Cancer development is a complex process that follows an intricate scenario with a dynamic interplay of selective and adaptive steps and an extensive cast of molecules and signaling pathways. Solid tumor initially grows as an avascular bulk of cells carrying oncogenic mutations until diffusion distances from the nearest functional blood vessels limit delivery of nutrients and oxygen on the one hand and removal of metabolic waste on the other one. These restrictions result in regional hypoxia and acidosis that select for adaptable tumor cells able to promote aberrant angiogenesis, remodel metabolism, acquire invasiveness and metastatic propensity, and gain therapeutic resistance. Tumor cells are thereby endowed with capability to survive and proliferate in hostile microenvironment, communicate with stroma, enter circulation, colonize secondary sites, and generate metastases. While the role of oncogenic mutations initializing and driving these processes is well established, a key contribution of non-genomic, landscaping molecular players is still less appreciated despite they can equally serve as viable targets of anticancer therapies. Carbonic anhydrase IX (CA IX) is one of these players: it is induced by hypoxia, functionally linked to acidosis, implicated in invasiveness, and correlated with therapeutic resistance. Here, we summarize the available experimental evidence supported by accumulating preclinical and clinical data that CA IX can contribute virtually to each step of cancer progression path via its enzyme activity and/or non-catalytic mechanisms. We also propose that targeting tumor cells that express CA IX may provide therapeutic benefits in various settings and combinations with both conventional and newly developed treatments.

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.

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.

Cells of the growing tumor tissue are exposed to physiological stresses connected with insufficient delivery of oxygen (hypoxia) and accumulation of acidic products of the glycolytic metabolism (acidosis). Adaptation to these microenvironmental stresses involves remodeling of the cellular expression program mediated by hypoxia-inducible factor (HIF), which activates broad array of genes functionally involved in angiogenesis, anaerobic glycolysis, de-adhesion, invasion etc. This leads to increased aggressiveness of tumors, metastatic spread and poor response to therapy. Genes coding for transmembrane carbonic anhydrase (CA) isoforms IX and XII are induced in response to low oxygen as a part of the hypoxic transcriptome. Moreover, CA IX is a direct target of HIF and serves as a surrogate marker of hypoxia and prognostic indicator. Its expression is strongly linked to different types of tumors with the HIF pathway activated due to genetic defect or physiological hypoxia. CA IX (and possibly also CA XII) is participates in pH regulation, which is important for survival of hypoxic cells. Both enzymes are therefore promising therapeutic molecules targetable by inhibitors of CA activity. Some of these sulfonamide compounds and their derivatives are capable to block CA-mediated pH regulation in hypoxia. This review summarizes research data related to distribution, regulation and functional aspects of CA IX and CA XII, and describes emerging possibilities for clinical exploitation of CA inhibitors as imaging tools and anticancer drugs.