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 (HIF1alpha). One of the consequences of HIF1alpha 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 CO(2) from cells. Expression of the enzyme carbonic anhydrase 9 on the tumor cell surface catalyses the extracellular trapping of acid by hydrating cell-generated CO(2) into [see text] 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 pH(i) among cells of large, unvascularized cell-clusters.

Cell survival is conditional on the maintenance of a favourable acid–base balance (pH). Owing to intensive respiratory CO 2 and lactic acid production, cancer cells are exposed continuously to large acid–base fluxes, which would disturb pH if uncorrected. The large cellular reservoir of H + -binding sites can buffer pH changes but, on its own, is inadequate to regulate intracellular pH. To stabilize intracellular pH at a favourable level, cells control trans-membrane traffic of H + -ions (or their chemical equivalents, e.g. ) using specialized transporter proteins sensitive to pH. In poorly perfused tumours, additional diffusion-reaction mechanisms, involving carbonic anhydrase (CA) enzymes, fine-tune control extracellular pH. The ability of H + -ions to change the ionization state of proteins underlies the exquisite pH sensitivity of cellular behaviour, including key processes in cancer formation and metastasis (proliferation, cell cycle, transformation, migration). Elevated metabolism, weakened cell-to-capillary diffusive coupling, and adaptations involving H + /H + -equivalent transporters and extracellular-facing CAs give cancer cells the means to manipulate micro-environmental acidity, a cancer hallmark. Through genetic instability, the cellular apparatus for regulating and sensing pH is able to adapt to extracellular acidity, driving disease progression. The therapeutic potential of disturbing this sequence by targeting H + /H + -equivalent transporters, buffering or CAs is being investigated, using monoclonal antibodies and small-molecule inhibitors.

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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 ϳ300 m) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH i ) and pH in the restricted extracellular spaces (pH e ). With no CA9 expression, spheroids developed very low pH i (ϳ6.3) and reduced pH e (ϳ6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pH i ‫؍‬ ϳ6.6), whereas extracellular acidity was enhanced (pH e ‫؍‬ ϳ6.6), so that radial pH i gradients were smaller and radial pH e gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pH e indicates the enzyme is facilitating CO 2 excretion from cells (by converting vented CO 2 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 HCO 3 ؊ , which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pH e will depend on the cellular CO 2 /lactic acid emission ratio (set by local oxygenation and membrane HCO 3 ؊ uptake). Our results suggest that CO 2 -producing tumors may express CA9 to facilitate CO 2 excretion, thus raising pH i and reducing pH e , which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.

Purpose Bevacizumab, an anti-VEGFA antibody, inhibits the developing vasculature of tumours, 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 tumour types and with worse outcome in Bevacizumab-treated metastatic colorectal cancer patients, malignant astrocytoma and recurrent malignant glioma. Experimental Design We knocked-down CAIX expression by shRNA in a colon cancer (HT29) and a glioblastoma (U87) cell line which have high hypoxic-induction of CAIX, and over-expressed CAIX in HCT116 cells which has low CAIX. We investigated the effect on growth rate in 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 tumours (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 2D culture in response to CAIX knockdown. CAIX knockdown enhanced the effect of Bevacizumab treatment, reducing tumour growth rate in vivo. Conclusion This work provides evidence that inhibition of the hypoxic adaptation to anti-angiogenic therapy enhances Bevacizumab treatment and highlights the value of developing small molecules or antibodies which inhibit CAIX for combination therapy.