Selective Tumor Hypoxia Targeting Using M75 Antibody Conjugated Photothermally Active MoOx Nanoparticles.

Photothermal therapy (PTT) mediated at the nanoscale has a unique advantage over currently used cancer treatments, by being spatially highly specific and minimally invasive. Although PTT combats traditional tumor treatment approaches, its clinical implementation has not yet been successful. The reasons for its disadvantage include an insufficient treatment efficiency or low tumor accumulation. Here, we present a promising new PTT platform combining a recently emerged two-dimensional (2D) inorganic nanomaterial, MoOx, and a tumor hypoxia targeting element, the monoclonal antibody M75. M75 specifically binds to carbonic anhydrase IX (CAIX), a hypoxia marker associated with many solid tumors with a poor prognosis. The as-prepared nanoconjugates showed highly specific binding to cancer cells expressing CAIX while being able to produce significant photothermal yield after irradiation with near-IR wavelengths. Small aminophosphonic acid linkers were recognized to be more effective over the combination of poly(ethylene glycol) chain and biotin-avidin-biotin bridge in constructing a PTT platform with high tumor-binding efficacy. The in vitro cellular uptake of nanoconjugates was visualized by high-resolution fluorescence microscopy and label-free live cell confocal Raman microscopy. The key to effective cancer treatment may be the synergistic employment of active targeting and noninvasive, tumor-selective therapeutic approaches, such as nanoscale-mediated PTT. The use of active targeting can streamline nanoparticle delivery increasing photothermal yield and therapeutic success.

Functional consequences of altered glycosylation of tumor-associated hypoxia biomarker carbonic anhydrase IX.

Glycosylation is a posttranslational modification of proteins affecting numerous cellular functions. A growing amount of evidence confirms that aberrant glycosylation is involved in pathophysiological processes, including tumor development and progression. Carbonic anhydrase IX (CAIX) is a transmembrane protein whose expression is strongly induced in hypoxic tumors, which makes it an attractive target for anti-tumor therapy. CAIX facilitates the maintenance of intracellular pH homeostasis through its catalytic activity, which is linked with extracellular pH acidification promoting a more aggressive phenotype of tumor cells. The involvement of CAIX in destabilizing cell-cell contacts and the focal adhesion process also contributes to tumor progression. Previous research shows that CAIX is modified with N-glycans, O-glycans, and glycosaminoglycans (GAG). Still, the impact of glycosylation on CAIX functions has yet to be fully elucidated. By preparing stably transfected cells expressing mutated forms of CAIX, unable to bind glycans at their defined sites, we have attempted to clarify the role of glycan structures in CAIX functions. All three types of prepared mutants exhibited decreased adhesion to collagen. By surface plasmon resonance, we proved direct binding between CAIX and collagen. Cells lacking glycosaminoglycan modification of CAIX also showed reduced migration and invasion, indicating CAIX glycosaminoglycans' involvement in these processes. Analysis of signaling pathways affected by the loss of GAG component from CAIX molecule revealed decreased phosphorylation of c-Jun, of p38α kinase, focal adhesion kinase, and reduced level of heat shock protein 60 in cells cultured in hypoxia. Cells expressing CAIX without GAG exhibited increased metabolon formation and increased extracellular pH acidification. We also observed reduced CAIX GAG glycans in the inflammatory environment in hypoxia, pathophysiological conditions reflecting in vivo tumor microenvironment. Understanding the glycan involvement in the characteristics and functions of possible targets of cancer treatment, such as cell surface localized CAIX, could improve the therapy, as many drugs target glycan parts of a protein.

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.

DNA Methylation Mediates EMT Gene Expression in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Due to abundant stroma and extracellular matrix, accompanied by lack of vascularization, pancreatic ductal adenocarcinoma (PDAC) is characterized by severe hypoxia. Epigenetic regulation is likely one of the mechanisms driving hypoxia-induced epithelial-to-mesenchymal transition (EMT), responsible for PDAC aggressiveness and dismal prognosis. To verify the role of DNA methylation in this process, we assessed gene expression and DNA methylation changes in four PDAC cell lines. BxPC-3, MIA PaCa-2, PANC-1, and SU.86.86 cells were exposed to conditioned media containing cytokines and inflammatory molecules in normoxic and hypoxic (1% O2) conditions for 2 and 6 days. Cancer Inflammation and Immunity Crosstalk and Human Epithelial to Mesenchymal Transition RT² Profiler PCR Arrays were used to identify top deregulated inflammatory and EMT-related genes. Their mRNA expression and DNA methylation were quantified by qRT-PCR and pyrosequencing. BxPC-3 and SU.86.86 cell lines were the most sensitive to hypoxia and inflammation. Although the methylation of gene promoters correlated with gene expression negatively, it was not significantly influenced by experimental conditions. However, DNA methyltransferase inhibitor decitabine efficiently decreased DNA methylation up to 53% and reactivated all silenced genes. These results confirm the role of DNA methylation in EMT-related gene regulation and uncover possible new targets involved in PDAC progression.

The effect of surface modification of TiMg composite on the in-vitro degradation response, cell survival, adhesion, and proliferation.

This study is aimed to evaluate the influence of mechanical surface treatment on the degradation response, cell survival, adhesion, and proliferation of a TiMg composite material. Two sets of the TiMg samples with different surface characteristics were studied: i) as-machined samples (TiMg-T) and ii) samples with a mechanically modified surface (TiMg-P). Surface roughness was determined using a confocal microscope. Degradation rates (DR) were evaluated in artificial Plasma, HBSS, and NaCl 0.9%. The cell viability was evaluated using an MTT assay. The initial cell adhesion and spreading were investigated using the direct contact assay. An xCELLigence system was employed to provide real-time cell proliferation. The focal adhesion and cell morphological changes were also examined. The DR of TiMg-P decreased by ⁓5 times compared with that of TiMg-T. Surface of the TiMg-P specimens after 72 h exposure to either HBSS or Plasma was passivated by a layer enriched with bioactive Ca/P species. The cell viability of L929 and Saos-2 after 72 h incubation for TiMg-P was 94.6% and 94.8% compared with 73.8% and 74.3% obtained for TiMg-T, respectively. The direct contact assay showed that the initial adhesion and spreading of the L929 cells incubated with TiMg-P was more pronounced compared with that of TiMg-T. The proliferation rate of Saos-2 cells incubated with TiMg-P was higher when compared with that of TiMg-T, and was almost comparable to that of the DMEM-blank between the 24 and 72 h interval. TiMg-P had a pronounced difference in the number and area of Focal Adhesions (FA) compared with that of TiMg-T. The morphology of cells incubated with TiMg-P was not altered. The results confirmed that the smooth and less strained surface of the TiMg-P samples effectively improved the in-vitro degradation response, cell survival, adhesion, and proliferation.

CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia.

Tumor hypoxia represents a severe microenvironmental stress that is frequently associated with acidosis. Cancer cells respond to these stresses with changes in gene expression that promote survival at least in part through pH regulation and metabolic reprogramming. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a critical adaptive role in response to hypoxic and acidic environments by catalytically hydrating extracellular CO2 to produce bicarbonate for buffering intracellular pH (pHi). We used proteome-wide profiling to study the cellular response to transient CA IX knockdown in hypoxia and found a decrease in the levels of key glycolytic enzymes and lactate dehydrogenase A (LDHA). Interestingly, the activity of LDH was also decreased as demonstrated by native in-gel activity assay. These changes led to a significant reduction in glycolytic flux and extracellular lactate levels in cancer cells in vitro, contributing to a decrease in proliferation. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate restored LDHA activity, extracellular acidification, pHi, and cellular proliferation. These results indicate that in the absence of CA IX, reduction of pHi disrupts LDHA activity and hinders the cellular capacity to regenerate NAD+ and secrete protons to the extracellular space. Hypoxia-induced CA IX therefore mediates adaptation to microenvironmental hypoxia and acidosis directly, by enzymatically converting extracellular CO2 to bicarbonate, and indirectly, by maintaining glycolysis-permissive intracellular milieu.

Clinical and Pre-Clinical Evidence of Carbonic Anhydrase IX in Pancreatic Cancer and Its High Expression in Pre-Cancerous Lesions.

