Unlocking the paracrine crosstalk: adipocyte-derived factors affect carbonic anhydrase IX expression in colon and breast cancer cells.

Obesity is a major public health concern because it increases the risk of several diseases, including cancer. Crosstalk between obesity and cancer seems to be very complex, and the interaction between adipocytes and cancer cells leads to changes in adipocytes' function and their paracrine signaling, promoting a microenvironment that supports tumor growth. Carbonic anhydrase IX (CA IX) is a tumor-associated enzyme that not only participates in pH regulation but also facilitates metabolic reprogramming and supports the migration, invasion, and metastasis of cancer cells. In addition, CA IX expression, predominantly regulated via hypoxia-inducible factor (HIF-1), serves as a surrogate marker of hypoxia. In this study, we investigated the impact of adipocytes and adipocyte-derived factors on the expression of CA IX in colon and breast cancer cells. We observed increased expression of CA9 mRNA as well as CA IX protein in the presence of adipocytes and adipocyte-derived conditioned medium. Moreover, we confirmed that adipocytes affect the hypoxia signaling pathway and that the increased CA IX expression results from adipocyte-mediated induction of HIF-1α. Furthermore, we demonstrated that adipocyte-mediated upregulation of CA IX leads to increased migration and decreased adhesion of colon cancer cells. Finally, we brought experimental evidence that adipocytes, and more specifically leptin, upregulate CA IX expression in cancer cells and consequently promote tumor progression.

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.

Hypoxia alters the immune response in mouse peritoneal macrophages infected with influenza a virus with truncated NS1 protein.

Macrophages are the most abundant cells in infected tissue and are involved in the clearing infection, and immunomodulation of the innate and adaptive immune response. NS80 virus of influenza A virus, which encodes only the first 80 aa of the NS1 protein, suppresses the immune host response and is associated with enhanced pathogenicity. Hypoxia promotes infiltration of peritoneal macrophages into the adipose tissue and production of cytokines. To understand the role of hypoxia in the regulation of immune response, macrophages were infected with A/WSN/33 (WSN) and NS80 virus, and transcriptional profiles of the RIG-I-like receptor signalling pathway and expression of cytokines were evaluated in normoxia and hypoxia. Hypoxia inhibited the proliferation of IC-21 cells, downregulated the RIG-I-like receptor signalling pathway, and inhibited transcriptional activity of IFN-α, IFN-ß, IFN-ε, and IFN-λ mRNA in infected macrophages. While transcription of IL-1ß and Casp-1 mRNAs were increased in infected macrophages in normoxia, hypoxia resulted in decreased transcription activity of IL-1ß and Casp-1 mRNAs. Hypoxia significantly affected expression of the translation factors IRF4, IFN-γ, and CXCL10 involved in regulation of immune response and polarization of the macrophages. The expression of pro-inflammatory cytokines such as sICAM-1, IL-1α, TNF-α, CCL2, CCL3, CXCL12, and M-CSF was to a large extent affected in uninfected and infected macrophages cultivated in hypoxia. The NS80 virus increased the expression of M-CSF, IL-16, CCL2, CCL3, and CXCL12, especially under hypoxia. The results show that hypoxia may play an important role in peritoneal macrophage activation, regulates the innate and adaptive immune response, changes production of pro-inflammatory cytokines, promotes macrophage polarization, and could affect the function of other immune cells.

ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non­redundantly to ADAM17.

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme participating in adaptive responses of tumors to hypoxia and acidosis. CA IX regulates pH, facilitates metabolic reprogramming, and supports migration, invasion and metastasis of cancer cells. Extracellular domain (ECD) of CA IX can be shed to medium and body fluids by a disintegrin and metalloproteinase (ADAM) 17. Here we show for the first time that CA IX ECD shedding can be also executed by ADAM10, a close relative of ADAM17, via an overlapping cleavage site in the stalk region of CA IX connecting its exofacial catalytic site with the transmembrane region. This finding is supported by biochemical evidence using recombinant human ADAM10 protein, colocalization of ADAM10 with CA IX, ectopic expression of a dominant­negative mutant of ADAM10 and RNA interference­mediated suppression of ADAM10. Induction of the CA IX ECD cleavage with ADAM17 and/or ADAM10 activators revealed their additive effect. Similarly, additive effect was observed with an ADAM17­inhibiting antibody and an ADAM10­preferential inhibitor GI254023X. These data indicated that ADAM10 is a CA IX sheddase acting on CA IX non­redundantly to ADAM17.

Seroprevalence of SARS-CoV-2 antibodies in the county town of Slovakia - a pilot study from the Trencin city.

Slovakia is a country with only 5.45 million inhabitants. However, the past two years of the COVID-19 pandemic have shown huge inter-regional differences. These were represented by different numbers of diagnosed SARS-CoV-2 cases and the vaccination rates in the regions, as well as by the willingness of the inhabitants to comply with anti-pandemic measures or to undergo testing. The occurrence of such regional disparities provided a rational basis for monitoring the epidemic situation within smaller areas, e.g. at city level. Trencin is a medium-sized Slovak county town with about 55 000 inhabitants. The city administration gave its residents the opportunity to assess their current level of antibodies against the SARS-CoV-2 virus, and received an additional benefit in the form of data on the real epidemic situation in the city, which helped in further management of anti-pandemic measures. The primary aim of the study, conducted in January and February 2022, was to determine the levels of antibodies against the SARS-CoV-2 virus in the inhabitants of Trencin. The results showed that 75% of the study participants, representing the adult population of the city, had detectable IgG antibodies against the SARS-CoV-2 spike protein. Noteworthy, at the time of the study, 13% of the Trencin city population who were unaware of overcoming COVID-19 had specific antibodies against the virus. Furthermore, the antibody levels in recovered unvaccinated subjects increased not only with the severity of their COVID-19 symptoms, but also after multiple recoveries from the disease. On the other hand, the severity of side effects after vaccination did not influence the antibody levels. The results of the study are in line with the current view that hybrid immunity (vaccination plus SARS-CoV-2 infection in any order) offers greater protection than immunity elicited by vaccination or COVID-19 separately. Keywords: SARS-CoV-2 coronavirus; COVID-19; ELISA; seroprevalence; antibodies; vaccination.

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.

Seroprevalence of SARS-CoV-2 IgG antibodies in the staff of the Slovak Academy of Sciences in response to COVID-19 and/or vaccination: situation in August 2021.

Cross-sectional seroprevalence study of SARS-CoV-2 IgG antibodies was accomplished in the Slovak Academy of Sciences to inform authorities of research institutions about the situation at their workplaces, to assess the risk of next exposure to SARS-CoV-2, and to guide decisions on institutional measures sustaining essential research in evolving epidemic situation. Study participants provided informed consent, anamnestic information, and self-collected dry blood spot samples that were analyzed by ELISA for SARS-CoV-2 S protein-specific IgG antibodies. Relative antibody levels detected in 1928 subjects showed seroprevalence of 84.13% and led to the following main findings consistent with the current knowledge: (1) mRNA-based vaccines induce better humoral response compared to adenovirus vaccines, (2) antibody levels reflect severity of COVID-19 symptoms, (3) post-COVID vaccination results in marked elevation of IgG levels particularly in asymptomatic and mild cases, (4) antibody levels decrease with increasing time elapsed from vaccination or COVID-19. In addition, data sorting to distinct research institutes and their clustering to three principal scientific sections of the Slovak Academy of Sciences revealed marked differences in seroprevalence, and allowed to identify workplaces with relatively high seropositivity and response rate that can potentially provide a safer working environment than those, where seroprevalence was low or unknown due to low participation. Thus, findings of this study can have direct implications on management decisions during the next pandemic development, with the necessity to keep in mind the phenomenon of time-dependent immunity waning and current spread of more contagious Delta variant of SARS-CoV-2. Keywords: SARS-CoV-2 coronavirus; COVID-19; spike protein; seroprevalence; antibodies; vaccination.

Targeting of carbonic anhydrase IX-positive cancer cells by glycine-coated superparamagnetic nanoparticles.

