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Abbreviations
HIF

hypoxia-inducible factor

CA

carbonic anhydrase

FIH

factor inhibiting HIF

PHD

prolyl hydroxylase

VHL

von Hippel–Lindau

HRE

hypoxia-response element

VEGF

vascular endothelial growth factor

GLUT

glucose transporter

LDH

lactate dehydrogenase

MCT

H+/monocarboxylate transporter

NHE

Na+/H+ exchanger

pHe

extracellular pH

AE

anion exchangers

NBC

Na+/bicarbonate cotransporters

mAb

monoclonal antibody

PG

proteoglycan-like

FITC-CAI

fluorescein-conjugated CA inhibitor.

INTRODUCTION

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

Hypoxia is a crucial factor in tumour physiology that leads to massive molecular and phenotypic changes associated with cancer progression and resistance to treatment. These changes are orchestrated by hypoxia-inducible factor (HIF) and include a shift to oncogenic metabolism, which produces acidosis in the tumour microenvironment. Acidosis has been traditionally attributed to accumulation of lactate and protons excessively produced by glycolysis, extruded from cells and poorly removed by the tumour vasculature. However, experiments with glycolysis-deficient cells indicate that CO2 is a significant source of acidity in tumours and suggest a role for carbonic anhydrase (CA), a zinc metalloenzyme catalysing the reversible conversion of CO2 to bicarbonate and proton. There are 15 human CA isoforms that regulate diverse physiological processes based on ion transport and pH balance. CAIX isoform is induced in different solid tumours in response to hypoxia or inactivating mutation of the von Hippel–Lindau (VHL) tumour suppressor gene, and serves as a hypoxic marker and prognostic indicator. It is an active enzyme with an extracellular catalytic site, and therefore is well positioned to act in control of tumour pH. Recent studies showed that CAIX cooperates with bicarbonate transporters, and participates in extracellular acidification in response to hypoxia. Moreover, its function can be inhibited by CAIX-selective sulphonamides. CAIX can also diminish the intracellular pH gradient in the hypoxic core of three-dimensional tumour spheroids. Reduced CAIX expression in tumour cells perturbs their in vitro survival under hypoxia. Deletion of the catalytic domain from CAIX delays tumour growth in vivo. Interestingly, mutations in intracellular tail of CAIX perturb its pH-regulating function exerted by the extracellular catalytic domain. Altogether, these data show direct functional involvement of CAIX in regulation of pH in tumour microenvironment and suggest novel strategies based on selective CAIX inhibitors for in vivo imaging and therapy of hypoxic tumours.

HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

Solid tumours often develop hypoxic areas due to insufficient supply of oxygen by irregular and functionally defective tumour vasculature. Hypoxia then creates selective pressure in favour of tumour cells that can adapt to this microenvironmental stress. Adaptation to diminished oxygenation has serious consequences: hypoxic tumour cells acquire oncogenic alterations in metabolism, gain increased resistance to conventional anticancer treatment, and are more prone to mutations. Finally, these adaptive changes result in expansion of cells with more aggressive phenotype and increased metastatic potential [1].

Cellular responses to hypoxia include shift to anaerobic glycolysis, angiogenesis, acidosis, reduced cell adhesion, decreased proliferation, and cell death (in severely hypoxic areas). These phenotypic modifications are principally determined at the molecular level by significant alterations of the transcriptional profile of hypoxic cells. The primary molecular response to low oxygen is the stabilization and activation of α subunit of HIF, a key transcriptional regulator of the genes involved in adaptation to hypoxia. In normoxia, HIF-α is modified by oxygen-dependent prolyl hydroxylases (PHDs) and an asparaginyl hydroxylase, (factors inhibiting HIF, named FIH), enzymes that all belong to the Fe(II) and 2-oxoglutarate dioxygenase superfamily. FIH hydroxylates aspragine 803 in the C-terminal activation domain of human HIF-1α thus preventing its interaction with transcriptional coactivators. Prolyl hydroxylases hydoxylate prolines 564 and 402 within the oxygen-dependent degradation domain thus enabling HIF-α recognition by the product of the VHL tumour suppressor gene, followed by its rapid ubiquitylation and proteasome degradation (Fig. 1). Loss or inactivating mutation in VHL, the main negative regulator of the hypoxic pathway, results in development of the hypoxic phenotype also under normoxic conditions in most clear cell RCCs [2,3].

image

Figure 1. Oxygen-dependent regulation of HIF and its downstream targets. In normoxia, HIF-α is post-translationally modified by PHDs 1–3 and FIH that all require oxygen as a substrate and Fe(II) and 2-oxoglutarate (2-OG) as cofactors. PHD-mediated hydroxylation of critical prolyl residues (Pro564 and Pro402 in HIF-1α) enables binding of the VHL tumour suppressor protein that leads to rapid ubiquitylation and degradation of HIF-α by the proteasome. FIH-mediated hydroxylation of a specific asparaginyl residue (Asp803 in HIF-1α) blocks binding of co-activators (p300/CPB) that are required for transactivation of HIF target genes. In hypoxia, hydroxylases cannot modify HIF-α, which escapes recognition by the VHL protein, accumulates in the cytoplasm, translocates to the nucleus, and dimerizes with HIF-β to form the HIF transcription factor. HIF binds to HREs, recruits co-activators and induces the transcription of target genes whose products facilitate adaptation to hypoxia by activation of various downstream pathways.

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In addition, increased levels and activation of HIF-α may be achieved under normoxic conditions by signal transduction through pathways regulated by activated oncogenes that increase transcription, translation and/or activity of HIF-α and thereby can contribute to or amplify the effects of hypoxia [4–6]. Hypoxia-like responses can be induced in normoxia also by genetic or metabolic events that perturb tricarboxylic acid cycle probably through the accumulation of Krebs cycle intermediates that compete with 2-oxoglutarate to inhibit the HIF hydroxylases [7].

In hypoxia and/or following oncogenic activation or loss of VHL function HIF-α escapes modifications by PHDs and FIH, enters the nucleus, dimerizes with a constitutive β subunit of HIF, interacts with coactivators and forms an active transcriptional complex. This complex binds DNA at sites containing the hypoxia-response element (HRE; 5′-RCGTG-3′), which is present in the promoters of a wide spectrum of target genes. HIF targets include genes encoding the haematopoietic growth factor, erythropoietin, mediators of angiogenesis such as vascular endothelial growth factor (VEGF) and VEGF receptors, enzymes of the glycolytic pathway such as hexokinase 2, lactate dehydrogenase, and glucose transporters (GLUT-1, GLUT-3), and many other genes (Fig. 1). Additional hypoxia-induced genes are involved in regulation of vascular remodelling and plasticity, cell proliferation and viability, cell adhesion, cell matrix metabolism, pH regulation and other cellular processes [8].

HIF-α exists in three basic isoforms (further diversified by alternative splicing). HIF-3α is the most distantly related isoform, which in one spliced form encodes an inhibitor of HRE-dependent gene expression termed IPAS. On the other hand, HIF-1α and HIF-2α are closely related and mediate transcriptional responses to hypoxia via HRE elements, albeit in apparently distinct temporal, tissue-specific and target-selective patterns [9,10]. This view is supported by phenotypes of knock-out mice and differential roles of HIF-2α vs HIF-1α in the progression of certain tumours. Moreover, many cell types concurrently express both isoforms, but preferentially use one of them to drive expression of certain hypoxia-induced pathways [11,12]. Thus, erythropoiesis and angiogenesis seem to be mediated preferentially by HIF-2α, whereas glycolysis is apparently driven by HIF-1α[13].

ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

Diminished oxygen supply to hypoxic tumour areas restricts energy production by oxidative phosphorylation and therefore metabolism of hypoxic cells is shifted to glycolysis, which can generate limited quantities of ATP in absence of oxygen. Underlying mechanism involves HIF-1 coordinated up-regulation of virtually all glycolytic enzymes and glucose transporters via HRE elements in the promoters of their genes [14]. HIF-1 also strongly induces both lactate dehydrogenase isoforms LDH-A and LHD-5, which convert pyruvate to lactate, and pyruvate dehydrogenase kinase 1, which prevents entry of pyruvate into the Krebs cycle, thereby reinforcing the switch to glycolysis over aerobic metabolism [13,15].

Glycolysis is less efficient than oxidative phosphorylation in energy yield; however, its metabolic intermediates can be utilized for biosynthetic reactions leading to production of certain amino acids, nucleotides and lipids. Therefore, glycolysis provides selective advantage to proliferating tumour cells in both anaerobic and aerobic conditions [16]. This provides a rationale for Warburg’s classical finding of high glucose consumption and high lactate production in tumour tissues independently of oxygen availability. Increased expression of the genes coding for glucose transporters and glycolytic enzymes can also be induced in normoxic tumour cells by the AKT pathway and the transcription factor MYC. The AKT serine-threonine kinase enhances glycolytic flux by mobilizing glucose transporters to cell surface as well as by activation of hexokinase 2 that catalyses initial step of glycolysis. The MYC transcription factor can transactivate glycolytic enzyme genes in close cooperation with HIF-1 [17]. Moreover, nonhypoxic oncogenic activation of HIF-1 can also stimulate glycolysis via activation of pyruvate dehydrogenase kinase PDK1 gene reinforcing the phenomenon of increased aerobic glycolysis that Warburg described [18].

Although lactate is the principal end-product of glycolysis, oncogenic metabolism produces also excess of protons and CO2, which have to be eliminated from tumour cells to maintain neutral intracellular pH that is critical for cell proliferation and survival ((Fig. 2). Lactate and protons are extruded by various molecules, including the H+/monocarboxylate transporter (MCT), the Na+/H+ exchanger (NHE), and the vacuolar H+/ATP pump, and accumulate in the extracellular space because of insufficient removal via the blood stream consequent on an inadequate tumour vasculature [19–22]. Thereby acid extrusion results in reduction of extracellular pH (pHe) that is a typical feature of the tumour microenvironment. On the other hand, anion exchangers (AE) and Na+/bicarbonate cotransporters (NBC) import bicarbonate ions that can react with intracellular protons generated by glycolysis, thus increasing cellular production of CO2[19]. CO2 diffuses across the plasma membrane to pericellular space and significantly contributes to extracellular acidosis as shown by experiments with lactate-deficient cells that are still capable of forming acidic tumours in vivo[23].

image

Figure 2. The main components of pH regulation in hypoxic tumour cells. HIF-1-mediated up-regulation of glucose transporters and glycolytic enzymes induces a metabolic shift to glycolysis that produces excess of lactate and protons. To prevent intracellular acidification that is incompatible with the cell growth and survival, these acidic products are extruded by the HIF-regulated MCT4 and the NHE1 and accumulate in the extracellular microenvironment due to insufficient removal via an inadequate vasculature. Oncogenic metabolism also produces high levels of CO2 that diffuses through the plasma membrane and contributes to the extracellular acidosis, which supports tumour cell invasion. Pericellular CO2 is hydrated to bicarbonate ions and protons in a reaction catalysed by hypoxia-activated CAIX. CAIX-facilitated production of bicarbonate ions is coupled to AE-mediated transport into the cytoplasm where these ions buffer protons, resulting in neutralization of intracellular pH, and further production of CO2 that leaves the cell by diffusion and may enter a new round of hydration. GLUT, glucose transporter; TCA, tricarboxylic acid.

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Extracellular acidification develops especially in tumour regions that are hypoxic as indicated on one hand by the correlation between the mean profiles of partial oxygen pressure and intratumoural pH values, and on the other by VHL/HIF pathway-controlled expression of several components of the pH regulating molecular machinery including AE2, NHE1, and MCT4 [24–26].

Acidic pHe has been associated with tumour progression via multiple effects including up-regulation of angiogenic factors and proteases, increased invasion, and impaired immune functions. Also, it can influence the uptake of anticancer drugs and modulate the response of tumour cells to conventional therapy [27].

CAs

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

The recently appreciated involvement of CO2 in generating microenvironmental acidosis in tumours has suggested a role for CAs. CAs are zinc metalloenzymes catalysing the reversible conversion of CO2 to bicarbonate and proton in a reaction that involves facilitated hydration of CO2 to H2CO3 followed by the spontaneous dissociation of H2CO3 to bicarbonate and proton. CAs are expressed in almost all living organisms and participate in diverse physiological processes based on ion transport and pH balance such as respiration, digestion, renal acidification, bone resorption, etc. The human genome encodes 15 CA isoforms that show variable levels of enzyme activity and differ in molecular features, tissue distribution, expression levels, kinetic properties and sensitivity to inhibitors. They also occupy various subcellular compartments (cytoplasm, mitochondrion, plasma membrane or secretory vesicles) and are engaged in various biochemical pathways. Twelve catalytically active isozymes (CA I–IV, VA, VB, VI, VII, IX, XII–XIV) possess a conserved active site that contains three histidine residues involved in the coordination of a zinc ion with the fourth histidine residue functioning as a proton shuttle [28]. Three acatalytic CA isoforms (VIII, X, XI) lack one of the three critical histidines. In addition, two CA-related proteins that principally function as receptor protein tyrosine phosphatases (RPTPβ/γ) have inactive CA domain pocket, which serves as a receptor site for neuronal adhesion molecule contactin.

Most of the active CA isoforms are present in differentiated cells of normal tissues and abnormal expression levels and/or activities are implicated in various noncancerous pathologies [29]. By contrast, expression of CAIX is strongly associated with solid tumours and therefore it is well suited to participate in control of tumour pH.

CAIX: IDENTIFICATION AND MOLECULAR FEATURES

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

The molecular identity of CAIX was resolved in 1994 by sequence analysis of its cDNA isolated using the monoclonal antibody (mAb) M75 specific for a plasma membrane antigen detected in human carcinoma cell line HeLa and increased at high cell density in culture [30]. Expression of this antigen, originally named ‘MN’, was found in various tumour cell lines and surgical tumour specimens, but not in the corresponding normal tissues, suggesting its potential usefulness as a tumour marker [31]. Primary structures of the MN cDNA and gene disclosed a large CA domain with a well-conserved active site [30,32]. Because it was the ninth mammalian CA identified, the MN protein was renamed CAIX. In an independent line of research, a RCC-associated antigen detected by mAb G250 was then found to be identical with MN/CAIX [33,34] (also reviewed by Oosterwijk elsewhere in this issue).

In addition to the CA domain that displays high enzyme activity, CAIX contains a C-terminal transmembrane anchor followed by a short cytoplasmic tail and an N-terminal extension containing a proteoglycan-like (PG) region that is absent from the other CA isozymes and thus represents a unique feature of CAIX [32]. Since M75 mAb binds to the repetitive epitope in the PG region, it allows selective detection of CAIX without cross-reactivity with other CAs [35]. CAIX is expressed in the form of a 153 kDa trimer composed of disulphide-linked 58/54 kDa monomers, which are glycosylated at asparagine 346 [30,31]. The cytoplasmic tail of CAIX contains three putative phosphorylation sites (T443, S448 and Y449). Of note, epidermal growth factor-induced phosphorylation of tyrosine 449 has recently been implicated in signal transduction via the AKT pathway [36].

CAIX exhibits distinct expression pattern characterized by limited expression in normal tissues contrasting with its broad distribution in many different tumours. Normal expression of CAIX is seen in the epithelia of gastrointestinal tract, particularly in the glandular gastric mucosa [37]. Different types of human tumour show high levels of ectopic expression of CAIX in significant proportion of specimens. These include carcinomas of the uterine cervix, kidney, oesophagus, lung, breast, colon, brain, vulva (reviewed by Parkkila in this issue). Comparison of the cDNA sequences of CAIX expressed in HeLa cervical carcinoma cell line and in the stomach showed no difference, indicating that mutations are not responsible for the association of CAIX with tumours and suggesting involvement of cancer-related regulatory pathways in the control of its expression [37].

CAIX REGULATION

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

Expression of CAIX is primarily regulated at the level of transcriptional activation. The promoter of the CA9 gene contains an HRE element (localized just in front of the transcription start site at position −3/−10) that binds HIF-1 and induces transcription of the CA9 gene in response to microenvironmental hypoxia and increased cell density [38]. In both conditions, mitogen-activated protein kinase and phosphatidyl inositol 3-kinase pathways mediate signalling to HIF-1 in an SP1-dependent fashion and cooperate in activation of CA9 transcription [39,40].

In most clear cell RCCs, CAIX is frequently expressed at a high level even in normoxia due to functional inactivation of VHL tumour suppressor gene that is unable to negatively regulate HIF-α[41]. In keeping with this, re-expression of the wild type VHL then leads to decreased level of CAIX in RCC cells [42]. Interestingly, CAIX appears to be an exclusive HIF-1α target in RCC and therefore its expression decreases at more advanced tumour stages together with expression of HIF-1α, which is replaced by HIF-2α as the tumour develops [12].

In addition to transcriptional regulation, control of CAIX expression involves alternative splicing of the CA9 transcript [43], which produces less abundant, hypoxia independent mRNA lacking the exons 8/9 and coding for truncated, cytoplasmic/secreted form of CAIX that displays diminished enzyme activity and behaves in a dominant negative fashion.

