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Long noncoding RNA EGFR-AS1 mediates epidermal growth factor receptor addiction and modulates treatment response in squamous cell carcinoma

Nature Medicine volume 23, pages 1167–1175 (2017)Cite this article

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Abstract

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

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Figure 1: EGFR p.Gln787Gln status correlates with sensitivity of patient-derived HNSCC cell lines to EGFR TKIs.
Figure 2: The lncRNA EGFR-AS1 modulates sensitivity to EGFR TKIs.
Figure 3: LncRNA EGFR-AS1 alters the expression of EGFR isoforms and modulates ligand-dependent EGFR addiction.
Figure 4: The EGFR genotype for p.Gln787Gln correlates with EGFR-AS1 levels, EGFR isoform ratio and response to EGFR TKI treatment in patients.

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Acknowledgements

We would like to extend our gratitude to all patients and families involved in this study. We would also like to thank C. Chuan Young Ng of the National Cancer Centre Singapore Genomics Service for technical support with the NanoString assays. D.S.W.T. and N.G.I. are both supported by National Medical Research Council (NMRC) (Singapore) clinician–scientist awards (D.S.W.T.: NMRC/CSA/007/2016; N.G.I.: NMRC/CSA/042/2012, NMRC/CSA-INV/011/2016). Further support for this project was obtained from NMRC (grant no. NMRC/1304/2011, NMRC/CIRG/1434/2015), National Cancer Centre Research Funds and a Singhealth Foundation Grant (SHF/FG496P/2012).

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Author notes

  1. Fui Teen Chong, Hui Sun Leong and Shen Yon Toh: These authors contributed equally to this work.

Authors and Affiliations

  1. Cancer Therapeutics Research Laboratory, National Cancer Centre Singapore, Singapore

    Daniel S W Tan, Fui Teen Chong, Hui Sun Leong, Shen Yon Toh, Dawn P Lau, Xue Lin Kwang & N Gopalakrishna Iyer

  2. Division of Medical Oncology, National Cancer Centre Singapore, Singapore

    Daniel S W Tan, Wan Teck Lim, Eng Huat Tan & Mei Kim Ang

  3. Genome Institute of Singapore, Agency for Science, Technology & Research (A*STAR), Singapore

    Daniel S W Tan, Xiaoqian Zhang, Mei Mei Chang, Anders J Skanderup & Ramanuj DasGupta

  4. Institute of Medical Biology, Agency for Science, Technology & Research (A*STAR), Singapore

    Gopinath M Sundaram & Prabha Sampath

  5. Department of Pathology, Singapore General Hospital, Singapore

    Gek San Tan & Tony K H Lim

  6. IMCB-NCC Singapore Oncogenome Program, Institute of Molecular and Cell Biology, Singapore

    Boon Tin Chua

  7. Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore

    Prabha Sampath & N Gopalakrishna Iyer

  8. Laboratory of Clinical Pharmacology, National Cancer Centre Singapore, Singapore

    Balram Chowbay

  9. Office of Clinical Sciences, Duke-NUS Medical School, Singapore

    Balram Chowbay

  10. Division of Surgical Oncology, National Cancer Centre Singapore, Singapore

    N Gopalakrishna Iyer

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Contributions

D.S.W.T. and N.G.I. conceived and designed the study. F.T.C., H.S.L., S.Y.T., D.P.L., X.L.K., X.Z., G.M.S. and G.S.T. performed experiments, with additional technical guidance and expertise from B.T.C., T.K.H.L., P.S., B.C. and R.D. M.M.C. and A.J.S. performed computational analysis. D.S.W.T., W.T.L., E.H.T. and M.K.A. conducted the co-clinical trials through the IMPACT protocol. F.T.C., H.S.L., S.Y.T. and N.G.I. performed statistical analyses. D.S.W.T. and N.G.I. wrote the manuscript, with extensive input from all authors.

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Correspondence to Daniel S W Tan or N Gopalakrishna Iyer.

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Tan, D., Chong, F., Leong, H. et al. Long noncoding RNA EGFR-AS1 mediates epidermal growth factor receptor addiction and modulates treatment response in squamous cell carcinoma. Nat Med 23, 1167–1175 (2017). https://doi.org/10.1038/nm.4401

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