Miriam Zatovicova

Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH

Acidic extracellular pH (pHe) is a typical attribute of a tumor microenviroment, which has an impact on cancer development and treatment outcome. It was believed to result from an accumulation of lactic acid excessively produced by glycolysis. However, metabolic profiles of glycolysis‐impaired tumors have revealed that CO2 is a significant source of acidity, thereby indicating a contribution of carbonic anhydrase (CA). The tumor‐associated CA IX isoform is the best candidate, because its extracellular enzyme domain is highly active, expression is induced by hypoxia and correlates with poor prognosis. This study provides the first evidence for the role of CA IX in the control of pHe. We show that CA IX can acidify the pH of the culture medium in hypoxia but not in normoxia. This acidification can be perturbed by deletion of the enzyme active site and inhibited by CA IX‐selective sulfonamides, which bind only to hypoxic cells containing CA IX. Our findings suggest that hypoxia regulates both expression and activity of CA IX in order to enhance the extracellular acidification, which may have important implications for tumor progression.

Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with β-catenin

Abstract Carbonic anhydrase IX (CA IX) is a cancer-associated transmembrane isoform of zinc metalloenzymes that catalyse interconversion between carbon dioxide and bicarbonate. CA IX is strongly induced by tumor hypoxia and has been proposed to participate in acidification of tumor microenvironment and in cell adhesion. To elucidate the cell adhesion-related role of CA IX, we investigated its subcellular localization and relationship to E-cadherin, a key adhesion molecule whose loss or destabilization is linked to tumor invasion. For this purpose, we generated MDCK cells with constitutive expression of human CA IX protein. During the monolayer formation, CA IX was localized to cell–cell contacts and its distribution in lateral membranes overlapped with E-cadherin. Calcium switch-triggered disruption and reconstitution of cell contacts resulted in relocalization of both CA IX and E-cadherin to cytoplasm and back to plasma membrane. A similar phenomenon was observed in hypoxia-treated and reoxygenated cells. Moreover, CA IX-expressing MDCK cells exhibited reduced cell adhesion capacity and lower levels of Triton-insoluble E-cadherin. Finally, CA IX was found to coprecipitate with β-catenin. We conclude that CA IX has a capacity to modulate E-cadherin-mediated cell adhesion via interaction with β-catenin, which could be of potential significance in hypoxia-induced tumor progression.

Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients

Tumour-associated protein carbonic anhydrase IX (CA IX) has two major forms. One is a cell-associated, transmembrane protein seen on Western blots as a twin band of 54/58 kDa, expressed in gastric mucosa and in several types of cancer. The other is a soluble protein s-CA IX of 50/54 kDa, which is released into the culture medium or into the body fluids, most likely by proteolytic cleavage of the extracellular part from transmembrane and intracellular sequences. While TC media of CA IX-positive tumour cell lines or short-term cultures of tumour explants contain a relatively high concentration of s-CA IX (20–50 ng ml−1), the level of this antigen in blood serum and urine of renal clear cell carcinoma patients is about 1000 × lower. The concentration of CA IX in the blood and in urine varies within wide limits and there is no obvious correlation with tumour size. After nephrectomy, s-CA IX is cleared from the blood within a few days. Only an extremely low concentration of CA IX was detectable in the sera and in urine of control individuals.

Carbonic Anhydrase IX Interacts with Bicarbonate Transporters in Lamellipodia and Increases Cell Migration via Its Catalytic Domain

Background: Hypoxia-induced CA IX contributes to pH control in tumor cells, and control of pH is important for cell migration. Results: CA IX increases migration through catalytic domain and interacts with bicarbonate transporters in lamellipodia. Conclusion: CA IX is an active component of the molecular machinery that facilitates migration of tumor cells through pH regulation at the leading edge membranes. Significance: This identifies CA IX as a target to suppress cell migration and reduce tumor aggressiveness. Carbonic anhydrase IX (CA IX) is a hypoxia-induced cell surface enzyme expressed in solid tumors, and functionally involved in acidification of extracellular pH and destabilization of intercellular contacts. Since both extracellular acidosis and reduced cell adhesion facilitate invasion and metastasis, we investigated the role of CA IX in cell migration, which promotes the metastatic cascade. As demonstrated here, ectopically expressed CA IX increases scattering, wound healing and transwell migration of MDCK cells, while an inactive CA IX variant lacking the catalytic domain (ΔCA) fails to do so. Correspondingly, hypoxic HeLa cells exhibit diminished migration upon inactivation of the endogenous CA IX either by forced expression of the dominant-negative ΔCA variant or by treatment with CA inhibitor, implying that the catalytic activity is indispensable for the CA IX function. Interestingly, CA IX improves cell migration both in the absence and presence of hepatocyte growth factor (HGF), an established inducer of epithelial-mesenchymal transition. On the other hand, HGF up-regulates CA IX transcription and triggers CA IX protein accumulation at the leading edge of lamellipodia. In these membrane regions CA IX co-localizes with sodium bicarbonate co-transporter (NBCe1) and anion exchanger 2 (AE2) that are both components of the migration apparatus and form bicarbonate transport metabolon with CA IX. Moreover, CA IX physically interacts with AE2 and NBCe1 in situ, as shown here for the first time. Thus, our findings suggest that CA IX actively contributes to cell migration via its ability to facilitate ion transport and pH control at protruding fronts of moving cells.

Phosphorylation of Carbonic Anhydrase IX Controls Its Ability to Mediate Extracellular Acidification in Hypoxic Tumors

In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment.

Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17

Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFα-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.

Alternative splicing variant of the hypoxia marker carbonic anhydrase IX expressed independently of hypoxia and tumour phenotype.

CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8–9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia- and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control.

Characterization of carbonic anhydrase IX interactome reveals proteins assisting its nuclear localization in hypoxic cells.

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface.

Intact intracellular tail is critical for proper functioning of the tumor-associated, hypoxia-regulated carbonic anhydrase IX

MINT‐7293982: E‐cadherin (uniprotkb:Q95LE0) and CA IX (genbank_protein_gi:223556027) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Tumor Microenvironmental Changes Induced by the Sulfamate Carbonic Anhydrase IX Inhibitor S4 in a Laryngeal Tumor Model

Background and Purpose Carbonic anhydrase IX (CAIX) plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3′(3″,5″-dimethylphenyl)-ureido)phenyl sulfamate (S4) on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding. Methods SCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole) and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR. Results CAIX ectodomain shedding decreased after treatment with S4 (p<0.01). S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole) and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed. Conclusion As the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth.