Despite extensive research, the molecular role of AGR2 in the progression and metastasis of colorectal cancer (CRC) has not been fully characterized. We used quantitative mass spectrometry (SWATH MS) to identify differentially expressed proteins in paired CRC cell models of the SW480 and SW620 cell lines in response to AGR2 protein level manipulation. Relying on the results from SWATH MS and subsequent immunochemical validation, we selected NMP3 as the top candidate protein associated with AGR2 in CRC tumour cells in our screen. RT‒qPCR and immunochemical analysis confirmed the involvement of AGR2-mediated regulation of NPM3 at the transcriptional and posttranscriptional levels. Since PD-L1 is a constituent of the NPM3 regulatory axis, we aimed to correlate the changes in PD-L1 to the differential expression of AGR2 in our cell models. We found that AGR2 positively regulates PD-L1 levels in both SW480 and SW620 cell lines; additionally, several different CRC patient transcriptome cohorts confirmed the association of AGR2 with PD-L1. Our work reveals a new AGR2-NPM3 regulatory axis and the involvement of AGR2 in the regulation of PD-L1, which paves the way for the association of AGR2 with immune evasion in CRC cells.

• Klíčová slova
• AGR2, Colorectal cancer, NPM3, PD-L1,
• MeSH
• antigeny CD274 * metabolismus   genetika   MeSH
• jaderné proteiny metabolismus   genetika   MeSH
• kolorektální nádory * genetika   metabolismus   patologie   MeSH
• lidé MeSH
• mukoproteiny * metabolismus   genetika   MeSH
• nádorové buněčné linie MeSH
• nukleofosmin * MeSH
• onkogenní proteiny * metabolismus   genetika   MeSH
• proteiny * metabolismus   genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• antigeny CD274 * MeSH
• CD274 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• mukoproteiny * MeSH
• NPM3 protein, human MeSH Prohlížeč
• nukleofosmin * MeSH
• onkogenní proteiny * MeSH
• proteiny * MeSH

In this paper we compare electrochemical behavior of two homolog proteins, namely anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), playing an important role in cancer cell biology. The slight variation in their protein structures has an impact on protein adsorption and orientation at charged surface and also enables AGR2 and AGR3 to form heterocomplexes. We confirm interaction between AGR2 and AGR3 (i) in vitro by immunochemical and constant current chronopotentiometric stripping (CPS) analysis and (ii) in vivo by bioluminescence resonance energy transfer (BRET) assay. Mutation of AGR2 in dimerization domain (E60A) prevents development of wild type AGR2 dimers and also negatively affects interaction with wild type AGR3 as shown by CPS analysis. Beside new information about AGR2 and AGR3 protein including their joint interaction, our work introduces possible applications of CPS in bioanalysis of protein complexes, including those relatively unstable, but important in the cancer research.

• Klíčová slova
• AGR2/AGR3 heterocomplex formation, Anterior gradient2 protein, Anterior gradient3 protein, BRET assay, Constant current chronopotentiometric stripping,
• MeSH
• adsorpce MeSH
• kvarterní struktura proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• mukoproteiny chemie   MeSH
• multimerizace proteinu * MeSH
• nádorové proteiny chemie   MeSH
• onkogenní proteiny chemie   MeSH
• proteinové domény MeSH
• transportní proteiny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• AGR3 protein, human MeSH Prohlížeč
• mukoproteiny MeSH
• nádorové proteiny MeSH
• onkogenní proteiny MeSH
• transportní proteiny MeSH

Anterior gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) known to be overexpressed in many human epithelial cancers and is involved in cell migration, cellular transformation, angiogenesis, and metastasis. This protein inhibits the activity of the tumor suppressor p53, and its expression levels can be used to predict cancer patient outcome. However, the precise network of AGR2-interacting partners and clients remains to be fully characterized. Herein, we used label-free quantification and also stable isotope labeling with amino acids in cell culture-based LC-MS/MS analyses to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 and its interaction partners are associated with processes in the ER that maintain intracellular metabolic homeostasis and participate in the unfolded protein response, including those associated with changes in cellular metabolism, energy, and redox states in response to ER stress. As a proof of concept, the interaction between AGR2 and PDIA3, another ER-resident PDI, was studied in more detail. Pathway analysis revealed that AGR2 and PDIA3 play roles in protein folding in ER, including post-translational modification and in cellular response to stress. We confirmed the AGR2-PDIA3 complex formation in cancer cells, which was enhanced in response to ER stress. Accordingly, molecular docking characterized potential quaternary structure of this complex; however, it remains to be elucidated whether AGR2 rather contributes to PDIA3 maturation in ER, the complex directly acts in cellular signaling, or mediates AGR2 secretion. Our study provides a comprehensive insight into the protein-protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells.

• Klíčová slova
• anterior gradient protein 2, mass spectrometry, protein disulfide isomerase, protein–protein interactions, secretory pathway,
• MeSH
• chromatografie kapalinová MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• mukoproteiny * metabolismus   MeSH
• nádory * MeSH
• onkogenní proteiny * metabolismus   MeSH
• proteindisulfidisomerasy * MeSH
• simulace molekulového dockingu MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• mukoproteiny * MeSH
• onkogenní proteiny * MeSH
• proteindisulfidisomerasy * MeSH

In the past decades, highly related members of the protein disulphide isomerase family, anterior gradient protein AGR2 and AGR3, attracted researchers' attention due to their putative involvement in developmental processes and carcinogenesis. While AGR2 has been widely demonstrated as a metastasis-related protein whose elevated expression predicts worse patient outcome, little is known about AGR3's role in tumour biology. Thus, we aim to confront the issue of AGR3 function in physiology and pathology in the following review by comparing this protein with the better-described homologue AGR2. Relying on available data and in silico analyses, we show that AGR proteins are co-expressed or uncoupled in context-dependent manners in diverse carcinomas and healthy tissues. Further, we discuss plausible roles of both proteins in tumour-associated processes such as differentiation, proliferation, migration, invasion and metastasis. This work brings new hints and stimulates further thoughts on hitherto unresolved conundrum of anterior gradient protein function.

• Klíčová slova
• AGR2, AGR3, Cancer, PDI family,
• MeSH
• buněčná diferenciace MeSH
• invazivní růst nádoru MeSH
• lidé MeSH
• mukoproteiny MeSH
• nádorové proteiny metabolismus   MeSH
• nádory metabolismus   patologie   MeSH
• onkogenní proteiny MeSH
• počítačová simulace MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• proteiny metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• AGR3 protein, human MeSH Prohlížeč
• mukoproteiny MeSH
• nádorové proteiny MeSH
• onkogenní proteiny MeSH
• proteiny MeSH
• transportní proteiny MeSH

Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.

• Klíčová slova
• AGR2, TMED2, endoplasmic reticulum, inflammation, proteostasis,
• MeSH
• endoplazmatické retikulum genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• homeostáze proteinů * MeSH
• lidé MeSH
• mukoproteiny genetika   metabolismus   MeSH
• multimerizace proteinu * MeSH
• myši MeSH
• onkogenní proteiny genetika   metabolismus   MeSH
• stres endoplazmatického retikula * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• Agr2 protein, mouse MeSH Prohlížeč
• mukoproteiny MeSH
• onkogenní proteiny MeSH

AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.

• MeSH
• adhezní molekula epiteliálních buněk genetika   metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• mukoproteiny MeSH
• onkogenní proteiny MeSH
• peptidy metabolismus   MeSH
• proteiny genetika   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adhezní molekula epiteliálních buněk MeSH
• AGR2 protein, human MeSH Prohlížeč
• EPCAM protein, human MeSH Prohlížeč
• MDM2 protein, human MeSH Prohlížeč
• mukoproteiny MeSH
• onkogenní proteiny MeSH
• peptidy MeSH
• proteiny MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• rekombinantní proteiny MeSH

The detection of cancer antigens is a major aim of cancer research in order to develop better patient management through early disease detection. Many cancers including prostate, lung, and ovarian secrete a protein disulfide isomerase protein named AGR2 that has been previously detected in urine and plasma using mass spectrometry. Here we determine whether a previously developed monoclonal antibody targeting AGR2 can be adapted from an indirect two-site ELISA format into a direct detector using solid-phase printed gold electrodes. The screen-printed gold electrode was surface functionalized with the anti-AGR2 specific monoclonal antibody. The interaction of the recombinant AGR2 protein and the anti-AGR2 monoclonal antibody functionalized electrode changed its electrochemical impedance spectra. Nyquist diagrams were obtained after incubation in an increasing concentration of purified AGR2 protein with a range of concentrations from 0.01 fg/mL to 10 fg/mL. In addition, detection of the AGR2 antigen can be achieved from cell lysates in medium or artificial buffer. These data highlight the utility of an AGR2-specific monoclonal antibody that can be functionalized onto a gold printed electrode for a one-step capture and quantitation of the target antigen. These platforms have the potential for supporting methodologies using more complex bodily fluids including plasma and urine for improved cancer diagnostics.

• Klíčová slova
• AGR2 protein, electrochemical impedance spectroscopy, screen-printed gold electrode, sensor,
• MeSH
• biosenzitivní techniky * MeSH
• elektrochemické techniky MeSH
• elektrody MeSH
• kovové nanočástice MeSH
• lidé MeSH
• limita detekce MeSH
• monoklonální protilátky MeSH
• mukoproteiny analýza   MeSH
• nádory MeSH
• onkogenní proteiny analýza   MeSH
• zlato MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• monoklonální protilátky MeSH
• mukoproteiny MeSH
• onkogenní proteiny MeSH
• zlato MeSH

The tumor suppressor p53 plays a key role in malignant transformation and tumor development. However, the frequency of p53 mutations within individual types of cancer is different, suggesting the existence of other mechanisms attenuating p53 tumor suppressor activity. Changes in upstream regulators of p53 such as MDM2 amplification and overexpression, expression of viral oncoproteins, estrogen receptor signaling, or changes in p53 transcriptional target genes were previously described in wild-type p53 tumors. We identified a novel pathway responsible for attenuation of p53 activity in human cancers. We demonstrate that AGR2, which is overexpressed in a variety of human cancers and provides a poor prognosis, up-regulates DUSP10 which subsequently inhibits p38 MAPK and prevents p53 activation by phosphorylation. Analysis of human breast cancers reveals that AGR2 specifically provides a poor prognosis in ER+ breast cancers with wild-type p53 but not ER- or mutant p53 breast cancers, and analysis of independent data sets show that DUSP10 levels also have prognostic significance in this specific sub-group of patients. These data not only reveal a novel pro-oncogenic signaling pathway mediating resistance to DNA damaging agents in human tumors, but also has implications for designing alternative strategies for modulation of wild-type p53 activity in cancer therapy.

• Klíčová slova
• AGR2, Breast cancer, DUSP10, Drug resistance, p38 MAPK, p53,
• MeSH
• aktivace enzymů účinky léků   MeSH
• chemorezistence MeSH
• dospělí MeSH
• fosfatasy MAP kinas metabolismus   MeSH
• fosfatasy s dvojí specifitou metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• mukoproteiny MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory prsu farmakoterapie   metabolismus   patologie   MeSH
• nádory farmakoterapie   metabolismus   patologie   MeSH
• onkogenní proteiny MeSH
• proteiny metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• prsy účinky léků   metabolismus   patologie   MeSH
• senioři MeSH
• signální transdukce * účinky léků   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• DUSP10 protein, human MeSH Prohlížeč
• fosfatasy MAP kinas MeSH
• fosfatasy s dvojí specifitou MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• mukoproteiny MeSH
• nádorový supresorový protein p53 MeSH
• onkogenní proteiny MeSH
• proteiny MeSH
• protinádorové látky MeSH

BACKGROUND: During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance. METHODS: The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay. RESULTS: Induction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2. CONCLUSION: Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.

• Klíčová slova
• AGR2, E-cadherin, EMT, Metastasis, TGF-β, Vimentin,
• MeSH
• buněčná adheze genetika   MeSH
• epitelo-mezenchymální tranzice účinky léků   genetika   MeSH
• genový knockdown MeSH
• kadheriny metabolismus   MeSH
• lidé MeSH
• mukoproteiny MeSH
• nádorové buněčné linie MeSH
• onkogenní proteiny MeSH
• pohyb buněk genetika   MeSH
• proteiny Smad metabolismus   MeSH
• proteiny genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• transformující růstový faktor beta farmakologie   MeSH
• vimentin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• kadheriny MeSH
• mukoproteiny MeSH
• onkogenní proteiny MeSH
• proteiny Smad MeSH
• proteiny MeSH
• transformující růstový faktor beta MeSH
• vimentin MeSH

Expression of the AGR2 oncogene was shown to be associated with estrogen receptor positive tumors. This gene contributes to enhanced cellular proliferation, drug resistance, metastasis development and may also serve as a predictor of poor prognosis. However, our analysis of AGR2 expression in a subset of estrogen-receptor negative tumors revealed that AGR2 could also be upregulated in hormone-independent manner. AGR2 expression was shown to be significantly increased in HER2 positive breast tumors on both the mRNA and the protein level. Moreover, in a subset of estrogen- and progesterone-receptor negative and simultaneously HER2-positive cases, increased AGR2 expression significantly correlated with worse patient prognosis. Subsequent analysis of independent data sets either collected in our institute or obtained from Oncomine cancer microarray database confirmed all these findings.

• Klíčová slova
• AGR2, Breast cancer, HER2,
• MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mukoproteiny MeSH
• nádorové buněčné linie MeSH
• nádory prsu diagnóza   genetika   MeSH
• onkogenní proteiny MeSH
• prognóza MeSH
• proliferace buněk MeSH
• proteiny genetika   metabolismus   MeSH
• receptor erbB-2 genetika   MeSH
• receptory pro estrogeny genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• upregulace MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• ERBB2 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• mukoproteiny MeSH
• onkogenní proteiny MeSH
• proteiny MeSH
• receptor erbB-2 MeSH
• receptory pro estrogeny MeSH