Tumor-associated Carbonic Anhydrase 9 Spatially Coordinates Intracellular pH in Three-dimensional Multicellular Growths

Abstract

CA9 is a membrane-tethered, carbonic anhydrase (CA) enzyme, expressed mainly at the external surface of cells, that catalyzes reversible CO 2 hydration. Expression is greatly enhanced in many tumors, particularly in aggressive carcinomas. The functional role of CA9 in tumors is not well established. Here we show that CA9, when expressed heterologously in cultured spheroids (0.5-mm diameter, ϳ25,000 cells) of RT112 cells (derived from bladder carcinoma), induces a nearuniform intracellular pH (pH i ) throughout the structure. Dynamic pH i changes during displacements of superfusate CO 2 concentration are also spatially coincident (within 2 s). In contrast, spheroids of wild-type RT112 cells lacking CA9 exhibit an acidic core (ϳ0.25 pH i reduction) and significant time delays (ϳ9 s) for pH i changes in core versus peripheral regions. pH i non-uniformity also occurs in CA9-expressing spheroids after selective pharmacological inhibition of the enzyme. In isolated RT112 cells, pH i regulation is unaffected by CA9 expression. The influence of CA9 on pH i is thus only evident in multicellular tissue. Diffusion-reaction modeling indicates that CA9 coordinates pH i spatially by facilitating CO 2 diffusion in the unstirred extracellular space of the spheroid. We suggest that pH i coordination may favor survival and growth of a tumor. By disrupting spatial pH i control, inhibition of CA9 activity may offer a novel strategy for the clinical treatment of CA9-associated tumors.Intracellular H ϩ ions are powerful modulators of physiological and biochemical processes, including cell growth, division, and development (1-3). Many metabolic reactions within the cell generate H ϩ ions as end-products (4). Because of their high chemical reactivity, particularly with intracellular proteins, these ions must be removed if normal cell function is to continue. Tumors display a notably high rate of H ϩ ion production, partly because their growth demands a ready supply of energy, but also because much of the metabolism that supports this is anaerobic (5). Energy production via extramitochondrial routes is associated with a larger metabolic flux of acid per ATP molecule (4). Indeed, growing tumors are typically hypoxic, resulting in stabilization of hypoxia-inducible factor 1␣, which upregulates a variety of proteins, including enzymes that control glycolytic lactic acid production (6). If intracellular H ϩ ions accumulate, intracellular pH (pH i ) 3 falls and cellular activity becomes compromised. One solution is for cells to express plasmalemmal ion transport proteins that extrude excess H ϩ equivalents (7). Extrusion is achieved either by expelling H ϩ ions directly (e.g. via Na ϩ -H ϩ exchangers, H ϩ -lactate co transporters, and H ϩ -ATPases), or by importing HCO 3 Ϫ anions (e.g. via Na ϩ -dependent HCO 3 Ϫ transporters), to neutralize intracellular H ϩ ions. The pH i of tumor cells, like that of most other eukaryotic cells, appears to be relatively alkaline (pH i ϳ 7.2), indicating that they possess an efficient system for eliminating...

Functional screening identifies MCT4 as a key regulator of breast cancer cell metabolism and survival

“…The regulation of pH(i) by carbonic anhydrase IX (CA9) can promote the ability of tumour cells to grow as three-dimensional (3D) spheroids [26]. These culture conditions recapitulate the oxygen, nutrient and pH gradients experienced by cancer cells within tumours [27].…”

Interfering with pH regulation in tumours as a therapeutic strategy

“…Oncogenic metabolism also generates an excess of protons and carbon dioxide, which are kept in equilibrium with carbonic acid by the enzyme carbonic anhydrase [3][4][5][6][7][8][9] . Thus, increased glucose metabolism in tumour cells leads to enhanced acidification of the extracellular milieu, which is frequently accompanied by various levels of hypoxia.…”

“…They eliminate acidic catabolites by ion transporters and pumps to preserve a slightly alkaline intracellular pH (pH i ), which is optimal for cell proliferation and tumour survival [3][4][5][6][7][8][9][10] . Acid export leads to a reduction in the extracellular pH (pH e ) to values as low as 6.0 (the usual pH e in tumours is in the range of 6.5-7.0) 3 , which is a salient feature of the tumour microenvironment [3][4][5][6][7][8][9] . As well as triggering the overexpression of many proteins involved in glucose metabolism -such as the glucose transporter GLUT1 (also known as SLC2A1) and pH-regulating proteins such as carbonic anhydrase 9 (CA9) 3,4,10 -hypoxia also constitutes a detrimental feature for radiotherapy, as oxygen is needed to oxidize the radiation-induced DNA free radicals that subsequently lead to tumour cell death 4 .…”

“…In tumour cells, these processes are even more complex owing to the internal compartment being slightly more alkaline (pH 7.4 or more) and the external compartment being more acidic than in normal cells [4][5][6][7][8][9] . A variation in the pH i /pH e ratio as low as 0.1 pH units may disrupt important biochemical and/or biological processes such as ATP synthesis, enzyme function and the proliferation, migration, invasion and metastasis of tumour cells 5 ; consequently, a tight regulation of these processes has evolved [5][6][7][8][9] . Changes in the pH i as low as 0.1-0.2 pH units also trigger mechanisms of alternative splicing of extracellular matrix components that generate different isoforms of tenascin and fibronectin, which are typical of tumour cells and not normal cells 11,12 .…”

“…Several sophisticated molecular mechanisms are responsible for maintaining the alkaline pH i and the acidic pH e in tumour cells [5][6][7][8][9][10] . These include proteins that import weak bases (such as the HCO 3 -ion) into the cells and proteins that export the weak acids generated during metabolism -such as carbon dioxide, carbonic acid or lactic acid -out of the cells 4 .…”