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.

Central European Institute for Technology Masaryk University Brno Czech Republic

Department of Biochemistry Faculty of Science Masaryk University Brno Czech Republic

Department of Human Protein Sciences Faculty of Medicine University of Geneva Geneva Switzerland

INSERM U1242 Chemistry Oncogenesis Stress Signaling Université Rennes 1 Rennes France

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Brno Czech Republic; International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Brno Czech Republic

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Brno Czech Republic; National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic

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Ryan D.P., Matthews J.M. Protein-protein interactions in human disease. Curr. Opin. Struct. Biol. 2005;15:441–446. PubMed

Maryas J., Faktor J., Dvorakova M., Struharova I., Grell P., Bouchal P. Proteomics in investigation of cancer metastasis: Functional and clinical consequences and methodological challenges. Proteomics. 2014;14:426–440. PubMed

Chevet E., Fessart D., Delom F., Mulot A., Vojtesek B., Hrstka R., Murray E., Gray T., Hupp T. Emerging roles for the pro-oncogenic anterior gradient-2 in cancer development. Oncogene. 2013;32:2499–2509. PubMed

Delom F., Mohtar M.A., Hupp T., Fessart D. The anterior gradient-2 interactome. Am. J. Physiol. Cell Physiol. 2020;318:C40–C47. PubMed

Persson S., Rosenquist M., Knoblach B., Khosravi-Far R., Sommarin M., Michalak M. Diversity of the protein disulfide isomerase family: Identification of breast tumor induced Hag2 and Hag3 as novel members of the protein family. Mol. Phylogenet. Evol. 2005;36:734–740. PubMed

Higa A., Mulot A., Delom F., Bouchecareilh M., Nguyen D.T., Boismenu D., Wise M.J., Chevet E. Role of pro-oncogenic protein disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) in the control of endoplasmic reticulum homeostasis. J. Biol. Chem. 2011;286:44855–44868. PubMed PMC

Brychtova V., Vojtesek B., Hrstka R. Anterior gradient 2: A novel player in tumor cell biology. Cancer Lett. 2011;304:1–7. PubMed

Hu R., Huffman K.E., Chu M., Zhang Y., Minna J.D., Yu Y. Quantitative secretomic analysis identifies extracellular protein factors that modulate the metastatic phenotype of non-small cell lung cancer. J. Proteome Res. 2016;15:477–486. PubMed PMC

Wayner E.A., Quek S.I., Ahmad R., Ho M.E., Loprieno M.A., Zhou Y., Ellis W.J., True L.D., Liu A.Y. Development of an ELISA to detect the secreted prostate cancer biomarker AGR2 in voided urine. Prostate. 2012;72:1023–1034. PubMed

Di Maro G., Salerno P., Unger K., Orlandella F.M., Monaco M., Chiappetta G., Thomas G., Oczko-Wojciechowska M., Masullo M., Jarzab B., Santoro M., Salvatore G. Anterior gradient protein 2 promotes survival, migration and invasion of papillary thyroid carcinoma cells. Mol. Cancer. 2014;13:160. PubMed PMC

Hrstka R., Bouchalova P., Michalova E., Matoulkova E., Muller P., Coates P.J., Vojtesek B. AGR2 oncoprotein inhibits p38 MAPK and p53 activation through a DUSP10-mediated regulatory pathway. Mol. Oncol. 2016;10:652–662. PubMed PMC

Wang Z., Hao Y., Lowe A.W. The adenocarcinoma-associated antigen, AGR2, promotes tumor growth, cell migration, and cellular transformation. Cancer Res. 2008;68:492–497. PubMed

Fletcher G.C., Patel S., Tyson K., Adam P.J., Schenker M., Loader J.A., Daviet L., Legrain P., Parekh R., Harris A.L., Terrett J.A. hAG-2 and hAG-3, human homologues of genes involved in differentiation, are associated with oestrogen receptor-positive breast tumours and interact with metastasis gene C4.4a and dystroglycan. Br. J. Cancer. 2003;88:579–585. PubMed PMC

Park S.W., Zhen G., Verhaeghe C., Nakagami Y., Nguyenvu L.T., Barczak A.J., Killeen N., Erle D.J. The protein disulfide isomerase AGR2 is essential for production of intestinal mucus. Proc. Natl. Acad. Sci. U. S. A. 2009;106:6950–6955. PubMed PMC

Maurel M., Obacz J., Avril T., Ding Y.P., Papadodima O., Treton X., Daniel F., Pilalis E., Horberg J., Hou W., Beauchamp M.C., Tourneur-Marsille J., Cazals-Hatem D., Sommerova L., Samali A., et al. Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation. EMBO Mol. Med. 2019;11:e10120. PubMed PMC