Hypoxia is a common phenomenon that occurs in most solid tumors. Regardless of tumor origin, the evolution of a hypoxia-adapted phenotype is critical for invasive cancer development. Pancreatic ductal adenocarcinoma is also characterized by hypoxia, desmoplasia, and the presence of necrosis, predicting poor outcome. Carbonic anhydrase IX (CAIX) is one of the most strict hypoxia regulated genes which plays a key role in the adaptation of cancer cells to hypoxia and acidosis. Here, we summarize clinical data showing that CAIX expression is associated with tumor necrosis, vascularization, expression of Frizzled-1, mucins, or proteins involved in glycolysis, and inevitably, poor prognosis of pancreatic cancer patients. We also describe the transcriptional regulation of CAIX in relation to signaling pathways activated in pancreatic cancers. A large part deals with the preclinical evidence supporting the relevance of CAIX in processes leading to the aggressive behavior of pancreatic tumors. Furthermore, we focus on CAIX occurrence in pre-cancerous lesions, and for the first time, we describe CAIX expression within intraductal papillary mucinous neoplasia. Our review concludes with a detailed account of clinical trials implicating that treatment consisting of conventionally used therapies combined with CAIX targeting could result in an improved anti-cancer response in pancreatic cancer patients.

CAIX-Mediated Control of LIN28/let-7 Axis Contributes to Metabolic Adaptation of Breast Cancer Cells to Hypoxia.

Solid tumors, including breast cancer, are characterized by the hypoxic microenvironment, extracellular acidosis, and chemoresistance. Hypoxia marker, carbonic anhydrase IX (CAIX), is a pH regulator providing a selective survival advantage to cancer cells through intracellular neutralization while facilitating tumor invasion by extracellular acidification. The expression of CAIX in breast cancer patients is associated with poor prognosis and metastases. Importantly, CAIX-positive hypoxic tumor regions are enriched in cancer stem cells (CSCs). Here we investigated the biological effects of CA9-silencing in breast cancer cell lines. We found that CAIX-downregulation in hypoxia led to increased levels of let-7 (lethal-7) family members. Simultaneously with the increase of let-7 miRNAs in CAIX-suppressed cells, LIN28 protein levels decreased, along with downstream metabolic pathways: pyruvate dehydrogenase kinase 1 (PDK1) and phosphorylation of its substrate, pyruvate dehydrogenase (PDH) at Ser-232, causing attenuation of glycolysis. In addition to perturbed glycolysis, CAIX-knockouts, in correlation with decreased LIN28 (as CSC reprogramming factor), also exhibit reduction of the further CSC-associated markers NANOG (Homeobox protein NANOG) and ALDH1 (Aldehyde dehydrogenase isoform 1). Oppositely, overexpression of CAIX leads to the enhancement of LIN28, ALDH1, and NANOG. In conclusion, CAIX-driven regulation of the LIN28/let-7 axis augments glycolytic metabolism and enhances stem cell markers expression during CAIX-mediated adaptation to hypoxia and acidosis in carcinogenesis.

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.

CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins.

Tumor metastasis is tightly linked with invasive membrane protrusions, invadopodia, formed by actively invading tumor cells. Hypoxia and pH modulation play a role in the invadopodia formation and in their matrix degradation ability. Tumor-associated carbonic anhydrase IX (CAIX), induced by hypoxia, is essential for pH regulation and migration, predisposing it as an active component of invadopodia. To investigate this assumption, we employed silencing and inhibition of CA9, invadopodia isolation and matrix degradation assay. Quail chorioallantoic membranes with implanted tumor cells, and lung colonization assay in murine model were used to assess efficiency of in vivo invasion and the impact of CAIX targeting antibodies. We showed that CAIX co-distributes to invadopodia with cortactin, MMP14, NBCe1, and phospho-PKA. Suppression or enzymatic inhibition of CAIX leads to impaired invadopodia formation and matrix degradation. Loss of CAIX attenuated phosphorylation of Y421-cortactin and influenced molecular machinery coordinating actin polymerization essential for invadopodia growth. Treatment of tumor cells by CAIX-specific antibodies against carbonic or proteoglycan domains results in reduced invasion and extravasation in vivo. For the first time, we demonstrated in vivo localization of CAIX within invadopodia. Our findings confirm the key role of CAIX in the metastatic process and gives rationale for its targeting during anti-metastatic therapy.

Carbonic Anhydrase IX-Mouse versus Human.

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.

Lactate stimulates CA IX expression in normoxic cancer cells.

Besides hypoxia, other factors and molecules such as lactate, succinate, and reactive oxygen species activate transcription factor hypoxia-inducible factor-1 (HIF-1) even in normoxia. One of the main target gene products of HIF-1 is carbonic anhydrase IX (CA IX). CA IX is overexpressed in many tumors and serves as prognostic factor for hypoxic, aggressive and malignant cancers. CA IX is also induced in normoxia in high cell density. In this study, we observed that lactate induces CA IX expression in normoxic cancer cells in vitro and in vivo. We further evidenced that participation of both HIF-1 and specificity protein 1 (SP1) transcription factors is crucial for lactate-driven normoxic induction of the CA9 gene. By inducing CA IX, lactate can facilitate the maintenance of cancer cell aggressive behavior in normoxia.

Sulforaphane-induced apoptosis involves the type 1 IP3 receptor.

In this study we show that anti-tumor effect of sulforaphane (SFN) is partially realized through the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). This effect was verified in vitro on three different stable cell lines and also in vivo on the model of nude mice with developed tumors. Early response (6 hours) of A2780 ovarian carcinoma cells to SFN treatment involves generation of mitochondrial ROS and increased transcription of NRF2 and its downstream regulated genes including heme oxygenase 1, NAD(P)H:quinine oxidoreductase 1, and KLF9. Prolonged SFN treatment (24 hours) upregulated expression of NRF2 and IP3R1. SFN induces a time-dependent phosphorylation wave of HSP27. Use of IP3R inhibitor Xestospongin C (Xest) attenuates both SFN-induced apoptosis and the level of NRF2 protein expression. In addition, Xest partially attenuates anti-tumor effect of SFN in vivo. SFN-induced apoptosis is completely inhibited by silencing of IP3R1 gene but only partially blocked by silencing of NRF2; silencing of IP3R2 and IP3R3 had no effect on these cells. Xest inhibitor does not significantly modify SFN-induced increase in the rapid activity of ARE and AP1 responsive elements. We found that Xest effectively reverses the SFN-dependent increase of nuclear content and decrease of reticular calcium content. In addition, immunofluorescent staining with IP3R1 antibody revealed that SFN treatment induces translocation of IP3R1 to the nucleus. Our results clearly show that IP3R1 is involved in SFN-induced apoptosis through the depletion of reticular calcium and modulation of transcription factors through nuclear calcium up-regulation.

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.

Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors.

Acidic tissue microenvironment contributes to tumor progression via multiple effects including the activation of angiogenic factors and proteases, reduced cell-cell adhesion, increased migration and invasion, etc. In addition, intratumoral acidosis can influence the uptake of anticancer drugs and modulate the response of tumors to conventional therapy. Acidification of the tumor microenvironment often develops due to hypoxia-triggered oncogenic metabolism, which leads to the extensive production of lactate, protons, and carbon dioxide. In order to avoid intracellular accumulation of the acidic metabolic products, which is incompatible with the survival and proliferation, tumor cells activate molecular machinery that regulates pH by driving transmembrane inside-out and outside-in ion fluxes. Carbonic anhydrase IX (CA IX) is a hypoxia-induced catalytic component of the bicarbonate import arm of this machinery. Through its catalytic activity, CA IX directly participates in many acidosis-induced features of tumor phenotype as demonstrated by manipulating its expression and/or by in vitro mutagenesis. CA IX can function as a survival factor protecting tumor cells from hypoxia and acidosis, as a pro-migratory factor facilitating cell movement and invasion, as a signaling molecule transducing extracellular signals to intracellular pathways (including major signaling and metabolic cascades) and converting intracellular signals to extracellular effects on adhesion, proteolysis, and other processes. These functional implications of CA IX in cancer are supported by numerous clinical studies demonstrating the association of CA IX with various clinical correlates and markers of aggressive tumor behavior. Although our understanding of the many faces of CA IX is still incomplete, existing knowledge supports the view that CA IX is a biologically and clinically relevant molecule, exploitable in anticancer strategies aimed at targeting adaptive responses to hypoxia and/or acidosis.

