title: Chronic treatment of non-small-cell lung cancer cells with gefitinib leads to an epigenetic loss of epithelial properties associated with reductions in microRNA-155 and -200c.
abstract: BACKGROUND
  The EGFR tyrosine kinase inhibitor gefitinib is used in therapy for non-small-cell lung cancer (NSCLC). However, its application is limited by resistance-accelerated disease progression, which is accompanied by the epithelial-to-mesenchymal transition (EMT). In the present study, we performed multiple expression analyses of microRNAs (miRNAs) and quantified the expression of several related EMT players in gefitinib-resistant NSCLC cells.
  
  
  RESULTS
  To establish gefitinib-resistant NSCLC cells, gefitinib-sensitive HCC827 cells, which exhibit an in-frame deletion [E746-A750] in EGFR exon 19, were exposed to gefitinib for at least 1.5 months. Next, to profile "gefitinib-resistant HCC827 (HCC827GR)" cells, which have a secondary T790M mutation in EGFR exon 20, a miRNA array analysis was performed in HCC827 and HCC827GR cells. The greatest differences were seen in the levels of miR-155 and miR-200c, which essentially disappeared in HCC827GR cells. In addition to these reductions, the levels of smad2 and zeb1, which are both key players in EMT and targets for miR-155 and miR-200c, respectively, were dramatically increased in HCC827GR cells. In HCC827GR cells, the expression of epithelial-cadherin (E-cadherin) was greatly reduced with repressive histone modifications, whereas vimentin, which is expressed in mesenchymal cells, was dramatically increased with active histone modifications. In another gefitinib-resistant NSCLC cell line (H1975 cells), similar to the findings in HCC827GR cells, both miR-155 and miR-200c were absent, and the EMT was induced along with epigenetic modifications. Interestingly, the inhibition of both miR-155 and miR-200c in HCC827 cells without gefitinib induced significant increases in smad2 and zeb1 along with a dramatic decrease in E-cadherin and a slight increase in vimentin. Furthermore, although the inhibition of these miRNAs in HCC827 cells decreased gefitinib sensitivity, this dual-inhibition in HCC827 cells without gefitinib did not produce a secondary T790M mutation in EGFR exon 20.
  
  
  CONCLUSIONS
  These results suggest that chronic treatment of NSCLC cells with gefitinib changes the expression of miRNAs, including dramatic reductions in miR-155 and miR-200c along with an EGFR mutation. Furthermore, this depletion of miR-155 and miR-200c may be associated with the EMT along with histone modifications, and may contribute to the decrease in the sensitivity to gefitinib independent of a secondary EGFR mutation.
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Narita
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Shimura
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Atsumi
authors.initials: A
authors.lastname: Nagasawa
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Aiuchi
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Suda
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hamada
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Daigo
authors.initials: D
authors.lastname: Ikegami
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Chizuru
authors.initials: C
authors.lastname: Iwasawa
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Arakawa
authors.affiliation: Life Science Tokyo Advanced Research Center (L-StaR), Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Katsuhide
authors.initials: K
authors.lastname: Igarashi
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Kuzumaki
authors.affiliation: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Yoshioka
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.|Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ochiya
authors.affiliation: Division of Epigenomics, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takeshima
authors.affiliation: Life Science Tokyo Advanced Research Center (L-StaR), Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.|Division of Epigenomics, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan.
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Ushijima
authors.affiliation: Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.|Life Science Tokyo Advanced Research Center (L-StaR), Hoshi University School of Pharmacy and Pharmaceutical Sciences, Ebara, Shinagawa-ku, Tokyo, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Narita
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D044127:Epigenesis, Genetic / Q000187:drug effects*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; C524942:MIRN155 microRNA, human; C533896:MIRN200 microRNA, human; D035683:MicroRNAs; D011799:Quinazolines; D000077156:Gefitinib