A novel library of human carbonic anhydrase (hCA) inhibitors based on the 2-sulfanilamido[1,2,4]triazolo[1,5-a]pyrimidine skeleton modified at its 7-position was prepared by an efficient convergent procedure. These derivatives were evaluated in vitro for their inhibition properties against a representative panel of hCA isoforms (hCA I, II, IV, IX, and XII). The target tumour-associated isoforms hCA IX and XII were potently inhibited with KIs in the low nanomolar range of 5-96 nM and 4-72 nM, respectively. Compounds 1d, 1j, 1v, and 1x were the most potent hCA IX inhibitors with KIs of 5.1, 8.6, 4.7, and 5.1 nM, respectively. Along with derivatives 1d and 1j, compounds 1r and 1ab potently inhibited hCA XII isoform with KIs in a single-digit nanomolar range of 8.8, 5.4, 4.3, and 9.0 nM, respectively. Compounds 1e, 1m, and 1p exhibited the best selectivity against hCA IX and hCA XII isoforms over off-target hCA II, with selectivity indexes ranging from 5 to 14.

• Klíčová slova
• Carbonic anhydrase inhibitors, [1,2,4]triazolo[15-a]pyrimidine, antiproliferative activity, structure–activity relationship, sulphanilamide,
• MeSH
• antigeny nádorové * MeSH
• inhibitory karboanhydras farmakologie   MeSH
• karboanhydrasa I metabolismus   MeSH
• karboanhydrasa II * metabolismus   MeSH
• karboanhydrasa IX metabolismus   MeSH
• lidé MeSH
• molekulární struktura MeSH
• protein - isoformy MeSH
• sulfanilamidy MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové * MeSH
• inhibitory karboanhydras MeSH
• karboanhydrasa I MeSH
• karboanhydrasa II * MeSH
• karboanhydrasa IX MeSH
• protein - isoformy MeSH
• sulfanilamidy MeSH

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.

• Klíčová slova
• LIN28/let-7 axis, carbonic anhydrase IX, hypoxia, metabolism,
• 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
• Názvy látek
• antigeny nádorové MeSH
• CA9 protein, human MeSH Prohlížeč
• karboanhydrasa IX MeSH
• Lin28A protein, human MeSH Prohlížeč
• mikro RNA MeSH
• mirnlet7 microRNA, human MeSH Prohlížeč
• proteiny vázající RNA 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
• Názvy látek
• karboanhydrasa IX MeSH
• protein ADAM17 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.

• Klíčová slova
• Apoptosis, Carbonic anhydrase IX, Chemotherapy, Ectodomain, Hypoxia, Metalloproteinase, Shedding,
• 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
• Názvy látek
• ADAM17 protein, human MeSH Prohlížeč
• cykloheximid MeSH
• karboanhydrasa IX MeSH
• monoklonální protilátky MeSH
• protein ADAM17 MeSH