Stark C., Breitkreutz B.J., Reguly T., Boucher L., Breitkreutz A., Tyers M. BioGRID: A general repository for interaction datasets. Nucleic Acids Res. 2006;34:D535–D539. PubMed PMC

Murray E., McKenna E.O., Burch L.R., Dillon J., Langridge-Smith P., Kolch W., Pitt A., Hupp T.R. Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2. Biochemistry. 2007;46:13742–13751. PubMed

Prochazkova I., Lenco J., Fucikova A., Dresler J., Capkova L., Hrstka R., Nenutil R., Bouchal P. Targeted proteomics driven verification of biomarker candidates associated with breast cancer aggressiveness. Biochim. Biophys. Acta Proteins Proteom. 2017;1865:488–498. PubMed

Bouchal P., Roumeliotis T., Hrstka R., Nenutil R., Vojtesek B., Garbis S.D. Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis. J. Proteome Res. 2009;8:362–373. PubMed

Shannon P., Markiel A., Ozier O., Baliga N.S., Wang J.T., Ramage D., Amin N., Schwikowski B., Ideker T. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–2504. PubMed PMC

Bindea G., Galon J., Mlecnik B. CluePedia cytoscape plugin: Pathway insights using integrated experimental and in silico data. Bioinformatics. 2013;29:661–663. PubMed PMC

Bindea G., Mlecnik B., Hackl H., Charoentong P., Tosolini M., Kirilovsky A., Fridman W.H., Pages F., Trajanoski Z., Galon J. ClueGO: A cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics. 2009;25:1091–1093. PubMed PMC

Mohtar M.A., Hernychova L., O'Neill J.R., Lawrence M.L., Murray E., Vojtesek B., Hupp T.R. The sequence-specific peptide-binding activity of the protein sulfide isomerase AGR2 directs its stable binding to the oncogenic receptor EpCAM. Mol. Cell Proteomics. 2018;17:737–763. PubMed PMC

Lamiable A., Thevenet P., Rey J., Vavrusa M., Derreumaux P., Tuffery P. PEP-FOLD3: Faster de novo structure prediction for linear peptides in solution and in complex. Nucleic Acids Res. 2016;44:W449–W454. PubMed PMC

Shen Y., Maupetit J., Derreumaux P., Tuffery P. Improved PEP-FOLD approach for peptide and miniprotein structure prediction. J. Chem. Theor. Comput. 2014;10:4745–4758. PubMed

Thevenet P., Shen Y., Maupetit J., Guyon F., Derreumaux P., Tuffery P. PEP-FOLD: An updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides. Nucleic Acids Res. 2012;40:W288–W293. PubMed PMC

van Zundert G.C.P., Rodrigues J., Trellet M., Schmitz C., Kastritis P.L., Karaca E., Melquiond A.S.J., van Dijk M., de Vries S.J., Bonvin A. The HADDOCK2.2 web server: User-friendly integrative modeling of biomolecular complexes. J. Mol. Biol. 2016;428:720–725. PubMed

Patel P., Clarke C., Barraclough D.L., Jowitt T.A., Rudland P.S., Barraclough R., Lian L.Y. Metastasis-promoting anterior gradient 2 protein has a dimeric thioredoxin fold structure and a role in cell adhesion. J. Mol. Biol. 2013;425:929–943. PubMed

Nguyen V.D., Biterova E., Salin M., Wierenga R.K., Ruddock L.W. Crystal structure of human anterior gradient protein 3. Acta Crystallogr. F Struct. Biol. Commun. 2018;74(Pt 7):425–430. PubMed PMC

Berman H.M., Westbrook J., Feng Z., Gilliland G., Bhat T.N., Weissig H., Shindyalov I.N., Bourne P.E. The protein data bank. Nucleic Acids Res. 2000;28:235–242. PubMed PMC

Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 1990;215:403–410. PubMed

Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. PubMed PMC

Park T., Won J., Baek M., Seok C. GalaxyHeteromer: Protein heterodimer structure prediction by template-based and ab initio docking. Nucleic Acids Res. 2021;49:W237–W241. PubMed PMC

Desta I.T., Porter K.A., Xia B., Kozakov D., Vajda S. Performance and its limits in rigid body protein-protein docking. Structure. 2020;28:1071–1081.e3. PubMed PMC

Dong G., Wearsch P.A., Peaper D.R., Cresswell P., Reinisch K.M. Insights into MHC class I peptide loading from the structure of the tapasin-ERp57 thiol oxidoreductase heterodimer. Immunity. 2009;30:21–32. PubMed PMC

Laskowski R.A., Jablonska J., Pravda L., Varekova R.S., Thornton J.M. PDBsum: Structural summaries of PDB entries. Protein Sci. 2018;27:129–134. PubMed PMC

Moretti R., Lyskov S., Das R., Meiler J., Gray J.J. Web-accessible molecular modeling with Rosetta: The Rosetta online server that includes everyone (ROSIE) Protein Sci. 2018;27:259–268. PubMed PMC

Marin-Lopez M.A., Planas-Iglesias J., Aguirre-Plans J., Bonet J., Garcia-Garcia J., Fernandez-Fuentes N., Oliva B. On the mechanisms of protein interactions: Predicting their affinity from unbound tertiary structures. Bioinformatics. 2018;34:592–598. PubMed PMC

Bouchal P., Dvorakova M., Scherl A., Garbis S.D., Nenutil R., Vojtesek B. Intact protein profiling in breast cancer biomarker discovery: Protein identification issue and the solutions based on 3D protein separation, bottom-up and top-down mass spectrometry. Proteomics. 2013;13:1053–1058. PubMed

Holden P., Horton W.A. Crude subcellular fractionation of cultured mammalian cell lines. BMC Res. Notes. 2009;2:243. PubMed PMC

Moser B., Hochreiter B., Herbst R., Schmid J.A. Fluorescence colocalization microscopy analysis can be improved by combining object-recognition with pixel-intensity-correlation. Biotechnol. J. 2017;12:1600332. PubMed PMC

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., Tinevez J.Y., White D.J., Hartenstein V., Eliceiri K., Tomancak P., et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. PubMed PMC

Ostatna V., Vargova V., Hrstka R., Durech M., Vojtesek B., Palecek E. Effect of His(6)-tagging of anterior gradient 2 protein on its electro-oxidation. Electrochimica Acta. 2014;150:218–222.

Sicari D., Centonze F.G., Pineau R., Le Reste P.J., Negroni L., Chat S., Mohtar M.A., Thomas D., Gillet R., Hupp T., Chevet E., Igbaria A. Reflux of endoplasmic reticulum proteins to the cytosol inactivates tumor suppressors. EMBO Rep. 2021;22 PubMed PMC

Bommiasamy H., Back S.H., Fagone P., Lee K., Meshinchi S., Vink E., Sriburi R., Frank M., Jackowski S., Kaufman R.J., Brewer J.W. ATF6alpha induces XBP1-independent expansion of the endoplasmic reticulum. J. Cell Sci. 2009;122(Pt 10):1626–1636. PubMed PMC

Schuck S., Prinz W.A., Thorn K.S., Voss C., Walter P. Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response. J. Cell Biol. 2009;187:525–536. PubMed PMC

Sriburi R., Bommiasamy H., Buldak G.L., Robbins G.R., Frank M., Jackowski S., Brewer J.W. Coordinate regulation of phospholipid biosynthesis and secretory pathway gene expression in XBP-1(S)-induced endoplasmic reticulum biogenesis. J. Biol. Chem. 2007;282:7024–7034. PubMed

Cernocka H., Vonka P., Kasalova V., Sommerova L., Vandova V., Hrstka R., Ostatna V. AGR2-AGR3 hetero-oligomeric complexes: Identification and characterization. Bioelectrochemistry. 2021;140:107808. PubMed

Pirneskoski A., Klappa P., Lobell M., Williamson R.A., Byrne L., Alanen H.I., Salo K.E., Kivirikko K.I., Freedman R.B., Ruddock L.W. Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase. J. Biol. Chem. 2004;279:10374–10381. PubMed

Lee E., Lee D.H. Emerging roles of protein disulfide isomerase in cancer. BMB Rep. 2017;50:401–410. PubMed PMC

Arumugam T., Deng D., Bover L., Wang H., Logsdon C.D., Ramachandran V. New blocking antibodies against novel AGR2-C4.4A pathway reduce growth and metastasis of pancreatic tumors and increase survival in mice. Mol. Cancer Ther. 2015;14:941–951. PubMed PMC

Dong A., Wodziak D., Lowe A.W. Epidermal growth factor receptor (EGFR) signaling requires a specific endoplasmic reticulum thioredoxin for the post-translational control of receptor presentation to the cell surface. J. Biol. Chem. 2015;290:8016–8027. PubMed PMC

Stelzl U., Worm U., Lalowski M., Haenig C., Brembeck F.H., Goehler H., Stroedicke M., Zenkner M., Schoenherr A., Koeppen S., Timm J., Mintzlaff S., Abraham C., Bock N., Kietzmann S., et al. A human protein-protein interaction network: A resource for annotating the proteome. Cell. 2005;122:957–968. PubMed

Tiemann K., Garri C., Lee S.B., Malihi P.D., Park M., Alvarez R.M., Yap L.P., Mallick P., Katz J.E., Gross M.E., Kani K. Loss of ER retention motif of AGR2 can impact mTORC signaling and promote cancer metastasis. Oncogene. 2019;38:3003–3018. PubMed PMC

