NR3C1, LAX1, and RCAN3 as Circulating Epigenetic Biomarkers for Prognosis and Chemotherapy Response Prediction in Metastatic Pancreatic Cancer

Abstract

Pancreatic cancer remains highly lethal, largely due to late diagnosis and limited efficacy of treatments. Improving first-line treatment selection and patient monitoring requires novel, non-invasive biomarkers beyond carbohydrate antigen 19-9 (CA19-9) and imaging. This study investigates epigenetic biomarkers from liquid biopsy with prognostic and predictive potential in metastatic pancreatic ductal adenocarcinoma (PDAC; mPDAC). Genome-wide methylation profiling of cell-free DNA (cfDNA) from healthy individuals and stage IV mPDAC patients identified 13 gene-associated CpG sites with significantly altered methylation patterns. ddPCR validation confirmed consistent methylation differences in lymphocyte transmembrane adaptor 1 (LAX1), nuclear receptor subfamily 3 group C member 1 (NR3C1), and RCAN3 between healthy and patient groups. Elevated LAX1 and RCAN3 methylation and reduced NR3C1 methylation at diagnosis were associated with poor prognosis and correlated with high-risk circulating biomarker profiles, including CA19-9 levels, RAS MAF (mutant allele fraction), cfDNA concentration, and cfDNA fragmentation. Notably, baseline NR3C1 methylation levels predicted response to first-line FOLFIRINOX-based treatment with an acceptable 75% sensitivity and a high specificity of 92.86%. These findings highlight the clinical significance of cfDNA methylation as a minimally invasive biomarker source, emphasizing LAX1, NR3C1, and RCAN3 as prognostic biomarkers in mPDAC. Specifically, baseline NR3C1 methylation emerges as a promising predictor of treatment response, supporting personalized therapeutic strategies in mPDAC.

Keywords

DNA methylation and response prediction / liquid biopsy / pancreatic ductal adenocarcinoma

References

[1] H. Wei and H. Ren, “Precision Treatment of Pancreatic Ductal Adenocarcinoma,” Cancer Letters 585 (2024): 216636.

[2] C. Bosetti, V. Rosato, D. Li, et al., “Diabetes, Antidiabetic Medications, and Pancreatic Cancer Risk: An Analysis From the International Pancreatic Cancer Case-Control Consortium,” Annals of Oncology 25, no. 10 (2014): 2065–2072.

[3] J. D. Mizrahi, R. Surana, J. W. Valle, and R. T. Shroff, “Pancreatic Cancer,” Lancet 395, no. 10242 (2020): 2008–2020.

[4] W. Park, A. Chawla, and E. M. O'Reilly, “Pancreatic Cancer: A Review,” Jama 326, no. 9 (2021): 851–862.

[5] J. X. Hu, C. F. Zhao, W. B. Chen, et al., “Pancreatic Cancer: A Review of Epidemiology, Trend, and Risk Factors,” World Journal of Gastroenterology 27, no. 27 (2021): 4298–4321.

[6] I. Garajová, M. Peroni, F. Gelsomino, and F. Leonardi, “A Simple Overview of Pancreatic Cancer Treatment for Clinical Oncologists,” Current Oncology 30, no. 11 (2023): 9587–9601.

[7] X. Y. He and Y. Z. Yuan, “Advances in Pancreatic Cancer Research: Moving Towards Early Detection,” World Journal of Gastroenterology 20, no. 32 (2014): 11241–11248.

[8] A. Carrato, A. Falcone, M. Ducreux, et al., “A Systematic Review of the Burden of Pancreatic Cancer in Europe: Real-World Impact on Survival, Quality of Life and Costs,” Journal of Gastrointestinal Cancer 46, no. 3 (2015): 201–211.

[9] M. F. Lansbergen, M. P. G. Dings, P. Manoukian, et al., “Transcriptome-Based Classification to Predict FOLFIRINOX Response in a Real-World Metastatic Pancreatic Cancer Cohort,” Translational Research 273 (2024): 137–147.

[10] T. Conroy, F. Desseigne, M. Ychou, et al., “FOLFIRINOX Versus Gemcitabine for Metastatic Pancreatic Cancer,” New England Journal of Medicine 364, no. 19 (2011): 1817–1825.

[11] E. N. Pijnappel, W. P. M. Dijksterhuis, and G. L. G. der, “First- and Second-Line Palliative Systemic Treatment Outcomes in a Real-World Metastatic Pancreatic Cancer Cohort,” Journal of the National Comprehensive Cancer Network 20, no. 5 (2021): 443–450.

[12] M. Taherian, H. Wang, and H. Wang, “Pancreatic Ductal Adenocarcinoma: Molecular Pathology and Predictive Biomarkers,” Cells 11, no. 19 (2022): 3068.

[13] R. A. Moffitt, R. Marayati, E. L. Flate, et al., “Virtual Microdissection Identifies Distinct Tumor- and Stroma-Specific Subtypes of Pancreatic Ductal Adenocarcinoma,” Nature Genetics 47, no. 10 (2015): 1168–1178.

[14] E. A. Collisson, P. Bailey, and D. K. Chang, “Biankin AV. Molecular Subtypes of Pancreatic Cancer,” Nature Reviews Gastroenterology & Hepatology 16, no. 4 (2019): 207–220.

[15] B. Zhao, B. Zhao, and F. Chen, “Diagnostic Value of Serum Carbohydrate Antigen 19-9 in Pancreatic Cancer: A Systematic Review and Meta-Analysis,” European Journal of Gastroenterology & Hepatology 34, no. 9 (2022): 891.

[16] E. D. Saad, M. C. Machado, D. Wajsbrot, et al., “Pretreatment CA 19-9 Level as a Prognostic Factor in Patients With Advanced Pancreatic Cancer Treated With Gemcitabine,” International Journal of Gastrointestinal Cancer 32, no. 1 (2002): 35–41.

[17] T. M. Bauer, B. F. El-Rayes, X. Li, et al., “Carbohydrate Antigen 19-9 Is a Prognostic and Predictive Biomarker in Patients With Advanced Pancreatic Cancer Who Receive Gemcitabine-Containing Chemotherapy: A Pooled Analysis of 6 Prospective Trials,” Cancer 119, no. 2 (2013): 285–292.

[18] A. J. Bronkhorst, V. Ungerer, and S. Holdenrieder, “The Emerging Role of Cell-Free DNA as a Molecular Marker for Cancer Management,” Biomolecular Detection and Quantification 17 (2019): 100087.

[19] M. Ilié and P. Hofman, “Pros: Can Tissue Biopsy be Replaced by Liquid Biopsy?” Translational Lung Cancer Research 5, no. 4 (2016): 420–423.

[20] M. Toledano-Fonseca, M. T. Cano, E. Inga, et al., “Circulating Cell-Free DNA-Based Liquid Biopsy Markers for the Non-Invasive Prognosis and Monitoring of Metastatic Pancreatic Cancer,” Cancers 12, no. 7 (2020): E1754.

[21] M. V. García-Ortiz, P. Cano-Ramírez, M. Toledano-Fonseca, et al., “Diagnosing and Monitoring Pancreatic Cancer Through Cell-Free DNA Methylation: Progress and Prospects,” Biomarker Research 11, no. 1 (2023): 88.

[22] M. V. García-Ortiz, P. Cano-Ramírez, M. Toledano-Fonseca, et al., “Circulating NPTX2 Methylation as a Non-Invasive Biomarker for Prognosis and Monitoring of Metastatic Pancreatic Cancer,” Clinical Epigenetics 15 (2023): 118.

[23] Q. Guo and W. Qin, “DKK3 Blocked Translocation of β-Catenin/EMT Induced by Hypoxia and Improved Gemcitabine Therapeutic Effect in Pancreatic Cancer Bxpc-3 Cell,” Journal of Cellular and Molecular Medicine 19, no. 12 (2015): 2832–2841.

[24] P. C. Mahon, P. Baril, V. Bhakta, et al., “S100A4 Contributes to the Suppression of BNIP3 Expression, Chemoresistance, and Inhibition of Apoptosis in Pancreatic Cancer,” Cancer Research 67, no. 14 (2007): 6786–6795.

[25] C. Zhang, S. Ou, Y. Zhou, et al., “m6A Methyltransferase METTL14-Mediated Upregulation of Cytidine Deaminase Promoting Gemcitabine Resistance in Pancreatic Cancer,” Frontiers in Oncology 11 (2021): 696371.

[26] M. E. Gómez García, F. Carbonell Castelló, A. Alberola Soler, et al., “‘Second-look’ en Adenocarcinoma de Páncreas Inicialmente Irresecable tras Quimioterapia Neoadyuvante,” Cirugía Española 91, no. 10 (2013): 683–685.

[27] J. Roessler, O. Ammerpohl, J. Gutwein, et al., “Quantitative Cross-Validation and Content Analysis of the 450k DNA Methylation Array From Illumina, Inc,” BMC Research Res Notes 5, no. 1 (2012): 210.

[28] L. Van Wesenbeeck, L. Janssens, H. Meeuws, et al., “Droplet Digital PCR Is an Accurate Method to Assess Methylation Status on FFPE Samples,” Epigenetics 13, no. 3 (2018): 207–213.

[29] B. Győrffy, G. Bottai, T. Fleischer, et al., “Aberrant DNA Methylation Impacts Gene Expression and Prognosis in Breast Cancer Subtypes,” International Journal of Cancer 138, no. 1 (2016): 87–97.

[30] J. Liu, J. Chen, S. Ehrlich, et al., “Methylation Patterns in Whole Blood Correlate With Symptoms in Schizophrenia Patients,” Schizophrenia Bulletin 40, no. 4 (2014): 769–776.

[31] H. Luo, H. Zhu, D. Bao, et al., “Genome-Wide DNA Methylation and mRNA Transcription Analysis Revealed Aberrant Gene Regulation Pathways in Patients With Dermatomyositis and Polymyositis,” Chinese Medical Journal 138, no. 1 (2025): 120.

[32] Broad Institute TCGA Genome Data Analysis Center. Correlation Between mRNA Expression and DNA Methylation. (Broad Institute of MIT and Harvard, 2016).

[33] F. Yin, S. Yi, L. Wei, et al., “Microarray-Based Identification of Genes Associated With Prognosis and Drug Resistance in Ovarian Cancer,” Journal of Cellular Biochemistry 120, no. 4 (2019): 6057–6070.

[34] H. Huang, C. Wu, A. Colaprico, et al., “Discovery of Oncogenic Mediator Genes in Rectal Cancer Chemotherapy Response Using Gene Expression Data From Matched Tumor and Patient-Derived Organoid,” preprint, medRxiv, January 30, 2024.

[35] M. Lapin, S. Oltedal, K. Tjensvoll, et al., “Fragment Size and Level of Cell-Free DNA Provide Prognostic Information in Patients With Advanced Pancreatic Cancer,” Journal of translational medicine 16 (2018): 300.

[36] Y. Liang, W. Diao, X. Yang, et al., “Regulator of Calcineurin 3 as a Novel Predictor of Diagnosis and Prognosis in Pan-Cancer,” Croatian Medical Journal 65, no. 4 (2024): 356–372.

[37] E. Serrano-Candelas, D. Farré, Á. Aranguren-Ibáñez, et al., “The Vertebrate RCAN Gene Family: Novel Insights Into Evolution, Structure and Regulation,” PLoS ONE 9, no. 1 (2014): e85539.

[38] S. Martínez-Høyer, S. Solé-Sánchez, F. Aguado, et al., “A Novel Role for an RCAN3-derived Peptide as a Tumor Suppressor in Breast Cancer,” Carcinogenesis 36, no. 7 (2015): 792–799.

[39] Z. Wang, Y. Li, Y. Zhong, Y. Wang, and M. Peng, “Comprehensive Analysis of Aberrantly Expressed Competitive Endogenous RNA Network and Identification of Prognostic Biomarkers in Pheochromocytoma and Paraganglioma,” OncoTargets and Therapy 13 (2020): 11377–11395.

[40] L. Li, W. Xing, L. Jiang, D. Chen, and G. Zhang, “NR3C1 Overexpression Regulates the Expression and Alternative Splicing of Inflammation-associated Genes Involved in PTSD,” Gene 859 (2023): 147199.

[41] G. E. Lind, K. Kleivi, G. I. Meling, et al., “ADAMTS1, CRABP1, and NR3C1 Identified as Epigenetically Deregulated Genes in Colorectal Tumorigenesis,” Cellular Oncology 28, no. 5-6 (2006): 259–272.

[42] L. M. Noureddine, O. Trédan, N. Hussein, et al., “Glucocorticoid Receptor: A Multifaceted Actor in Breast Cancer,” International Journal of Molecular Sciences 22, no. 9 (2021): 4446.

[43] S. Khadka, S. R. Druffner, B. C. Duncan, and J. T. Busada, “Glucocorticoid Regulation of Cancer Development and Progression,” Frontiers in Endocrinology 14 (2023): 1161768.

[44] T. Wu and Y. Shao, “NR3C1/Glucocorticoid Receptor Activation Promotes Pancreatic β-Cell Autophagy Overload in Response to Glucolipotoxicity,” Autophagy 19, no. 9 (2023): 2538–2557.

[45] N. Bakour, F. Moriarty, G. Moore, et al., “Prognostic Significance of Glucocorticoid Receptor Expression in Cancer: A Systematic Review and Meta-Analysis,” Cancers 13, no. 7 (2021): 1649.

[46] L. C. Matthews, A. A. Berry, D. J. Morgan, et al., “Glucocorticoid Receptor Regulates Accurate Chromosome Segregation and Is Associated With Malignancy,” PNAS 112, no. 17 (2015): 5479–5484.

[47] S. Prekovic, K. Schuurman, I. Mayayo-Peralta, et al., “Glucocorticoid Receptor Triggers a Reversible Drug-tolerant Dormancy State With Acquired Therapeutic Vulnerabilities in Lung Cancer,” Nature Communications 12 (2021): 4360.

[48] E. Ayroldi, L. Cannarile, D. V. Delfino, and C. Riccardi, “A Dual Role for Glucocorticoid-Induced Leucine Zipper in Glucocorticoid Function: Tumor Growth Promotion or Suppression?,” Cell Death & Disease 9, no. 5 (2018): 1–12.

[49] H. Snider, B. Villavarajan, Y. Peng, et al., “Region-Specific Glucocorticoid Receptor Promoter Methylation Has Both Positive and Negative Prognostic Value in Patients With Estrogen Receptor-Positive Breast Cancer,” Clinical Epigenetics 11, no. 1 (2019): 155.

[50] Y. Deng, X. Xia, Y. Zhao, et al., “Glucocorticoid Receptor Regulates PD-L1 and MHC-I in Pancreatic Cancer Cells to Promote Immune Evasion and Immunotherapy Resistance,” Nature Communications 12, no. 1 (2021): 7041.

[51] J. Yu, M. Chen, Q. Sang, et al., “Super-Enhancer Activates Master Transcription Factor NR3C1 Expression and Promotes 5-FU Resistance in Gastric Cancer,” Advanced Science 12, no. 7 (2024): 2409050.

[52] Q. Jia, Y. Zhu, H. Yao, et al., “Oncogenic GALNT5 Confers FOLFIRINOX Resistance via Activating the MYH9/NOTCH/ DDR Axis in Pancreatic Ductal Adenocarcinoma,” Cell Death & Disease 15, no. 10 (2024): 1–14.

[53] K. M. Hanssen, M. Haber, and J. I. Fletcher, “Targeting Multidrug Resistance-Associated Protein 1 (MRP1)-Expressing Cancers: Beyond Pharmacological Inhibition,” Drug Resistance Updates 59 (2021): 100795.

[54] K. L. Aung, S. E. Fischer, R. E. Denroche, et al., “Genomics-Driven Precision Medicine for Advanced Pancreatic Cancer: Early Results From the COMPASS Trial,” Clinical Cancer Research 24, no. 6 (2018): 1344–1354.

[55] Z. Ni, P. Zhu, L. Liu, et al., “Single-Cell Transcriptomic Analysis Reveals Epithelial-Mesenchymal Transition and Key Gene AGRN as a Universal Programme in Gastrointestinal Tumours by an Artificial Intelligence-Derived Prognostic Index,” Med Research (2025).

[56] Y. Zhao, Y. Zheng, Y. Zhu, et al., “M1 Macrophage-Derived Exosomes Loaded With Gemcitabine and Deferasirox Against Chemoresistant Pancreatic Cancer,” Pharmaceutics 13, no. 9 (2021): 1493.

[57] A. Vivancos, E. Aranda, M. Benavides, et al., “Comparison of the Clinical Sensitivity of the Idylla Platform and the OncoBEAM RAS CRC Assay for KRAS Mutation Detection in Liquid Biopsy Samples,” Scientific Reports 9, no. 1 (2019): 8976.

[58] K. Kataoka, T. Yamada, M. Shiozawa, et al., “Monitoring ctDNA RAS Mutational Status in Metastatic Colorectal Cancer: A Trial Protocol of RAS-Trace and RAS-Trace-2 Studies,” Journal of the Anus, Rectum and Colon 8, no. 2 (2024): 132.

[59] T. J. Morris, L. M. Butcher, A. Feber, et al., “ChAMP: 450k Chip Analysis Methylation Pipeline,” Bioinformatics 30, no. 3 (2014): 428–430.

[60] A. E. Teschendorff, F. Marabita, M. Lechner, et al., “A Beta-mixture Quantile Normalization Method for Correcting Probe Design Bias in Illumina Infinium 450 k DNA Methylation Data,” Bioinformatics 29, no. 2 (2013): 189–196.

[61] Y. Li, D. Ge, and C. Lu, “The SMART App: An Interactive Web Application for Comprehensive DNA Methylation Analysis and Visualization,” Epigenetics & Chromatin 12, no. 1 (2019): 71.

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