The effect of carbonic anhydrase IX on focal contacts during cell spreading and migration.

Carbonic anhydrase IX is a hypoxia-induced transmembrane enzyme linked with solid tumors. It catalyzes the reversible hydration of CO2 providing bicarbonate ions for intracellular neutralization and protons for extracellular acidosis, thereby supporting tumor cell survival and invasiveness. CA IX is the only human CA isoform containing the proteoglycan (PG) domain in its extracellular part. The PG domain appears to enhance the catalytic activity of CA IX and mediate its binding to the extracellular matrix. Moreover, manipulation of the CA IX level by siRNA or overexpression modulates cell adhesion pathway so that in the presence of CA IX, cells display an increased rate of adhesion and spreading. Here we show that deletion of the PG domain as well as treatment with the PG-binding monoclonal antibody M75 can impair this CA IX effect. Accordingly, CA IX-expressing cells show more prominent and elongated maturing paxillin-stained focal contacts (FC) than CA IX-negative controls, proving the role of CA IX in cell spreading. However, during active cell movement, CA IX is relocalized to lamellipodia and improves migration via its catalytic domain. Thus, we examined the influence of CA IX on FC turnover in these structures. While the lamellipodial regions lacking CA IX display dash-like adhesions, the CA IX-enriched neighboring regions exhibit dynamic dot-like FCs. These results suggest that CA IX can promote initial adhesion through its PG domain, but at the same time it facilitates formation of nascent adhesions at the leading edge of moving cells. Thereby it may allow for transmission of large forces and enhanced migration rate, presumably through catalytic activity and impact of pHe on FC dynamics. Thus, we provide the first evidence that CA IX protein localizes directly in focal adhesion (FA) structures and propose its functional relationship with the proteins involved in the regulation of FC turnover and maturation.

Suppression of carbonic anhydrase IX leads to aberrant focal adhesion and decreased invasion of tumor cells.

Carbonic anhydrase IX (CA IX) is a well-recognized hypoxia marker with promising diagnostic and therapeutic value. CA IX regulates the pH in hypoxic tumor cells and, thereby, contributes to microenvironmental acidosis and cell migration. To gain a better insight into the molecular processes driven by CA IX, we performed gene expression profiling of HT-1080 fibrosarcoma cells subjected to CA IX depletion by shRNA silencing. We identified the focal adhesion pathway as being significantly inhibited in the absence of CA IX and confirmed this finding by functional assays. Thus, we obtained the first direct evidence for the role of CA IX in focal adhesion.

Carbonic anhydrase IX: a hypoxia-controlled "catalyst" of cell migration.

Cell migration can be principally viewed as a chain of well-orchestrated morphological events that lead to dynamic reshaping of the cell body. However, behind the scene of such a "morphological theater" there are very complex, interrelated molecular and physiological processes that drive the cell movement. Among them, ion transport and pH regulation play a key role, with carbonic anhydrase IX (CA IX) emerging as one of the important "molecular actors." CA IX is a highly active cell surface enzyme expressed in a broad range of solid tumors in response to hypoxia and explored as a clinically useful biomarker of hypoxia and as a therapeutic target. Its biological role is to protect tumor cells from hypoxia and acidosis in the tumor microenvironment. The study published recently by our group showed that CA IX actively contributes to cell migration and invasion. For the first time, we demonstrated CA IX accumulation in lamellipodia of migrating cells and its direct in situ interaction with bicarbonate transporters. Our findings indicate that tumor cells need CA IX not only as a pro-survival factor in hypoxia and acidosis, but also as a pro-migratory component of the cellular apparatus driving epithelial-mesenchymal transition.

Carbonic anhydrase IX interacts with bicarbonate transporters in lamellipodia and increases cell migration via its catalytic domain.

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.

Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors.

In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment.