|Year : 2020 | Volume
| Issue : 3 | Page : 97-104
Carbonic anhydrase IX as a marker of hypoxia in gliomas: A narrative review
Roger E McLendon
Department of Pathology, Preston Robert Tisch Brain Tumor Center, Duke University, Durham, NC, USA
|Date of Submission||09-Jul-2020|
|Date of Decision||22-Aug-2020|
|Date of Acceptance||28-Aug-2020|
|Date of Web Publication||17-Oct-2020|
Dr. Roger E McLendon
Department of Pathology, Preston Robert Tisch Brain Tumor Center, Duke University, DUMC Box: 3712, Durham, NC 27710
Source of Support: None, Conflict of Interest: None
Hypoxia is a powerful driver of the malignant phenotype in solid tumors including gliomas. A major, though not sole, driver of this effect is the hypoxia-inducible factors (HIF) which promote the expression of hundreds of downstream genes through binding with hypoxia-responsive elements in the promoter regions of targeted genes. HIF-2α drives the cancer stem cell phenotype that has been shown to promote chemo- and radioresistance. HIF-1α drives the transcription of a number of genes, the most prolific and important of which appears to be that of CAIX, but also drives the transcription of VEGF and a number of glycolytic enzymes, thus participating in driving the Warburg effect. This brief review introduces how the localization of CAIX by immunohistochemistry has, though still in its early phases, allowed the identification of gliomas with worse prognosis, an application of significant importance in diagnostic neuropathology. The future of hypoxia research will manipulate these downstream pathways to provide further biomarkers through which the presence of hypoxia and its effects can be established, analyzed, and exploited.
Keywords: Biomarker, carbonic anhydrase, glioma, hypoxia, review
|How to cite this article:|
McLendon RE. Carbonic anhydrase IX as a marker of hypoxia in gliomas: A narrative review. Glioma 2020;3:97-104
| Introduction|| |
Malignant human gliomas continue to be a cause of significant morbidity and mortality. Despite aggressive surgery, chemotherapy, radiation therapy, and innovative immunotherapeutic approaches,, the overall survival (OS) of glioblastoma, the most common and most malignant form of human glioma, has only slightly improved. Advances in molecular techniques have identified biomarkers such as mutations in isocitrate dehydrogenase (IDH) 1/2 and telomerase, the co-deletion of 1p, 19q, and the promoter methylation of methylguanine methyltransferase, which have allowed a more precise reclassification and refinement of diagnoses. Indeed, the characterization of the effects of the IDH1 mutations has increased the profile of studies in metabolomics. Along these lines, the role of hypoxia in mediating chemo- and radiation insensitivity, genomic instability, angiogenesis, stem cell regulation, and immunologic inhibition has further stimulated an interest in this area of oncology in the search for biomarkers on which to base precision medicine. However, the application of biomarkers of hypoxia in pathology has not been thoroughly explored. The goal of this report is a brief overview of the biomarkers associated with the hypoxic response mediated by hypoxia-inducible factor-1α (HIF-1α) with a focus on the use of carbonic anhydrase IX (CAIX) as a biomarker of hypoxia.
| Retrieval Strategy|| |
Currently, a PubMed search using the keywords “Carbonic anhydrase IX and glioma” retrieved 57 papers, of which a careful reading of the papers includes only 18 papers related to the histopathologic application of CAIX on gliomas. The author focused the paper to include those 18 papers as well as a brief sketch of background to refamiliarize the reader, who may have an incomplete knowledge base of this very broad subject.
| Background|| |
Glucose is the food source of life including its most primitive representatives. Anaerobic glycolysis is a process that long predates the presence of atmospheric oxygen, takes one molecule of glucose, produces two molecules of pyruvate, and stores energy by phosphorylating two molecules of adenosine diphosphate. Alternatively, pyruvate can be converted either to lactate or to CO2 and ethanol (fermentation). Under the evolutionary pressure of atmospheric oxygen liberated by cyanobacteria via photosynthesis, cells develop the tricarboxylic acid cycle, which metabolizes glucose to form pyruvate and uses it to produce three molecules of nicotinamide adenine dinucleotide (NADH), one of flavin adenine dinucleotide (FADH2), and one of guanosine-5'-triphosphate [Figure 1]. NADH and FADH2 are then transferred to the mitochondria in which oxidative phosphorylation produces ~36–38 molecules of adenosine triphosphate (ATP) [Figure 1].
|Figure 1: Glucose is metabolized by glycolysis into 3 carbon pyruvates that can be directed to produce lactate, ethanol, and CO2or enter the Krebs cycle, which uses it to produce three molecules of nicotinamide adenine dinucleotide, one of flavin adenine dinucleotide (FADH2) and 1 of guanosine-5'-triphosphate. Nicotinamide adenine dinucleotide and FADH2are then transferred to the mitochondria in which oxidative phosphorylation produces ~36–38 molecules of adenosine triphosphate|
Click here to view
| Warburg Effect and Aerobic Glycolysis|| |
In 1924, Otto Warburg noted that solid cancers manifest excessive lactate production that he attributed to dysfunctional mitochondria and a dependence of cancer cells on anaerobic respiration., He postulated that because cancer cells depended primarily on glycolysis to produce energy, this conversion to anaerobic glycolysis was a carcinogenic event. Subsequent research found that, rather than being the cause of cancer, genomic events activated pathways in which oxidative respiration is bypassed as the primary source of energy even in the presence of oxygen, called aerobic glycolysis. In doing so, the cancer cells manifest alterations that promoted biosynthesis uncoupled from NADH production resulting in the production of lactate from either glycolysis or glutaminolysis. Further research identified that lactate was subsequently used by cancer in promoting its own survival, namely angiogenesis, immune surveillance escape, cell migration, and self-sufficient metabolism.,, While the mechanisms whereby lactate mediated these processes escaped understanding, Zhang et al. implicated lactate in the lysine lactylation of histones resulting in altered transcriptional activity in both benign and neoplastic cells.
A number of hypotheses have been tested to explain the switch to aerobic glycolysis, a switch found in both proliferating and benign cells, for example, lymphocytes and macrophages,,, as well as in cancer. One hypothesis suggests that glycolysis is a faster producer of ATP as long as glucose is abundant. Another implicates the generation of reactive oxygen species in the mitochondria of hyperfunctioning cells, thereby altering mitochondrial function. Regardless, the key to understanding the Warburg effect is the switch to lactate production and away from pyruvate and acetyl-CoA.
This intracellular production of excessive amounts of lactate, carbon dioxide, and acids (H+), predominately in the form of lactic acid, is a major manifestation of most solid cancers, including gliomas. Under resting aerobic conditions, glycolysis results in the production of lactate from glucose that is converted to pyruvate and then converted to acetyl-CoA for entrance into the tricarboxylic acid cycle [Figure 1]. Under stress or low oxygen levels, lactate is preserved and is used as a building block for other proteins needed in rapid proliferation.
| Hypoxic Response|| |
HIF-1α is a major transcription factor driving the switch from utilizing lactate in the tricarboxylic acid cycle to aerobic glycolysis. This HIF-1α-dependent metabolic switch to aerobic glycolysis results in the binding of the transcription factor to hypoxia response elements in target genes such as various glycolytic enzymes including lactate dehydrogenase, which mediates the production of lactate, and increases the levels of pyruvate dehydrogenase kinase, inhibiting the conversion of pyruvate to acetyl-CoA.
This process results in the formation of excess intracellular lactic acid and CO2. In this way, the switch to aerobic glycolysis results in a trend toward an acidotic intracellular environment. Survival of the cell depends on maintaining an optimal intracellular pH by neutralizing and/or exporting these metabolic byproducts by monocarboxylate transporter 1 and the HIF-dependent monocarboxylate transporter 4 along with its chaperone, CD147/basigin. The result is the acidification of the extracellular environment, which cannot be adequately cleared due to the dysfunctional vascularity found in cancers, a result that has the effect of blunting the anticancer immune response.
A HIF-1α-targeted gene, CAIX, mediates this alkalization of the intracellular environment. Membrane-bound CAIX hydrates CO2 to form the anion, bicarbonate. Spatially adjacent anion transporters produce an influx of bicarbonate anions from the extracellular tumor microenvironment (TME). Bicarbonate binds with H + to produce CO2 and water. CO2 then diffuses through the cell membrane to the extracellular environment, raising the intracellular pH and lowering that of the TME. Given the role of pH deviations in solid cancers, the influence of pH on gliomas has received much attention, not only to understand its role in glioma detection and progression but also for its utility as a prognostic biomarker. Hubesch et al., using magnetic resonance (MR) spectroscopic imaging of human brain tumors, documented intracellular alkalinity. Extracellular acidity was confirmed by García-Espinosa et al. also using MR spectroscopy, confirming previous microelectrode and miniature optical probe studies. The histologic documentation of these cancer-induced pH changes has also been undertaken, with variable results. The primary focus has been on CAIX with various studies supplementing the analyses by including HIF-1α, vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), and osteopontin.
| Hypoxia-Inducible Factors|| |
The HIFs have important, though distinctive, roles in facilitating important changes in hypoxia., In the normoxic state, expression of the HIF-α genes, Hif-1α and Hif-2α, are under the control of the von Hippel–Lindau protein, pVHL., pVHL targets the destruction of both HIF-1α and HIF-2α by ubiquitinating the proteins, leading to their subsequent degradation. Hydroxylated proline residues found in the oxygen-dependent degradation domain of HIF-1α are bound by pVHL., In hypoxic states, the oxygen-dependent degradation domain prolines are dehydroxylated and pVHL loses its binding partners; HIF-αs are allowed to persist, migrate into the nucleus, and bind to the constitutively expressed HIF-β., Interestingly, prolyl-hydroxylases are dependent on α-ketoglutaric acid (the product of IDH). Loss of α-ketoglutarate and the resultant production of 2-hydroxyglutarate, as a result of mutated IDH1/2, results in stabilization of HIF-1α, creating what has been called a pseudohypoxic environment.
Alternatively, factor inhibiting HIF-1 or FIH1 controls HIF-1α expression. FIH1 binds to HIF-1α interfering with its forming a complex with HIF-β. In low-oxygen states, FIH1 frees HIF-1α to bind HIF-β. In contrast to gliomas, in renal cell carcinoma, and in the von Hippel–Lindau associated tumor, capillary hemangioblastoma, VHL exhibits a germline mutation and the alternate allelic VHL is somatically mutated leading to a loss of its transcriptional control function and constitutive expression of HIF-1α.
