Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib

Cancer Sci. 2015 Oct;106(10):1377-84. doi: 10.1111/cas.12749. Epub 2015 Sep 30.


Afatinib is an irreversible epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR-TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non-small cell lung cancer. In this study, we established various afatinib-resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib-resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827-ACR, was established from one of the MET amplified-cell lines. Several afatinib-resistant cell lines including HCC827-ACR displayed epithelial-to-mesenchymal transition (EMT) features and epigenetic silencing of miR-200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib-resistant cells and found that MET amplification, EMT, and stem cell-like features are observed in cells with acquired resistance to EGFR-TKIs. This finding may provide clues to overcoming resistance to EGFR-TKIs.

Keywords: Afatinib; EGFR-TKI; cancer stem cells; drug resistance; non-small cell lung cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B / biosynthesis
  • Afatinib
  • Aldehyde Dehydrogenase / biosynthesis
  • Aldehyde Dehydrogenase 1
  • Animals
  • Antineoplastic Agents / pharmacology
  • Base Sequence
  • Carcinoma, Non-Small-Cell Lung / genetics
  • Carcinoma, Non-Small-Cell Lung / pathology*
  • Cell Line, Tumor
  • Cell Proliferation
  • Crizotinib
  • DNA Methylation / genetics
  • Docetaxel
  • Drug Resistance, Neoplasm*
  • Epithelial-Mesenchymal Transition / drug effects
  • ErbB Receptors / antagonists & inhibitors
  • Female
  • Humans
  • Lung Neoplasms / genetics
  • Lung Neoplasms / pathology*
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • MicroRNAs / genetics
  • Mutation / drug effects
  • Neoplastic Stem Cells / drug effects*
  • Neoplastic Stem Cells / pathology
  • Protein Kinase Inhibitors / pharmacology*
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Pyrazoles / pharmacology
  • Pyridines / pharmacology
  • Quinazolines / pharmacology*
  • Retinal Dehydrogenase
  • Sequence Analysis, DNA
  • Taxoids / pharmacology


  • ABCB1 protein, human
  • ATP Binding Cassette Transporter, Subfamily B
  • Aldehyde Dehydrogenase 1
  • Antineoplastic Agents
  • MIRN200 microRNA, human
  • MicroRNAs
  • Protein Kinase Inhibitors
  • Pyrazoles
  • Pyridines
  • Quinazolines
  • Taxoids
  • Docetaxel
  • Afatinib
  • Crizotinib
  • Aldehyde Dehydrogenase
  • ALDH1A1 protein, human
  • Retinal Dehydrogenase
  • EGFR protein, human
  • ErbB Receptors
  • Protein-Tyrosine Kinases