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Carbonic anhydrase IX as a target for metastatic disease
Bioorganic & Medicinal Chemistry 21 (2013) 1470–1476
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Carbonic anhydrase IX as a target for metastatic disease Roben G. Gieling ⇑, Kaye J. Williams Hypoxia and Therapeutics Group, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Stopford Building, Oxford Road, Rm. 3.125, M13 9PL Manchester, United Kingdom
a r t i c l e
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Article history: Available online 11 October 2012 Keywords: Carbonic anhydrase IX Tumour Metastasis Metastatic dissemination Migration Invasion Hypoxia Hypoxia-inducible factor Acidosis Anticancer therapy
a b s t r a c t Metastatic disease is responsible for the majority of cancer related deaths. Tumour-associated carbonic anhydrase IX (CA IX) is a powerful marker to diagnose various types of metastatic cancers including those of cervical, renal, breast and head & neck origin. The precise prognostic role of CA IX in determining local control versus overall survival is complex, although the majority of reports favour CA IX as a marker for poor prognosis in patients with metastatic cancer. Preclinical studies in cell cultures clearly demonstrate that CA IX stimulates features that enhance metastatic properties of cancer cells for example, reducing cell adhesion, increasing motility and migration, inducing vascularisation and activating proteases, in which CA IX-induced acidification of the microenvironment of the tumour is essential. As most findings are consistent with the idea that CA IX is important in metastatic dissemination, small molecular CA IX inhibitors (including fluorescent-tagged or radiolabelled) and monoclonal antibodies targeting the CA IX isoform have been developed. Studies in tumour xenograft models showed that these CA IX-specific inhibitors and antibodies can be very effective in therapy and imaging of a variety of different metastatic cancers. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
2. CA IX expression correlates with metastasis in patients
A key part in the development of effective anticancer therapy is to target metastatic dissemination as metastasis is responsible for 90% of deaths from solid tumours.1 Basically metastatic dissemination is a multi-step process involving reduced cell adhesion, increased motility and migration, local tissue and lymph node invasion and distant metastasis formation. The deprivation of oxygen (termed hypoxia) by the growth of the tumour beyond its blood vessel supply is a key inducer of hypoxia-dependent factors such as carbonic anhydrase (CA, EC 4.2.1.1) IX (CA IX) (see Section 3.1), although the regulation of CA IX via hypoxia-independent mechanisms has been reported.2,3 CA IX is expressed in invasive tumours and metastases but not in benign tumours. In normal tissue CA IX expression is low, except for within the mucosa of the glandular stomach, secreting H+ and HCO 3 into the lumen of the stomach as part of the normal biology.4–6 In tumours CA IX is involved in pH regulation facilitating acidification of the microenvironment enhancing cell growth and migration.7 It is therefore not surprising that CA IX is a pivotal target in anticancer therapy. It is less obvious from reports in the literature whether CA IX and acidosis also promote metastatic dissemination.7
2.1. Cervical cancer
⇑ Corresponding author. Tel.: +44 0 161 275 7004; fax: +44 0 161 275 8360. E-mail address: [email protected] (R.G. Gieling). 0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2012.09.062
In metastatic cervical cancer the expression of CA IX was a strong independent diagnostic8 and prognostic factor9–15 associated with poorer prognosis, the presence of metastases in lymph nodes, disease-free and metastasis-free survival. A correlation between CA IX expression in primary cervical cancer and in lymph node metastases also indicated that CA IX expressing-clones in primary tumours are highly metastatic.11,12 Hedley et al.,16 however did not find a significant correlation between CA IX in primary cervical tumours and clinicopathological features including tumour size, presence of lymph node metastases, and histological subtype. That they failed to find a correlation may be due to sampling error using single biopsies with intratumoral heterogeneity which accounted for 41% of the total variance in the data set.16 The use of computer-assisted image analysis of tumour CA IX staining16 as compared to a histological grading system in the other studies9,10,12–15 can also be a cause for the different outcome. 2.2. Renal cancer In renal cell carcinoma (RCC) the expression of CA IX is a powerful marker for the diagnosis and appears specific for clear cell RCC (ccRCC) histology.17 In the study of Liao (1997) high expression of CA IX was seen in all ccRCC specimens but none in those
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of benign renal lesions.18 Li et al.,19 concluded that CA IX expression is a positive marker for micrometastatic ccRCC based on their work with the G250/CA IX antibody. Furthermore, the detection of CA IX-expressing epithelial cells in blood samples from patients with renal cortical tumours was associated with an increased risk of recurrence after partial or radical nephrectomy.20 The high diagnostic value of CA IX can be explained by frequent early inactivation of the von Hippel-Lindau (VHL) gene in ccRCC stimulating the activation of the hypoxia inducible factor (HIF) pathway and CA IX expression.21,22 Dorai et al.,23 demonstrated that the high expression of CA IX in ccRCC tumours may also be the result of a positive feed-back loop induced by epidermal growth factor receptor (EGFR) which is frequently overexpressed in ccRCC. According to their results EGFR phosphorylates CA IX in its intracellular domain at a tyrosine side (CAIX-pY). CAIX-pY activates Akt which promotes the expression of Hif-1a which in turn enhances the expression of CA IX facilitating acidosis and tumour cell invasion.23 The importance of the EGFR pathway in CA IX signalling in primary renal tumours is still unclear. The prognostic value of CA IX in ccRCC gave controversial results which may vary in clinically localised versus metastatic ccRCC.22 In this regard, semiquantitative assessment of CA IX stained tissue samples revealed that decreased intratumoral levels of CA IX are independently associated with a more aggressive ccRCC subtype.24,25 This finding is supported by the observation that the expression of CA IX in ccRCC is a positive independent predictor of metastasis-free survival and may be used to predict early metastasis after nephrectomy.26 The gradual loss of CA IX in ccRCC patients with high grade tumours and poorer prognosis might be a consequence of a gradual switch from a HIF-1a to a HIF-2a response,27 the tumour becoming more tolerant to hypoxia or more effective neovascularisation. Never the less CA IX peptide vaccination in a phase 1 trial in patients with ccRCC induced an antigen-specific immune response which led to disappearance and shrinkage of lung metastatic lesions in 3 out of 23 patients.28 Furthermore, additional therapeutic trials in ccRCC patients with the monoclonal antibody cG250, the chimeric variant of murine G250, targeting CA IX are in phases 1 and 2.29 2.3. Breast cancer In invasive breast cancer CA IX expression in the primary tumour is an independent predictive factor for overall survival30 and is associated with an unfavourable outcome.31 In line with this breast metastases in skin show an expansive growth pattern correlated with increased angiogenesis and hypoxia-induced CA IX expression.32 Van den Eynden et al.,33 also observed that high CA IX and HIF expression, is determined by the microenvironment in which the cells reside within the primary tumour and lymph node, explaining why a substantial number of lymph node metastasis were CA IX and HIF-1a negative despite their primary tumours being positive for the same proteins. Interestingly, preclinical studies in mice revealed that breast carcinoma cells in blood display an altered response to hypoxia compared with the parental cells from which they were derived, which resulted in a greater aggression.34 The different phenotypes can be the result of selection pressure in the primary tumour by the shortage of oxygen and nutrition and/or CA IX expressing cells are more likely to metastasis into the lymph node. In addition to hypoxia and the activation of HIF-1 as the lead force behind the expression of CA IX in most solid tumours, other microenvironmental conditions as acidosis, glucose deprivation and cell density can also regulate CA IX expression in tumour cells. In this respect glioblastoma cells grown under normoxic conditions in acidified medium (pH 6.4 as compared to pH 7.4 normally) upregulated their CA IX expression.35 Glucose deprivation abolished the induction of CA IX in fibrosarcoma cells under anoxic conditions and high cell density caused CA IX induction in aerobic