Once in the nucleus, free HIF-1α and HIF-2α form complexes with HIF-β, a complex that recognizes the hypoxia response elements in the promoter regions of various genes. In the severe disorganization found in the cell masses of solid cancers including gliomas, regions in the TME become profoundly hypoxic. In such an environment, deregulated HIF1-α promotes the malignant phenotype of gliomas via its downstream targets, CAIX being one of its prime targets. Alternatively, HIF-2α's effects are greatly limited and confined to glioma stem cells.
| Carbonic Anhydrase Ix|| |
CAIX (EC 220.127.116.11) is one of the most prominently expressed molecules under control of the activated HIF-1α and, as such, is one of the most powerful markers of hypoxia. CAIX was first isolated in HeLa cells and named MN antigen. In benign tissue, CAIX is expressed by gastrointestinal tract epithelium, choroid plexus cells, ovarian surface epithelium, and hair follicle cells. Even in these benign tissues, CAIX is also under the transcriptional control of HIF-1α.,,
The protein was subsequently isolated, using monoclonal antibody M75, cloned, and sequenced, revealing a 257 amino acid catalytic domain qualifying it as a CA., Ivanov et al. followed up with the identification of representatives of the CA family in a number of cancers. Functionally, CAIX is a transmembrane protein that catalyzes the reversible hydration of carbon dioxide: CO2+H2O > HCO3−+ H+. Aprelikova, Svastová et al., then implicated CAIX in the control of the intracellular pH by noting CAIX's expression in many cancers, but few benign cells, placing the enzyme in key neoplastic activities. Its location in the plasma membrane provided a favorable vantage point with its extracellularly exposed enzyme site. It also exhibited the highest proton transfer rate among the CAs. Finally, the enzyme's enhanced expression in hypoxia, and thus appearance in a highly acidified environment, implicated it in a pH moderating role.
As noted, many solid tumors, including malignancies of the brain, head/neck, breast, lung, bladder, cervix uteri, colon/rectum, and kidney,, express the MN antigen (CAIX). Rather than being a marker of a specific cancer type, CAIX expression is, rather, a general marker of tumor hypoxia in these tumors under the control of HIF-1α. No activating mutations in CAIX have been described with its overexpression driven by hypoxia and acidosis. While VHL mutations predominate in clear cell renal cell carcinoma and capillary hemangioblastomas, rare examples of VHL mutations have been described in gliomas. Mutations in VHL result in a lack of binding to HIF-1α allowing its constitutive expression via the mechanism discussed earlier.
The acidification of the TME facilitates tumor progression and aggression via multiple effects including upregulation of angiogenic factors and proteases,, increased invasion, and impaired immune function., Although previously attributed to the accumulation of lactic acid produced by the excessive dependence on glycolysis and poorly removed by inadequate tumor vasculature, experiments subsequently implicated the effects of CAIX on lowered extracellular pH., Membrane-bound anion transporters produce an influx of bicarbonate anions from the TME. Bicarbonate anions react with H + to produce CO2 and water, lowering the intracellular pH. CO2 then diffuses through the cell membrane to the TME. CAIX catalytic activity thus contributes to extracellular acidification by elevating the extracellular levels of carbon dioxide and H + and to intracellular neutralization.
| Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor 2|| |
Hypoxia-related markers under the control of HIF-1 complex include other proteins as well. VEGF is another example of a gene that has been used to identify regions of hypoxia as will be discussed later. In the glioblastoma, VEGF expression significantly contributes to the neoangiogenesis and vascular remodeling that form one of the histologic hallmarks of cancer, vascular endothelial proliferation. In applying VEGF as a biomarker of prognosis, VEGFR2 is considered the main receptor mediating VEGF family-induced angiogenesis and is the most practical biomarker to use., While the new vessels should contribute to the improved oxygenation of the TME of glioblastoma, the vascular responsein vivo is dysregulated and thus, dysfunctional, failing to improve the microcirculation, perpetuating the hypoxia and acidosis. While VEGF is under the control of the HIF complex, VEGFR2 is not. Rather, with VEGF (and another ligand) binding, VEGFR2 translocates to the nucleus and binds to the Sp1-responsive region of the VEGFR2 proximal promoter. In this fashion, VEGF and VEGFR2 binding directly links to the transcriptional activation of the VEGFR2 promoter. Therefore, VEGF expression promotes the synthesis of VEGFR2. VEGFR2 ligand binding and activation alone are sufficient for inducing endothelial cell proliferation, vessel formation, and vascular permeability.
| Histopathologic Analysis of Carbonic Anhydrase Ix in Glioma|| |
CAIX has become accepted as a biomarker of hypoxia in formalin-fixed, paraffin-embedded tissue.,, The identification by immunohistochemistry of hypoxia-related tissue factors including HIF-1α, VEGF [Figure 2]A and [Figure 2]B, VEGFR2 [Figure 2]C, and CAIX [Figure 3]A and [Figure 3]B has been associated with patient outcome in retrospective studies involving various glioma types.,,,,, The methods have focused on the application of immunohistochemistry, which allows not only the semi-quantitative detection of the biomarker but also its cyto/histologic localization. In this regard, perinecrotic staining in astrocytomas reveals the highest localization, while in oligodendrogliomas, individual cell staining is the rule. Most studies focused on the utility of CAIX as an independent marker of prognosis; however, some studies combined markers of hypoxia to resolve various hypoxia scores. Some studies focused on glioblastomas, while others examined the spectrum of grades of infiltrating gliomas. To date, infiltrating astrocytomas, oligodendrogliomas, and ependymomas have been studied.
