scholarly journals Type 1 Sodium Calcium Exchanger Forms a Complex with Carbonic Anhydrase IX and Via Reverse Mode Activity Contributes to pH Control in Hypoxic Tumors

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1139 ◽  
Author(s):  
Veronika Liskova ◽  
Sona Hudecova ◽  
Lubomira Lencesova ◽  
Filippo Iuliano ◽  
Marta Sirova ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Ph Regulation ◽  
Ph Control ◽  
Carbonic Anhydrase Ix ◽  
Proximity Ligation Assay ◽  
Reverse Mode ◽  
Ca Ix

Hypoxia and acidosis are among the key microenvironmental factors that contribute to cancer progression. We have explored a possibility that the type 1Na+/Ca2+ exchanger (NCX1) is involved in pH control in hypoxic tumors. We focused on changes in intracellular pH, co-localization of NCX1, carbonic anhydrase IX (CA IX), and sodium proton exchanger type 1 (NHE1) by proximity ligation assay, immunoprecipitation, spheroid formation assay and migration of cells due to treatment with KB-R7943, a selective inhibitor of the reverse-mode NCX1. In cancer cells exposed to hypoxia, reverse-mode NCX1 forms a membrane complex primarily with CA IX and also with NHE1. NCX1/CA IX/NHE1 assembly operates as a metabolon with a potent ability to extrude protons to the extracellular space and thereby facilitate acidosis. KB-R7943 prevents formation of this metabolon and reduces cell migration. Thus, we have shown that in hypoxic cancer cells, NCX1 operates in a reverse mode and participates in pH regulation in hypoxic tumors via cooperation with CAIX and NHE1.

scholarly journals A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells

2019 ◽  
Vol 476 (10) ◽  
pp. 1497-1513 ◽  
Author(s):  
Mam Y. Mboge ◽  
Zhijuan Chen ◽  
Daniel Khokhar ◽  
Alyssa Wolff ◽  
Lingbao Ai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Expression Patterns ◽  
Extracellular Ph ◽  
Carbonic Anhydrase Ix ◽  
Monocarboxylate Transporter ◽  
Proton Efflux ◽  
Ca Ix

AbstractThe most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+ shuttle system in CA IX to facilitate proton efflux through MCT4.

scholarly journals A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells

2018 ◽  
Author(s):  
Mam Y. Mboge ◽  
Zhijuan Chen ◽  
Daniel Khokhar ◽  
Alyssa Wolff ◽  
Lingbao Ai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Expression Patterns ◽  
Extracellular Ph ◽  
Carbonic Anhydrase Ix ◽  
Monocarboxylate Transporter ◽  
Proton Efflux ◽  
Ca Ix

ABSTRACTThe most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX cooperates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple negative breast cancer (TNBC), appears to coordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+shuttle system in CA IX to facilitate proton efflux through MCT4.

Novel Humanized Monoclonal Antibodies for Targeting Hypoxic Human Tumors via Two Distinct Extracellular Domains of Carbonic Anhydrase IX

2021 ◽  
Author(s):  
Miriam Zatovicova ◽  
Ivana Kajanova ◽  
Monika Barathova ◽  
Martina Takacova ◽  
Martina Labudova ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Cancer Progression ◽  
Standard Therapy ◽  
Catalytic Domain ◽  
Conformational Epitope ◽  
Carbonic Anhydrase Ix ◽  
Acidic Environment ◽  
Primary Tumors ◽  
Ca Ix

Abstract Background Hypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy. Methods Parental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma we obtained 25 clones. Each clone was tested for ADCC and CDC activity, affinity, extracellular pH measurement, multicellular aggregation analysis and real-time monitoring of invasion with xCELLigence system. ResultsBoth CA9hu-1 and CA9hu-2 are IgG1 antibodies and they were both examined in vivo. Here we describe anti-CAIX antibodies that can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME. CA9hu-1 is directed at the conformational epitope of the catalytic domain, while CA9hu-2 targets the sequential epitope of the proteo-glycan domain. They are both able to induce an immune response, have high affinity, as well as ADCC and CDC activity. While the first one internalizes after binding to the antigen, the second one is able to reduce metastases formation. More importantly, they have both proved the ability to block the acidification of the extracellular environment. ConclusionCA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.

scholarly journals A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX

2015 ◽  
Vol 71 (10) ◽  
pp. 1352-1358 ◽  
Author(s):  
Melissa A. Pinard ◽  
Mayank Aggarwal ◽  
Brian P. Mahon ◽  
Chingkuang Tu ◽  
Robert McKenna
Keyword(s):  
Carbonic Anhydrase ◽  
Binding Site ◽  
Cancer Progression ◽  
Active Site ◽  
Ph Regulation ◽  
Transmembrane Protein ◽  
Specific Inhibitor ◽  
Site Directed Mutagenesis ◽  
Structure Based Drug Design ◽  
Ca Ix

Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3 −, thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an R cryst of 18.0% and an R free of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.

scholarly journals Binding of Carbonic Anhydrase IX to 45S rDNA Genes Is Prevented by Exportin-1 in Hypoxic Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Emanuele Sasso ◽  
Monica Vitale ◽  
Francesca Monteleone ◽  
Francesca Ludovica Boffo ◽  
Margherita Santoriello ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Nuclear Export ◽  
Ph Regulation ◽  
Surrogate Marker ◽  
Carbonic Anhydrase Ix ◽  
Gene Promoters ◽  
45S Rdna ◽  
Rdna Genes ◽  
Rdna Gene ◽  
Ca Ix

Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45S rDNA gene promoters.

scholarly journals Carbonic Anhydrase IX—Mouse versus Human

2019 ◽  
Vol 21 (1) ◽  
pp. 246
Author(s):  
Martina Takacova ◽  
Monika Barathova ◽  
Miriam Zatovicova ◽  
Tereza Golias ◽  
Ivana Kajanova ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Carbonic Anhydrase ◽  
Functional Properties ◽  
Ph Regulation ◽  
Tumor Hypoxia ◽  
Data Interpretation ◽  
Tissue Expression ◽  
Carbonic Anhydrase Ix ◽  
Ca Ix ◽  
Cell Adhesion And Migration

In contrast to human carbonic anhydrase IX (hCA IX) that has been extensively studied with respect to its molecular and functional properties as well as regulation and expression, the mouse ortholog has been investigated primarily in relation to tissue distribution and characterization of CA IX-deficient mice. Thus, no data describing transcriptional regulation and functional properties of the mouse CA IX (mCA IX) have been published so far, despite its evident potential as a biomarker/target in pre-clinical animal models of tumor hypoxia. Here, we investigated for the first time, the transcriptional regulation of the Car9 gene with a detailed description of its promoter. Moreover, we performed a functional analysis of the mCA IX protein focused on pH regulation, cell–cell adhesion, and migration. Finally, we revealed an absence of a soluble extracellular form of mCA IX and provided the first experimental evidence of mCA IX presence in exosomes. In conclusion, though the protein characteristics of hCA IX and mCA IX are highly similar, and the transcription of both genes is predominantly governed by hypoxia, some attributes of transcriptional regulation are specific for either human or mouse and as such, could result in different tissue expression and data interpretation.

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

2011 ◽  
Vol 286 (18) ◽  
pp. 15789-15796 ◽  
Author(s):  
Ying Li ◽  
Chingkuang Tu ◽  
Hai Wang ◽  
David N. Silverman ◽  
Susan C. Frost
Keyword(s):  
Breast Cancer ◽  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Total Concentration ◽  
Ph Control ◽  
Carbonic Anhydrase Activity ◽  
Carbonic Anhydrase Ix ◽  
Intact Cells ◽  
Membrane Inlet Mass Spectrometry

Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related 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 18O 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 CO2 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.

scholarly journals Catalysis of carbonic anhydrase IX and pH control in MDA‐MB‐231 breast cancer cells

2011 ◽  
Vol 25 (S1) ◽  
Author(s):  
Susan Cooke Frost ◽  
Ying Li ◽  
Chingkuang Tu ◽  
Hai Wang ◽  
David N Silverman
Keyword(s):  
Breast Cancer ◽  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Ph Control ◽  
Carbonic Anhydrase Ix

scholarly journals Distinct Expression Patterns of Carbonic Anhydrase IX in Clear Cell, Microcystic, and Angiomatous Meningiomas

2019 ◽  
Vol 78 (12) ◽  
pp. 1081-1088
Author(s):  
Rati Chkheidze ◽  
Patrick J Cimino ◽  
Kimmo J Hatanpaa ◽  
Charles L White ◽  
Manuel Ferreira ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Clear Cell ◽  
Expression Patterns ◽  
Carbonic Anhydrase Ix ◽  
World Health ◽  
Loss Of Function ◽  
Ca Ix ◽  
Hypoxia Marker ◽  
Other World ◽  
Health Organization

Abstract Clear cell, microcytic, and angiomatous meningiomas are 3 vasculature-rich variants with overlapping morphological features but different prognostic and treatment implications. Distinction between them is not always straightforward. We compared the expression patterns of the hypoxia marker carbonic anhydrase IX (CA-IX) in meningiomas with predominant clear cell (n = 15), microcystic (n = 9), or angiomatous (n = 11) morphologies, as well as 117 cases of other World Health Organization recognized histological meningioma variants. Immunostaining for SMARCE1 protein, whose loss-of-function has been associated with clear cell meningiomas, was performed on all clear cell meningiomas, and selected variants of meningiomas as controls. All clear cell meningiomas showed absence of CA-IX expression and loss of nuclear SMARCE1 expression. All microcystic and angiomatous meningiomas showed diffuse CA-IX immunoreactivity and retained nuclear SMARCE1 expression. In other meningioma variants, CA-IX was expressed in a hypoxia-restricted pattern and was highly associated with atypical features such as necrosis, small cell change, and focal clear cell change. In conclusion, CA-IX may serve as a useful diagnostic marker in differentiating clear cell, microcystic, and angiomatous meningiomas.

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