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conditions.36 The significance of intratumoral acidosis and glucose deprivation as regulator of CA IX expression in cancer progression is unknown. 2.4. Head and neck cancer CA IX expression in metastatic head and neck cancer has in most cases been associated with a poorer prognosis. In this respect, high levels of CA IX in primary head and neck cancer is associated with a more aggressive metastatic phenotype, decreased local relapse-free survival and decreased overall and disease-free survival.37–39 In contrary Eriksen et al.,40 did not find a correlation between CA IX expression and prognosis. Furthermore, patients with high CA IX positivity in head and neck tumours treated with accelerated radiotherapy with carbogen and nicotinamide (ARCON), had a better prognosis based on the locoregional control of the primary tumour and freedom from distant metastases.41 That high CA IX levels cannot be used to identify a cohort of patients that would benefit from ARCON therapy suggests for instance that other mechanisms beside hypoxia may regulate CA IX expression in head and neck cancer. Hypoxia-independent regulation of the CA IX promoter gene has been found in experimental studies to explain CA IX expression in high density cell cultures with intermediate oxygen levels (i.e., O2 levels >1–<10%).42 Intermediate levels of oxygen are likely more available in lower than higher grade head and neck tumours. Secondly CA IX expression in histological samples may be more a reflection of cumulative hypoxia than acute hypoxia. In this respect the half-life of CA IX protein is several days and CA IX expression may still be detected long after the area becomes reoxygenated. 2.5. Miscellaneous cancers Based on biopsy material and clinicopathological data CA IX expression has also been correlated with metastasis formation and prognosis in a variety of other hypoxic tumour types. Reports on CA IX expression in metastatic tumours originating from bladder, brain, colorectal, gastric, lung, sarcoma, thyroid and vulvar cancer are summarised in Table 1. In most instances a correlation between CA IX expression and poor prognosis was reported. In the case of gastric cancer a low expression or even loss of CA IX expression compared to normal gastric mucosa has been reported in most tumours.6,43,44 This is probably associated with the normal biological role of CA IX in pH regulation in the stomach. Histology on gastric cancers showed the restoration of CA IX expression only in cancer cells at the invasion front.44 Hypomethylation of CpG sites in the CA IX gene promoter after treatment with 5-aza-20 deoxycytidine (5-aza-dC) induced reexpression of CA IX in gastric cancer cell lines.44 Hypomethylation in the CA IX gene promoter also resulted in increased migration of gastric cancer cells. The regulation of CA IX promoter activity by means of hypomethylation is also observed in renal cancer.45 In conclusion: High CA IX levels is a good diagnostic marker in the majority of solid cancers except in gastric cancer were the gradual loss of CA IX expression is associated with a lower survival. There also seems to be a high prognostic value for CA IX with a poorer prognosis for cancer patients with high CA IX levels. An exception is advanced renal cancer were the gradual loss of CA IX expression correlates with an overall poorer prognosis. The predictive value of CA IX in the selection of patients which would benefit more from CA IX-based anticancer treatment remains uncertain. CA IX expression cannot be used to select head and neck cancer patients which may benefit more from ARCON therapy (Section 2.4). As in the case of in patients with ccRCC, high dose IL-2 therapy is shown to be more effective in patients that express CA IX in their primary tumour.58 The importance of hypoxia-independent CA IX regulatory pathways
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Table 1 Miscellaneous cancers Type of cancer
Side analysed
Prognosis associated with CA IX expression
Reference
Bladder
Primary
Poorer prognosis in both invasive and noninvasive bladder cancer (strong predictor of recurrence, progression and overall survival) Poorer prognosis in both invasive and superficial bladder cancer (median survival) Poorer prognosis (malignancy grade and survival) Poorer prognosis (grade of malignancy) Poorer prognosis (overall survival) Poorer prognosis (higher CA IX levels at tumour margin and liver metastases)
Klatte46
Poorer prognosis (invasion depth, lymph node metastasis and lymphatic invasion) Reexpression is associated with poor prognosis (reduced post-operative survival time; high CA IX levels at invasion front and in lymph nodes) Poorer prognosis Poor outcome (overall survival) Poor prognosis (disease-specific and overall survival) Poorer prognosis (more aggressive disease phenotype) Poorer prognosis (tumour size, depth of invasion, lymph node metastases)
Kato52 Chen44
Brain
Colorectal Gastric
Lung Sarcoma Thyroid Vulvar
Primary Primary Primary Primary Primary and metastases Primary Primary lymph node Primary Primary Primary Primary Primary
in early versus advanced tumour stage has been able to explain at least in some occasions the differences in outcome. 3. CA IX in tumour cell malignant behaviour 3.1. Regulation of CA IX in tumour cells The HIF pathway is central in the transactivation of CA IX via binding of the HIF transcription factor to the hypoxia-responsive element (HRE) in the CA IX promoter.21 Other pathways for example phosphatidylinositol 3-kinase (PI-3K), factors (VHL, iron chelation 2-oxoglutarate analogues) and environmental conditions (hypoxia, acidosis) associated with CA IX expression also target the HIF pathway supporting its role as master regulator.21 Hypoxia is by far the most important stimulator behind the expression of CA IX in tumours. In line with this CA IX in head and neck squamous cell carcinoma (HNSCC) is located at a constant distance from blood vessels (i.e., 40–140 lm) outside areas of necrosis which would be predicted to be hypoxic.59 However, oxygen-independent factors such as oncogenes: Rat sarcoma ‘Ras’, Sarcoma ‘Src’ and PI-3K and tumour suppressor Phosphatase and tensin homolog ‘PTEN’ and direct tyrosine phosphorylation of CA IX (CAIX-pY, see Section 2.2) can also increase HIF-1a and expression of CA IX.21,60 Next to this direct binding of intracellular proteins to the CA IX gene promoter have been reported. Cell density dependent induction of CA IX is regulated by both the mitogen-activated protein kinase (MAPK) and PI-3K pathways by direct activation of the CA IX gene promoter in the presence of a minimal level of Hif-1a.42,61 Oxygen-independent activation of HIF-1a and direct binding of factors to the CA IX promoter gene help to explain discrepancies reported on several occasions between oxygen levels and CA IX expression in tumours. A diagrammatic overview of pathways used by CA IX to stimulate metastasis formation is shown in Figure 1.
Hussain47 Haapasalo48 Proescholdt49 Nordfors50 Rajaganeshan51
Swinson53 Giatromanolaki54 Maseide55 Burrows56 Choschzick57
other hypoxia-induced processes.63,64 In this respect pericellular acidosis around tumour cells led to the redistribution to the cell surface and an increased secretion of active cathepsin B.65,66 On the tumour cell surface this lysosomal cysteine protease is involved in degradative processes associated with tumour invasion.65 Secondly, CA IX is able to destabilise adherent junctions between cells by directly modulating E-cadherin thereby affecting E-cadherin binding to b-catenin and increasing cell dissociation.67 CA IX actively contributes to migration by facilitating ion transport in the leading edge of lamellipodia generating a reverse pH gradient at the cell front (acidic extracellular pH (pHe) and neutral alkaline intracellular pH (pHi)).63 The loss of phenotype of transformed cells in response to hypoxia and hypoxia-induced proteins are similar to those associated with epithelial-mesenchymal transition (EMT) in embryonic development and enhance metastatic properties.68 Many of the changes in phenotype associated with a more invasive and metastatic behaviour of CA IX-transfected cervical carcinoma cells could be attributed to CA IX-induced interference with the Rho/ROCK (Rho-associated kinase) signalling pathway.69 Interestingly Liao et al.,70 introduced the idea that CA IX may be a marker for stem cells in certain tissues based on the colocalisation of CA IX expressing cells with sites normally corresponding to stem cell niches (e.g., Müllerian-type columnar and reserve cells of the cervix). The use of genetic knockdown of CA IX has produced contradictory results in literature. On the one hand CA IX gene silencing with RNA interference (RNAi) failed to show a convincing relationship between CA IX expression and migration or invasion potential in breast carcinoma cells.71 On the other hand CA IX gene silencing with short hairpin RNA (shCAIX) did result in regression of primary breast tumours and increased the survival rate of shCAIX as compared to scramble shRNA inoculated mice.72 4. CA IX as a diagnostic and therapeutic target 4.1. CA IX expression in serum
3.2. CA IX in the metastatic tumour Preclinical studies have clearly shown that CA IX in response to hypoxia regulates pH, resulting in acidification of the microenvironment (termed acidosis) in the tumour. To regulate pH CA IX has to cooperate with the sodium bicarbonate co-transporter (NBCe1) at the membrane of the tumour cell (see Fig. 1).62 There is strong evidence that acidosis in the tumour supports its progression by reducing cell adhesion, increasing motility and migration, inducing neo-vascularization, activating proteases and enhancing
CA IX expression in serum can potentially be used to select patients which are most likely to benefit from CA IX-based therapy and to monitor the response. The use of CA IX levels in serum, released after proteolytic cleavage at the cellular transmembrane form of CA IX, as marker for intratumoral CA IX expression is unclear as some studies were able to correlate these two parameters whereas others did not find a correlation.15,73,74 Consequently the use of CA IX levels in serum of cancer patients as marker for disease progression is under debate. In several instances the presence of
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Figure 1. Signalling pathways that link carbonic anhydrase IX (CA IX) expression with tumour cell invasion/metastasis formation. CA IX catalyse the reversible hydration + (H2O) of carbon dioxide (CO2) that has passively diffused to the extracellular space, to bicarbonate (HCO 3 ) and protons (H ). The coupled bicarbonate transporter (NBCe1) + transports HCO 3 back into the cell. The net export of H leads to acidification of the extracellular space. CA IX-induced acidosis can enhance cathepsin B secretion into the extracellular space where it is involved in degradation of the extracellular matrix (ECM) and the release of growth factors facilitating tumour cell invasion and metastasis formation. CA IX-induced acidosis also targets the hypoxia inducible factor (HIF) pathway enhancing CA IX expression. Hypoxia-induced HIF expression is the master regulator of CA IX gene (CA9). The HIF pathway can also be affected by the phosphoinositide 3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) pathway and oncogenes (Src, Phosphatase and tensin homolog (PTEN)). Direct stimulation of the CA IX promoter gene involving the PI3K and MAPK pathways has been reported in the case of high density cell cultures with minimal Hif-1a levels. In renal cell carcinoma (RCC) the overexpression of epithelial growth factor receptor (EGFR) and inactivation of the von Hippel-Lindau (VHL) gene are also involved in upregulation of HIF/CA IX expression. CA IX is able to destabilise adherent junctions (E-cadherin-b-catenin binding) reducing cell adhesion. To this CA IX may interfere with the Rho/Rho-associated protein kinase (ROCK) signalling pathway affecting b-catenin. Reduced cell adhesion is associated with increased tumour cell invasion and metastasis formation.
the soluble isoform of CA IX in serum correlated with disease status. High serum levels of CA IX in renal,75,76 breast,77 cervical15 and vulvar cancer78 correlated with circulating tumour cells (CTCs), metastasis and disease-free survival. However, Woelber et al.,79 did not observe a correlation between CA IX levels in serum and those in ovarian tumours of cancer patients. The discrepancy between serum levels of CA IX and diagnosis or prognosis can partly be explained by a restricted number of patients analysed in some studies. In addition CA IX in serum is very effectively cleared from the blood by the kidneys keeping the CA IX levels in serum low.65 4.2. CA IX in imaging of metastasis Improvements are also being made in the detection of lymph node and distant metastasis using the expression of CA IX on tumour cells. Intratumoural injection of a CA IX specific monoclonal antibody conjugated to fluorescent dye (i.e., CA9Ab-680) marked the primary breast tumour and the tumour cells in the nearby lymph nodes and was detectable with non-invasive fluorescent imaging.80 Fluorescent-labelled CA IX inhibitors of the sulfonamide class are also synthesised and in particular the membrane-impermeant derivatives, with the physicochemical property adding to more selectivity for the membrane-associated CA IX over the cytosolic CA I and II isoforms, are of interest.81 Furthermore fluorescent-labelled CA IX inhibitors of the sulfonamide class can be effectively used in tumour xenograft studies to visualise subcutaneously transplanted colorectal carcinoma cells.82 Radioactive io-
dine-labelled cG250/CA IX monoclonal antibody is successfully used to visualise renal cell carcinoma (RCC) tumours as well as metastases in positron emission tomography (PET) with studies progressed to phase 3 clinical trials.29 4.3. Targeting CA IX in immunotherapy The use of monoclonal antibodies targeting CA IX in the treatment of RCC has progressed into clinical trials. The use of CA IX immunotherapy in non-RCC tumours such as mouse monoclonal antibody VII/20 in colorectal carcinoma shows a potential anticancer effect in mice.83 However, it is beyond the scope of this paper to give a complete overview on CA IX in immunotherapy in RCC patients as it is an intensively studied field. Readers are referred to the review by Stillebroer and colleagues.29
Figure 2. Chemical structure of carbonic anhydrase IX inhibitor S4.
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Figure 3. Overview of the role of carbonic anhydrase IX in local, regional and metastatic spreading of cancer.
4.4. Small molecular CA IX inhibitors in anticancer therapy There is a major advantage for the use of small molecular CA IX inhibitors in anticancer therapy. As the target enzyme is expressed on the plasma membrane there is no need to penetrate and diffuse into the cell to build up an adequate concentration to be effective, especially as the vasculature in tumours is mostly of poor quality which can affect tumour drug distributions, particularly to CA IX expressing tumour cells that are distal to the vasculature. The carbonic anhydrase inhibitors that have been developed can be grouped into four main classes, that is, sulfonamides, sulfamates, sulfamides and coumarins.84 As the synthesis of CA inhibitors specifically targeting the tumour-specific CA isoforms (i.e., CA IX and CA XII) has improved more studies have reported finding a pivotal role for CA IX in metastatic dissemination. Small molecular inhibitors belonging to the sulfonamide72,85 and sulfamate86–88 classes of CA inhibitors have a high selectivity at nanomolar (nM) level for CA IX and CA XII and have a low toxicity in mice. Experiments with CA IX selective inhibitors ureidosulfonamide 25 and 104 and glycosyl coumarins 204 and 205 showed that these are targeting breast cancer metastasis in mice improving metastasis-free survival.72,85 Recently, we presented evidence that sulfamates can inhibit metastasis formation.86 From a panel of 11 sulfamate CA IX inhibitors 3 showed inhibition of cell migration and spreading of hypoxic tumour cells in a dose-dependent manner. One of them, S4 (Fig. 2), was taken forward into in vivo tests using a breast cancer model for spontaneous metastatic dissemination. Mice were given a 10 mg of S4 per kg body
weight (10 mg/kg) maintenance dosage, daily on a ‘5 days on, 2 days off’ regimen which reduced metastatic tumour burden in the lung while not affecting primary tumour growth or mouse condition.86 In summary, the majority of reports on CA IX in metastatic disease based on biopsy material and clinicopathological data are consistent with the idea that CA IX expression in tumours is a sign of poorer prognosis (Fig. 3). Preclinical studies in various hypoxic tumour cell lines show that CA IX expression is a pivotal inducer of a more migratory and invasive phenotype. In support of this in vivo xenograft models of breast cancer metastasis indicated a role for CA IX in tumour growth and metastasis formation. CA IX-based therapy has advanced in clinical trials in some instances as in the treatment of renal cell carcinoma. Clinical trials have yet to reveal whether CA IX-based anticancer therapy will be similar effective in other types of cancer. 5. Notes The authors declare the following competing financial interest(s): Professor Williams is author of a patent (Carbonic Anhydrase Inhibitors, PCT/EP2011/052156). Acknowledgments We thank the European Commission for financial supporting the research of the Hypoxia and Therapeutics Group (EU FP7 Metoxia, Grant Agreement No. 222741).
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Svastova, E.; Zilka, N.; Zat’ovicova, M.; Gibadulinova, A.; Ciampor, F.; Pastorek, J.; Pastorekova, S. Exp. Cell Res. 2003, 290, 332. 68. Brahimi-Horn, M. C.; Bellot, G.; Pouyssegur, J. Curr. Opin. Genet. Dev. 2011, 21, 67. 69. Shin, H. J.; Rho, S. B.; Jung, D. C.; Han, I. O.; Oh, E. S.; Kim, J. Y. J. Cell Sci. 2011, 124, 1077. 70. Liao, S. Y.; Lerman, M. I.; Stanbridge, E. J. BMC Dev. Biol. 2009, 9, 22. 71. Robertson, N.; Potter, C.; Harris, A. L. Cancer Res. 2004, 64, 6160. 72. Lou, Y.; Mc Donald, P. C.; Oloumi, A.; Chia, S.; Ostlund, C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.; Leung, S.; Huntsman, D., et al Cancer Res. 2011, 71, 3364. 73. Zhou, G. X.; Ireland, J.; Rayman, P.; Finke, J.; Zhou, M. Urology 2010, 75, 257. 74. Hyrsl, L.; Zavada, J.; Zavadova, Z.; Kawaciuk, I.; Vesely, S.; Skapa, P. Neoplasma 2009, 56, 298. 75. Zavada, J.; Zavadova, Z.; Zat’ovicova, M.; Hyrsl, L.; Kawaciuk, I. Br. J. Cancer 2003, 89, 1067. 76. Li, G.; Feng, G.; Gentil-Perret, A.; Genin, C.; Tostain, J. 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Carbonic anhydrase IX as a target for metastatic disease Roben G. Gieling ⇑, Kaye J. Williams Hypoxia and Therapeutics Group, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Stopford Building, Oxford Road, Rm. 3.125, M13 9PL Manchester, United Kingdom
a r t i c l e
i n f o
Article history: Available online 11 October 2012 Keywords: Carbonic anhydrase IX Tumour Metastasis Metastatic dissemination Migration Invasion Hypoxia Hypoxia-inducible factor Acidosis Anticancer therapy
a b s t r a c t Metastatic disease is responsible for the majority of cancer related deaths. Tumour-associated carbonic anhydrase IX (CA IX) is a powerful marker to diagnose various types of metastatic cancers including those of cervical, renal, breast and head & neck origin. The precise prognostic role of CA IX in determining local control versus overall survival is complex, although the majority of reports favour CA IX as a marker for poor prognosis in patients with metastatic cancer. Preclinical studies in cell cultures clearly demonstrate that CA IX stimulates features that enhance metastatic properties of cancer cells for example, reducing cell adhesion, increasing motility and migration, inducing vascularisation and activating proteases, in which CA IX-induced acidification of the microenvironment of the tumour is essential. As most findings are consistent with the idea that CA IX is important in metastatic dissemination, small molecular CA IX inhibitors (including fluorescent-tagged or radiolabelled) and monoclonal antibodies targeting the CA IX isoform have been developed. Studies in tumour xenograft models showed that these CA IX-specific inhibitors and antibodies can be very effective in therapy and imaging of a variety of different metastatic cancers. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
2. CA IX expression correlates with metastasis in patients
A key part in the development of effective anticancer therapy is to target metastatic dissemination as metastasis is responsible for 90% of deaths from solid tumours.1 Basically metastatic dissemination is a multi-step process involving reduced cell adhesion, increased motility and migration, local tissue and lymph node invasion and distant metastasis formation. The deprivation of oxygen (termed hypoxia) by the growth of the tumour beyond its blood vessel supply is a key inducer of hypoxia-dependent factors such as carbonic anhydrase (CA, EC 4.2.1.1) IX (CA IX) (see Section 3.1), although the regulation of CA IX via hypoxia-independent mechanisms has been reported.2,3 CA IX is expressed in invasive tumours and metastases but not in benign tumours. In normal tissue CA IX expression is low, except for within the mucosa of the glandular stomach, secreting H+ and HCO 3 into the lumen of the stomach as part of the normal biology.4–6 In tumours CA IX is involved in pH regulation facilitating acidification of the microenvironment enhancing cell growth and migration.7 It is therefore not surprising that CA IX is a pivotal target in anticancer therapy. It is less obvious from reports in the literature whether CA IX and acidosis also promote metastatic dissemination.7
2.1. Cervical cancer
⇑ Corresponding author. Tel.: +44 0 161 275 7004; fax: +44 0 161 275 8360. E-mail address: [email protected] (R.G. Gieling). 0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2012.09.062
In metastatic cervical cancer the expression of CA IX was a strong independent diagnostic8 and prognostic factor9–15 associated with poorer prognosis, the presence of metastases in lymph nodes, disease-free and metastasis-free survival. A correlation between CA IX expression in primary cervical cancer and in lymph node metastases also indicated that CA IX expressing-clones in primary tumours are highly metastatic.11,12 Hedley et al.,16 however did not find a significant correlation between CA IX in primary cervical tumours and clinicopathological features including tumour size, presence of lymph node metastases, and histological subtype. That they failed to find a correlation may be due to sampling error using single biopsies with intratumoral heterogeneity which accounted for 41% of the total variance in the data set.16 The use of computer-assisted image analysis of tumour CA IX staining16 as compared to a histological grading system in the other studies9,10,12–15 can also be a cause for the different outcome. 2.2. Renal cancer In renal cell carcinoma (RCC) the expression of CA IX is a powerful marker for the diagnosis and appears specific for clear cell RCC (ccRCC) histology.17 In the study of Liao (1997) high expression of CA IX was seen in all ccRCC specimens but none in those
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of benign renal lesions.18 Li et al.,19 concluded that CA IX expression is a positive marker for micrometastatic ccRCC based on their work with the G250/CA IX antibody. Furthermore, the detection of CA IX-expressing epithelial cells in blood samples from patients with renal cortical tumours was associated with an increased risk of recurrence after partial or radical nephrectomy.20 The high diagnostic value of CA IX can be explained by frequent early inactivation of the von Hippel-Lindau (VHL) gene in ccRCC stimulating the activation of the hypoxia inducible factor (HIF) pathway and CA IX expression.21,22 Dorai et al.,23 demonstrated that the high expression of CA IX in ccRCC tumours may also be the result of a positive feed-back loop induced by epidermal growth factor receptor (EGFR) which is frequently overexpressed in ccRCC. According to their results EGFR phosphorylates CA IX in its intracellular domain at a tyrosine side (CAIX-pY). CAIX-pY activates Akt which promotes the expression of Hif-1a which in turn enhances the expression of CA IX facilitating acidosis and tumour cell invasion.23 The importance of the EGFR pathway in CA IX signalling in primary renal tumours is still unclear. The prognostic value of CA IX in ccRCC gave controversial results which may vary in clinically localised versus metastatic ccRCC.22 In this regard, semiquantitative assessment of CA IX stained tissue samples revealed that decreased intratumoral levels of CA IX are independently associated with a more aggressive ccRCC subtype.24,25 This finding is supported by the observation that the expression of CA IX in ccRCC is a positive independent predictor of metastasis-free survival and may be used to predict early metastasis after nephrectomy.26 The gradual loss of CA IX in ccRCC patients with high grade tumours and poorer prognosis might be a consequence of a gradual switch from a HIF-1a to a HIF-2a response,27 the tumour becoming more tolerant to hypoxia or more effective neovascularisation. Never the less CA IX peptide vaccination in a phase 1 trial in patients with ccRCC induced an antigen-specific immune response which led to disappearance and shrinkage of lung metastatic lesions in 3 out of 23 patients.28 Furthermore, additional therapeutic trials in ccRCC patients with the monoclonal antibody cG250, the chimeric variant of murine G250, targeting CA IX are in phases 1 and 2.29 2.3. Breast cancer In invasive breast cancer CA IX expression in the primary tumour is an independent predictive factor for overall survival30 and is associated with an unfavourable outcome.31 In line with this breast metastases in skin show an expansive growth pattern correlated with increased angiogenesis and hypoxia-induced CA IX expression.32 Van den Eynden et al.,33 also observed that high CA IX and HIF expression, is determined by the microenvironment in which the cells reside within the primary tumour and lymph node, explaining why a substantial number of lymph node metastasis were CA IX and HIF-1a negative despite their primary tumours being positive for the same proteins. Interestingly, preclinical studies in mice revealed that breast carcinoma cells in blood display an altered response to hypoxia compared with the parental cells from which they were derived, which resulted in a greater aggression.34 The different phenotypes can be the result of selection pressure in the primary tumour by the shortage of oxygen and nutrition and/or CA IX expressing cells are more likely to metastasis into the lymph node. In addition to hypoxia and the activation of HIF-1 as the lead force behind the expression of CA IX in most solid tumours, other microenvironmental conditions as acidosis, glucose deprivation and cell density can also regulate CA IX expression in tumour cells. In this respect glioblastoma cells grown under normoxic conditions in acidified medium (pH 6.4 as compared to pH 7.4 normally) upregulated their CA IX expression.35 Glucose deprivation abolished the induction of CA IX in fibrosarcoma cells under anoxic conditions and high cell density caused CA IX induction in aerobic
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conditions.36 The significance of intratumoral acidosis and glucose deprivation as regulator of CA IX expression in cancer progression is unknown. 2.4. Head and neck cancer CA IX expression in metastatic head and neck cancer has in most cases been associated with a poorer prognosis. In this respect, high levels of CA IX in primary head and neck cancer is associated with a more aggressive metastatic phenotype, decreased local relapse-free survival and decreased overall and disease-free survival.37–39 In contrary Eriksen et al.,40 did not find a correlation between CA IX expression and prognosis. Furthermore, patients with high CA IX positivity in head and neck tumours treated with accelerated radiotherapy with carbogen and nicotinamide (ARCON), had a better prognosis based on the locoregional control of the primary tumour and freedom from distant metastases.41 That high CA IX levels cannot be used to identify a cohort of patients that would benefit from ARCON therapy suggests for instance that other mechanisms beside hypoxia may regulate CA IX expression in head and neck cancer. Hypoxia-independent regulation of the CA IX promoter gene has been found in experimental studies to explain CA IX expression in high density cell cultures with intermediate oxygen levels (i.e., O2 levels >1–<10%).42 Intermediate levels of oxygen are likely more available in lower than higher grade head and neck tumours. Secondly CA IX expression in histological samples may be more a reflection of cumulative hypoxia than acute hypoxia. In this respect the half-life of CA IX protein is several days and CA IX expression may still be detected long after the area becomes reoxygenated. 2.5. Miscellaneous cancers Based on biopsy material and clinicopathological data CA IX expression has also been correlated with metastasis formation and prognosis in a variety of other hypoxic tumour types. Reports on CA IX expression in metastatic tumours originating from bladder, brain, colorectal, gastric, lung, sarcoma, thyroid and vulvar cancer are summarised in Table 1. In most instances a correlation between CA IX expression and poor prognosis was reported. In the case of gastric cancer a low expression or even loss of CA IX expression compared to normal gastric mucosa has been reported in most tumours.6,43,44 This is probably associated with the normal biological role of CA IX in pH regulation in the stomach. Histology on gastric cancers showed the restoration of CA IX expression only in cancer cells at the invasion front.44 Hypomethylation of CpG sites in the CA IX gene promoter after treatment with 5-aza-20 deoxycytidine (5-aza-dC) induced reexpression of CA IX in gastric cancer cell lines.44 Hypomethylation in the CA IX gene promoter also resulted in increased migration of gastric cancer cells. The regulation of CA IX promoter activity by means of hypomethylation is also observed in renal cancer.45 In conclusion: High CA IX levels is a good diagnostic marker in the majority of solid cancers except in gastric cancer were the gradual loss of CA IX expression is associated with a lower survival. There also seems to be a high prognostic value for CA IX with a poorer prognosis for cancer patients with high CA IX levels. An exception is advanced renal cancer were the gradual loss of CA IX expression correlates with an overall poorer prognosis. The predictive value of CA IX in the selection of patients which would benefit more from CA IX-based anticancer treatment remains uncertain. CA IX expression cannot be used to select head and neck cancer patients which may benefit more from ARCON therapy (Section 2.4). As in the case of in patients with ccRCC, high dose IL-2 therapy is shown to be more effective in patients that express CA IX in their primary tumour.58 The importance of hypoxia-independent CA IX regulatory pathways
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Table 1 Miscellaneous cancers Type of cancer
Side analysed
Prognosis associated with CA IX expression
Reference
Bladder
Primary
Poorer prognosis in both invasive and noninvasive bladder cancer (strong predictor of recurrence, progression and overall survival) Poorer prognosis in both invasive and superficial bladder cancer (median survival) Poorer prognosis (malignancy grade and survival) Poorer prognosis (grade of malignancy) Poorer prognosis (overall survival) Poorer prognosis (higher CA IX levels at tumour margin and liver metastases)
Klatte46
Poorer prognosis (invasion depth, lymph node metastasis and lymphatic invasion) Reexpression is associated with poor prognosis (reduced post-operative survival time; high CA IX levels at invasion front and in lymph nodes) Poorer prognosis Poor outcome (overall survival) Poor prognosis (disease-specific and overall survival) Poorer prognosis (more aggressive disease phenotype) Poorer prognosis (tumour size, depth of invasion, lymph node metastases)
Kato52 Chen44
Brain
Colorectal Gastric
Lung Sarcoma Thyroid Vulvar
Primary Primary Primary Primary Primary and metastases Primary Primary lymph node Primary Primary Primary Primary Primary
in early versus advanced tumour stage has been able to explain at least in some occasions the differences in outcome. 3. CA IX in tumour cell malignant behaviour 3.1. Regulation of CA IX in tumour cells The HIF pathway is central in the transactivation of CA IX via binding of the HIF transcription factor to the hypoxia-responsive element (HRE) in the CA IX promoter.21 Other pathways for example phosphatidylinositol 3-kinase (PI-3K), factors (VHL, iron chelation 2-oxoglutarate analogues) and environmental conditions (hypoxia, acidosis) associated with CA IX expression also target the HIF pathway supporting its role as master regulator.21 Hypoxia is by far the most important stimulator behind the expression of CA IX in tumours. In line with this CA IX in head and neck squamous cell carcinoma (HNSCC) is located at a constant distance from blood vessels (i.e., 40–140 lm) outside areas of necrosis which would be predicted to be hypoxic.59 However, oxygen-independent factors such as oncogenes: Rat sarcoma ‘Ras’, Sarcoma ‘Src’ and PI-3K and tumour suppressor Phosphatase and tensin homolog ‘PTEN’ and direct tyrosine phosphorylation of CA IX (CAIX-pY, see Section 2.2) can also increase HIF-1a and expression of CA IX.21,60 Next to this direct binding of intracellular proteins to the CA IX gene promoter have been reported. Cell density dependent induction of CA IX is regulated by both the mitogen-activated protein kinase (MAPK) and PI-3K pathways by direct activation of the CA IX gene promoter in the presence of a minimal level of Hif-1a.42,61 Oxygen-independent activation of HIF-1a and direct binding of factors to the CA IX promoter gene help to explain discrepancies reported on several occasions between oxygen levels and CA IX expression in tumours. A diagrammatic overview of pathways used by CA IX to stimulate metastasis formation is shown in Figure 1.
Hussain47 Haapasalo48 Proescholdt49 Nordfors50 Rajaganeshan51
Swinson53 Giatromanolaki54 Maseide55 Burrows56 Choschzick57
other hypoxia-induced processes.63,64 In this respect pericellular acidosis around tumour cells led to the redistribution to the cell surface and an increased secretion of active cathepsin B.65,66 On the tumour cell surface this lysosomal cysteine protease is involved in degradative processes associated with tumour invasion.65 Secondly, CA IX is able to destabilise adherent junctions between cells by directly modulating E-cadherin thereby affecting E-cadherin binding to b-catenin and increasing cell dissociation.67 CA IX actively contributes to migration by facilitating ion transport in the leading edge of lamellipodia generating a reverse pH gradient at the cell front (acidic extracellular pH (pHe) and neutral alkaline intracellular pH (pHi)).63 The loss of phenotype of transformed cells in response to hypoxia and hypoxia-induced proteins are similar to those associated with epithelial-mesenchymal transition (EMT) in embryonic development and enhance metastatic properties.68 Many of the changes in phenotype associated with a more invasive and metastatic behaviour of CA IX-transfected cervical carcinoma cells could be attributed to CA IX-induced interference with the Rho/ROCK (Rho-associated kinase) signalling pathway.69 Interestingly Liao et al.,70 introduced the idea that CA IX may be a marker for stem cells in certain tissues based on the colocalisation of CA IX expressing cells with sites normally corresponding to stem cell niches (e.g., Müllerian-type columnar and reserve cells of the cervix). The use of genetic knockdown of CA IX has produced contradictory results in literature. On the one hand CA IX gene silencing with RNA interference (RNAi) failed to show a convincing relationship between CA IX expression and migration or invasion potential in breast carcinoma cells.71 On the other hand CA IX gene silencing with short hairpin RNA (shCAIX) did result in regression of primary breast tumours and increased the survival rate of shCAIX as compared to scramble shRNA inoculated mice.72 4. CA IX as a diagnostic and therapeutic target 4.1. CA IX expression in serum
3.2. CA IX in the metastatic tumour Preclinical studies have clearly shown that CA IX in response to hypoxia regulates pH, resulting in acidification of the microenvironment (termed acidosis) in the tumour. To regulate pH CA IX has to cooperate with the sodium bicarbonate co-transporter (NBCe1) at the membrane of the tumour cell (see Fig. 1).62 There is strong evidence that acidosis in the tumour supports its progression by reducing cell adhesion, increasing motility and migration, inducing neo-vascularization, activating proteases and enhancing
CA IX expression in serum can potentially be used to select patients which are most likely to benefit from CA IX-based therapy and to monitor the response. The use of CA IX levels in serum, released after proteolytic cleavage at the cellular transmembrane form of CA IX, as marker for intratumoral CA IX expression is unclear as some studies were able to correlate these two parameters whereas others did not find a correlation.15,73,74 Consequently the use of CA IX levels in serum of cancer patients as marker for disease progression is under debate. In several instances the presence of
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Figure 1. Signalling pathways that link carbonic anhydrase IX (CA IX) expression with tumour cell invasion/metastasis formation. CA IX catalyse the reversible hydration + (H2O) of carbon dioxide (CO2) that has passively diffused to the extracellular space, to bicarbonate (HCO 3 ) and protons (H ). The coupled bicarbonate transporter (NBCe1) + transports HCO 3 back into the cell. The net export of H leads to acidification of the extracellular space. CA IX-induced acidosis can enhance cathepsin B secretion into the extracellular space where it is involved in degradation of the extracellular matrix (ECM) and the release of growth factors facilitating tumour cell invasion and metastasis formation. CA IX-induced acidosis also targets the hypoxia inducible factor (HIF) pathway enhancing CA IX expression. Hypoxia-induced HIF expression is the master regulator of CA IX gene (CA9). The HIF pathway can also be affected by the phosphoinositide 3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) pathway and oncogenes (Src, Phosphatase and tensin homolog (PTEN)). Direct stimulation of the CA IX promoter gene involving the PI3K and MAPK pathways has been reported in the case of high density cell cultures with minimal Hif-1a levels. In renal cell carcinoma (RCC) the overexpression of epithelial growth factor receptor (EGFR) and inactivation of the von Hippel-Lindau (VHL) gene are also involved in upregulation of HIF/CA IX expression. CA IX is able to destabilise adherent junctions (E-cadherin-b-catenin binding) reducing cell adhesion. To this CA IX may interfere with the Rho/Rho-associated protein kinase (ROCK) signalling pathway affecting b-catenin. Reduced cell adhesion is associated with increased tumour cell invasion and metastasis formation.
