CA9 gene was first cloned by Pastorek et al. (1994) and found to encode a 466 amino acid-long protein composed of a proteoglycan domain, a central catalytic M.A. Hayat (ed.), Tumors of the Central Nervous System, Volume 5, 65
DOI 10.1007/978-94-007-2019-0_7, © Springer Science+Business Media B.V. 2012
CA domain, a transmembrane part, and a short COOH- terminal cytoplasmic tail. Recently, Hilvo et al. (2008) reported a detailed characterization of human CA IX protein. Stopped-flow spectrophotometry experiments demonstrated that, in the excess of certain metal ions, the recombinant CA IX protein exhibits the highest catalytic activity ever measured for any CA isozyme.
Investigations on the oligomerization revealed that CA IX protein forms dimers that are stabilized by intermolecular disulfide bond(s).
Expression of Carbonic Anhydrase IX in Normal Tissue
When the expression of CA IX in mouse tissue was investigated, the highest immunoreactivity for CA IX was described in the gastric mucosa, moderate signals were seen in the colon and brain, and low expression was detectable in the pancreas and various segments of the small intestine (Hilvo et al., 2004). Similarly, high expression of human CA IX has been reported in the gastrointestinal tract, in which the epithelia of the gastric and gallbladder mucosa were strongly stained (Pastorek et al.,1994; Pastoreková et al.,1997;
Saarnio et al.,1998b). Intensive signals have also been reported in the intestinal epithelium, the duodenum and jejunum, having stronger expression than in the more distal segments (Saarnio et al.,1998b). Moreover, CA IX expression has been shown in the mesothelium, and in the epithelial cells of the esophagus, pancre- atic and biliary ducts (Turner et al.,1997; Pastoreková et al.,1997; Ivanov et al.,2001). CA IX has not been detected in the normal human brain tissue except for the weak immunostaining in the epithelial cells of the choroid plexus (Ivanov et al.,2001; Proescholdt et al., 2005). This is in accordance with the recent observa- tions that CA IX mRNA levels are low in a normal brain compared to malignant tissues (Said et al.,2007).
Expression of Carbonic Anhydrase IX in Neoplastic Tissue
CA IX is clearly the most predominant CA isozyme in various tumors. Its expression has been frequently observed in tumors derived from different segments
of the gastrointestinal tract, beginning from oral and ending to colorectal cancers (Pastoreková et al.,2006).
The expression and localisation of CA IX have been examined in head and neck squamous cell carcinoma (HNSCC) (Beasley et al., 2001). The enzyme was related to the location of tumor microvessels, angio- genesis, necrosis, and tumor stage, and was considered a potential target for future therapy in HNSCC. Turner et al. (1997) suggested that tumor-associated CA IX may play a role in the proliferation and regeneration in esophageal squamous epithelium, and loss of its expression may be related to cancer progression in Barrett’s-associated adenocarcinomas.
Ectopic expression is a characteristic feature for CA IX. Based on this phenomenon, CA IX is usually most highly expressed in tumors that originate from CA IX-negative tissues. Because the normal gastric mucosa contains the highest levels of CA IX among normal tissues, it was not surprising that relatively low CA IX expression was reported in gastric carcinomas (Chen et al.,2005). Interestingly, a subgroup of gastric cancers retain CA IX expression in malignant cells at the invasion front (Chen et al.,2005), suggesting that increased CA IX expression may contribute to a more advanced disease and tumor progression in a subset of gastric cancers.
Many colorectal tumors also overexpress CA IX (Saarnio et al., 1998a). It seems that CA IX expres- sion is quite heterogenous among different categories of colorectal cancer. The recent results have shown, however, that CA IX expression is most prominent in hereditary non-polyposis colorectal cancer (HNPCC) (Niemelä et al., 2007). Furthermore, its high expres- sion in premalignant lesions has suggested that it might be a useful marker in early diagnosis of colorectal tumors (Saarnio et al.,1998a).
Some CA IX positivity has been reported in both biliary and pancreatic tumors. Saarnio et al. (2001) showed that immunostaining for CA IX was mainly localised on the basolateral surface of the epithe- lial cells in the biliary epithelial tumors, similar to the normal biliary mucosa. The presence of CA IX in neoplastic hepatobiliary cells and its absence in hepatocellular carcinomas suggests that CA IX could be used as a marker for biliary differentiation in hepatobiliary neoplasms. The expression of CA IX in a relatively low number of malignant pancreatic tumor specimens suggests that it may have a limited value in diagnostic evaluation of pancreatic carcinoma.
However, the increased expression of CA IX in hyper- plastic ductal epithelium may contribute to pancreatic tumorigenesis.
CA IX may also serve as a valuable marker to pre- dict the prognosis of certain cancers. Its expression, for instance, has predicted poor survival rate in lung cancer (Kim et al.,2005). The presence of CA IX has specifically been linked to the expression of proteins that are involved in angiogenesis, apoptosis inhibition, and cell-cell adhesion disruption, which explains the strong association of this enzyme with poor clinical outcome of lung cancer (Giatromanolaki et al.,2001).
There are also several other publications demonstrat- ing the association between the CA IX expression and disease prognosis. In cervical cancer, Loncaster et al.
(2001) showed clinical evidence that CA IX expres- sion correlates with the levels of tumor hypoxia, and also associates with a poor prognosis of the disease.
Interestingly, it was suggested that the level of CA IX expression may be used to select patients who would benefit most from hypoxia-modification therapies or bio-reductive drugs.
Breast cancer is a very common tumor type in which CA IX might be useful as a tumor biomarker.
The main conclusion from several expression studies is that CA IX correlates with a poor prognosis, even though Span et al. (2007) recently pointed out that CA IX is more predictive than prognostic in this cancer type.
Some human renal cancer cell lines and renal can- cers have shown high CA IX expression at mRNA or protein level (McKiernan et al., 1997). Recently, Sandlund et al. (2007) assessed CA IX expression in different subtypes of renal cell cancer. They found that expression is higher in conventional cancer types rather than in other renal cell cancers, and patients with both conventional renal cell cancer and low CA IX expres- sion had a less favourable prognosis. According to the extensive literature available on CA IX in renal can- cer, the enzyme may indeed represent an ideal marker for clear cell renal cancer, as well as a promising therapeutic target for novel oncological applications, including immunotherapy and radioisotopic methods (Pastorekova et al.,2006).
Recent studies have also clearly demonstrated that CA IX is highly expressed in some malignant gliomas (Haapasalo et al., 2006). The immunohistochemical results showing positive signal for CA IX in glioma cells have been confirmed at mRNA level. When CA9
mRNA was studied in human malignant glioma cell lines, distinct patterns of hypoxic expression of CA IX were observed (Said et al.,2007), suggesting that the low oxygen concentration is probably the driving force for the increased CA IX expression due to the pres- ence of a hypoxia responsive element (HRE) in the CA9 promoter (Pastorekova et al.,2006). The role and prognostic significance of CA IX in malignant gliomas is also discussed in detail in another chapter of this series.
Role of Carbonic Anhydrase IX in Carcinogenesis
The distribution of CA IX in tumors is related to a transcriptional activation of the CA9 gene by a hypoxia-inducible factor 1 (HIF-1), which binds to HRE as a response to low oxygen supply (Pastorekova et al.,2006). This is a common feature in solid tumors in which irregular and functionally defective tumor vasculature results in hypoxic regions. HIF-1 plays a central role by activating genes that change the expres- sion profile of tumor cells suffering from hypoxia;
thus, either leading to adaptation to the hypoxic stress or resulting in cell death. Furthermore, the surviving tumor cell population is associated with worse prog- nosis and resistance to anti-cancer treatment due to increasingly aggressive behaviour involving invasion and metastases. This mechanism is supported by var- ious immunohistochemical studies in which the CA IX expression is located in perinecrotic regions of solid tumors (Haapasalo et al., 2006). CA IX isoform is also induced in cells with an inactivating mutation of the von Hippel–Lindau (VHL) tumor suppressor gene, which is a common feature e.g., in renal carcinomas.
Loss of functional VHL protein causes stabilization of HIF-1, leading to concomitant up-regulation of CAs with loss of regulation by hypoxia (Pastorekova et al., 2004,2006).
Many reports have been published to assess the physiology of CA IX in neoplastic tissue. CA IX increases extracellular acidification by shifting the site of CO2hydration from intra- to extracellular, and this process can be disturbed by inhibiting CA IX with selective sulfonamide inhibitors (Pastorekova et al., 2004,2006). The authors suggest that this could lead to increased capability of tumor cells to survive and
invade. Furthermore, CA IX has also been proposed to diminish the intracellular pH gradient in the hypoxic core of three-dimensional tumor spheroids grown from cancer-derived cell lines (Swietach et al.,2008).
Another potential mechanism for the function of CA IX was proposed by the finding that CA IX modulates E-cadherin mediated cell adhesion by decreasing the binding of this cell adhesion molecule to beta-catenin (Pastorekova et al.,2004,2006). The disruption of the link between these adhesion molecules would possibly promote cell motility and invasion. Acetazolamide, a potent CA inhibitor, suppresses invasion of renal can- cer cells in vitro. However, when the inhibition of CA IX in RNAi-treated breast cancer cells was studied, the invasion capacity of the cells appeared to be slightly reduced compared with the control, but this difference was not considered significant (Robertson et al.,2004).