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.
- MeSH
- antigeny nádorové genetika MeSH
- glykolýza MeSH
- hypoxie buňky MeSH
- karboanhydrasa IX genetika MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- mikro RNA genetika MeSH
- nádorové buněčné linie MeSH
- nádorové kmenové buňky metabolismus MeSH
- nádory prsu genetika metabolismus MeSH
- přeprogramování buněk MeSH
- proteiny vázající RNA genetika MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- fenotyp MeSH
- invazivní růst nádoru patologie MeSH
- karboanhydrasa IX metabolismus MeSH
- karcinogeneze metabolismus patologie MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory metabolismus patologie MeSH
- protein ADAM17 metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Publikační typ
- časopisecké články MeSH
Diagnosis of oncological diseases remains at the forefront of current medical research. Carbonic Anhydrase IX (CA IX) is a cell surface hypoxia-inducible enzyme functionally involved in adaptation to acidosis that is expressed in aggressive tumors; hence, it can be used as a tumor biomarker. Herein, we propose a nanoscale graphene oxide (GO) platform functionalized with magnetic nanoparticles and a monoclonal antibody specific to the CA IX marker. The GO platforms were prepared by a modified Hummers and Offeman method from exfoliated graphite after several centrifugation and ultrasonication cycles. The magnetic nanoparticles were prepared by a chemical precipitation method and subsequently modified. Basic characterization of GO, such as the degree of oxidation, nanoparticle size and exfoliation, were determined by physical and chemical analysis, including X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDX), and atomic force microscopy (AFM). In addition, the size and properties of the poly-L-lysine-modified magnetic nanoparticles were characterized. The antibody specific to CA IX was linked via an amidic bond to the poly-L-lysine modified magnetic nanoparticles, which were conjugated to GO platform again via an amidic bond. The prepared GO-based platform with magnetic nanoparticles combined with a biosensing antibody element was used for a hypoxic cancer cell targeting study based on immunofluorescence.
- Publikační typ
- časopisecké články MeSH
- Klíčová slova
- endosialin, CD248, TEM1, HIF-2,
- MeSH
- antigeny nádorové MeSH
- CD antigeny * farmakologie genetika chemie účinky léků MeSH
- cílená molekulární terapie MeSH
- exprese genu MeSH
- extracelulární matrix MeSH
- látky modulující angiogenezi MeSH
- lidé MeSH
- nádorové biomarkery MeSH
- nádory MeSH
- patologická angiogeneze MeSH
- pericyty MeSH
- regulace genové exprese MeSH
- remodelace cév * MeSH
- sekvence nukleotidů MeSH
- transkripční faktory klasifikace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
S100P belongs to the S100 family of calcium-binding proteins regulating diverse cellular processes. Certain S100 family members (S100A4 and S100B) are associated with cancer and used as biomarkers of metastatic phenotype. Also S100P is abnormally expressed in tumors and implicated in migration-invasion, survival, and response to therapy. Here we show that S100P binds the tumor suppressor protein p53 as well as its negative regulator HDM2, and that this interaction perturbs the p53-HDM2 binding and increases the p53 level. Paradoxically, the S100P-induced p53 is unable to activate its transcriptional targets hdm2, p21WAF, and bax following the DNA damage. This appears to be related to reduced phosphorylation of serine residues in both N-terminal and C-terminal regions of the p53 molecule. Furthermore, the S100P expression results in lower levels of pro-apoptotic proteins, in reduced cell death response to cytotoxic treatments, followed by stimulation of therapy-induced senescence and increased clonogenic survival. Conversely, the S100P silencing suppresses the ability of cancer cells to survive the DNA damage and form colonies. Thus, we propose that the oncogenic role of S100P involves binding and inactivation of p53, which leads to aberrant DNA damage responses linked with senescence and escape to proliferation. Thereby, the S100P protein may contribute to the outgrowth of aggressive tumor cells resistant to cytotoxic therapy and promote cancer progression.
- MeSH
- chemorezistence fyziologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny metabolismus MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- proteiny vázající vápník metabolismus MeSH
- protoonkogenní proteiny c-mdm2 metabolismus MeSH
- stárnutí buněk fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- antigeny nádorové imunologie metabolismus MeSH
- buněčné sféroidy metabolismus MeSH
- karboanhydrasa IX imunologie metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- lékové transportní systémy metody MeSH
- lidé MeSH
- mikrosféry * MeSH
- monoklonální protilátky aplikace a dávkování imunologie farmakokinetika MeSH
- nádorové biomarkery imunologie metabolismus MeSH
- nádorové buňky kultivované MeSH
- nádory metabolismus patologie MeSH
- PEG-DMA hydrogel * MeSH
- protinádorové látky aplikace a dávkování MeSH
- uvolňování léčiv MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- aktivace transkripce účinky léků MeSH
- antikarcinogenní látky farmakologie terapeutické užití MeSH
- antioxidační responzivní elementy MeSH
- apoptóza účinky léků MeSH
- buněčné jádro metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- faktor 2 související s NF-E2 metabolismus MeSH
- hemoxygenasa-1 metabolismus MeSH
- inositol-1,4,5-trisfosfát - receptory antagonisté a inhibitory metabolismus MeSH
- isothiokyanatany farmakologie terapeutické užití MeSH
- lidé MeSH
- makrocyklické sloučeniny farmakologie MeSH
- mitochondrie účinky léků metabolismus MeSH
- myši nahé MeSH
- myši MeSH
- NAD(P)H dehydrogenasa (chinon) metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory vaječníků farmakoterapie patologie MeSH
- oxazoly farmakologie MeSH
- oxidační stres účinky léků MeSH
- reaktivní formy kyslíku metabolismus MeSH
- transkripční faktory Krüppel-like metabolismus MeSH
- upregulace MeSH
- vápník metabolismus MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- apoptóza účinky léků genetika MeSH
- cykloheximid aplikace a dávkování MeSH
- epitelo-mezenchymální tranzice genetika MeSH
- HeLa buňky MeSH
- hypoxie buňky genetika MeSH
- karboanhydrasa IX aplikace a dávkování genetika metabolismus MeSH
- lidé MeSH
- monoklonální protilátky aplikace a dávkování MeSH
- nádory genetika patologie MeSH
- protein ADAM17 genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- antigeny nádorové imunologie metabolismus MeSH
- biokatalýza účinky léků MeSH
- cílená molekulární terapie metody MeSH
- hypoxie enzymologie MeSH
- inhibitory enzymů terapeutické užití MeSH
- karboanhydrasy imunologie metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- monoklonální protilátky imunologie terapeutické užití MeSH
- nádory farmakoterapie enzymologie patologie MeSH
- pohyb buněk * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
