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

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

Shinsuke Hashida  1   2 , Hiromasa Yamamoto  1 , Kazuhiko Shien  1   2 , Yuichiro Miyoshi  1 , Tomoaki Ohtsuka  1 , Ken Suzawa  1 , Mototsugu Watanabe  1 , Yuho Maki  1 , Junichi Soh  1 , Hiroaki Asano  1 , Kazunori Tsukuda  1 , Shinichiro Miyoshi  1 , Shinichi Toyooka  1   2
Affiliations

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

Shinsuke Hashida et al. Cancer Sci. 2015 Oct.

Abstract

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.

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Figures

Figure 1
Figure 1
Copy number gains of MET and EGFR in EGFR-mutant lung cancer cell lines and their afatinib-resistant sublines. (a) The copy numbers of MET examined by real-time PCR were significantly amplified in HCC827-AR2, -AR3 and -ACR cells and slightly amplified in HCC4011-AR1 cells. (b) The copy number of EGFR in HCC827-ACR cells was approximately half of that in the parental cell line. All experiments were performed at least three times, and error bars indicate standard deviations.
Figure 2
Figure 2
Epithelial to mesenchymal transition in afatinib-resistant cell lines. (a) HCC827-AR1, -ACR, -AR4, HCC4006-AR1, and -AR2 displayed downregulation of E-cadherin and upregulation of vimentin. (b) Immunofluorescence cytochemistry of E-cadherin and vimentin on HCC827 and its several sublines are shown. HCC827 displayed epithelial phenotype although HCC827-AR1 and ACR displayed mesenchymal phenotype. Scale bars, 10 μm.
Figure 3
Figure 3
Stem cell-like properties of afatinib-resistant cell lines. (a) The putative stem cell markers, ALDHA1 and ABCB1 were overexpressed in HCC827-AR1, HCC827-ACR, and HCC4006-AR1 cells. The error bars indicate standard deviations. (b) The images of colonies of HCC827, -AR1 and –ACR were represented and time-lapse change of occupied area of the colonies in soft agar colony formation assays were shown. Scale bars, 100 μm. (c) In immunohistochemical staining of mouse xenograft models, HCC827 cells were negative for ALDH1 expression, but HCC827-AR1 cells displayed ALDH1 expression. Scale bars, 100 μm.
Figure 4
Figure 4
The changes of expressions of stem cell-related markers in HCC827 sublines. The result of target mRNAseq for 100 stem cell-related markers in in HCC827 sublines was shown. The fold changes of gene expressions from parental cell line (HCC827) were displayed as heatmap.
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