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Tumor Biology |
Oxford Centre for Head and Neck Oncology, Radcliffe Infirmary, Oxford OX2 6HE, United Kingdom [N. J. P. B., G. J. C.]; ICRF Molecular Oncology Group, Institute of Molecular Medicine [C. C. W., P. H. W., R. L., H. T., A. L. H.] and Nuffield Department of Clinical Laboratory Sciences [K. G.], John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic [J. P.]; and Welcome Trust Centre for Human Genetics, Oxford OX3 7BN, United Kingdom [P. R.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-CA, ß-CA, and 
-CA. CA92
is one of the -CA isoenzymes. Carbonic anhydrases catalyze the reversible conversion of carbon dioxide to carbonic acid and are involved in respiration, calcification, acid-base balance, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. The different carbonic anhydrases have different tissue distribution, subcellular localization, biological function, kinetic properties, and susceptibility to inhibitors (1)
. CA9 is a novel member of the carbonic anhydrase family which codes for a transmembrane glycoprotein that possesses an extracellular catalytic domain with weak enzymatic activity. It has homology also to basic helix-loop-helix domain proteins. Transfection into murine fibroblast NIH mouse fibroblast cells promotes proliferation, and it may be involved in control of cell proliferation and oncogenesis (1 , 2) .
There is abundant expression of CA IX protein in normal human upper GI mucosa and GI-associated structures, such as pancreas, gallbladder, and liver (3, 4, 5) . Expression is most prominent on the basolateral surfaces of the crypt enterocytes in the duodenum and jejunum. This suggests that it might serve as a ligand or a receptor for another protein that regulates intercellular communication or cell proliferation (6) . Interestingly, expression is lost in gastric adenocarcinoma (5) . Normal human heart, lung, kidney, prostate, peripheral blood, brain, placenta, and muscle do not express CA IX (3) .
CA IX overexpression has been identified in a number of solid tumors, including renal carcinoma and particularly clear cell adenocarcinoma (7, 8, 9, 10) , cervical squamous carcinoma (11 , 12) , ovarian carcinoma (13) , colorectal carcinoma (14) , esophageal carcinoma (15) , bladder carcinoma (16) , and non-small cell lung carcinoma (17) . In some epithelial tissues, expression has been observed in areas of severe dysplasia, e.g., cervix (12) , but in most, no expression is present until malignant invasion occurs, where it is often an early indicator of malignancy, e.g., lung carcinoma (17) . There appears to be an inverse correlation between CA IX expression and stage and grade in some tumors (7 , 11 , 14 , 15) , and low expression of CA IX has been correlated with poor prognostic factors, such as lymph node metastases and depth of invasion (11 , 14 , 15) .
Clear cell renal carcinoma where CA IX expression is particularly high is almost always associated with mutation of the VHL tumor suppressor gene and loss of function of pVHL (18) . CA IX is also overexpressed in mutant VHL renal cell carcinoma cell lines. This overexpression is reversed by transfection of the wt VHL gene back into the cell (19) .
After the recent description of VHL regulation of HIF-1 (20)
, we investigated the hypoxic regulation of CA IX and have shown it is inducible by hypoxia. A HIF binding site in the 5' promoter region of CA9 was found, and we showed that a hypoxia response element and HIF-1 were essential for CA9 transcription under hypoxia.
HNSCC is known to be a particularly hypoxic tumor with the degree of hypoxia having a significant impact on its response to radiotherapy chemotherapy and prognosis (22, 23, 24, 25) . The aim of this study was to examine the induction of CA IX by hypoxia in HNSCC cells lines and to analyze its expression and localization in HNSCC. Expression has been examined in relation to MVD as a measure of angiogenesis and necrosis as an indicator of the effects of hypoxia. Additionally, the distance of CA IX expression from blood vessels was analyzed as a surrogate assessment of the relation to hypoxia.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primary renal adenocarcinoma cell lines expressing pVHL (RCC4/VHL) or empty vector (RCC4) were a gift from C. H. C. M. Buys (University of Groningen) and used as positive and negative controls as described (20) . Cells were maintained in DMEM, 10% heat-inactivated FCS, 2 mM fresh glutamine, and 1 mg/ml G418 (G418 was removed from the medium 24 h before collection of samples). Cells were harvested on ice and prepared in 8 M urea lysis buffer as above. Whole cell preparations for immunostaining were harvested and fixed in 10% neutral buffered formalin overnight. Cell pellets were embedded in paraffin and sectioned onto silanized glass slides.
