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.

Department of Experimental Biology Faculty of Science 117204 Masaryk University Kamenice 5 Brno 62500 Czech Republic

International Centre for Cancer Vaccine Science University of Gdansk Kladki 24 Gdansk 80 822 Poland

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 Brno 62500 Czech Republic

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Zluty Kopec 7 Brno 65653 Czech Republic

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Robinson, P. J. & Bulleid, N. J. Mechanisms of disulfide bond formation in nascent polypeptides entering the secretory pathway. PubMed PMC

Beatty, G. L. & Gladney, W. L. Immune escape mechanisms as a guide for cancer immunotherapy. PubMed PMC

Boisteau, E. et al. Anterior gradient proteins in gastrointestinal cancers: From cell biology to pathophysiology. PubMed

Sommerova, L. et al. ZEB1/miR-200c/AGR2: a New Regulatory Loop modulating the epithelial-mesenchymal transition in lung adenocarcinomas. PubMed PMC

Sommerova, L., Ondrouskova, E., Vojtesek, B. & Hrstka, R. Suppression of AGR2 in a TGF-beta-induced smad regulatory pathway mediates epithelial-mesenchymal transition. PubMed DOI PMC

Delom, F., Nazaraliyev, A. & Fessart, D. The role of protein disulphide isomerase AGR2 in the tumour niche. PubMed

Sicari, D. et al. Reflux of endoplasmic reticulum proteins to the cytosol inactivates tumor suppressors. PubMed PMC

Hrstka, R. et al. AGR2 oncoprotein inhibits p38 MAPK and p53 activation through a DUSP10-mediated regulatory pathway. PubMed DOI PMC

Alsereihi, R. et al. Leveraging the role of the metastatic associated protein anterior gradient homologue 2 in unfolded protein degradation: A novel therapeutic biomarker for cancer. PubMed DOI PMC

Okuwaki, M. et al. Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling. PubMed PMC

Huang, N., Negi, S., Szebeni, A. & Olson, M. O. J. Protein NPM3 interacts with the multifunctional nucleolar protein B23/nucleophosmin and inhibits ribosome biogenesis. PubMed

Wang, H. et al. Pumilio1 regulates NPM3/NPM1 axis to promote PD-L1-mediated immune escape in gastric cancer. PubMed

Wei, S., Xing, J., Lu, K., Wang, K. & Yu, W. NPM3 as a novel oncogenic factor and poor prognostic marker contributes to cell proliferation and migration in lung adenocarcinoma. PubMed PMC

Han, Y., Liu, D. & Li, L. PD-1/PD-L1 pathway: Current researches in cancer. PubMed PMC

El-Sayes, N., Vito, A. & Mossman, K. Tumor heterogeneity: A great barrier in the age of cancer immunotherapy. PubMed PMC

Hewitt, R. E. et al. Validation of a model of colon cancer progression. PubMed

Dumartin, L. et al. ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation. PubMed DOI PMC

Rouillard, A. D. et al. The harmonizome: A collection of processed datasets gathered to serve and mine knowledge about genes and proteins. PubMed DOI PMC

Ciribilli, Y., Singh, P., Inga, A. & Borlak, J. c-Myc targeted regulators of cell metabolism in a transgenic mouse model of papillary lung adenocarcinoma. PubMed PMC

Adomavicius, T. et al. The structural basis of translational control by eIF2 phosphorylation. PubMed PMC

Costa-Mattioli, M. & Walter, P. The integrated stress response: From mechanism to disease. PubMed PMC

Bouchalova, P. et al. Characterization of the AGR2 interactome uncovers new players of protein disulfide Isomerase Network in Cancer cells. PubMed PMC

Higa, A. et al. Role of pro-oncogenic protein disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) in the control of endoplasmic reticulum homeostasis. PubMed DOI PMC

Garcia-Diaz, A. et al. Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression. PubMed PMC

Antonangeli, F. et al. Regulation of PD-L1 expression by NF-κB in Cancer. PubMed DOI PMC

Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. PubMed PMC

de Bruijn, I. et al. Analysis and visualization of longitudinal genomic and clinical data from the AACR Project GENIE Biopharma Collaborative in cBioPortal. PubMed PMC

Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. PubMed PMC

Muzny, D. M. et al. Comprehensive molecular characterization of human colon and rectal cancer. PubMed PMC

Roelands, J. et al. An integrated tumor, immune and microbiome atlas of colon cancer. PubMed PMC

Vanderlaag, K. E. et al. Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-alpha and survivin. PubMed DOI PMC

Gadad, S. S., Shandilya, J., Kishore, A. H. & Kundu, T. K. NPM3, a member of the nucleophosmin/nucleoplasmin family, enhances activator-dependent transcription. PubMed

Wei, Q. et al. Pan-cancer analysis of the prognostic and immunological role of nucleophosmin/nucleoplasmin 3 (NPM3) and its potential significance in lung adenocarcinoma. PubMed PMC

