the accumulation of HIF-1a in normoxic conditions.68 Given these data, we speculate that HIF-1 is involved in tumorigenesis through a number of different mechanisms, as suggested by the results of our study. On the one hand, hypoxia, in the case of GBM, induces the activation of HIF-1, which increases the expression of several glycolytic enzymes and hence the glycolysis rate for cells’ adaptive modifications to hypoxia. On the other hand, HIF-1 is involved in the establishment of vascular supply, and neoangiogenesis can be triggered by tumor cells becoming hypoxic while enlarging, as in cases of meningioma. 5. Conclusion We conclude that HIF-1a is elevated in both TMs and GBMs, suggesting that although hypoxia is one of the most important and powerful stimuli for HIF-1a elevation and consequent angiogenesis, other mechanisms may play roles in HIF-1a stimulation in benign brain tumors such as TM. References 1. Chan DA, Sutphin PD, Denko NC, et al. Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1a. J Biol Chem 2002;277:40112–7. 2. Chun YS, Choi E, Kim TY, et al. A dominant-negative isoform lacking exons 11 and 12 of the human hypoxiainducible factor-1a gene. Biochem J 2002;362:71–9. 3. Rapisarda A, Uranchimeg B, Scudiero DA, et al. Identification of small molecule inhibitors of hypoxia-inducible factor-1 transcriptional activation pathway. Cancer Res 2002;62:4316–24. 4. Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995;92:5510–4. 5. Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 1995;270:1230–7. 6. Chun YS, Choi E, Kim GT, et al. Cadmium blocks hypoxia-inducible factor (HIF)-1-mediated response to hypoxia by stimulating the proteasome-dependent degradation of HIF-1a. Eur J Biochem 2000;267:4198–204. 7. Narravula S, Colgan SP. Hypoxia-Inducible Factor-1-mediated inhibition of peroxisome proliferator-activated receptor a expression during hypoxia. J Immunol 2001;166:7543–8.
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