Finally, the amount of the full-length transmembrane CAIX protein is regulated post-translationally by metalloproteinase-mediated shedding of the ectodomain, which can be induced by treatment with modulators of phosphorylation signalling [44].

Hypoxia-regulated expression of CAIX provides an explanation for its broad distribution in different types of solid tumours and further strengthens the idea of the involvement of CAIX in the molecular machinery regulating the pH of tumour cells.

CAIX ROLE IN pH CONTROL

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

From the functional point of view, CAIX behaves as an adhesion molecule, which on the one hand destabilizes E-cadherin mediated cell–cell contacts by competitive interaction with β-catenin, and on the other mediates attachment of cells to solid support [35,45]. Such behaviour is compatible with known progression-associated attributes of malignant tumour cells that need to disconnect from primary tumour mass and then re-attach to secondary sites.

However, CAIX is primarily functioning as an enzyme with the catalytic domain localized at the extracellular face of the plasma membrane, and this position supports its role in pericellular metabolism of CO2. Interestingly, the enzymatic activity of CAIX is insensitive to high lactate concentrations (in contrast to the other CA isozymes) thus allowing CAIX to work efficiently in the hypoxic tumour microenvironment, which is rich in lactate produced by anaerobic glycolysis [46]. On the other hand, CAIX activity is inhibited by bicarbonate suggesting that it can preferentially catalyse the CO2 hydration arm of the reaction producing bicarbonate ions in pericellular tumour regions loaded with CO2 and deprived of bicarbonate. Because bicarbonate ions are unable to diffuse across the plasma membrane, they cannot contribute to the regulation of intracellular pH unless they are actively transported to cell interior by bicarbonate transporters (i.e. AE and NBC mentioned above). These circumstances create the basis for meaningful spatial and functional cooperation between bicarbonate transporter(s) and CAIX on the surface of tumour cells.

The concept of such a cooperative complex, named a ‘metabolon’, which is composed of bicarbonate transporter communicating with CA, has been proposed by Vince and Reithmeier [47]. Although the issue of direct physical interaction between the metabolon components remains controversial, their functional cooperation has been proven in various cellular/tissue contexts (e.g. in erythrocytes and renal epithelium) and for different combinations of transporters and CA isozymes (AE1-3, NBC and CAII, CAIV, reviewed in [48]). The main advantage of the bicarbonate metabolon relies in locally concentrated production of bicarbonate immediately coupled with its transport thereby resulting in accelerated flux of bicarbonate ions from one side of the plasma membrane to the other one. The improved flux is particularly important for physiological situations that require excessive pH regulation and/or ion movement, and indeed, tumour hypoxia constitutes such a situation.

We have now two important arguments supporting the functional involvement of CAIX in pH regulation in hypoxic cells with ectopic, constitutive expression of CAIX. First, CAIX contributes to the acidification of the extracellular microenvironment of hypoxic cells [49], and second, CAIX minimizes the intracellular pH gradient in the core of three-dimensional tumour spheroids indicating that it can help to neutralize intracellular pH of hypoxic tumour cells [50]. These pH-modulating effects of CAIX are apparently related to its catalytic activity producing bicarbonate ions imported to cytoplasm where they enter into a dehydration reaction that titrates intracellular protons and helps to maintain neutral intracellular pH. On the other hand, extracellular hydration of CO2 results in the net production of protons, which remain outside of the cells and contribute to acidosis (Fig. 2). This may have important implications for cancer progression, because maintenance of neutral intracellular pH is vital for cell proliferation and survival, whereas microenvironmental acidosis contributes to aggressive tumour phenotype by promoting invasion and metastasis [51]. In accord with this view, RNA interference-mediated reduction in CAIX decreased the clonogenic survival of hypoxic tumour cells and expression of a dominant-negative variant of CA with deleted CA domain led to delayed tumour growth in vivo[52 and unpublished results].

Reports that CAIX-mediated pH regulation is increased by hypoxia in cellular models with constitutive, hypoxia-independent level of CAIX can be translated to a conclusion that hypoxia activates the catalytic performance of CAIX. On this basis, we propose that in the natural context, hypoxia can lead to increased expression of CAIX, enhanced catalytic activity, assembly of transport metabolon and increased bicarbonate transport. Interestingly, CAIX can co-immunoprecipitate with anion transporters and to some extent increase the bicarbonate transport even under normoxia [53]. Unfortunately, co-immunoprecipitation data as well as direct measurements of bicarbonate transport under hypoxia are missing and therefore it is not possible to evaluate how tight the interaction of CAIX with AE is, and how strongly induced is the CAIX-facilitated performance of the hypoxic metabolon. Nevertheless, based on pHe values measured in culture medium with high buffering capacity, we can assume that the increase in bicarbonate transport under hypoxia greatly exceeds the normoxic transport. The underlying mechanism remains to be elucidated, but it might involve either hypoxia-triggered incorporation of an additional metabolon component, or functional cooperation of diverse metabolic pathways (such as bicarbonate transport and lactate export) leading to formation of a larger metabolon complex. Alternatively, post-translational modifications of the cytoplasmic domains of CAIX and/or AE might result in their increased activity. It is also conceivable that all these events can occur simultaneously and that they are mutually interconnected.

SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

The role of CAIX in pH regulation has been confirmed using a CAIX-deletion variant lacking the catalytic domain, which increased the pHe in hypoxia. Similar effects were achieved with selective inhibitors of CAIX enzyme activity [49].

Notably, a fluorescein-conjugated homosulfanilamide inhibitor (FITC-CAI) could bind only to hypoxic cells that expressed CAIX, but neither to hypoxic cells lacking CAIX nor to normoxic cells. This finding evoked the idea that hypoxia induces the catalytic activity of CAIX (it is well established that inhibitors can bind only to active CAs) possibly via modulated CAIX folding that opens the active site and makes it accessible to the inhibitor. Although the mechanism behind the proposed folding alteration is not known, it has been suggested that the highly acidic PG domain could communicate with a highly basic residues around the conserved zinc-binding histidines and serve as a cover closing or opening the entrance to the catalytic site depending on hypoxia. In favour of this assumption, a CAIX variant lacking the PG domain showed increased (albeit not complete) binding of inhibitor even in normoxia [54]. Based on this ability to recognize CAIX in hypoxic and not in normoxic cells, FITC-CAI became an excellent tool for more detailed investigations of molecular determinants of CAIX catalytic activity under hypoxia. Thorough analysis of a whole series of deletion and mutation variants of CAIX using the FITC-CAI binding assay and correlation of this with acidification capacity led to several important findings indicating an intracellular tail as an important part of the CAIX molecule whose integrity is needed for full enzyme activity (unpublished). It is quite plausible that the intracellular tail mediates an interaction of CAIX with intracellular proteins that might then modulate the function of the extracellular domain. However, further experimentation is needed to better understand this phenomenon.

TRANSLATION INTO THE CLINIC

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

CAIX is not only a subject of intense basic research, but also is attracting considerable attention as a clinically useful molecule. Its strong link to cancer and tight regulation by the HIF pathway offers an opportunity to use CAIX as a surrogate marker of hypoxia in solid tumours, as an indicator of VHL mutation in renal cancer, as a prognostic indicator, and also as a target for immunotherapy and pH-modulating therapy [55,56].

The CAIX-specific mAb M75 that works well in immunohistochemistry is suitable for the routine survey of tumour specimens for CAIX expression for prediction of treatment outcome and patient stratification. Numerous studies clearly support the prognostic/predictive value of increased expression of CAIX that indicates a good prognosis in clear cell RCC due to its link with HIF-1α[12] and a poor prognosis in other tumour types [57]. Moreover, a combination of M75 mAb binding to the N-terminal PG domain and the V/10 mAb recognizing the central CA domain allows for detection of extracellular domain of CAIX shed from tumour cells to body fluids and appears promising for screening/monitoring of patients with cancer [44,58].

The CAIX-specific mAb G250 that binds to a non-denatured form of CAIX within the CA domain (as indicated by its competitive binding with the other CA domain specific MAbs, unpublished) has been quite thoroughly investigated as a tool for in vivo imaging as well as for immunotherapy of RCC [59,60] (for more details see the articles published in this issue).

However, recent experimental data from the study of CAIX-mediated pH regulation indicate that CAIX can also serve as a functional target for in vivo imaging and treatment of hypoxic tumours using inhibitors of the enzyme. These compounds, mostly represented by sulphonamides, sulphanilamides, sulphamates and their derivatives, have already been proposed as anticancer drugs based on their antiproliferative effects in tumour cell lines although due to nonselective binding to CAs, the isoform target of these inhibitors was not specified [61]. Therefore, current efforts are focused on increasing the selectivity of the inhibitors towards CAIX by modulating the physical and chemical properties of the compounds via attachment of different side chains and other modifications as described elsewhere [62]. Certain alterations can introduce or improve the membrane impermeability, whereas other changes can modify the size or surface topology that fits better into active site cavity of CAIX than into other isoforms. Some types of modifications have increased the efficiency of inhibitors so that they can inhibit a recombinant catalytic domain of CAIX at subnanomolar concentrations and show a reasonable selectivity for cancer-related CAIX compared with the ubiquitous CAII isoform [63].

As mentioned above, some sulphonamide derivatives represented by the fluorescein-conjugated thioureido-homosulphanilamide, bind only to hypoxic cells that express CAIX [49,64]. Moreover, this FITC-CAI accumulates in the central hypoxic areas of tumour spheroids (unpublished) supporting the view that labelled CAIX-selective inhibitors can be potentially used as tools for in vivo imaging of hypoxic tumours. While CAIX-specific mAbs bind to CAIX protein, which is very stable (with a half life of ≈40 h) and remains expressed at the cell surface for a relatively long time after reoxygenation [65], CAIX inhibitors are expected to bind only to enzymatically active CAIX expressed on the surface of hypoxic cells and therefore would image only currently hypoxic tumour areas. Thus, the imaging with CAIX inhibitors might provide different imaging/prognostic information than the mAbs and might be useful particularly in non-RCC tumours in which hypoxia plays an important role in driving tumour progression.

CAIX inhibitors can be potentially useful also in cancer therapy when applied alone or in combination with other modulators of pH control, and/or with conventional chemotherapeutic drugs [66]. Via blocking the CAIX-mediated pH regulation, the inhibitors could prevent intracellular neutralization and simultaneously reduce extracellular acidosis and thus could decrease tumour cell survival and invasion. Even more pronounced effects could possibly be achieved by concurrent treatment with inhibitors of bicarbonate import as well as with inhibitors of lactate and proton-extruding pathways [67]. CAIX inhibition-induced changes in pH gradient across the plasma membrane might also modulate therapeutic responses by influencing the uptake of weakly electrolytic anticancer drugs. It has been already shown that reduction of extracellular acidosis can increase the cytotoxic effects of weakly basic drugs, including doxorubicin and that acetazolamide (a general inhibitor of CA activity) reduces in vivo growth of tumour xenografts when given alone and produced additive tumour growth delays when administered in combination with various chemotherapeutic compounds [68]. However, our current knowledge in this area is insufficient and requires further preclinical experimentation.

CONCLUSION

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

Although a role for CAIX in control of tumour pH has been predicted for almost a decade, only recently obtained experimental data has provide clear experimental support for this. CAIX is now perceived as an active cell surface component of the pH-regulating machinery of hypoxic tumour cells with a functional relevance for cancer progression. As a direct transcription target of the HIF/VHL pathway, it strongly responds to hypoxia by elevated expression and increased catalytic activity. These attributes together with the tight link to various types of tumours make CAIX a promising subject for different types of anticancer therapies. Ongoing research is expected to shed more light into the molecular mechanisms underlying the role of CAIX in cancer progression and to facilitate further analysis of diagnostic and therapeutic strategies based on its detection or inactivation.

ACKNOWLEDGEMENTS

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES

The research of the authors is supported by grants from the 6th Framework Program of the European Commission (Integrated project EUROXY, LSHC-CT-2003-502932), from WILEX A.G., from the Slovak Research and Development Support Agency (APVV-51-024805), from the Slovak Government (BITCET SP 337/2003) and the Wellcome Trust.

REFERENCES

  1. Top of page
  2. INTRODUCTION
  3. HYPOXIA AND THE ROLE OF HIF TRANSCRIPTION FACTOR
  4. ONCOGENIC METABOLISM AND pH REGULATION IN TUMOURS
  5. CAs
  6. CAIX: IDENTIFICATION AND MOLECULAR FEATURES
  7. CAIX REGULATION
  8. CAIX ROLE IN pH CONTROL
  9. SELECTIVE INHIBITORS AS TOOLS TO DETECT AND STUDY HYPOXIA-ACTIVATED CAIX
  10. TRANSLATION INTO THE CLINIC
  11. CONCLUSION
  12. ACKNOWLEDGEMENTS
  13. CONFLICT OF INTEREST
  14. REFERENCES