Fresh Tissue Samples.
Three paired tumor and normal tissue samples from primary HNSCC were snap frozen in liquid nitrogen. Tissue samples were frozen sectioned and stained with H&E to ensure that material contained normal or tumor tissue as appropriate. Samples were sectioned on ice and homogenized in 8 M urea lysis buffer as above.
CA IX Immunoblotting.
Cell and tissue extracts were protein quantified using the Bio-Rad detergent-compatible protein assay (Bio-Rad, UK) to ensure even protein loading between lanes. Samples were diluted in phosphate buffered saline to give 50 µg of protein/well. RCC4 and RCC4/VHL extracts were used as positive and negative controls, respectively. Proteins were resolved in NuPage Bis Tris 4–12% gels (Novex, UK) and transferred with a wet blotter (Novex) to Immobilon-P membrane (Millipore, Bedford, MA) in 25 mM Tris base, 190 mM glycine, and 15% methanol. Membranes were developed using the Western Breeze chemiluminescent immunodetection system (Novex) with mouse MoAb M75, as described (27)
, at a dilution of 1:50 in assay diluent.
Archived Tumor Specimens.
Previously untreated patients (79) with HNSCC presented to the Oxford Center for Head and Neck Oncology between 1995 and 1999 were studied (Table 1)
. All had surgery as their first line of management, with some receiving postoperative radiotherapy. Specimens of complete resections rather than biopsies were selected so that both normal and tumor tissue were present on each slide. Seven samples of lymph node metastases from patients in this series were also selected.
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Immunostaining.
Sections (4 µm) of formalin-fixed, paraffin-embedded tissue were cut onto silanized glass slides. They were cleared of paraffin in Citroclear (HD Supplies, UK) and rehydrated through graded alcohol baths. After a rinse in tap water, they were placed in 3% hydrogen peroxide for 15 min.
CA IX.
Slides were blocked in 10% normal human serum for 15 min, then incubated with 1:50 MoAb M75, as described (27)
, in TBS and 5% normal human serum for 30 min. They were rinsed twice in TBS and then developed using the Horse Radish Peroxidase Envision System (DAKO). Slides were counterstained with hematoxylin (Sigma Chemical Co. diagnostics, St. Louis, MO) and mounted with Aquamount (BDH, Poole, UK). Slides were examined at low (x40) and medium (x100) power by two observers (P. W. and N. B.). The percentage of tumor cells positive for CA IX in the whole tumor section was determined. Formalin-fixed, paraffin-embedded RCC4 and RCC4/VHL cell pellets were used as positive and negative controls, respectively.
CD34.
Slides were pressure cooked for 3 min in Tris/EDTA lysis buffer (pH 9.0) and incubated with 1:100 MoAb Qbend 10 (DAKO) in TBS for 60 min. After two rinses in TBS, they were incubated with goat antimouse IgG (PO447, DAKO) for 30 min, washed in TBS, and then incubated with alkaline phosphatase anti-alkaline phosphatase for 30 min. The last two steps were repeated twice with 10-min incubations as described (29)
. New Fucsin Red substrate was applied for 15 min, and slides were counterstained with hematoxylin and mounted with Aquamount. MVD was determined in tumor microvessel hotspots using a Chalkley point counting grid at high power (x250) by two observers (R. L. and N. B.). The average of the vessel counts in the three most vascular areas per section was taken (30)
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Double Staining CA IX, CD34.
Two representative slides demonstrating overexpression of CA IX and areas of necrosis were selected. CA IX immunostaining was carried out as above using the Horse Radish Peroxidase Envision System (DAKO) without counterstaining. Slides were then washed for 15 min in TBS, and immunostaining for CD34 was carried out as above. Slides were counterstained and mounted as above.
