Hypoxia Inducible Carbonic Anhydrase IX,Marker of Tumour: Hypoxia,Survival Pathway and Therapy Target

Over 50 genes are inducible by hypoxia, via hypoxia inducible factor 1a (HIF-1a). Carbonic anhydrase IX (CAIX) is one of the most inducible and most uniformly induced genes and because of its stability and membrane location provides a reliable histochemical marker of hypoxia. Recent studies have shown the importance of pH in cell death under hypoxia, thus mechanisms of pH regulation are likely to be vital pathways for survival. Carbonic anhydrases have a widespread role in normal tissues in regulating pH, with 14 isoforms described, so inhibition may have substantial normal tumour toxicity. Selective non-membrane permeable inhibitors are available and may synergise with chemotherapy agents more active in acid conditions. CAIX has a major role in regulating hydrogen ion (H+) flux and blockade of CAIX results in increased cell death under hypoxia, indicating that it is one mechanism of hypoxic adaptation. As it is commonly expressed in tumours with the worst prognosis it is a potential target for therapy.     

Role of hypoxia and EGF on expression, activity, localization and phosphorylation of carbonic anhydrase IX in MDA-MB-231 breast cancer cells

Carbonic anhydrase IX (CAIX) is a zinc metalloenzyme that catalyzes the reversible hydration of CO₂. CAIX is overexpressed in many types of cancer, including breast cancer, but is most frequently absent in corresponding normal tissues. CAIX expression is strongly induced by hypoxia and is significantly associated with tumor grade and poor survival. Herein, we show that hypoxia induces a significant increase in CAIX protein in MDA-MB-231 breast cancer cells. Using a unique mass spectrophotometric assay, we demonstrate that CAIX activity in plasma membranes isolated from MDA-MB-231 is correlated with CAIX content. We also show that CAIX exists predominantly as a dimeric, high-mannose N-linked glycoprotein. While there is some evidence that the dimeric form resides specifically in lipid rafts, our data do not support this hypothesis. EGF, alone, did not affect the distribution of CAIX into lipid rafts. However, acute EGF treatment in the context of hypoxia increased the amount of CAIX in lipid rafts by about 5-fold. EGF did not stimulate tyrosine phosphorylation of CAIX, although EGFR and down-stream signaling pathways were activated by EGF. Interestingly, hypoxia activated Akt independent of EGF action. Together, these data demonstrate that the active form of CAIX in the MDA-MB-231 breast cancer cell line is dimeric but that neither lipid raft localization nor phosphorylation are likely required for its dimerization or activity.     

CAIX furthers tumour progression in the hypoxic tumour microenvironment of esophageal carcinoma and is a possible therapeutic target

The hypoxic tumour microenvironment of solid tumours represents an important starting point for modulating progression and metastatic spread. Carbonic anhydrase IX (CAIX) is a known HIF-1α-dependent key player in maintaining cell pH conditions under hypoxia. We show that CAIX is strongly expressed in esophageal carcinoma tissues. We hypothesize that a moderate CAIX expression facilitates metastases and thereby worsens prognosis. Selective inhibition of CAIX by specific CAIX inhibitors and a CAIX knockdown effectively inhibit proliferation and migration in vitro. In the orthotopic esophageal carcinoma model, the humanized HER2 antibody trastuzumab down-regulates CAIX, possibly through CAIX’s linkage with HER2 in the hypoxic microenvironment. Our results show CAIX to be an essential part of the tumour microenvironment and a possible master regulator of tumour progression. This makes CAIX a highly effective and feasible therapeutic target for selective cancer treatment.     

Tumor Microenvironmental Changes Induced by the Sulfamate Carbonic Anhydrase IX Inhibitor S4 in a Laryngeal Tumor Model

Background and Purpose

Carbonic anhydrase IX (CAIX) plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3′(3″,5″-dimethylphenyl)-ureido)phenyl sulfamate (S4) on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding.

Methods

SCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole) and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR.

Results

CAIX ectodomain shedding decreased after treatment with S4 (p<0.01). S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole) and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed.

Conclusion

As the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth.     

Unique Biological Properties of Catalytic Domain Directed Human Anti-CAIX Antibodies Discovered through Phage-Display Technology

Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.     

Targeting ion transport in cancer

The metabolism of cancer cells differs substantially from normal cells, including ion transport. Although this phenomenon has been long recognized, ion transporters have not been viewed as suitable therapeutic targets. However, the acidic pH values present in tumours which are well outside of normal limits are now becoming recognized as an important therapeutic target. Carbonic anhydrase IX (CAIX) is fundamental to tumour pH regulation. CAIX is commonly expressed in cancer, but lowly expressed in normal tissues and that presents an attractive target. Here, we discuss the possibilities of exploiting the acidic, hypoxic tumour environment as possible target for therapy. Additionally, clinical experience with CAIX targeting in cancer patients is discussed.     

Design,synthesis & biological evaluation of ferulic acid-based small molecule inhibitors against tumor-associated carbonic anhydrase IX

Carbonic anhydrase IX (CAIX) is an emerging drug target for hypoxia associated cancers. To identify potent and selective inhibitors of CAIX, a small library of ferulic acid (FA) derivatives bearing triazole moiety has been designed, synthesized and evaluated against different human CA isoforms (CAII, CAVA & CAIX). Though most of the compounds showed CAIX inhibition in the micromolar range, compound 7i selectively inhibits CAIX in the nanomolar range (IC₅₀ = 24 nM). In silico analysis revealed binding of 7i with the catalytically important amino acid residues of CAIX. Further, cell-based studies indicate that 7i inhibits the activity of CAIX, decreases the epithelial to mesenchymal transitions, induces apoptosis, inhibits cell migration and colonization potential of cancer cells. Taken together, these results emphasized the use of 7i as a prospective pharmacological lead molecule in CAIX targeted anticancer therapeutics.     

Targeting carbonic anhydrase IX and XII isoforms with small molecule inhibitors and monoclonal antibodies

Carbonic anhydrases IX and CAXII (CAIX/CAXII) are transmembrane zinc metalloproteins that catalyze a very basic but crucial physiological reaction: the conversion of carbon dioxide into bicarbonate with a release of the proton. CA, especially CAIX and CAXII isoforms gained the attention of many researchers interested in anticancer drug design due to pivotal functions of enzymes in the cancer cell metastasis and response to hypoxia, and their expression restricted to malignant cells. This offers an opportunity to develop new targeted therapies with fewer side effects. Continuous efforts led to the discovery of a series of diverse compounds with the most abundant sulphonamide derivatives. Here we review current knowledge considering small molecule and antibody-based targeting of CAIX/CAXII in cancer.     

Catalysis and pH control by membrane-associated carbonic anhydrase IX in MDA-MB-231 breast cancer cells

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 (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.     

HIF-1alpha, HIF-2alpha and VHL mRNA expression in different cell lines during hypoxia

Hypoxia inducible factor-1alpha (HIF-1alpha) mRNA expression is significantly decreased under hypoxia in different cell lines exposed directly to hypoxia or treated with dimethyloxalylglycine which mimics hypoxic effects under normoxic conditions. However, the decreased expression of HIF-1alpha mRNA is accompanied by an increase of HIF-1alpha protein (pHIF-1alpha) level as well as by overexpression of known HIF-dependent genes (VEGF, Glut1, PFKFB-3 and PFKFB-4) under hypoxic conditions or with the use of dimethyloxalylglycine. Expression of HIF-1alpha mRNA also depends on iron because desferrioxamine and cobalt chloride produce similar to hypoxia effects on the levels of this mRNA. It was shown that HIF-1alpha mRNA expression did not change significantly in some cell lines (SKBR3, MDA-MB468 and BT549) under hypoxia. However, in these cell lines hypoxia decreases expression of HIF-2alpha mRNA, another member of HIF-alpha gene family, as a result of cell specific regulation of HIF-alpha genes under hypoxia. Moreover, hypoxia slightly induces expression of PFKFB-4 mRNA in SKBR3, MDA-MB468 and BT549 as compared to other cell lines where this effect of hypoxia was much stronger and adaptation to hypoxia is controlled by HIF-1alpha. Hypoxia slightly reduces expression of tumor suppressor VHL which targets HIF-1alpha for ubiquitination. Thus, our results clearly demonstrated down regulation of HIF-1alpha or HIF-2alpha in different cell lines by hypoxia.     