Antibody-modified magnetic nanoparticles were prepared to study their cellular uptake in 3D multicellular spheroidal cell cultures. For this purpose, carbonic anhydrase IX specific monoclonal antibody VII/20 was selected to conjugate on the surface of positively charged glycine coated magnetic nanoparticles in a form of a stable magnetic fluid. In this work, glycine-functionalized magnetic nanoparticles were characterized by different methods. X-ray photoelectron analysis confirmed the binding of glycine to the magnetic nanoparticles, and quantification of the glycine coating on the surface of the magnetic nanoparticles was conducted by thermogravimetric analysis. The optimal weight ratio of glycine to magnetic nanoparticles was determined to be 5 showing good colloid stability due to the high surface charge density of protonated glycine coating shown by the great zeta potential (⁓40 mV). The antibody conjugation to the functionalized magnetic nanoparticles was performed at an antibody to magnetic nanoparticles weight ratio equal to 0.5. Applications of antibody-modified magnetic nanoparticles in cancer therapy rely on their ability to specifically target cancer tissues and enter the tumour intracellular space. Here, we show that antibody coupled nanoparticle internalization was triggered by selective binding to tumour cells expressing hypoxic marker carbonic anhydrase IX. Moreover, our results confirmed specific penetration of conjugated nanoparticles into the tumour cell spheroids.

Understanding metabolic alterations and heterogeneity in cancer progression through validated immunodetection of key molecular components: a case of carbonic anhydrase IX.

Cancer metabolic heterogeneity develops in response to both intrinsic factors (mutations leading to activation of oncogenic pathways) and extrinsic factors (physiological and molecular signals from the extracellular milieu). Here we review causes and consequences of metabolic alterations in cancer cells with focus on hypoxia and acidosis, and with particular attention to carbonic anhydrase IX (CA IX). CA IX is a cancer-associated enzyme induced and activated by hypoxia in a broad range of tumor types, where it participates in pH regulation as well as in molecular mechanisms supporting cancer cells' invasion and metastasis. CA IX catalyzes reversible conversion of carbon dioxide to bicarbonate ion plus proton and cooperates with a spectrum of molecules transporting ions or metabolites across the plasma membrane. Thereby CA IX contributes to extracellular acidosis as well as to buffering intracellular pH, which is essential for cell survival, metabolic performance, and proliferation of cancer cells. Since CA IX expression pattern reflects gradients of oxygen, pH, and other intratumoral factors, we use it as a paradigm to discuss an impact of antibody quality and research material on investigating metabolic reprogramming of tumor tissue. Based on the validation, we propose the most reliable CA IX-specific antibodies and suggest conditions for faithful immunohistochemical analysis of molecules contributing to heterogeneity in cancer progression.

Bratislava Statement: consensus recommendations for improving pancreatic cancer care.

Pancreatic cancer is one of the most lethal tumours, and it is the fourth cause of cancer death in Europe. Despite its important public health impact, no effective treatments exist, nor are there high-visibility research efforts to improve care. This alarming situation is emblematic of a larger group of cancer diseases, known as neglected cancers. To address the impact of these diseases, the European Commission-supported Innovative Partnership for Action Against Cancer launched a multi-stakeholder initiative to determine key steps that healthcare systems can rapidly implement to improve their response. A working group comprising 20 representatives from European medical societies, patient associations, cancer plan organisations and other relevant European healthcare stakeholders was organised. A consensus process based on the results of different studies, discussion of research outcomes, and development and endorsement of draft statements resulted in 22 consensus recommendations (the Bratislava Statement). The statement argues that substantial improvements can be achieved in patient outcomes by centralising pancreatic cancer care around state-of-the-art reference centres, staffed by expert multidisciplinary teams capable of providing high-quality care. This organisational model requires a specific care framework encompassing primary, palliative and survivorship care, and a policy environment prioritising the use of quality criteria and performance assessments as well as research investments dedicated to prevention, risk prediction, early detection and diagnosis. In order to address the challenges posed by neglected cancers in general and pancreatic cancer in particular, a specific control strategy tailored to this reality is required.

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.

Molecular targeting of bioconjugated graphene oxide nanocarriers revealed at a cellular level using label-free Raman imaging.

Two-dimensional materials as graphene oxide (GO) are able to accommodate labels as well as toxins for diagnostics and therapy, respectively. The transmembrane protein carbonic anhydrase (CA IX) is one of the molecules selectively expressed by tumor cells. Here, we demonstrate bioconjugation of GO to biotinylated M75 antibody highly selective towards CA IX. Based on a model system, binding between the bioconjugated GO-M75 and Madin-Darby Canine Kidney (MDCK) cells was evaluated. As proven by fluorescence-activated cell sorting, higher intake was observed for GO-M75 towards MDCK cells ectopically expressing CA IX protein on their surface when compared to control MDCK. In particular, we were able to localize GO nanocarrier crossing the membrane during endocytosis, thanks to the optical cross-sectioning of living cells in real-time employed the label-free confocal Raman microscopy. The increased affinity of the prepared GO-M75 molecular complexes validates the use of two-dimensional materials for future strategies of targeted cancer treatment.

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.

A bioconjugated MoS2 based nanoplatform with increased binding efficiency to cancer cells.

We evaluate the application of surfactant-free liquid-phase exfoliated MoS2 nanosheets as a nanoplatform for a cancer detection and treatment system equipped with an antibody-antigen based recognition element. Employing antigen-antibody binding, we increased the probability of the endocytosis of MoS2 nanosheets into CAIX expressing cells by 30%. The nanosheets are functionalized with a specific antibody M75, which forms an antigen-antibody complex with CAIX. The bioconjugation of MoS2 nanosheets involves biocompatible components with low cytotoxicity, verified in the tested cell lines by fluorescence-based cell viability assay. The cellular internalization is quantified by flow cytometry, while the internalization is confirmed by label-free confocal Raman imaging. Raman measurements show increased lysosomal activity in the proximity of the internalized nanoplatforms.

Annual Meeting of the International Society of Cancer Metabolism (ISCaM): Metabolic Adaptations and Targets in Cancer.

The metabolism of cancer cells differs from that of their normal counterparts in a spectrum of attributes, including imbalances in diverse metabolic arms and pathways, metabolic plasticity and extent of adaptive responses, levels, and activities of metabolic enzymes and their upstream regulators and abnormal fluxes of metabolic intermediates and products. These attributes endow cancer cells with the ability to survive stressors of the tumor microenvironment and enable them to landscape and exploit the host terrain, thereby facilitating cancer progression and therapy resistance. Understanding the molecular and physiological principles of cancer metabolism is one of the key prerequisites for the development of better anticancer treatments. Therefore, various aspects of cancer metabolism were addressed at the 5th annual meeting of the International Society of Cancer Metabolism (ISCaM) in Bratislava, Slovakia, on October 17-20, 2018. The meeting presentations and discussions were traditionally focused on mechanistic, translational, and clinical characteristics of metabolism and pH control in cancer, at the level of molecular pathways, cells, tissues, and organisms. In order to reflect major healthcare challenges of the current era, ISCaM has extended its scope to metabolic disorders contributing to cancer, as well as to opportunities for their prevention, intervention, and therapeutic targeting.

Extracellular AGR3 regulates breast cancer cells migration via Src signaling.

Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes.

Type 1 Sodium Calcium Exchanger Forms a Complex with Carbonic Anhydrase IX and Via Reverse Mode Activity Contributes to pH Control in Hypoxic Tumors.

Hypoxia and acidosis are among the key microenvironmental factors that contribute to cancer progression. We have explored a possibility that the type 1Na+/Ca2+ exchanger (NCX1) is involved in pH control in hypoxic tumors. We focused on changes in intracellular pH, co-localization of NCX1, carbonic anhydrase IX (CA IX), and sodium proton exchanger type 1 (NHE1) by proximity ligation assay, immunoprecipitation, spheroid formation assay and migration of cells due to treatment with KB-R7943, a selective inhibitor of the reverse-mode NCX1. In cancer cells exposed to hypoxia, reverse-mode NCX1 forms a membrane complex primarily with CA IX and also with NHE1. NCX1/CA IX/NHE1 assembly operates as a metabolon with a potent ability to extrude protons to the extracellular space and thereby facilitate acidosis. KB-R7943 prevents formation of this metabolon and reduces cell migration. Thus, we have shown that in hypoxic cancer cells, NCX1 operates in a reverse mode and participates in pH regulation in hypoxic tumors via cooperation with CAIX and NHE1.

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.