|Figure 2: Hematoxylin and eosin staining of glioblastoma and immunohistochemistry of hypoxia-related tissue factors VEGF and VEGF receptor 2 (VEGFR2). (A) A high magnification of a glioblastoma demonstrating extensive, net-like vascular proliferation (×40; hematoxylin and eosin staining). (B) A high magnification photograph of tumor shown in 2A demonstrating strong anti-VEGF immunoreactivity (×40; anti-VEGF immunohistochemistry). (C) A high magnification photograph of tumor shown in 2A demonstrating strong anti-VEGFR2 immunoreactivity (×40; anti-VEGFR2 immunohistochemistry). Figure 2 is sourced from the author's unpublished data. VEGF: Vascular endothelial growth factor|
Click here to view
|Figure 3: Hematoxylin and eosin staining of glioblastoma and immunohistochemistry of hypoxia-related tissue factor CAIX. (A) A high magnification photograph of a glioblastoma demonstrating palisading necrosis (×40; hematoxylin and eosin staining). (B) A high magnification photograph of tumor shown in 3a demonstrating strong anti-CAIX immunoreactivity (×40; anti-CAIX immunohistochemistry). Figure 3 is sourced from the author's unpublished data. CAIX: Carbonic anhydrase IX|
Click here to view
Haapasalo et al. used the M75 anti-CAIX monoclonal antibody to analyze 362 astrocytomas. In this study, criteria for grading CAIX IHC in glioma were stated as intensity varying from none (0) to strong (4+) and cellular expression in quartiles of 0 (negative), <25% (1+), 25%–50% (2+), and > 50% (3+). Using these criteria, immunoreactivity varied by both intensity and distribution according to histologic grade of astrocytoma with World Health Organization (WHO) Grade II tumors exhibiting the weakest and fewest cells positive to WHO Grade IV tumors exhibiting strongest reactivity in a perinecrotic distribution. Haapsalo also considered nuclear staining on a binary, present/absent scale. Strong cytoplasmic staining was confined to anaplastic astrocytomas, Grade III and glioblastomas, Grade IV tumors with the predominant location being perinecrotically. As subsequently confirmed by others, the necrotic regions strongly correlated with CAIX reactivity. As could perhaps be anticipated, CAIX intensity and distribution correlated with survival.
Birner et al., in a study of the vascular patterns of 114 primary glioblastomas (those arising de novo with no previous history of low-grade glioma), divided the vascular patterns into three styles that were eventually collapsed into two: classic and bizarre. The finding of a predominantly classic vascular was associated with diffuse (nonfocal) HIF-1α and high VEGF expression and of a longer OS.
Korkolopoulou et al., tested 84 astrocytic gliomas of Grades II–IV and found that both HIF-1α and VEGF implied a poor prognosis by univariate analysis but were not independent of grade. However, the combination of HIF-1α with grade was a significant prognostic indicator by multivariate analysis and was associated with the presence of factors analyzed in grading including Ki-67 labeling index, vascular density, and necrosis. Similar findings were made by Yoo et al. who also noted that while CAIX and VEGF were prognostic, their expression showed no correlation with each other.
Flynn et al. found no statistical correlation between CAIX expression and prognosis with glioblastomas nor did they find a correlation between CAIX expression and tumor grade when Grade II and III astrocytomas were included in the analysis. However, patients with CAIX-positive tumors trended toward a worse prognosis. To be clear, staining methods and size of the sample called their results into question.
The studies of Proescholdt, also reported robust staining in glioblastomas, again near necrotic areas, and confirmed a positive correlation between CAIX expression and high tumor grade. In some cases, the staining was found diffusely in the tumors, including those cells located near blood vessels, a finding that suggested CAIX induction in gliomas may involve hypoxia-independent mechanisms. In fact, acidosis has also been implicated in inducing CAIX expression independently of hypoxia in glioblastoma cell lines. In a drug trial including bevacizumab and irinotecan, Sathornsumetee et al., reported that high expression of CAIX was associated with poor survival outcome. In addition, when both CAIX and HIF-2α (as a marker of glioma stem cells) were simultaneously included in a Cox model as two separate factors, only CAIX remained as a statistically significant factor.
Recently, in a study of 66 patients with glioblastoma using both clinical data and CAIX staining, Cetin et al. found CAIX staining to be significantly associated with a poor prognosis. In multivariate analysis, preoperative Karnofsky performance scale score, CAIX overexpression, incomplete adjuvant temozolomide treatment, and gross-total resection (HR, 1.956; P = 0.034) were independently associated with OS.
Mutations in IDH1/2 have drawn interest from hypoxia researchers. As mentioned previously, loss of α-ketoglutarate, as a result of mutated IDH1/2, results in stabilization of HIF-1α, creating what has been called a pseudohypoxic environment in gliomas, thereby linking IDH status with hypoxia or, at the least, expression of hypoxic markers. This linkage was not found to be operative in 33 WHO Grade II and III gliomas examined for expression of CAIX, HIF-1α and glucose transporter 1, however, the absence of Grade IV tumors (glioblastomas), which are strongly associated with hypoxic regions, brings the findings into question. This point is further supported by Makela et al. who studied tissue microarray samples of 295 Grade II–IV astrocytomas, finding a close correlation between HIF-1α expression and IDH1 mutation status. Further, in IDH1 mutant tumors, positive HIF-1α staining correlated with CAIX expression, although, perhaps in agreement with the study by Metellus et al., no such correlation was found in IDH1 wild-type tumors.
Osteopontin is a hypoxia marker whose expression is driven by the phosphoinositol-3-kinase/Akt pathway, independent of HIF-1α. Recent reports implicate osteopontin in glioma biology through its ability to affect immune responses,, macrophage migration, and neutrophils and have been found to be significantly expressed in glioblastoma tissues over normal. It is of interest in that it plays a role in angiogenesis, glioma migration,,, and tumor growth. Said et al. examined mRNA expression of CAIX, osteopontin, VEGF, and HIF-1α in both human glioma cell lines and in 30 patients, half with glioblastoma (rich in hypoxic regions) and half with low-grade gliomas (low in hypoxic regions). Cell lines growing in progressively more hypoxic environments elevated their expressions of CAIX and osteopontin. The study on human biopsies indicated that CAIX and osteopontin were elevated in glioblastoma versus the low-grade gliomas, further supporting a role related to hypoxia.
In a study of 92 patients with high-grade gliomas, Erpolat et al. studied the expression patterns of CAIX, HIF-1α, and osteopontin and their correlation with OS. They combined the results from these stains to produce a hypoxic score (low score <2 markers positive and high score >1 marker positive). Patients whose tumors had a high hypoxic score correlated with glioblastoma histology and a significantly poorer OS by multivariate analysis.
In studies performed on oligodendrogliomas, contradictory results on survival have been reported, with one study indicating a prolonged survival and other studies indicating poor survival, Birner et al. tested a hypoxia score composed of IHC for HIF-1α and CAIX as well as an in situ hybridization analysis for VEGF mRNA. 0–1 positive markers indicated a low hypoxia score; 2–3 positive markers indicated a high score. Applied to oligodendrogliomas with 1p deletion, the high hypoxia score was an independent predictor of a poor survival, a finding at odds with the findings of Sathornsumetee et al., whose patient sample was admittedly small.
In an analysis of ependymomas, various laboratories, have measured vascular proliferation status, as well as the immunohistochemical detection of the proliferation marker, MIB-1, vascular markers (including tenascin C, CD34, and VEGF), and CAIX status. Strong perinecrotic CAIX localization correlated with progression-free survival (PFS) and OS. All patients with a CAIX ≤5% total area localization exhibited prolonged survival. Perinecrotic CAIX strong expression was also associated with vascular proliferation, though not with VEGF expression. Ki-67 determined proliferation rate and perinecrotic strong CAIX localization individually correlated with PFS that remained when grade was added to a Cox model predicting PFS. In contrast, Korshunov et al. found VEGF to be significant in delineating high grade from low-grade ependymomas
| Summary|| |
Hypoxia is a powerful driver of the malignant phenotype in solid tumors including gliomas. The effect is driven through the HIFs which promote the expression of hundreds of downstream genes through binding with hypoxia-responsive elements in the promoter regions of targeted genes. HIF-2α drives the cancer stem cell phenotype, that has been shown to promote chemo- and radioresistance. HIF-1α drives the transcription of a number of genes, the most prolific and important of which appears to be that of CAIX, but it also drives the transcription of VEGF and a number of glycolytic enzymes, thus participating in driving the Warburg effect. These downstream pathways provide biomarkers through which the presence of hypoxia can be established, analyzed, and measured.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Desjardins A, Gromeier M, Herndon JE 2nd
, Beaubier N, Bolognesi DP, Friedman AH, et al
. Recurrent glioblastoma treated with recombinant poliovirus. N
Engl J Med 2018;379:150-61.
Mitchell DA, Batich KA, Gunn MD, Huang MN, Sanchez-Perez L, Nair SK, et al
. Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients. Nature 2015;519:366-9.
Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et al
. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009;10:459-66.
Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, et al
. An integrated genomic analysis of human glioblastoma multiforme. Science 2008;321:1807-12.
Killela PJ, Reitman ZJ, Jiao Y, Bettegowda C, Agrawal N, Diaz LA Jr., et al
. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci U S A 2013;110:6021-6.
Reifenberger J, Reifenberger G, Liu L, James CD, Wechsler W, Collins VP. Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and 1p. Am J Pathol 1994;145:1175-90.
Friedman HS, McLendon RE, Kerby T, Dugan M, Bigner SH, Henry AJ, et al
. DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma. J Clin Oncol 1998;16:3851-7.
Secomb TW, Hsu R, Ong ET, Gross JF, Dewhirst MW. Analysis of the effects of oxygen supply and demand on hypoxic fraction in tumors. Acta Oncol 1995;34:313-6.
Cancer Genome Atlas Research Network, Brat DJ, Verhaak RG, Aldape KD, Yung WK, Salama SR, et al
. Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. N
Engl J Med 2015;372:2481-98.
Sathornsumetee S, Cao Y, Marcello JE, Herndon JE 2nd
, McLendon RE, Desjardins A, et al
. Tumor angiogenic and hypoxic profiles predict radiographic response and survival in malignant astrocytoma patients treated with bevacizumab and irinotecan. J Clin Oncol 2008;26:271-8.
Bache M, Rot S, Keßler J, Güttler A, Wichmann H, Greither T, et al
. mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma. Oncol Rep 2015;33:3155-61.
Multhoff G, Vaupel P. Hypoxia compromises anti-cancer immune responses. Adv Exp Med Biol 2020;1232:131-43.
Lyons TW, Reinhard CT, Planavsky NJ. The rise of oxygen in Earth's early ocean and atmosphere. Nature 2014;506:307-15.
Warburg O. On the origin of cancer cells. Science 1956;123:309-14.
Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. J Gen Physiol 1927;8:519-30.
Semenza GL. HIF-1 mediates metabolic responses to intratumoral hypoxia and oncogenic mutations. J Clin Invest 2013;123:3664-71.
San-Millán I, Brooks GA. Reexamining cancer metabolism: Lactate production for carcinogenesis could be the purpose and explanation of the Warburg Effect. Carcinogenesis 2017;38:119-33.
Panisova E, Kery M, Sedlakova O, Brisson L, Debreova M, Sboarina M, et al
. Lactate stimulates CA IX expression in normoxic cancer cells. Oncotarget 2017;8:77819-35.
Zhang D, Tang Z, Huang H, Zhou G, Cui C, Weng Y, et al
. Metabolic regulation of gene expression by histone lactylation. Nature 2019;574:575-80.
Donnelly RP, Finlay DK. Glucose, glycolysis and lymphocyte responses. Mol Immunol 2015;68:513-9.
Abraham S, Hu N, Jensen R. Hypoxia-inducible factor-1-regulated protein expression and oligodendroglioma patient outcome: Comparison with established biomarkers and preoperative UCSF low-grade scoring system. J Neurooncol 2012;108:459-68.
Kelly B, O'Neill LA. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res 2015;25:771-84.
Fernandez-de-Cossio-Diaz J, Vazquez A. Limits of aerobic metabolism in cancer cells. Sci Rep 2017;7:13488.
Rodic S, Vincent MD. Reactive oxygen species (ROS) are a key determinant of cancer's metabolic phenotype. Int J Cancer 2018;142:440-8.
Nagao A, Kobayashi M, Koyasu S, Chow CCT, Harada H. HIF-1-dependent reprogramming of glucose metabolic pathway of cancer cells and its therapeutic significance. Int J Mol Sci 2019;20:238-51.
Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter mct4, but not mct1, is up-regulated by hypoxia through a hif-1alpha-dependent mechanism. J Biol Chem 2006;281:9030-7.
Zagzag D, Zhong H, Scalzitti JM, Laughner E, Simons JW, Semenza GL. Expression of hypoxia-inducible factor 1alpha in brain tumors: Association with angiogenesis, invasion, and progression. Cancer 2000;88:2606-18.
Hubesch B, Sappey-Marinier D, Roth K, Meyerhoff DJ, Matson GB, Weiner MW. P-31 MR spectroscopy of normal human brain and brain tumors. Radiology 1990;174:401-9.
García-Espinosa MA, Rodrigues TB, Sierra A, Benito M, Fonseca C, Gray HL, et al
. Cerebral glucose metabolism and the glutamine cycle as detected byin vivo
13C NMR spectroscopy. Neurochem Int 2004;45:297-303.
Engin K, Leeper DB, Cater JR, Thistlethwaite AJ, Tupchong L, McFarlane JD. Extracellular pH distribution in human tumours. Int J Hyperthermia 1995;11:211-6.
Jayasundar R, Hall LD, Bleehen NM. Comparison of pH measurements made using 31P NMR and a fibreoptic pH meter. NMR Biomed 1992;5:360-3.
Wang GL, Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem 1993;268:21513-8.
Nakazawa MS, Keith B, Simon MC. Oxygen availability and metabolic adaptations. Nat Rev Cancer 2016;16:663-73.
Aprelikova O, Chandramouli GV, Wood M, Vasselli JR, Riss J, Maranchie JK, et al
. Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors. J Cell Biochem 2004;92:491-501.
Yang H, Kaelin WG Jr. Molecular pathogenesis of the von Hippel-Lindau hereditary cancer syndrome: Implications for oxygen sensing. Cell Growth Differ 2001;12:447-55.
Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, et al
. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing. Science 2001;292:464-8.
Kaluz S, Kaluzová M, Liao SY, Lerman M, Stanbridge EJ. Transcriptional control of the tumor- and hypoxia-marker carbonic anhydrase 9: A one transcription factor (HIF-1) show? Biochim Biophys Acta 2009;1795:162-72.
Kaelin WG Jr. The VHL tumor suppressor gene: Insights into oxygen sensing and cancer. Trans Am Clin Climatol Assoc 2017;128:298-307.
Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, et al
. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2009;462:739-44.
Yao L, Barontini M, Niederle B, Jech M, Pfragner R, Dahia PL. Mutations of the metabolic genes idh1, idh2, and sdhaf2 are not major determinants of the pseudohypoxic phenotype of sporadic pheochromocytomas and paragangliomas. J Clin Endocrinol Metab 2010;95:1469-72.
Zhang N, Fu Z, Linke S, Chicher J, Gorman JJ, Visk D, et al
. The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator of metabolism. Cell Metab 2010;11:364-78.
Tostain J, Li G, Gentil-Perret A, Gigante M. Carbonic anhydrase 9 in clear cell renal cell carcinoma: A marker for diagnosis, prognosis and treatment. Eur J Cancer 2010;46:3141-8.
Semenza GL, Agani F, Booth G, Forsythe J, Iyer N, Jiang BH, et al
. Structural and functional analysis of hypoxia-inducible factor 1. Kidney Int 1997;51:553-5.
Hu CJ, Iyer S, Sataur A, Covello KL, Chodosh LA, Simon MC. Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells. Mol Cell Biol 2006;26:3514-26.
Pastoreková S, Závadová Z, Kostál M, Babusíková O, Závada J. A novel quasi-viral agent, MaTu, is a two-component system. Virology 1992;187:620-6.
Pastoreková S, Parkkila S, Parkkila AK, Opavský R, Zelník V, Saarnio J, et al
. Carbonic anhydrase IX, MN/CA IX: Analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts. Gastroenterology 1997;112:398-408.
Liao SY, Brewer C, Závada J, Pastorek J, Pastorekova S, Manetta A, et al
. Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas. Am J Pathol 1994;145:598-609.
Benej M, Pastorekova S, Pastorek J. Carbonic anhydrase IX: Regulation and role in cancer. Subcell Biochem 2014;75:199-219.
Pastorek J, Pastoreková S, Callebaut I, Mornon JP, Zelník V, Opavský R, et al
. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene 1994;9:2877-88.
Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, et al
. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J Pathol 2001;158:905-19.
Hilvo M, Baranauskiene L, Salzano AM, Scaloni A, Matulis D, Innocenti A, et al
. Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes. J Biol Chem 2008;283:27799-809.
Svastová E, Hulíková A, Rafajová M, Zat'ovicová M, Gibadulinová A, Casini A, et al
. Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH. FEBS Lett 2004;577:439-45.
Pastorekova S, Gillies RJ. The role of carbonic anhydrase IX in cancer development: Links to hypoxia, acidosis, and beyond. Cancer Metastasis Rev 2019;38:65-77.
Kanno H, Shuin T, Kondo K, Yamamoto I, Ito S, Shinonaga M, et al
. Somatic mutations of the von Hippel-Lindau tumor suppressor gene and loss of heterozygosity on chromosome 3p in human glial tumors. Cancer Res 1997;57:1035-8.
Fukumura D, Xu L, Chen Y, Gohongi T, Seed B, Jain RK. Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo
. Cancer Res 2001;61:6020-4.
Korkolopoulou P, Patsouris E, Konstantinidou AE, Pavlopoulos PM, Kavantzas N, Boviatsis E, et al
. Hypoxia-inducible factor 1alpha/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis. Neuropathol Appl Neurobiol 2004;30:267-78.
Debreova M, Csaderova L, Burikova M, Lukacikova L, Kajanova I, Sedlakova O, et al
. CAIX regulates invadopodia formation through both a pH-dependent mechanism and interplay with actin regulatory proteins. Int J Mol Sci 2019;20:2745.
Preusser M, Wolfsberger S, Haberler C, Breitschopf H, Czech T, Slavc I, 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.
Ihnatko R, Kubes M, Takacova M, Sedlakova O, Sedlak J, Pastorek J, et al
. Extracellular acidosis elevates carbonic anhydrase ix in human glioblastoma cells via transcriptional modulation that does not depend on hypoxia. Int J Oncol 2006;29:1025-33.
Sakurai Y, Ohgimoto K, Kataoka Y, Yoshida N, Shibuya M. Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice. Proc Natl Acad Sci U S A 2005;102:1076-81.
Domingues I, Rino J, Demmers JA, de Lanerolle P, Santos SC. VEGFR2 translocates to the nucleus to regulate its own transcription. PLoS One 2011;6:e25668.
Nieminen T, Toivanen PI, Rintanen N, Heikura T, Jauhiainen S, Airenne KJ, et al
. The impact of the receptor binding profiles of the vascular endothelial growth factors on their angiogenic features. Biochim Biophys Acta 2014;1840:454-63.
Moon EJ, Brizel DM, Chi JT, Dewhirst MW. The potential role of intrinsic hypoxia markers as prognostic variables in cancer. Antioxid Redox Signal 2007;9:1237-94.
Ahlskog JK, Schliemann C, Mårlind J, Qureshi U, Ammar A, Pedley RB, et al
. Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours. Br J Cancer 2009;101:645-57.
van Kuijk SJ, Yaromina A, Houben R, Niemans R, Lambin P, Dubois LJ. Prognostic significance of carbonic anhydrase IX expression in cancer patients: A meta-analysis. Front Oncol 2016;6:69.
Haapasalo JA, Nordfors KM, Hilvo M, Rantala IJ, Soini Y, Parkkila AK, et al
. Expression of carbonic anhydrase IX in astrocytic tumors predicts poor prognosis. Clin Cancer Res 2006;12:473-7.
Järvelä S, Parkkila S, Bragge H, Kähkönen M, Parkkila AK, Soini Y, et al
. Carbonic anhydrase IX in oligodendroglial brain tumors. BMC Cancer 2008;8:1.
Karjalainen SL, Haapasalo HK, Aspatwar A, Barker H, Parkkila S, Haapasalo JA. Carbonic anhydrase related protein expression in astrocytomas and oligodendroglial tumors. BMC Cancer 2018;18:584.
Simi L, Venturini G, Malentacchi F, Gelmini S, Andreani M, Janni A, et al
. Quantitative analysis of carbonic anhydrase IX mRNA in human non-small cell lung cancer. Lung Cancer 2006;52:59-66.
Yoo H, Sohn S, Nam BH, Min HS, Jung E, Shin SH, et al
. The expressions of carbonic anhydrase 9 and vascular endothelial growth factor in astrocytic tumors predict a poor prognosis. Int J Mol Med 2010;26:3-9.
Birner P, Piribauer M, Fischer I, Gatterbauer B, Marosi C, Ambros PF, et al
. Vascular patterns in glioblastoma influence clinical outcome and associate with variable expression of angiogenic proteins: Evidence for distinct angiogenic subtypes. Brain Pathol 2003;13:133-43.
Korkolopoulou P, Perdiki M, Thymara I, Boviatsis E, Agrogiannis G, Kotsiakis X, et al
. Expression of hypoxia-related tissue factors in astrocytic gliomas. A multivariate survival study with emphasis upon carbonic anhydrase IX. Hum Pathol 2007;38:629-38.
Flynn JR, Wang L, Gillespie DL, Stoddard GJ, Reid JK, Owens J, et al
. Hypoxia-regulated protein expression, patient characteristics, and preoperative imaging as predictors of survival in adults with glioblastoma multiforme. Cancer 2008;113:1032-42.