Qin, G. et al. NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer. PubMed PMC

Qin, G. et al. Targeting the NAT10/NPM1 axis abrogates PD-L1 expression and improves the response to immune checkpoint blockade therapy. PubMed PMC

Chen, Z. et al. Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cord-derived mesenchymal stem cells by increasing PD-L1 expression. PubMed PMC

Ohmori, Y., Schreiber, R. D. & Hamilton, T. A. Synergy between interferon-gamma and tumor necrosis factor-alpha in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor kappaB. PubMed

Borgo, C., D’Amore, C., Sarno, S., Salvi, M. & Ruzzene, M. Protein kinase CK2: A potential therapeutic target for diverse human diseases. PubMed DOI PMC

de Bie, P. et al. Characterization of COMMD protein-protein interactions in NF-kappaB signalling. PubMed DOI PMC

Okazaki, T. & Honjo, T. The PD-1-PD-L pathway in immunological tolerance. PubMed

Dong, Y., Sun, Q. & Zhang, X. PD-1 and its ligands are important immune checkpoints in cancer. PubMed PMC

Li, Y. et al. Efficacy and safety of anti-PD-1/PD-L1 therapy in the treatment of advanced colorectal cancer: A meta-analysis. PubMed PMC

Lin, K. X. et al. PD-1 and PD-L1 inhibitors in cold colorectal cancer: Challenges and strategies. PubMed PMC

Rosenbaum, M. W., Bledsoe, J. R., Morales-Oyarvide, V., Huynh, T. G. & Mino-Kenudson, M. PD-L1 expression in colorectal cancer is associated with microsatellite instability, BRAF mutation, medullary morphology and cytotoxic tumor-infiltrating lymphocytes. PubMed

Williams, D. S. et al. Nonsense mediated decay resistant mutations are a source of expressed mutant proteins in colon cancer cell lines with microsatellite instability. PubMed PMC

Chevet, E. et al. AGR2 protein expression in colorectal tumour epithelialcompartment. PubMed DOI PMC

Fessart, D. et al. Anterior Gradient-2 (AGR2) is overexpressed in colon cancer and is a potential biomarker of microsatellite instability (MSI) tumors.

Boland, C. R. & Goel, A. Microsatellite instability in colorectal cancer. PubMed PMC

Zheng, Z. et al. T cells in colorectal cancer: Unravelling the function of different T cell subsets in the tumor microenvironment. PubMed PMC

Berg, K. C. G. et al. Multi-omics of 34 colorectal cancer cell lines—A resource for biomedical studies. PubMed DOI PMC

Cai, L., Chen, A. & Tang, D. A new strategy for immunotherapy of microsatellite-stable (MSS)-type advanced colorectal cancer: Multi-pathway combination therapy with PD-1/PD-L1 inhibitors. PubMed DOI

Ohm, J. E. et al. VEGF inhibits T-cell development and may contribute to tumor-induced immune suppression. PubMed

Sullivan, K. M. et al. Blockade of interleukin 10 potentiates antitumour immune function in human colorectal cancer liver metastases. PubMed PMC

Tauriello, D. V. F. et al. TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis. PubMed

Wang, F. et al. Combined anti-PD-1, HDAC inhibitor and anti-VEGF for MSS/pMMR colorectal cancer: A randomized phase 2 trial. PubMed

Zhang, Q. et al. Control of cyclin D1 and breast tumorigenesis by the EglN2 prolyl hydroxylase. PubMed PMC

Martisova, A. et al. AGR2 silencing contributes to metformin-dependent sensitization of colorectal cancer cells to chemotherapy. PubMed DOI PMC

Wiśniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. PubMed

Bouchal, P. et al. 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. PubMed DOI

Faktor, J. & Bouchal, P. Building mass spectrometry spectral libraries of human cancer cell lines. PubMed DOI

Bouchal, P. et al. Breast cancer classification based on proteotypes obtained by SWATH mass spectrometry. PubMed DOI PMC

Kong, A. T., Leprevost, F. V., Avtonomov, D. M., Mellacheruvu, D. & Nesvizhskii, A. I. MSFragger: Ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics. PubMed DOI PMC

MacLean, B. et al. Skyline: An open source document editor for creating and analyzing targeted proteomics experiments. PubMed DOI PMC

Reiter, L. et al. mProphet: Automated data processing and statistical validation for large-scale SRM experiments. PubMed DOI

Choi, M. et al. MSstats: An R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments. PubMed DOI

Villanueva, R. A. M. & Chen, Z. J. ggplot2: Elegant graphics for data analysis (2nd ed.). DOI

Galili, T., O’Callaghan, A., Sidi, J. & Sievert, C. Heatmaply: An R package for creating interactive cluster heatmaps for online publishing. PubMed DOI PMC

PCAtools, P. C. A. Everything Principal Components Analysis. R package version 2.16.0 (2024). https://github.com/kevinblighe/PCAtools.