Antibody-specific detection of CAIX in breast and prostate cancers

Carbonic anhydrase IX (CAIX) is frequently expressed in human tumors and serves as a marker for hypoxia. Further, CAIX expression is considered a predictor of poor survival in many, but not all, cancer types. Herein, we compare the specificity of two CAIX antibodies: the M75, monoclonal antibody which recognizes an epitope in the N-terminus and a commercially available polyclonal antibody generated against a C-terminal peptide (NB100-417). Western blot analysis of multiple breast cell lines revealed that the polyclonal antibody detected both membrane-bound and soluble proteins. The M75 antibody recognized only the membrane-bound species, which is presumed to be CAIX. These data were confirmed in an aggressive prostate cell line. We further compared these antibodies in prostate tumors by immunohistochemistry. Staining with NB100 was comparable to that of the M75 antibody, but only at high dilution. Otherwise, cytoplasmic staining was also noted. Two-dimensional gel electrophoresis followed by mass spectrometric analysis revealed that the cytoplasmic protein detected by NB100 is β-tubulin. This cross-reactivity could lead to false-positives for CAIX expression in samples where cytosolic proteins are present.     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Virtual screening,molecular dynamics,and binding free energy calculations on human carbonic anhydrase IX catalytic domain for deciphering potential leads

Carbonic anhydrase IX is a tumor-associated membrane-bound metallo-enzyme which catalyzes the reversible hydration of carbon dioxide (CO₂) to bicarbonate (HCO₃^?) and proton (H⁺) ions. It is a hypoxia-inducible enzyme and plays a critical role in tumor pH homeostasis favoring tumor cell invasiveness and drug resistance. Over expression of CAIX is documented in cancers of breast, lung, kidney, colon/rectum, etc. Chemical inhibition of CAIX activity has proven to be an effective therapeutic modality towards targeting cancer. Hence, in this study, we intend to identify potential molecules from NCI (National Cancer Institute) and Maybridge databases implementing high-throughput virtual screening. CAIX co-crystallized with acetazolamide (a known inhibitor of CAIX) (PDB ID: 3IAI) was used for reference-guided docking protocol. The potential inhibitors among the coupled data sets were finalized based on Glide docking score, Prime/MMGBSA scoring, significant intermolecular interactions, ADMET (absorption, distribution, metabolism and excretion, toxicity) prediction and stability of complex formation, molecular dynamics simulation, and comparative analysis. By this study, we propose NSC_93618, NSC_170253, NSC_93618, JFD03677, SEW06488, and BTB09372 to be highly significant, as all these compounds were found to qualify as potential leads surpassing all the stringent filtering process. However, NSC_93618 was found to be the most potential, as it featured with higher complex stability with strong bonded interactions, binding affinity synonymous to acetazolamide. Hence, these proposed compounds shall prove to be effective in targeting CAIX towards modulating carcinogenesis.     

Hypoxia induces the expression of the pro-apoptotic gene BNIP3

It has been shown that oxygen deprivation results in apoptotic cell death, and that hypoxia inducible factor 1 (HIF1) and the tumor suppressor p53 play key roles in this process. However, the molecular mechanism through which hypoxia and HIF1 induce apoptosis is not clear. Here we show that the expression of pro-apoptotic gene BNIP3 is dramatically induced by hypoxia in various cell types, including primary rat neonatal cardiomyocytes. Overexpression of HIF1alpha, but not p53, induces the expression of BNIP3. Overexpression of BNIP3 leads to a rather unusual type of apoptosis, as no cytochrome c leakage from mitochondria was detected and inhibitors of caspases were unable to prevent cell death. Taken together, these data suggest that HIF1-dependent induction of BNIP3 may play a significant role during hypoxia-induced cell death.