Worfolk J.C., Bell S., Simpson L.D., Carne N.A., Francis S.L., Engelbertsen V., Brown A.P., Walker J., Viswanath Y.K., Benham A.M. Elucidation of the AGR2 interactome in esophageal adenocarcinoma cells identifies a redox-sensitive chaperone Hub for the quality control of MUC-5AC. Antioxid. Redox Signal. 2019;31:1117–1132. PubMed

Ryu J., Park S.G., Lee P.Y., Cho S., Lee D.H., Kim G.H., Kim J.H., Park B.C. Dimerization of pro-oncogenic protein anterior gradient 2 is required for the interaction with BiP/GRP78. Biochem. Biophys. Res. Commun. 2013;430:610–615. PubMed

Pobre K.F.R., Poet G.J., Hendershot L.M. The endoplasmic reticulum (ER) chaperone BiP is a master regulator of ER functions: Getting by with a little help from ERdj friends. J. Biol. Chem. 2019;294:2098–2108. PubMed PMC

Gold L.I., Eggleton P., Sweetwyne M.T., Van Duyn L.B., Greives M.R., Naylor S.M., Michalak M., Murphy-Ullrich J.E. Calreticulin: Non-endoplasmic reticulum functions in physiology and disease. FASEB J. 2010;24:665–683. PubMed PMC

Kielbik M., Szulc-Kielbik I., Klink M. Calreticulin-multifunctional chaperone in immunogenic cell death: Potential significance as a prognostic biomarker in ovarian cancer patients. Cells. 2021;10:130. PubMed PMC

Sommerova L., Ondrouskova E., Martisova A., Zoumpourlis V., Galtsidis S., Hrstka R. ZEB1/miR-200c/AGR2: A new regulatory loop modulating the epithelial-mesenchymal transition in lung adenocarcinomas. Cancers (Basel) 2020;12:1614. PubMed PMC

Diaz P., Sandoval-Borquez A., Bravo-Sagua R., Quest A.F.G., Lavandero S. Perspectives on organelle interaction, protein dysregulation, and cancer disease. Front. Cell Dev. Biol. 2021;9:613336. PubMed PMC

Shishkin S.S., Eremina L.S., Kovalev L.I., Kovaleva M.A. AGR2, ERp57/GRP58, and some other human protein disulfide isomerases. Biochemistry (Mosc) 2013;78:1415–1430. PubMed

Coe H., Michalak M. ERp57, a multifunctional endoplasmic reticulum resident oxidoreductase. Int. J. Biochem. Cell Biol. 2010;42:796–799. PubMed

Groenendyk J., Peng Z., Dudek E., Fan X., Mizianty M.J., Dufey E., Urra H., Sepulveda D., Rojas-Rivera D., Lim Y., Kim D.H., Baretta K., Srikanth S., Gwack Y., Ahnn J., et al. Interplay between the oxidoreductase PDIA6 and microRNA-322 controls the response to disrupted endoplasmic reticulum calcium homeostasis. Sci. Signal. 2014;7:ra54. PubMed PMC

Fessart D., Domblides C., Avril T., Eriksson L.A., Begueret H., Pineau R., Malrieux C., Dugot-Senant N., Lucchesi C., Chevet E., Delom F. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties. Elife. 2016;5:e13887. PubMed PMC

Shental-Bechor D., Levy Y. Folding of glycoproteins: Toward understanding the biophysics of the glycosylation code. Curr. Opin. Struct. Biol. 2009;19:524–533. PubMed

Marjon P.L., Bobrovnikova-Marjon E.V., Abcouwer S.F. Expression of the pro-angiogenic factors vascular endothelial growth factor and interleukin-8/CXCL8 by human breast carcinomas is responsive to nutrient deprivation and endoplasmic reticulum stress. Mol. Cancer. 2004;3:4. PubMed PMC

Mihailidou C., Panagiotou C., Kiaris H., Kassi E., Moutsatsou P. Crosstalk between C/EBP homologous protein (CHOP) and glucocorticoid receptor in lung cancer. Mol. Cell Endocrinol. 2016;436:211–223. PubMed

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D.J., Inuganti A., Griss J., Mayer G., Eisenacher M., Perez E., Uszkoreit J., Pfeuffer J., Sachsenberg T., Yilmaz S., et al. The PRIDE database and related tools and resources in 2019: Improving support for quantification data. Nucleic Acids Res. 2019;47:D442–D450. PubMed PMC

Characterization of the AGR2-NPM3 axis uncovers the AGR2 involvement in PD-L1 regulation in colorectal cancer

Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition