Juraj Kopacek

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.

Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors

Acidic tissue microenvironment contributes to tumor progression via multiple effects including the activation of angiogenic factors and proteases, reduced cell-cell adhesion, increased migration and invasion, etc. In addition, intratumoral acidosis can influence the uptake of anticancer drugs and modulate the response of tumors to conventional therapy. Acidification of the tumor microenvironment often develops due to hypoxia-triggered oncogenic metabolism, which leads to the extensive production of lactate, protons, and carbon dioxide. In order to avoid intracellular accumulation of the acidic metabolic products, which is incompatible with the survival and proliferation, tumor cells activate molecular machinery that regulates pH by driving transmembrane inside-out and outside-in ion fluxes. Carbonic anhydrase IX (CA IX) is a hypoxia-induced catalytic component of the bicarbonate import arm of this machinery. Through its catalytic activity, CA IX directly participates in many acidosis-induced features of tumor phenotype as demonstrated by manipulating its expression and/or by in vitro mutagenesis. CA IX can function as a survival factor protecting tumor cells from hypoxia and acidosis, as a pro-migratory factor facilitating cell movement and invasion, as a signaling molecule transducing extracellular signals to intracellular pathways (including major signaling and metabolic cascades) and converting intracellular signals to extracellular effects on adhesion, proteolysis, and other processes. These functional implications of CA IX in cancer are supported by numerous clinical studies demonstrating the association of CA IX with various clinical correlates and markers of aggressive tumor behavior. Although our understanding of the many faces of CA IX is still incomplete, existing knowledge supports the view that CA IX is a biologically and clinically relevant molecule, exploitable in anticancer strategies aimed at targeting adaptive responses to hypoxia and/or acidosis.

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.

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.

Carbonic Anhydrase Inhibitors and the Management of Cancer

Recent progress in understanding the role of catalytically active carbonic anhydrases in tumors has opened new possibilities for diagnostic and/or therapeutic applications of carbonic anhydrase inhibitors selectively blocking the enzyme activity of cancer-related isoforms, namely CA IX and CA XII. Different classes of inhibitors have been investigated in order to evaluate their usefulness as in vivo imaging tools, as modulators of intratumoral pH that influences uptake of conventional chemotherapeutics, or as drugs impeding survival of tumor cells exposed to physiological stresses including hypoxia and acidosis. Here we summarize the most important data related to expression, regulation and functional aspects of cancer-related carbonic anhydrases and discuss advances in synthesis and preclinical studies of isozyme-selective and highly efficient carbonic anhydrase inhibitors.

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

The effect of carbonic anhydrase IX on focal contacts during cell spreading and migration

Carbonic anhydrase IX is a hypoxia-induced transmembrane enzyme linked with solid tumors. It catalyzes the reversible hydration of CO2 providing bicarbonate ions for intracellular neutralization and protons for extracellular acidosis, thereby supporting tumor cell survival and invasiveness. CA IX is the only human CA isoform containing the proteoglycan (PG) domain in its extracellular part. The PG domain appears to enhance the catalytic activity of CA IX and mediate its binding to the extracellular matrix. Moreover, manipulation of the CA IX level by siRNA or overexpression modulates cell adhesion pathway so that in the presence of CA IX, cells display an increased rate of adhesion and spreading. Here we show that deletion of the PG domain as well as treatment with the PG-binding monoclonal antibody M75 can impair this CA IX effect. Accordingly, CA IX-expressing cells show more prominent and elongated maturing paxillin-stained focal contacts (FC) than CA IX-negative controls, proving the role of CA IX in cell spreading. However, during active cell movement, CA IX is relocalized to lamellipodia and improves migration via its catalytic domain. Thus, we examined the influence of CA IX on FC turnover in these structures. While the lamellipodial regions lacking CA IX display dash-like adhesions, the CA IX-enriched neighboring regions exhibit dynamic dot-like FCs. These results suggest that CA IX can promote initial adhesion through its PG domain, but at the same time it facilitates formation of nascent adhesions at the leading edge of moving cells. Thereby it may allow for transmission of large forces and enhanced migration rate, presumably through catalytic activity and impact of pHe on FC dynamics. Thus, we provide the first evidence that CA IX protein localizes directly in focal adhesion (FA) structures and propose its functional relationship with the proteins involved in the regulation of FC turnover and maturation.

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)

Induction of carbonic anhydrase IX by hypoxia and chemical disruption of oxygen sensing in rat fibroblasts and cardiomyocytes

CA IX is an active transmembrane carbonic anhydrase isoform functionally implicated in cell adhesion and pH control. Human CA IX is strongly induced by hypoxia and frequently associated with various tumors. In this study, we investigated the expression of the rat CA IX in response to chronic hypoxia and to treatment with chemical compounds that disrupt oxygen sensing, including dimethyloxalylglycine, dimethylester succinate, diazoxide, and tempol. We brought the evidence that expression of CA IX is regulated by hypoxia and hypoxia-mimicking compounds in immortalized Rat2 fibroblasts and BP6 rat fibrosarcoma cells in a cell-type-specific manner. We also demonstrated, for the first time, that CA IX is expressed in hypoxic primary rat cardiomyocytes and in immortalized H9c2 cardiomyocytes exposed to physiological or chemical hypoxia and that CA IX expression is increased in hypoxic rat tissues in vivo. Our findings suggest that CA IX expression is not limited to cancer but may be also induced in other pathological situations associated with ischemia or metabolic disturbances leading to activation of the HIF pathway. These data support the view that rats can represent useful model for studies of CA IX as a component of endogenous protection mechanisms associated with hypoxia or perturbed oxygen sensing.

Expression Pattern of Carbonic Anhydrase IX in Medullary Thyroid Carcinoma Supports a Role for RET-Mediated Activation of the HIF Pathway

Medullary thyroid carcinoma is a relatively rare tumor with poor prognosis and therapy response. Its phenotype is determined by both genetic alterations (activating RET oncoprotein) and physiological stresses, namely hypoxia [activating hypoxia-inducible factor (HIF)]. Here, we investigated the cooperation between these two mechanisms. The idea emerged from the immunohistochemical analysis of carbonic anhydrases (CA) IX and XII expression in thyroid cancer. Although CAXII was present in all types of thyroid carcinomas, CAIX, a direct HIF target implicated in tumor progression, was associated with aggressive medullary and anaplastic carcinomas, and its expression pattern in medullary thyroid carcinomas suggested contribution of both hypoxic and oncogenic signaling. Therefore, we analyzed the CA9 promoter activity in transfected tumor cells expressing RET and/or the HIF-α subunit. We showed that overexpression of both wild-type and mutant RET can increase the CA9 promoter activity induced by HIF-1 (but not HIF-2) in hypoxia. Similar results were obtained with another HIF-1-regulated promoter derived from the lactate dehydrogenase A gene. Moreover, inhibition of the major kinase pathways, which transmit signals from RET and regulate HIF-1, abrogated their cooperative effect on the CA9 promoter. Thus, we brought the first experimental evidence for the crosstalk between RET and HIF-1 that can explain the increased expression of CAIX in medullary thyroid carcinoma and provide a rationale for therapy simultaneously targeting both pathways.