Can hypoxia marker carbonic anhydrase IX serve as a potential new diagnostic marker and therapeutic target of non-small cell lung cancer?

Lung cancer represents the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is a molecularly heterogeneous disease with intratumoral heterogeneity and a significant mutational burden associated with clinical outcome. Tumor microenvironment (TME) plays a fundamental role in the initiation and progression of primary de novo lung cancer and significantly influences the response of tumor cells to therapy. Hypoxia, an integral part of the tumor microenvironment and a serious clinical phenomenon, is associated with increased genetic instability and a more aggressive phenotype of NSCLC, which correlates with the risk of metastasis. Low oxygen concentration influences all components of TME including the immune microenvironment. Hypoxia-inducible pathway activated in response to low oxygen supply mediates the expression of genes important for the adaptation of tumor cells to microenvironmental changes. A highly active transmembrane hypoxia-induced metalloenzyme - carbonic anhydrase IX (CAIX), as a part of transport metabolon, contributes to the maintenance of intracellular pH within physiological values and to the acidification of the extracellular space. CAIX supports cell migration and invasion and plays an important role in NSCLC tumor tissue and pleural effusion. Due to its high expression, it also represents a potential diagnostic differential biomarker and therapeutic target in NSCLC. To test new potential targeted therapeutic compounds, suitable models are required that more faithfully simulate tumor tissue, TME components, and spatial architecture.

Propranolol, Promising Chemosensitizer and Candidate for the Combined Therapy through Disruption of Tumor Microenvironment Homeostasis by Decreasing the Level of Carbonic Anhydrase IX.

Resistance to chemotherapy represents a persisting medical problem, ranking among main causes of chemotherapy failure and cancer mortality. There is a possibility to utilize and repurpose already existing therapeutics which were not primarily intended for oncological treatment. Overactivation of adrenergic receptors and signaling dysregulation promotes tumor progression, metastatic potential, immune system evasion, tumor angiogenesis and drug resistance. The non-selective beta-blocker propranolol, approved in infantile haemangioma treatment, has a high potential for use in cancer therapy. We analyzed the effects of propranolol and 5-fluorouracil combination on sensitive and resistant cells derived from colorectal carcinoma in monolayers, single-component and co-culture spheroids and in vivo mouse models. Our results revealed that propranolol is able to exert its effect not only in chemosensitive colorectal cells, but also in 5-fluorouracil resistant cells. Propranolol disrupts the hypoxic adaptation machinery by inhibiting HIF1α, carbonic anhydrase IX, and activates apoptosis, which may be important in the management of chemo-resistant patients. We showed that propranolol slows down the growth of xenografts formed from colorectal cancer cells, even from cells already adapted to the ß-blocker. We provide clear evidence that blockade of ß-adrenergic receptors affects essential signaling pathways modulating tumor microenvironment and thus the response to anticancer therapy. Our findings indicate that propranolol could be repurposed to serve as chemosensitizer in combined therapy aimed at disrupting homeostasis of tumor microenvironment.

Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX.

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 antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, affinity, extracellular pH measurement, multicellular aggregation analysis, and real-time monitoring of invasion with the xCELLigence system. RESULTS: Based on the results from in vivo experiments, we have selected mouse monoclonal antibodies VII/20 and IV/18. The first one is directed at the conformational epitope of the catalytic domain, internalizes after binding to the antigen, and halts tumor growth while blocking extracellular acidification. The second targets the sequential epitope of the proteo-glycan domain, does not internalize, and is able to block the attachment of cancer cells to the matrix preventing metastasis formation. In vitro experiments prove that humanized versions of the parental murine antibodies, CA9hu-1 and CA9hu-2, have preserved these characteristics. They can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME, and both are able to induce an immune response and have high affinity, as well as ADCC and CDC activity. CONCLUSION: CA9hu-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.

Hypoxia Marker Carbonic Anhydrase IX Is Present in Abdominal Aortic Aneurysm Tissue and Plasma.

Abdominal aortic aneurysms (AAA) are a significant cause of premature deaths worldwide. Since there is no specific treatment for reducing AAA progression, it is crucial to understand the pathogenesis leading to aneurysm wall weakening/remodeling and identify new proteins involved in this process which could subsequently serve as novel therapeutic targets. In this study, we analyzed the presence of the hypoxia-related proteins carbonic anhydrase IX (CA IX), hypoxia-inducible factor 1α (HIF-1α), and AKT as the key molecule in the phosphoinositide-3-kinase pathway in the AAA wall. Additionally, we used a blood-based assay to examine soluble CA IX (s-CA IX) levels in the plasma of AAA patients. Using western blotting, we detected CA IX protein in 12 out of 15 AAA tissue samples. Immunohistochemistry staining proved CA IX expression in the media of the aneurysmal wall. Evaluation of phosphorylated (p-AKT) and total AKT showed elevated levels of both forms in AAA compared to normal aorta. Using ELISA, we determined the concentration of s-CA IX >20 pg/mL in 13 out of 15 AAA patients. Results obtained from in silico analysis of CA9 and aneurysm-associated genes suggest a role for CA IX in aneurysmal wall remodeling. Our results prove the presence of hypoxia-related CA IX in AAA tissues and indicate a possible role of CA IX in hypoxia-associated cardiovascular diseases.

PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity.

Human carbonic anhydrase IX (CAIX), a unique member of the α carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function.

Impairment of Hypoxia-Induced CA IX by Beta-Blocker Propranolol-Impact on Progression and Metastatic Potential of Colorectal Cancer Cells.

The coexistence of cancer and other concomitant diseases is very frequent and has substantial implications for treatment decisions and outcomes. Beta-blockers, agents that block the beta-adrenergic receptors, have been related also to cancers. In the model of multicellular spheroids formed by colorectal cancer cells we described a crosstalk between beta-blockade by propranolol and tumour microenvironment. Non-selective beta-blocker propranolol decreased ability of tumour cells to adapt to hypoxia by reducing levels of HIF1α and carbonic anhydrase IX in 3D spheroids. We indicated a double action of propranolol in the tumour microenvironment by inhibiting the stability of HIF1α, thus mediating decrease of CA IX expression and, at the same time, by its possible effect on CA IX activity by decreasing the activity of protein kinase A (PKA). Moreover, the inhibition of ß-adrenoreceptors by propranolol enhanced apoptosis, decreased number of mitochondria and lowered the amount of proteins involved in oxidative phosphorylation (V-ATP5A, IV-COX2, III-UQCRC2, II-SDHB, I-NDUFB8). Propranolol reduced metastatic potential, viability and proliferation of colorectal cancer cells cultivated in multicellular spheroids. To choose the right treatment strategy, it is extremely important to know how the treatment of concomitant diseases affects the superior microenvironment that is directly related to the efficiency of anti-cancer therapy.

Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells. METHODS: We generated a non-shed CA IX mutant by deletion of amino acids 393-402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. CONCLUSIONS: Our results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.

Hypoxic marker CA IX and adhesion mediator ß-catenin are downregulated by lymphocytic choriomeningitis virus persistent infection.

Renal cell carcinoma is one of the most frequent cancer diseases with high resistance to radio- and chemotherapy. Mutation of VHL gene is frequent in these tumors leading to simulation of hypoxic conditions. Lymphocytic choriomeningitis virus, belonging to RNA viruses, is a neglected human pathogen and teratogen. We have found that infection of renal cell carcinoma cells by lymphocytic choriomeningitis virus strain MX causes a decrease of carbonic anhydrase IX protein and RNA level. Lower expression of carbonic anhydrase IX on the cell surface provides less target for carbonic anhydrase IX-targeted immunotherapy. What more, reduced levels of adhesion mediating protein ß-catenin as well as E-cadherin, as a consequence of infection, suggest a possible increase in metastatic potential of cells infected by lymphocytic choriomeningitis virus strain MX. These results might help elucidate differences in patients susceptibility to immunotherapy directed against carbonic anhydrase IX or in developing new therapeutical strategies. Our data indicate that presence of infection can significantly affect patient response to cancer therapy.

Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

Carbonic anhydrase enzymes II, VII, IX and XII in colorectal carcinomas.

AIM: To investigate expression of four alpha-carbonic anhydrases (CAs) in colorectal carcinomas (CRC) and compare the results with patients' survival. METHODS: Colorectal carcinoma samples from 539 CRC patients and control tissues were arranged as tissue microarrays and analyzed with antibodies against CA II, CA VII, CA IX, and CA XII. Intensity and extent of staining were both scored from 0 to 3 in each sample. These enzyme expression levels were then correlated to patients' survival and clinicopathological parameters, which were tumor differentiation grade and stage, site of tumor, patients' age, and gender. Kaplan-Meier analysis and Cox regression hazard ratio model were used to analyze survival data. RESULTS: CA II and CA XII staining intensities correlated with patients' survival in that higher expression indicated poorer prognosis. In Cox regression analysis one unit increase in the CA II intensity increased the hazard ratio to 1.19 fold (CI: 1.04-1.37, P = 0.009). A significant correlation was also found when comparing CA XII staining intensity with survival of CRC patients (HR = 1.18, 95%CI: 1.01-1.38, P = 0.036). The extent of CA XII immunostaining did not correlate to the patients' survival (P = 0.242, Kaplan-Meier analysis). A significant interaction between age group and extent of the CA II staining was found. Increased extent of CA II had a significant hazard ratio among patients 65 years and older (1.42, 95%CI: 1.16-1.73, P = 0.0006). No correlations were found between CA VII (intensity P = 0.566, extent P = 0.495, Kaplan-Meier analysis), or CA IX (intensity P = 0.879, extent P = 0.315, Kaplan-Meier analysis) immunostaining results and survival, or the other parameters. CONCLUSION: The present findings indicate that CA II and CA XII could be useful in predicting survival in CRC.

Dexamethasone downregulates expression of carbonic anhydrase IX via HIF-1α and NF-κB-dependent mechanisms.

Dexamethasone is a synthetic glucocorticoid frequently used to suppress side-effects of anticancer chemotherapy. In the present study, we showed that dexamethasone treatment leads to concentration-dependent downregulation of cancer-associated marker, carbonic anhydrase IX (CA IX), at the level of promoter activity, mRNA and protein expression in 2D and 3D cancer cell models. The effect of dexamethasone on CA IX expression under hypoxic conditions is predominantly mediated by impaired transcriptional activity and decreased protein level of the main hypoxic transcription factor HIF-1α. In addition, CA9 downregulation can be caused by protein-protein interactions between activated glucocorticoid receptors, major effectors of glucocorticoid action, and transcription factors that trigger CA9 transcription (e.g. AP-1). Moreover, we identified a potential NF-κB binding site in the CA9 promoter and propose the involvement of NF-κB in the dexamethasone-mediated inhibition of CA9 transcription. As high level of CA IX is often linked to aggressive tumor behavior, poor prognosis and chemo- and radiotherapy resistance, uncovering its reduction after dexamethasone treatment and implication of additional regulatory mechanisms can be relevant for the CA IX-related clinical applications.

Encapsulation of anti-carbonic anhydrase IX antibody in hydrogel microspheres for tumor targeting.

Encapsulation is a well-established method of biomaterial protection, controlled release, and efficient delivery. Here we evaluated encapsulation of monoclonal antibody M75 directed to tumor biomarker carbonic anhydrase IX (CA IX) into alginate microbeads (SA-beads) or microcapsules made of sodium alginate, cellulose sulfate, and poly(methylene-co-guanidine) (PMCG). M75 antibody release was quantified using ELISA and its binding properties were assessed by immunodetection methods. SA-beads showed rapid M75 antibody release in the first hour, followed by steady release during the whole experiment of 7 days. In contrast, the M75 release from PMCG capsules was gradual, reaching the maximum concentration on the 7th day. The release was more efficient at pH 6.8 compared to pH 7.4. The released antibody could recognize CA IX, and target the CA IX-positive cells in 3D spheroids. In conclusion, SA-beads and PMCG microcapsules can be considered as promising antibody reservoirs for targeting of cancer cells.

Carbonic anhydrase IX deposits are associated with increased ascending aortic dilatation.

OBJECTIVES: Carbonic anhydrase IX (CA IX) expression is induced by local hypoxia. We studied whether CA IX deposits associate with ascending aortic dilatation. DESIGN: Aortic wall histology, CA IX expression, presence of leukocytes, plasma cells, macrophages, endothelial cells, smooth muscle cells, cell proliferation, elastin and collagen were studied in histological specimens collected from 30 patients who underwent surgery for ascending aorta. The samples were grouped according to presence of CA IX deposits. RESULTS: Twenty out of 30 patients had CA IX-positive deposits within the adventitia, whereas 10 specimens remained negative. Adventitial inflammation was increased in CA IX-positive samples as compared with CA IX-negative ones (p < 0.01). The mean diameter of the ascending aorta at the sinotubular junction increased significantly in patients with CA IX-positive staining as compared with CA IX-negative cases (63 ± 3 vs 53 ± 2 mm, p < 0.02). Receiver operating characteristic curve analysis confirmed the association of CA IX positivity with increased ascending aortic dilatation (AUC 0.766; S.E. 0.090; p = 0.020; 95% C.I. 0.590-0.941). CONCLUSIONS: Positive CA IX staining in certain aortic specimens suggests that increased CA activity may contribute to ascending aortic dilatation.

Apoptosis-induced ectodomain shedding of hypoxia-regulated carbonic anhydrase IX from tumor cells: a double-edged response to chemotherapy.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin. METHODS: Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay. RESULTS: We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness. CONCLUSIONS: These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.

Antiviral Effect of Interferon Lambda Against Lymphocytic Choriomeningitis Virus.

Lambda interferons inhibit replication of many viruses, but their role in the inhibition of lymphocytic choriomeningitis virus (LCMV) infection remains unclear. In this study, we examined the antiviral effects of interferon (IFN)-λ2 and IFN-λ3 against LCMV in A549 cells. We found that IFN-λ2 is a more potent inhibitor of LCMV strain MX compared with IFN-λ3, whereas both cytokines have similar antiviral effects against an immunosuppressive variant of LCMV, clone-13. We also demonstrated that the antiviral activity of IFN-λ2 is more effective if it is delivered early rather than after establishment of a long-term infection, suggesting that virus replication is only partially responsive to the cytokine. In agreement with this observation, we showed that LCMV infection significantly reduces IFNLR1 mRNA expression in infected cells. In addition, LCMV infection, to some extent, compromises the signal transduction pathway of IFN-λ2. This implies that IFN receptors as well as their downstream signaling components could be selectively targeted either directly by LCMV proteins or indirectly by cellular factor(s) that are induced or activated by LCMV infection.

Hypoxia-induced carbonic anhydrase IX as a target for cancer therapy: from biology to clinical use.

The tumor microenvironment includes a complicated network of physiological gradients contributing to plasticity of tumor cells and heterogeneity of tumor tissue. Hypoxia is a key component generating intratumoral oxygen gradients, which affect the cellular expression program and lead to therapy resistance and increased metastatic propensity of weakly oxygenated cell subpopulations. One of the adaptive responses of tumor cells to hypoxia involves the increased expression and functional activation of carbonic anhydrase IX (CA IX), a cancer-related cell surface enzyme catalyzing the reversible conversion of carbon dioxide to bicarbonate ion and proton. Via its catalytic activity, CA IX participates in regulation of intracellular and extracellular pH perturbations that result from hypoxia-induced changes in cellular metabolism producing excess of acid. Through the ability to regulate pH, CA IX also facilitates cell migration and invasion. In addition, CA IX has non-catalytic function in cell adhesion and spreading. Thus, CA IX endows tumor cells with survival advantages in hypoxia/acidosis and confers an increased ability to migrate, invade and metastasize. Accordingly, CA IX is expressed in a broad range of tumors, where it is associated with prognosis and therapy outcome. Its expression pattern and functional implications in tumor biology make CA IX a promising therapeutic target, which can be hit either by immunotherapy with monoclonal antibodies or with compounds inhibiting its enzyme activity. The first strategy has already reached the clinical trials, whereas the second one is still in preclinical testing. Both strategies indicate that CA IX can become a clinically useful anticancer target, but urge further efforts toward better selection of patients for immunotherapy and deeper understanding of tumor types, clinical situations and synthetic lethality interactions with other treatment approaches.

Monoclonal antibody G250 targeting CA â ¨: Binding specificity, internalization and therapeutic effects in a non-renal cancer model.

G250 (Girentuximab) is a chimeric IgG1 monoclonal antibody (MAb) currently being evaluated as an immunotherapy for kidney cancer. It targets carbonic anhydrase protein (CA Ⅸ), a transmembrane carbonic anhydrase (CA) isoform, which is regulated by VHL/HIF pathway and hence expressed in the majority of renal cell carcinomas (RCCs) as well as in hypoxic non­RCC tumours. CA Ⅸ functions in pH regulation and cell migration/invasion, and supports tumour cell survival in hypoxia and/or acidosis. It contains a highly active extracellular catalytic domain (CA) extended N-terminally with a proteoglycan-like region and C-terminally with short transmembrane and intracellular regions. Here we characterize the binding and internalization properties of G250, as well as its therapeutic effects in animal model, and discuss the impact of G250­mediated immunotherapy in non­RCC tumours. We demonstrated that G250 MAb recognizes a conformational epitope in the CA domain, detects the soluble CA Ⅸ ectodomain (ECD), but not the splicing variant, and does not cross-react with CA Ⅰ, Ⅱ, and Ⅻ isoforms. We showed that G250 internalizes via clathrin-coated vesicles, escapes degradation in lysosomes and enters the recycling pathway via the perinuclear compartment. This results in long intracellular persistence and enables consecutive internalization cycles. Moreover, the recycled antibody maintains an intact Fc portion potentially capable of continuous induction of antibody-dependent cell-mediated cytotoxicity (ADCC) response, thus explaining its therapeutic efficacy. Finally, we showed that G250 treatment is effective against HT-29 colorectal carcinoma xenografts that differ from RCC by more heterogeneous, hypoxia-related expression of CA Ⅸ. These results suggest potential therapeutic usefulness of the G250 MAb in non-RCC tumours.

Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts.

BACKGROUND: 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 (ß-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-1α and CA IX and/or altered CA IX function. METHODS: 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-1α 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. RESULTS: Carnosine increased the expression levels of HIF-1α 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. CONCLUSIONS: 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.

Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms.

Purpose of the study was to evaluate the presence of hypoxia-inducible, tumour-associated carbonic anhydrases IX and XII in normal skin and a series of cutaneous tumours. Human tumour samples were taken during surgical operations performed on 245 patients and were immunohistochemically stained. A histological score value was calculated for statistical analyses which were performed using SPSS for Windows, versions 17.0 and 20.0. In normal skin, the highest expression of CA IX was detected in hair follicles, sebaceous glands, and basal parts of epidermis. CA XII was detected in all epithelial components of skin. Both CA IX and CA XII expression levels were significantly different in epidermal, appendigeal, and melanocytic tumour categories. Both CA IX and XII showed the most intense immunostaining in epidermal tumours, whereas virtually all melanocytic tumours were devoid of CA IX and XII immunostaining. In premalignant lesions, CA IX expression significantly increased when the tumours progressed to more severe dysplasia forms. Both CA IX and XII are highly expressed in different epithelial components of skin. They are also highly expressed in epidermal tumours, in which CA IX expression levels also correlate with the dysplasia grade. Interestingly, both isozymes are absent in melanocytic tumours.

Expression pattern of carbonic anhydrase IX in Medullary thyroid carcinoma supports a role for RET-mediated activation of the HIF pathway.

Medullary thyroid carcinoma is a relatively rare tumor with poor prognosis and therapy response. Its phenotype is determined by both genetic alterations (activating RET oncoprotein) and physiological stresses, namely hypoxia [activating hypoxia-inducible factor (HIF)]. Here, we investigated the cooperation between these two mechanisms. The idea emerged from the immunohistochemical analysis of carbonic anhydrases (CA) IX and XII expression in thyroid cancer. Although CAXII was present in all types of thyroid carcinomas, CAIX, a direct HIF target implicated in tumor progression, was associated with aggressive medullary and anaplastic carcinomas, and its expression pattern in medullary thyroid carcinomas suggested contribution of both hypoxic and oncogenic signaling. Therefore, we analyzed the CA9 promoter activity in transfected tumor cells expressing RET and/or the HIF-α subunit. We showed that overexpression of both wild-type and mutant RET can increase the CA9 promoter activity induced by HIF-1 (but not HIF-2) in hypoxia. Similar results were obtained with another HIF-1-regulated promoter derived from the lactate dehydrogenase A gene. Moreover, inhibition of the major kinase pathways, which transmit signals from RET and regulate HIF-1, abrogated their cooperative effect on the CA9 promoter. Thus, we brought the first experimental evidence for the crosstalk between RET and HIF-1 that can explain the increased expression of CAIX in medullary thyroid carcinoma and provide a rationale for therapy simultaneously targeting both pathways.