The distance from blood vessels marked with CD34 to the start of CA IX expression and necrosis was assessed using an eyepiece graticule calibrated against a graduated slide. Measurements were taken from both tumors in three different areas carefully selected to represent cross sections of a tumor cord, avoiding oval or longitudinal sections. Measurements were done in three different directions from a single vessel.
Statistics.
Correlation between the level of CA IX expression in sections of primary tumor and nodal metastases was examined using Spearman’s rank correlation. The difference in CA IX expression with age (two categories at the median age), MVD (two categories at the highest third), percentage of tumor necrosis (three categories: 0–5%, 5–25%, and >25%), sex, T stage (T1–4), N stage (N0 and N+), grade of tumor, and margin of invasion was compared using a Mann-Whitney U test or Kruskall Wallis test as appropriate. The difference in necrosis score with MVD (two categories at the highest third) was examined using the Mann-Whitney U test. For multivariate analysis, bivariate logistic regression was used and an odds ratio calculated. For this test, CA IX expression was divided at the median into two categories of high expression (>20%) and low expression (
20%), necrosis into two categories present (>5%) or absent (5%), and T stage into early (T1/2) and advanced (T3/4). All statistics were done using SPSS software v9.0.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, controls for CA IX expression were extracts from the renal cell line RCC4 with VHL mutation. They showed marked up-regulation of CA IX in normoxia, in contrast to the control RCC4/VHL cell line transfected with wt VHL, where CA IX was minimal in normoxia. It appeared as two bands related to glycosylation. CA IX was up-regulated in all three head and neck cell lines exposed to hypoxia (0.1% O2 for 16 h) with little or no expression in normoxia (Fig. 1a)
. CA IX expression was clearly up-regulated in tumor samples compared with paired normal tissue taken at operation from patients with HNSCC (Fig. 1b)
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. It was absent or very low in the normal epithelium overlying tumor tissue (Fig. 2b)
. Expression was confined to the cell membrane (Fig. 2c)
. In the eight tumor sections where no CA IX expression was seen, these was little or no tumor necrosis observed (0% necrosis in four cases, 5% necrosis in two cases, 20% necrosis in two cases). The level of expression was similar in both the primary and lymph node metastases from the same patient in all seven cases, although no significant correlation could be demonstrated because of the small numbers [primary median CA IX 50% (range, 5–60%), secondary median CA IX 35% (range, 20–80%), n = 7, P = 0.093, Spearman’s correlation].
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. Using a formula published by Tomlinson and Gray (31)
, which makes assumptions about oxygen diffusion and consumption on the basis of experiments done on squamous cell carcinoma of the lung, the partial pressure of oxygen at a given distance from the nearest vessel can be calculated (Fig. 3)
. In the HNSCC sections examined here, the median distance from a blood vessel to the start of CA IX expression of 80 µm (range, 40–140 µm) gives a tissue pO2 of 1% (range, 0–2.8%). These results are only approximate because of the shrinkage of tissue on formalin fixation, the variation in sectioning of the tumor, and the assumptions made about oxygen diffusion and consumption.
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), as shown in Fig. 4
. CA IX expression was also higher as the percentage of tumor necrosis increased (P = 0.001, Kruskall Wallis; Table 1
), as shown in Fig. 5
. There was a significantly higher level of CA IX expression in more advanced tumors (T2–4) compared with T1 tumors (P = 0.033, Kruskall Wallis; Table 1
). There was no significant difference in CA IX expression between categories of age, sex, N-stage, grade, or margin of invasion (Table 1)
. There was no significant difference in percentage of tumor necrosis with increasing T stage (P = 0.37, Kruskall Wallis, data not shown) or high and low MVD (P = 0.47, Mann-Whitney, data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study demonstrates that CA IX is overexpressed in HNSCC. It was clearly induced by hypoxia in cell lines in vitro, and on immunostaining expression, it was clearly localized to the perinecrotic regions of the tumor, which are known to be hypoxic. CA IX expression was seen at a median distance from blood vessels of 80 µm correlating with a calculated pO2 in the range of 1% at the edge nearest the blood vessels. At the edge nearest the area of necrosis, the pO2 would be 
0.1%, which in vitro also gave strong induction of CA IX. There was a significant increase in CA IX expression as tumor necrosis increased. These observations suggest that CA IX is regulated by hypoxia in vivo. There was variability between tumors in distance from blood vessels to necrosis, which may reflect the susceptibility of the tumor cells to hypoxia-induced death, the heterogeneity of oxygen distribution within the tumor (39)
, or O2 consumption by the tumor contributing to the final effect of necrosis. However, these variations also appear to affect CA IX expression concomitantly. CA IX expression was only seen in the cell membrane on immunostaining. It is a transmembrane glycoprotein that makes CA IX a potentially useful indicator of tissue hypoxia, as the protein cannot diffuse away from its point of origin. This is in contrast to vascular endothelial growth factor, which does not correlate with biomarkers of hypoxia, such as Pimonidazole (40)
. Although secreted proteins may be useful peripheral blood markers of hypoxia (41)
, CA IX is induced at the same oxygen tension at which HIF-1 and its downstream target genes are induced and provides a measure of the percentage of the tumor population that is hypoxic (42)
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The correlation between CA IX and MVD is likely to be attributable to the overexpression of CA IX at the same oxygen tension as hypoxia-induced proangiogenic cytokines, such as vascular endothelial growth factor. The lack of correlation between MVD and necrosis may be because necrosis indicates both severe hypoxia, metabolic O2 consumption by the tumor, and the ability of a cell to withstand hypoxia rather than being a simple measure of hypoxia, although one study in breast carcinoma did find a correlation between MVD and necrosis (43) .
Tumor cells can undergo apoptosis in hypoxia, and therefore, there is strong selection for pathways to escape this fate, e.g., p53 mutation (44) . Selection of these cells in the hypoxic microenvironment is an important mechanism for malignant progression (45) . Hypoxic apoptosis is substantially mediated by an acidotic pHe, which occurs because of the switch from aerobic to anaerobic metabolism in hypoxia, in vitro studies (46) . In that study, buffering the pH changes in hypoxia either by changing the medium or more concentrated buffers allowed cell survival under hypoxia. Thus, induction of genes able to regulate extracellular or pHi may help hypoxic cells survive. Several mechanisms exist at a cellular level to generate an acidotic pHe and maintain the pHi. It has been proposed that increased activity of the mitogen-sensitive Na+/H+ exchanger, increased function or expression of H+ pumping ATPases, or an interaction between the tumor cell and its immediate environment may maintain the pHi at a normal level while lowering the pHe. Carbonic anhydrases have been suggested as one mechanism whereby the cell could maintain a difference in pH across its membrane (19 , 47 , 48) .
Carbonic anhydrases catalyze the reversible conversion of H2O and CO2 to carbonic acid. During aerobic metabolism, CO2 is generated. This moves out of the cell down a diffusion gradient as the extracellular CO2 is maintained at a lower level by conversion to carbonic acid, which dissociates into H+ and HCO3-. The bicarbonate ions are pumped back into the cell in exchange for chloride ions while the H+ ions remain in the extracellular environment and lower the pHe. The induction by hypoxia of CA IX with its active extracellular enzyme site could theoretically help to lower the extracellular CO2 and maintain the pHi at a normal level, preventing apoptosis and giving the cell a major survival advantage. A reduction in the pHe has advantages to tumor cells as it helps in the breakdown of the extracellular matrix, migration and invasion of tumor cells, induction of expression of growth factors, and reduction of the viability of nearby cells (49) . This potential ability of hypoxia-induced CA IX to affect the pHi and pHe in vitro and in vivo is currently under investigation. The pH of a tumor is one of the most significant factors in mathematical models of tumor survival. (50) . Chronic lowering of the pHi by inhibitors of the Na+/H+ exchanger or Na+-dependent HCO3-/Cl- exchanger is directly cytotoxic to tumor cells and inhibits tumor growth (48) .
