Monika Barathova

Carbonic Anhydrase IX Interacts with Bicarbonate Transporters in Lamellipodia and Increases Cell Migration via Its Catalytic Domain

Background: Hypoxia-induced CA IX contributes to pH control in tumor cells, and control of pH is important for cell migration. Results: CA IX increases migration through catalytic domain and interacts with bicarbonate transporters in lamellipodia. Conclusion: CA IX is an active component of the molecular machinery that facilitates migration of tumor cells through pH regulation at the leading edge membranes. Significance: This identifies CA IX as a target to suppress cell migration and reduce tumor aggressiveness. Carbonic anhydrase IX (CA IX) is a hypoxia-induced cell surface enzyme expressed in solid tumors, and functionally involved in acidification of extracellular pH and destabilization of intercellular contacts. Since both extracellular acidosis and reduced cell adhesion facilitate invasion and metastasis, we investigated the role of CA IX in cell migration, which promotes the metastatic cascade. As demonstrated here, ectopically expressed CA IX increases scattering, wound healing and transwell migration of MDCK cells, while an inactive CA IX variant lacking the catalytic domain (ΔCA) fails to do so. Correspondingly, hypoxic HeLa cells exhibit diminished migration upon inactivation of the endogenous CA IX either by forced expression of the dominant-negative ΔCA variant or by treatment with CA inhibitor, implying that the catalytic activity is indispensable for the CA IX function. Interestingly, CA IX improves cell migration both in the absence and presence of hepatocyte growth factor (HGF), an established inducer of epithelial-mesenchymal transition. On the other hand, HGF up-regulates CA IX transcription and triggers CA IX protein accumulation at the leading edge of lamellipodia. In these membrane regions CA IX co-localizes with sodium bicarbonate co-transporter (NBCe1) and anion exchanger 2 (AE2) that are both components of the migration apparatus and form bicarbonate transport metabolon with CA IX. Moreover, CA IX physically interacts with AE2 and NBCe1 in situ, as shown here for the first time. Thus, our findings suggest that CA IX actively contributes to cell migration via its ability to facilitate ion transport and pH control at protruding fronts of moving cells.

Alternative splicing variant of the hypoxia marker carbonic anhydrase IX expressed independently of hypoxia and tumour phenotype.

CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8–9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia- and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control.

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

Detection of carbonic anhydrase 9–expressing tumor cells in the lymph nodes of vulvar carcinoma patients by RT‐PCR

Regional lymph node status is an important prognostic factor for vulvar cancer. The goal of our study was to elaborate a reliable test for detecting micrometastases, undetectable by traditional methods, in the lymph nodes of vulvar squamous carcinoma patients. For this purpose, carbonic anhydrase‐9 (CA9) was investigated as a cancer‐related marker by RT‐PCR. Firstly, primary carcinoma specimens were examined for CA9 expression by immunohistochemistry with M75 monoclonal antibody. All 19 tissues exhibited a variable degree of staining, which was mostly confined to the plasma membranes of tumor cells. Correspondingly, all primary tumor specimens and the control A‐431 vulvar cancer cell line gave a positive signal in the nested RT‐PCR assay designed to detect CA9‐expressing cells with a high sensitivity. Analysis of 77 lymph node specimens from 20 patients revealed a full correlation between RT‐PCR results and standard hematoxylin–eosin staining in 75% of samples, whereas 25% of specimens were negative by the standard method and positive for CA9 mRNA, accounting for 28% of all histologically negative lymph nodes. There were no false‐negatives with RT‐PCR. A positive inguinal lymph node with a negative sentinel node was observed in the same groin only once in 38 specimens. Our findings clearly indicate potential value of CA9 as a molecular marker for the assessment of regional lymph node status in vulvar cancer patients and support a possible utility of our RT‐PCR assay in the detection of micrometastases.

Sulforaphane reduces molecular response to hypoxia in ovarian tumor cells independently of their resistance to chemotherapy

One of the recently emerging anticancer strategies is the use of natural dietary compounds, such as sulforaphane, a cancer-chemopreventive isothiocyanate found in broccoli. Based on the growing evidence, sulforaphane acts through molecular mechanisms that interfere with multiple oncogenic pathways in diverse tumor cell types. Herein, we investigated the anticancer effects of bioavailable concentrations of sulforaphane in ovarian carcinoma cell line A2780 and its two derivatives, adriamycin-resistant A2780/ADR and cisplatin-resistant A2780/CP cell lines. Since tumor microenvironment is characterized by reduced oxygenation that induces aggressive tumor phenotype (such as increased invasiveness and resistance to chemotherapy), we evaluated the effects of sulforaphane in ovarian cancer cells exposed to hypoxia (2% O2). Using the cell-based reporter assay, we identified several oncogenic pathways modulated by sulforaphane in hypoxia by activating anticancer responses (p53, ARE, IRF-1, Pax-6 and XRE) and suppressing responses supporting tumor progression (AP-1 and HIF-1). We further showed that sulforaphane decreases the level of HIF-1α protein without affecting its transcription and stability. It can also diminish transcription and protein level of the HIF-1 target, CA IX, which protects tumor cells from hypoxia-induced pH imbalance and facilitates their migration/invasion. Accordingly, sulforaphane treatment leads to diminished pH regulation and reduced migration of ovarian carcinoma cells. These effects occur in all three ovarian cell lines suggesting that sulforaphane can overcome the chemoresistance of cancer cells. This offers a path potentially exploitable in sensitizing resistant cancer cells to therapy, and opens a window for the combined treatments of sulforaphane either with conventional chemotherapy, natural compounds, or with other small molecules.

Novel monoclonal antibodies specific for CTLD-SSC and sialomucin domains of endosialin, a mural cell marker of tumor vasculature.

Endosialin, alternatively named tumor endothelial marker or CD248, was originally discovered as an antigen selectively expressed in tumor blood vessels. Subsequent studies showed that it is confined to stromal fibroblasts and pericytes of tumor vasculature rather than to tumor endothelium. Endosialin levels are upregulated in different tumor types including those derived from the brain, colon and breast. Expression of endosialin is associated with tumor growth, progression and correlates with a pro-proliferative and pro-migratory phenotype. However, the function of endosialin and mechanisms of its regulation are still incompletely understood. To facilitate further study of endosialin in angiogenesis, its interaction with the potential binding partners and other aspects of endosialin function, we generated six new domain-specific anti-endosialin monoclonal antibodies. Two of them recognize the C-type lectin-like domain-Sushi/SCR/CCP and four antibodies are directed to the sialomucin domain. The antibodies are suitable for various immunodetection methods including immunoblotting and immunohistochemistry. They represent important tools for improving our understanding of endosialin regulation, biological role and contribution of its extracellular domains to the tumor phenotype.

Hypoxic marker CA IX and adhesion mediator β-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 β-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.