the soluble isoform of CA IX in serum correlated with disease status. High serum levels of CA IX in renal,75,76 breast,77 cervical15 and vulvar cancer78 correlated with circulating tumour cells (CTCs), metastasis and disease-free survival. However, Woelber et al.,79 did not observe a correlation between CA IX levels in serum and those in ovarian tumours of cancer patients. The discrepancy between serum levels of CA IX and diagnosis or prognosis can partly be explained by a restricted number of patients analysed in some studies. In addition CA IX in serum is very effectively cleared from the blood by the kidneys keeping the CA IX levels in serum low.65 4.2. CA IX in imaging of metastasis Improvements are also being made in the detection of lymph node and distant metastasis using the expression of CA IX on tumour cells. Intratumoural injection of a CA IX specific monoclonal antibody conjugated to fluorescent dye (i.e., CA9Ab-680) marked the primary breast tumour and the tumour cells in the nearby lymph nodes and was detectable with non-invasive fluorescent imaging.80 Fluorescent-labelled CA IX inhibitors of the sulfonamide class are also synthesised and in particular the membrane-impermeant derivatives, with the physicochemical property adding to more selectivity for the membrane-associated CA IX over the cytosolic CA I and II isoforms, are of interest.81 Furthermore fluorescent-labelled CA IX inhibitors of the sulfonamide class can be effectively used in tumour xenograft studies to visualise subcutaneously transplanted colorectal carcinoma cells.82 Radioactive io-
dine-labelled cG250/CA IX monoclonal antibody is successfully used to visualise renal cell carcinoma (RCC) tumours as well as metastases in positron emission tomography (PET) with studies progressed to phase 3 clinical trials.29 4.3. Targeting CA IX in immunotherapy The use of monoclonal antibodies targeting CA IX in the treatment of RCC has progressed into clinical trials. The use of CA IX immunotherapy in non-RCC tumours such as mouse monoclonal antibody VII/20 in colorectal carcinoma shows a potential anticancer effect in mice.83 However, it is beyond the scope of this paper to give a complete overview on CA IX in immunotherapy in RCC patients as it is an intensively studied field. Readers are referred to the review by Stillebroer and colleagues.29
Figure 2. Chemical structure of carbonic anhydrase IX inhibitor S4.
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Figure 3. Overview of the role of carbonic anhydrase IX in local, regional and metastatic spreading of cancer.
4.4. Small molecular CA IX inhibitors in anticancer therapy There is a major advantage for the use of small molecular CA IX inhibitors in anticancer therapy. As the target enzyme is expressed on the plasma membrane there is no need to penetrate and diffuse into the cell to build up an adequate concentration to be effective, especially as the vasculature in tumours is mostly of poor quality which can affect tumour drug distributions, particularly to CA IX expressing tumour cells that are distal to the vasculature. The carbonic anhydrase inhibitors that have been developed can be grouped into four main classes, that is, sulfonamides, sulfamates, sulfamides and coumarins.84 As the synthesis of CA inhibitors specifically targeting the tumour-specific CA isoforms (i.e., CA IX and CA XII) has improved more studies have reported finding a pivotal role for CA IX in metastatic dissemination. Small molecular inhibitors belonging to the sulfonamide72,85 and sulfamate86–88 classes of CA inhibitors have a high selectivity at nanomolar (nM) level for CA IX and CA XII and have a low toxicity in mice. Experiments with CA IX selective inhibitors ureidosulfonamide 25 and 104 and glycosyl coumarins 204 and 205 showed that these are targeting breast cancer metastasis in mice improving metastasis-free survival.72,85 Recently, we presented evidence that sulfamates can inhibit metastasis formation.86 From a panel of 11 sulfamate CA IX inhibitors 3 showed inhibition of cell migration and spreading of hypoxic tumour cells in a dose-dependent manner. One of them, S4 (Fig. 2), was taken forward into in vivo tests using a breast cancer model for spontaneous metastatic dissemination. Mice were given a 10 mg of S4 per kg body
weight (10 mg/kg) maintenance dosage, daily on a ‘5 days on, 2 days off’ regimen which reduced metastatic tumour burden in the lung while not affecting primary tumour growth or mouse condition.86 In summary, the majority of reports on CA IX in metastatic disease based on biopsy material and clinicopathological data are consistent with the idea that CA IX expression in tumours is a sign of poorer prognosis (Fig. 3). Preclinical studies in various hypoxic tumour cell lines show that CA IX expression is a pivotal inducer of a more migratory and invasive phenotype. In support of this in vivo xenograft models of breast cancer metastasis indicated a role for CA IX in tumour growth and metastasis formation. CA IX-based therapy has advanced in clinical trials in some instances as in the treatment of renal cell carcinoma. Clinical trials have yet to reveal whether CA IX-based anticancer therapy will be similar effective in other types of cancer. 5. Notes The authors declare the following competing financial interest(s): Professor Williams is author of a patent (Carbonic Anhydrase Inhibitors, PCT/EP2011/052156). Acknowledgments We thank the European Commission for financial supporting the research of the Hypoxia and Therapeutics Group (EU FP7 Metoxia, Grant Agreement No. 222741).
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