Proescholdt MA, Mayer C, Kubitza M, Schubert T, Liao SY, Stanbridge EJ, et al
. Expression of hypoxia-inducible carbonic anhydrases in brain tumors. Neuro Oncol 2005;7:465-75.
Proescholdt MA, Merrill MJ, Stoerr EM, Lohmeier A, Pohl F, Brawanski A. Function of carbonic anhydrase IX in glioblastoma multiforme. Neuro Oncol 2012;14:1357-66.
Sathornsumetee S, Hjelmeland AB, Keir ST, McLendon RE, Batt D, Ramsey T, et al
. AAL881, a novel small molecule inhibitor of RAF and vascular endothelial growth factor receptor activities, blocks the growth of malignant glioma. Cancer Res 2006;66:8722-30.
Cetin B, Gonul II, Gumusay O, Bilgetekin I, Algin E, Ozet A, et al
. Carbonic anhydrase IX is a prognostic biomarker in glioblastoma multiforme. Neuropathology 2018;38:457-62.
Zhao S, Lin Y, Xu W, Jiang W, Zha Z, Wang P, et al
. Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha. Science 2009;324:261-5.
Metellus P, Colin C, Taieb D, Guedj E, Nanni-Metellus I, de Paula AM, et al
. IDH mutation status impact onin vivo
hypoxia biomarkers expression: New insights from a clinical, nuclear imaging and immunohistochemical study in 33 glioma patients. J Neurooncol 2011;105:591-600.
Wang Y, Yan W, Lu X, Qian C, Zhang J, Li P, et al
. Overexpression of osteopontin induces angiogenesis of endothelial progenitor cells via the avβ3/PI3K/AKT/eNOS/NO signaling pathway in glioma cells. Eur J Cell Biol 2011;90:642-8.
Cantor H, Shinohara ML. Regulation of T-helper-cell lineage development by osteopontin: The inside story. Nat Rev Immunol 2009;9:137-41.
Renkl AC, Wussler J, Ahrens T, Thoma K, Kon S, Uede T, et al
. Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype. Blood 2005;106:946-55.
Wei J, Marisetty A, Schrand B, Gabrusiewicz K, Hashimoto Y, Ott M, et al
. Osteopontin mediates glioblastoma-associated macrophage infiltration and is a potential therapeutic target. J Clin Invest 2019;129:137-49.
Atai NA, Bansal M, Lo C, Bosman J, Tigchelaar W, Bosch KS, et al
. Osteopontin is up-regulated and associated with neutrophil and macrophage infiltration in glioblastoma. Immunology 2011;132:39-48.
Matusan-Ilijas K, Behrem S, Jonjic N, Zarkovic K, Lucin K. Osteopontin expression correlates with angiogenesis and survival in malignant astrocytoma. Pathol Oncol Res 2008;14:293-8.
Jan HJ, Lee CC, Shih YL, Hueng DY, Ma HI, Lai JH, et al
. Osteopontin regulates human glioma cell invasiveness and tumor growth in mice. Neuro Oncol 2010;12:58-70.
Lu DY, Yeh WL, Huang SM, Tang CH, Lin HY, Chou SJ. Osteopontin increases heme oxygenase-1 expression and subsequently induces cell migration and invasion in glioma cells. Neuro Oncol 2012;14:1367-78.
Yan W, Qian C, Zhao P, Zhang J, Shi L, Qian J, et al
. Expression pattern of osteopontin splice variants and its functions on cell apoptosis and invasion in glioma cells. Neuro Oncol 2010;12:765-75.
Lamour V, Le Mercier M, Lefranc F, Hagedorn M, Javerzat S, Bikfalvi A, et al
. Selective osteopontin knockdown exerts anti-tumoral activity in a human glioblastoma model. Int J Cancer 2010;126:1797-805.
Said HM, Hagemann C, Staab A, Stojic J, Kühnel S, Vince GH, et al
. Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1alpha in human gliomain vitro
and in vivo
. Radiother Oncol 2007;83:398-405.
Erpolat OP, Gocun PU, Akmansu M, Ozgun G, Akyol G. Hypoxia-related molecules HIF-1α, CA9, and osteopontin : Predictors of survival in patients with high-grade glioma. Strahlenther Onkol 2013;189:147-54.
Sathornsumetee S, Cheunsuchon P, Sangruchi T. High carbonic anhydrase-9 expression identifies a subset of 1p/19q Co-deletion and favorable prognosis in oligodendroglioma. World Neurosurg 2016;91:518-230.
McLendon RE, Lipp E, Satterfield D, Ehinger M, Austin A, Fleming D, et al
. Prognostic marker analysis in pediatric intracranial ependymomas. J Neurooncol 2015;122:255-61.
Korshunov A, Golanov A, Timirgaz V. Immunohistochemical markers for intracranial ependymoma recurrence. An analysis of 88 cases. J Neurol Sci 2000;177:72-82.
Mohlin S, Wigerup C, Jögi A, Påhlman S. Hypoxia, pseudohypoxia and cellular differentiation. Exp Cell Res 2017;356:192-6.
Rosenberg T, Aaberg-Jessen C, Petterson SA, Kristensen BW. Heterogenic expression of stem cell markers in patient-derived glioblastoma spheroid cultures exposed to long-term hypoxia. CNS Oncol 2018;7:CNS15.
Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al
. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006;444:756-60.
[Figure 1], [Figure 2], [Figure 3]