Targeting carbonic anhydrase IX activity and expression.
ABSTRACT: Metastatic tumors are often hypoxic exhibiting a decrease in extracellular pH (~6.5) due to a metabolic transition described by the Warburg Effect. This shift in tumor cell metabolism alters the tumor milieu inducing tumor cell proliferation, angiogenesis, cell motility, invasiveness, and often resistance to common anti-cancer treatments; hence hindering treatment of aggressive cancers. As a result, tumors exhibiting this phenotype are directly associated with poor prognosis and decreased survival rates in cancer patients. A key component to this tumor microenvironment is carbonic anhydrase IX (CA IX). Knockdown of CA IX expression or inhibition of its activity has been shown to reduce primary tumor growth, tumor proliferation, and also decrease tumor resistance to conventional anti-cancer therapies. As such several approaches have been taken to target CA IX in tumors via small-molecule, anti-body, and RNAi delivery systems. Here we will review recent developments that have exploited these approaches and provide our thoughts for future directions of CA IX targeting for the treatment of cancer.
Project description:Antibody-decorated liposomes can facilitate the precise delivery of chemotherapeutic drugs to the lung by targeting a recognition factor present on the surface of lung tumor cells. Carbonic anhydrase IX (CA IX) is an enzyme expressed on the surface of lung cancer cells with a restricted expression in normal lungs. Here, we explored the utility of anti-carbonic anhydrase IX (CA IX) antibody, conjugated to the surface of triptolide (TPL)-loaded liposomes (CA IX-TPL-Lips), to promote the therapeutic effects for lung cancer via pulmonary administration. It was found that the CA IX-TPL-Lips significantly improved the cellular uptake efficiency in both CA IX-positive human non-small cell lung cancer cells (A549) and A549 tumor spheroids, resulting in the efficient cell killing compared with free TPL and non-targeted TPL-Lips. In vivo, CA IX-Lips via pulmonary delivery showed specificity and a sustained release property resided up to 96?h in the lung, both of which improved the efficiency of TPL formulations in restraining tumor growth and significantly prolonged the lifespan of mice with orthotopic lung tumors. The results suggest that CA IX-decorated liposomes can potentially be used as an effective therapeutic strategy for lung cancer.
Project description:Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%-70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6-24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.
Project description:The microenvironment within a solid tumor is heterogeneous with regions being both acidic and hypoxic. As a result of this, cancer cells upregulate genes that allow survival in such environments. Some of these genes are pH regulatory factors, including carbonic anhydrase IX (CA IX) and in some cases XII (CA XII). CA IX helps to maintain normal cytoplasmic pH (pHi) while simultaneously contributing to the extracellular pH (pHe). CA XII is also thought to be responsible for stabilizing pHe at physiological conditions. Extracellular acidification of the tumor microenvironment promotes local invasion and metastasis while decreasing the effectiveness of adjuvant therapies, thus contributing to poor cancer clinical outcomes. In this review, we describe the properties of CA IX and CA XII that substantiate their potential use as anticancer targets. We also discuss the current status of CA isoform-selective inhibitor development and patents of CA IX/XII targeted inhibitors that show potential for treating aggressive tumors. Some of the recently published patents discussed include sulfonamide-based small molecule inhibitors including derivatives of boron cluster compounds; metal complexes of poly(carboxyl)amine-containing ligands; nitroi-midazole-, ureidosulfonamide-, and coumarin-based compounds; as well as G250 and A610 monoclonal antibodies for cancer treatment.
Project description:Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (?-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1? and CA IX and/or altered CA IX function.The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1? and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively.Carnosine increased the expression levels of HIF-1? and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine.Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CA IX.
Project description:Proof-of-principle studies in ovarian, lung, and brain cancer patients have shown that fluorescence-guided surgery can enable removal of otherwise undetectable malignant lesions, decrease the number of cancer-positive margins, and permit identification of disease-containing lymph nodes that would have normally evaded resection. Unfortunately, the current arsenal of tumor-targeted fluorescent dyes does not permit identification of all cancers, raising the need to design new tumor-specific fluorescent dyes to illuminate the currently undetectable cancers. In an effort to design a more universal fluorescent cancer imaging agent, we have undertaken to synthesize a fluorophore that could label all hypoxic regions of tumors. We report here the synthesis, in vitro binding, and in vivo imaging of a near-infrared (NIR) fluorescent dye that is targeted to carbonic anhydrase IX (CA IX), i.e., a widely accepted marker of hypoxic tissues. The low molecular weight NIR probe, named Hypoxyfluor, is shown to bind CA IX with high affinity and accumulate rapidly and selectively in CA IX positive tumors. Because nearly all human cancers contain hypoxic regions that express CA IX abundantly, this NIR probe should facilitate surgical resection of a wide variety of solid tumors.
Project description:Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.
Project description:Carbonic anhydrase IX (CA IX) expression is important for the regulation of pH in hypoxic tumors and is emerging as a therapeutic target for the treatment of various cancers. Recent studies have demonstrated the selectivity of sucrose, saccharin, and acesulfame potassium for CA IX over other CA isoforms. Reported here is the X-ray crystal structure of CA IX-mimic in complex with sucralose determined to ?1.5 Å resolution. Furthermore, this structure is compared to the aforementioned sweetener/carbohydrate structural studies in order to determine active site properties of CA IX that promote selective binding. This structural analysis provides a further understanding of CA IX isoform specific inhibition to facilitate the design of new inhibitors and anticancer drugs.
Project description:Extracellular acidification, a mandatory feature of several malignancies, has been mainly correlated with metabolic reprogramming of tumor cells toward Warburg metabolism, as well as to the expression of carbonic anydrases or proton pumps by malignant tumor cells. We report herein that for aggressive prostate carcinoma, acknowledged to be reprogrammed toward an anabolic phenotype and to upload lactate to drive proliferation, extracellular acidification is mainly mediated by stromal cells engaged in a molecular cross-talk circuitry with cancer cells. Indeed, cancer-associated fibroblasts, upon their activation by cancer delivered soluble factors, rapidly express carbonic anhydrase IX (CA IX). While expression of CAIX in cancer cells has already been correlated with poor prognosis in various human tumors, the novelty of our findings is the upregulation of CAIX in stromal cells upon activation. The de novo expression of CA IX, which is not addicted to hypoxic conditions, is driven by redox-based stabilization of hypoxia-inducible factor-1. Extracellular acidification due to carbonic anhydrase IX is mandatory to elicit activation of stromal fibroblasts delivered metalloprotease-2 and -9, driving in cancer cells the epithelial-mesenchymal transition epigenetic program, a key event associated with increased motility, survival and stemness. Both genetic silencing and pharmacological inhibition of CA IX (with sulfonamide/sulfamides potent inhibitors) or metalloprotease-9 are sufficient to impede epithelial-mesenchymal transition and invasiveness of prostate cancer cells induced by contact with cancer-associated fibroblasts. We also confirmed in vivo the upstream hierarchical role of stromal CA IX to drive successful metastatic spread of prostate carcinoma cells. These data include stromal cells, as cancer-associated fibroblasts as ideal targets for carbonic anhydrase IX-directed anticancer therapies.
Project description:Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membrane-bound CA isoform IX (CA IX). The role of CA IX in the context of breast cancer is to regulate the pH of the tumor microenvironment. In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The ability of these sulfonamides to block CA IX activity in breast cancer cells is less effective than their ability to block activity of the recombinant protein (by one to two orders of magnitude depending on the inhibitor). The same is true for CA XII activity but now they are two to three orders of magnitude less effective. Thus, there is significantly greater specificity for CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this again occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII activity in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells where CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth potentiation.
Project description:Carbonic anhydrase IX (CA IX) is an extracellular transmembrane homodimeric zinc metalloenzyme that has been validated as a prognostic marker and therapeutic target for several types of aggressive cancers. CA IX shares a close homology with other CA isoforms, making the design of CA IX isoform selective inhibitors challenging. In this paper, we describe the development of a new class of CA IX inhibitors that comprise a sulfamate as the zinc binding group, a variable linker, and a carbohydrate "tail" moiety. Seven compounds inhibited CA IX with low nM Ki values of 1-2 nM and also exhibited permeability profiles to preferentially target the binding of extracellular CA IX over cytosolic CAs. The crystal structures of two of these compounds in complex with a CA IX-mimic (a variant of CA II, with active site residues that mimic CA IX) and one compound in complex with CA II have been determined to 1.7 Å resolution or better and demonstrate a selective mechanism of binding between the hydrophilic and hydrophobic pockets of CA IX versus CA II. These compounds present promising candidates for anti-CA IX drugs and the treatment for several aggressive cancer types.