Catalysis and pH Control by Membrane-associated Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells

Abstract

Carbonic anhydrase IX (CAIX) is a membrane-bound, tumorrelated enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using 18 O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO 2 species at 25 mM and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.The carbonic anhydrase (CA) 2 family of proteins are metalloenzymes that catalyze the reversible hydration of carbon dioxide:This reaction is fundamental to a variety of biological processes, including respiration, renal tubular acidification, and fluid secretion (1). The mammalian CAs belong to a single gene family that is referred to as the ␣-CAs. There are 16 isozymes or CA-related proteins in this group that differ in their kinetic and inhibitory properties, cell and tissue distribution, and function (2). Two of the catalytically active members of this family, carbonic anhydrase IX (CAIX) and carbonic anhydrase XII, are specifically tumorrelated (3). Of these two, CAIX has garnered more interest because of its limited normal expression (4, 5) and its apparent role in cell proliferation and migration (6), cell adhesion (7), and pH control (8 -10). CAIX is a transmembrane glycoprotein whose catalytic domain is oriented toward the extracellular milieu (11). In breast cancer, CAIX is a marker for hypoxic regions of tumors (12), is associated with poor prognosis (13,14), and is linked to acidification of the tumor microenvironment (10) that favors cancer cell survival and resistance to chemotherapeutic agents (15). CAIX expression has also been linked to the basal B, triple-negative phenotype (16, 17), an aggressive breast cancer for which there are few treatment options. Thus, CAIX may represent a new and valuable target in the visualization, diagnosis, and treatment of ...

Phosphorylation of Carbonic Anhydrase IX Controls Its Ability to Mediate Extracellular Acidification in Hypoxic Tumors

“…In contrast, Tyr449 phosphorylation was shown to occur in kidney cancer cells and was linked to downstream activation of the PI3K/Akt pathway (18). However, this phenomenon was not reproduced in breast cancer cells (9), indicating that it is cell-type specific. Moreover, information on the pertinent kinase and whether phosphorylation of Tyr449 affects CA IX function is still missing.…”

“…24; and this work). (vi) Hypoxia increases the catalytic activity of CA IX and its ability to acidify pHe (7,9).…”

“…Transcription of the CA IX-encoding gene is strongly activated by the hypoxia-inducible HIF-1 transcription factor that binds an HRE element localized next to the transcription initiation site (6). Hypoxia and/or acidosis also induce the catalytic activity of CA IX thus providing an effective adaptive cellular response to microenvironmental stresses, including pH regulation and cell migration/invasion (7)(8)(9)(10). Because hypoxia is associated with poor prognosis and worse response to treatment, CA IX as its surrogate marker and functional component possesses clinical potential as a prognostic/predictive factor and target for anticancer treatment (11).…”

Prognostic relevance of carbonic anhydrase IX expression is distinct in various subtypes of breast cancer and its silencing suppresses self-renewal capacity of breast cancer cells

“…(2) it is located on the surface of cells [21,22]; (3) CAIXknockout mice show only a mild phenotype related to gastric pit cell hyperplasia and glandular atrophy [23]; and (4) its overexpression in cancers confers a survival advantage in hypoxia and facilitates cell migration and metastasis [16,[24][25][26]. Depletion of CAIX in cancer cell lines has been shown to result in attenuated growth of tumour xenografts and inhibition of spontaneous metastasis formation [16,26].…”

Inhibition of carbonic anhydrase activity modifies the toxicity of doxorubicin and melphalan in tumour cellsin vitro

“…Microelectrode and non-invasive measurements of tumour pH have revealed that human tumours have acidic extracellular pH (pH e ), but the intracellular pH (pH i ) remains neutral to alkali 24,25 . CA IX has been shown to be a key contributor to the acidification process in tumours with the use of membrane ghost preparations 26 and tumour cell lines in which CA IX activity was either silenced or overexpressed 27 . Tumour acidification is positively correlated with enhance proliferation, invasiveness and metastasis 27,28 .…”