Pawel Swietach

Regulation of tumor pH and the role of carbonic anhydrase 9

The high metabolic rate required for tumor growth often leads to hypoxia in poorly-perfused regions. Hypoxia activates a complex gene expression program, mediated by hypoxia inducible factor 1 (HIF1α). One of the consequences of HIF1α activation is up-regulation of glycolysis and hence the production of lactic acid. In addition to the lactic acid-output, intracellular titration of acid with bicarbonate and the engagement of the pentose phosphate shunt release CO2 from cells. Expression of the enzyme carbonic anhydrase 9 on the tumor cell surface catalyses the extracellular trapping of acid by hydrating cell-generated CO2 into

The Role of Carbonic Anhydrase 9 in Regulating Extracellular and Intracellular pH in Three-dimensional Tumor Cell Growths

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

and H+. These mechanisms contribute towards an acidic extracellular milieu favoring tumor growth, invasion and development. The lactic acid released by tumor cells is further metabolized by the tumor stroma. Low extracellular pH may adversely affect the intracellular milieu, possibly triggering apoptosis. Therefore, primary and secondary active transporters operate in the tumor cell membrane to protect the cytosol from acidosis. We review mechanisms regulating tumor intracellular and extracellular pH, with a focus on carbonic anhydrase 9. We also review recent evidence that may suggest a role for CA9 in coordinating pHi among cells of large, unvascularized cell-clusters.

Carbonic Anhydrase IX Promotes Tumor Growth and Necrosis In Vivo and Inhibition Enhances Anti-VEGF Therapy

Intracellular pH (pHi) is an important modulator of cardiac excitation and contraction, and a potent trigger of electrical arrhythmia. This review outlines the intracellular and membrane mechanisms that control pHi in the cardiac myocyte. We consider the kinetic regulation of sarcolemmal H+, OH- and HCO3- transporters by pH, and by receptor-coupled intracellular signalling systems. We also consider how activity of these pHi effector proteins is coordinated spatially in the myocardium by intracellular mobile buffer shuttles, gap junctional channels and carbonic anhydrase enzymes. Finally, we review the impact of pHi regulatory proteins on intracellular Ca2+ signalling, and their participation in clinical disorders such as myocardial ischaemia, maladaptive hypertrophy and heart failure. Such multiple effects emphasise the fundamental role that pHi regulation plays in the heart.

The chemistry, physiology and pathology of pH in cancer

We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of ∼300 μm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pHi) and pH in the restricted extracellular spaces (pHe). With no CA9 expression, spheroids developed very low pHi (∼6.3) and reduced pHe (∼6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pHi = ∼6.6), whereas extracellular acidity was enhanced (pHe = ∼6.6), so that radial pHi gradients were smaller and radial pHe gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pHe indicates the enzyme is facilitating CO2 excretion from cells (by converting vented CO2 to extracellular H+), rather than facilitating membrane H+ transport (such as H+ associated with metabolically generated lactic acid). This latter process requires titration of exported H+ ions with extracellular HCO3−, which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pHe will depend on the cellular CO2/lactic acid emission ratio (set by local oxygenation and membrane HCO3− uptake). Our results suggest that CO2-producing tumors may express CA9 to facilitate CO2 excretion, thus raising pHi and reducing pHe, which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.

Measuring intracellular pH in the heart using hyperpolarized carbon dioxide and bicarbonate: a 13C and 31P magnetic resonance spectroscopy study.

In this review, we discuss the role of the tumour-associated carbonic anhydrase isoform IX (CAIX) in the context of pH regulation. We summarise recent experimental findings on the effect of CAIX on cell growth and survival, and present a diffusion-reaction model to help in the assessment of CAIX function under physiological conditions. CAIX emerges as an important facilitator of acid diffusion and acid transport, helping to overcome large cell-to-capillary distances that are characteristic of solid tumours. The source of substrate for CAIX catalysis is likely to be CO2, generated by adequately oxygenated mitochondria or from the titration of metabolic acids with HCO−3 taken up from the extracellular milieu. The relative importance of these pathways will depend on oxygen and metabolite availability, the spatiotemporal patterns of the cells exposure to hypoxia and on the regulation of metabolism by genes. This is now an important avenue for further investigation. The importance of CAIX in regulating tumour pH highlights the protein as a potential target for cancer therapy.

S0859, an N-cyanosulphonamide inhibitor of sodium-bicarbonate cotransport in the heart

CA9 is a membrane-tethered, carbonic anhydrase (CA) enzyme, expressed mainly at the external surface of cells, that catalyzes reversible CO2 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, ∼25,000 cells) of RT112 cells (derived from bladder carcinoma), induces a near-uniform intracellular pH (pHi) throughout the structure. Dynamic pHi changes during displacements of superfusate CO2 concentration are also spatially coincident (within 2 s). In contrast, spheroids of wild-type RT112 cells lacking CA9 exhibit an acidic core (∼0.25 pHi reduction) and significant time delays (∼9 s) for pHi changes in core versus peripheral regions. pHi non-uniformity also occurs in CA9-expressing spheroids after selective pharmacological inhibition of the enzyme. In isolated RT112 cells, pHi regulation is unaffected by CA9 expression. The influence of CA9 on pHi is thus only evident in multicellular tissue. Diffusion-reaction modeling indicates that CA9 coordinates pHi spatially by facilitating CO2 diffusion in the unstirred extracellular space of the spheroid. We suggest that pHi coordination may favor survival and growth of a tumor. By disrupting spatial pHi control, inhibition of CA9 activity may offer a novel strategy for the clinical treatment of CA9-associated tumors.

Functional diversity of electrogenic Na+–HCO3− cotransport in ventricular myocytes from rat, rabbit and guinea pig

Purpose: Bevacizumab, an anti-VEGFA antibody, inhibits the developing vasculature of tumors, but resistance is common. Antiangiogenic therapy induces hypoxia and we observed increased expression of hypoxia-regulated genes, including carbonic anhydrase IX (CAIX), in response to bevacizumab treatment in xenografts. CAIX expression correlates with poor prognosis in most tumor types and with worse outcome in bevacizumab-treated patients with metastatic colorectal cancer, malignant astrocytoma, and recurrent malignant glioma. Experimental Design: We knocked down CAIX expression by short hairpin RNA in a colon cancer (HT29) and a glioblastoma (U87) cell line which have high hypoxic induction of CAIX and overexpressed CAIX in HCT116 cells which has low CAIX. We investigated the effect on growth rate in three-dimensional (3D) culture and in vivo, and examined the effect of CAIX knockdown in combination with bevacizumab. Results: CAIX expression was associated with increased growth rate in spheroids and in vivo. Surprisingly, CAIX expression was associated with increased necrosis and apoptosis in vivo and in vitro. We found that acidity inhibits CAIX activity over the pH range found in tumors (pK = 6.84), and this may be the mechanism whereby excess acid self-limits the build-up of extracellular acid. Expression of another hypoxia inducible CA isoform, CAXII, was upregulated in 3D but not two-dimensional culture in response to CAIX knockdown. CAIX knockdown enhanced the effect of bevacizumab treatment, reducing tumor growth rate in vivo. Conclusion: This work provides evidence that inhibition of the hypoxic adaptation to antiangiogenic therapy enhances bevacizumab treatment and highlights the value of developing small molecules or antibodies which inhibit CAIX for combination therapy. Clin Cancer Res; 18(11); 3100–11. ©2012 AACR.