The pH of a tumor may significantly alter the uptake of chemotherapy drugs, particularly if they are weak electrolytes (51) . Chlorambucil and 5FU, both weak acids, have increased toxicity and are retained in tumors cells when there is a low pHe (52 , 53) . Doxorubicin and mitoxantrone, both weak bases, have reduced intracellular accumulation with low pHe (54 , 55) . Doxorubicin toxicity has been enhanced in an animal model by raising the pHe with bicarbonate in the drinking water (49) .
Carbonic anhydrase inhibitors have been shown to inhibit tumor cell invasion in vitro (47) , and in xenograft experiments, carbonic anhydrase inhibitors as part of a chemotherapy regimen enhanced the effect of chemotherapy drugs and helped delay tumor growth (56) .
Thus, our demonstration of up-regulation of CA IX in vivo in a perinecrotic pattern suggests this may be an important pathway in hypoxia, possibly regulating pHe to allow survival of a viable rim of cells under hypoxic conditions. This subpopulation of cells may be a suitable target for inhibitors of carbonic anhydrase. Use of CA IX as a target for radioimmunotherapy with MoAbs or use of CA IX to convert a pro-drug to an active drug may have potential problems because of its abundant expression normal human upper GI mucosa and GI-associated structures.
CA IX expression correlates with the oxygen diffusion distance and is expressed in a perinecrotic manner; this may be a marker for hypoxia in HNSCC. It is induced by hypoxia in HNSCC cells and is up-regulated in HNSCC. Up-regulation correlates with tumor necrosis and MVD. Overexpression may help to maintain the pHi, give tumor cells a survival advantage, and enhance resistance to radiotherapy and chemotherapy. CA IX provides a potential target for future therapy.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at ICRF Molecular Oncology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. E-mail: aharris.lab{at}icrf.icnet.uk 
2 The abbreviations used are: CA9, carbonic anhydrase 9 (gene); CA IX, carbonic anhydrase IX (protein); HIF-1, hypoxia inducible factor 1 ; HNSCC, head and neck squamous cell carcinoma; MVD, microvessel density; TBS, Tris-buffered saline; VHL, von Hippel Lindau; MoAb, monoclonal antibody; wt, wild type; pHi, intracellular pH; pHe, extracellular pH; GI, gastrointestinal.
Received 1/16/01. Accepted 4/24/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E. P. Hui, A. T. C. Chan, F. Pezzella, H. Turley, K.-F. To, T. C. W. Poon, B. Zee, F. Mo, P. M. L. Teo, D. P. Huang, et al. Coexpression of Hypoxia-inducible Factors 1{alpha} and 2{alpha}, Carbonic Anhydrase IX, and Vascular Endothelial Growth Factor in Nasopharyngeal Carcinoma and Relationship to Survival Clin. Cancer Res., August 1, 2002; 8(8): 2595 - 2604. [Abstract] [Full Text] [PDF]
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 E. L. Engelhardt, R. F. Schneider, S. H. Seeholzer, C. C. Stobbe, and J. D. Chapman The Synthesis and Radiolabeling of 2-Nitroimidazole Derivatives of Cyclam and Their Preclinical Evaluation as Positive Markers of Tumor Hypoxia J. Nucl. Med., June 1, 2002; 43(6): 837 - 850. [Abstract] [Full Text] [PDF]
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C. N. Coleman, J. B. Mitchell, and K. Camphausen Tumor Hypoxia: Chicken, Egg, or a Piece of the Farm? J. Clin. Oncol., February 1, 2002; 20(3): 610 - 615. [Full Text] [PDF]
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P. L. Olive, C. Aquino-Parsons, S. H. MacPhail, S.-Y. Liao, J. A. Raleigh, M. I. Lerman, and E. J. Stanbridge Carbonic Anhydrase 9 as an Endogenous Marker for Hypoxic Cells in Cervical Cancer Cancer Res., December 1, 2001; 61(24): 8924 - 8929. [Abstract] [Full Text] [PDF]
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