Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Epiregulin confers EGFR-TKI resistance via EGFR/ErbB2 heterodimer in non-small cell lung cancer


Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective against non-small cell lung cancer (NSCLC) with EGFR-activating mutations. The mechanisms underlying EGFR-TKI resistance are not fully understood. This study aimed to analyze the effects of seven EGFR ligands on EGFR-TKI sensitivity in NSCLC cells and patients. Cells with EGFR E746-A750del mutation were treated with recombinant EGFR ligands, and analyzed for cell viability, proliferation, and apoptosis. shRNA knockdown of endogenous Epiregulin (EREG) or overexpression of exogenous EREG and immunofluorescence experiments were carried out. Public gene expression datasets were used for tumor microenvironment and clinical assessment. Among the EGFR ligands, EREG significantly diminished cellular sensitivity to TKIs and was associated with decreased response to erlotinib in NSCLC patients. EREG induced AKT phosphorylation and attenuated TKI-induced cellular apoptosis in an ErbB2-dependent manner. EREG induced the formation of the EGFR/ErbB2 heterodimer regardless of gefitinib treatment. However, overexpression or knockdown of EREG in cancer cells had little impact on TKI sensitivity. Single-cell RNA sequencing data revealed that EREG was predominantly expressed in macrophages in the tumor microenvironment. In addition, EREG-enriched macrophage conditional medium induced EGFR-TKI resistance. These findings shed new light on the mechanism underlying EGFR-TKI resistance, and suggest macrophage-produced intratumoral EREG as a novel regulator and biomarker for EGFR-TKI therapy in NSCLC.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: EREG confers EGFR-TKI resistance in NSCLC cells and patients.
Fig. 2: EREG induces EGFR-TKI resistance by preventing apoptosis.
Fig. 3: EREG-conferred EGFR-TKI resistance depends on ErbB2.
Fig. 4: The mechanism of ErbB2 affecting EREG-mediated TKI resistance.
Fig. 5: Cancer cell-derived EREG does not affect EGFR-TKI sensitivity.
Fig. 6: Impact of EREG on EGFR-TKI sensitivity is based on a paracrine effect.


  1. 1.

    Recondo G, Facchinetti F, Olaussen KA, Besse B, Friboulet L. Making the first move in EGFR-driven or ALK-driven NSCLC: first-generation or next-generation TKI? Nat Rev Clin Oncol. 2018;15:694–708.

    CAS  Article  Google Scholar 

  2. 2.

    Westover D, Zugazagoitia J, Cho BC, Lovly CM, Paz-Ares L. Mechanisms of acquired resistance to first- and second-generation EGFR tyrosine kinase inhibitors. Ann Oncol. 2018;29:I10–9.

    CAS  Article  Google Scholar 

  3. 3.

    Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee KH, et al. Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer. N Engl J Med. 2018;378:113–25.

    CAS  Article  Google Scholar 

  4. 4.

    Santoni-Rugiu E, Melchior LC, Urbanska EM, Jakobsen JN, de Stricker K, Grauslund M, et al. Intrinsic resistance to EGFR-tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer: differences and similarities with acquired resistance. Cancers. 2019;11:57.

    Article  Google Scholar 

  5. 5.

    Nagano T, Tachihara M, Nishimura Y. Mechanism of resistance to epidermal growth factor receptor-tyrosine kinase inhibitors and a potential treatment strategy. Cells. 2018;7:16.

    Article  Google Scholar 

  6. 6.

    Freed DM, Bessman NJ, Kiyatkin A, Salazar-Cavazos E, Byrne PO, Moore JO, et al. EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics. Cell. 2017;171:683–95.e618.

    CAS  Article  Google Scholar 

  7. 7.

    Chen JC, Zeng FH, Forrester SJ, Eguchi S, Zhang MZ, Harris RC Expression and function of the epidermal growth factor receptor in physiology and disease. Physiol Rev. 2016;96:1025–69.

    CAS  Article  Google Scholar 

  8. 8.

    Du ZF, Lovly CM. Mechanisms of receptor tyrosine kinase activation in cancer. Mol Cancer. 2018;17:13.

    Article  Google Scholar 

  9. 9.

    Kakiuchi S, Daigo Y, Ishikawa N, Furukawa C, Tsunoda T, Yano S, et al. Prediction of sensitivity of advanced non-small cell lung cancers to gefitinib (Iressa, ZD1839). Hum Mol Genet. 2004;13:3029–43.

    CAS  Article  Google Scholar 

  10. 10.

    Ishikawa N, Daigo Y, Takano A, Taniwaki M, Kato T, Hayama S, et al. Increases of amphiregulin and transforming growth factor-alpha in serum as predictors of poor response to gefitinib among patients with advanced non-small cell lung cancers. Cancer Res. 2005;65:9176–84.

    CAS  Article  Google Scholar 

  11. 11.

    Vollebergh MA, Kappers I, Klomp HM, Buning-Kager JC, Korse CM, Hauptmann M, et al. Ligands of epidermal growth factor receptor and the insulin-like growth factor family as serum biomarkers for response to epidermal growth factor receptor inhibitors in patients with advanced non-small cell lung cancer. J Thorac Oncol. 2010;5:1939–48.

    Article  Google Scholar 

  12. 12.

    Kim TM, Song A, Kim DW, Kim S, Ahn YO, Keam B, et al. Mechanisms of acquired resistance to AZD9291: a mutation-selective, irreversible EGFR inhibitor. J Thorac Oncol. 2015;10:1736–44.

    CAS  Article  Google Scholar 

  13. 13.

    De Luca A, Carotenuto A, Rachiglio A, Gallo M, Maiello MR, Aldinucci D, et al. The role of the EGFR signaling in tumor microenvironment. J Cell Physiol. 2008;214:559–67.

    Article  Google Scholar 

  14. 14.

    Poole JA, Nordgren TM, Heires AJ, Nelson AJ, Katafiasz D, Bailey KL, et al. Amphiregulin modulates murine lung recovery and fibroblast function following exposure to agriculture organic dust. Am J Physiol Lung Cell Mol Physiol. 2020;318:L180–91.

    CAS  Article  Google Scholar 

  15. 15.

    Wang Y, Jing Y, Ding L, Zhang X, Song Y, Chen S, et al. Epiregulin reprograms cancer-associated fibroblasts and facilitates oral squamous cell carcinoma invasion via JAK2-STAT3 pathway. J Exp Clin Cancer Res. 2019;38:274.

    Article  Google Scholar 

  16. 16.

    Vlaicu P, Mertins P, Mayr T, Widschwendter P, Ataseven B, Högel B, et al. Monocytes/macrophages support mammary tumor invasivity by co-secreting lineage-specific EGFR ligands and a STAT3 activator. BMC Cancer. 2013;13:197.

    CAS  Article  Google Scholar 

  17. 17.

    Roepstorff K, Grandal MV, Henriksen L, Knudsen SL, Lerdrup M, Grovdal L, et al. Differential effects of EGFR ligands on endocytic sorting of the receptor. Traffic. 2009;10:1115–27.

    CAS  Article  Google Scholar 

  18. 18.

    Bauer AK, Velmurugan K, Xiong KN, Alexander CM, Xiong J, Brooks R. Epiregulin is required for lung tumor promotion in a murine two-stage carcinogenesis model. Mol Carcinog. 2017;56:94–105.

    CAS  Article  Google Scholar 

  19. 19.

    Riese DJ, Cullum RL. Epiregulin: roles in normal physiology and cancer. Semin Cell Dev Biol. 2014;28:49–56.

    CAS  Article  Google Scholar 

  20. 20.

    Zhang J, Iwanaga K, Choi KC, Wislez M, Raso MG, Wei W, et al. Intratumoral epiregulin is a marker of advanced disease in non-small cell lung cancer patients and confers invasive properties on EGFR-mutant cells. Cancer Prev Res. 2008;1:201–7.

    CAS  Article  Google Scholar 

  21. 21.

    Sunaga N, Kaira K. Epiregulin as a therapeutic target in non-small-cell lung cancer. Lung Cancer. 2015;6:91–98.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Takezawa K, Pirazzoli V, Arcila ME, Nebhan CA, Song XL, de Stanchina E, et al. HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFR(T790M) mutation. Cancer Disco. 2012;2:922–33.

    CAS  Article  Google Scholar 

  23. 23.

    Hsu CC, Liao BC, Liao WY, Markovets A, Stetson D, Thress K, et al. Exon 16-skipping HER2 as a novel mechanism of osimertinib resistance in EGFR L858R/T790M-positive non-small cell lung cancer. J Thorac Oncol. 2020;15:50–61.

    CAS  Article  Google Scholar 

  24. 24.

    Lim B, Lin Y, Navin N. Advancing cancer research and medicine with single-cell genomics. Cancer Cell. 2020;37:456–70.

    CAS  Article  Google Scholar 

  25. 25.

    Zilionis R, Engblom C, Pfirschke C, Savova V, Zemmour D, Saatcioglu HD, et al. Single-cell transcriptomics of human and mouse lung cancers reveals conserved myeloid populations across individuals and species. Immunity. 2019;50:1317–34.e1310.

    CAS  Article  Google Scholar 

  26. 26.

    Massip-Copiz M, Clauzure M, Valdivieso AG, Santa-Coloma TA. Epiregulin (EREG) is upregulated through an IL-1 autocrine loop in Caco-2 epithelial cells with reduced CFTR function. J Cell Biochem. 2018;119:2911–22.

    CAS  Article  Google Scholar 

  27. 27.

    Ono T, Igawa S, Kurahayashi S, Okuma Y, Sugimoto A, Kusuhara S, et al. Impact of neutrophil-to-lymphocyte ratio in patients with EGFR-mutant NSCLC treated with tyrosine kinase inhibitors. Invest. New Drugs. 2020;38:885–93.

    CAS  Article  Google Scholar 

  28. 28.

    Mok T, Ladrera G, Srimuninnimit V, Sriuranpong V, Yu CJ, Thongprasert S, et al. Tumor marker analyses from the phase III, placebo-controlled, FASTACT-2 study of intercalated erlotinib with gemcitabine/platinum in the first-line treatment of advanced non-small-cell lung cancer. Lung Cancer. 2016;98:1–8.

    Article  Google Scholar 

  29. 29.

    Byers LA, Diao L, Wang J, Saintigny P, Girard L, Peyton M, et al. An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clin Cancer Res. 2013;19:279–90.

    CAS  Article  Google Scholar 

  30. 30.

    Kim ES, Herbst RS, Wistuba II, Lee JJ, Blumenschein GR, Jr, Tsao A, et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Disco. 2011;1:44–53.

    CAS  Article  Google Scholar 

Download references


We would like to thank Dr. Changmin Chen for his help with cell viability analysis.


This work was supported by the National Natural Science Foundation of China (Grant No. 81602731), the Science and Technology Department of Sichuan Province (Grant Nos. 2019YJ0573 and 2020YJ0453), and the Innovation Program of Sichuan Medical Association Science for Youth (Grant No. Q18009).

Author information



Corresponding authors

Correspondence to Jinyi Lang or Bangrong Cao.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ma, S., Zhang, L., Ren, Y. et al. Epiregulin confers EGFR-TKI resistance via EGFR/ErbB2 heterodimer in non-small cell lung cancer. Oncogene 40, 2596–2609 (2021).

Download citation


Quick links