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

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 multip...

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Published in:	PloS one Vol. 12; no. 2; p. e0172115
Main Authors:	Narita, Michiko, Shimura, Eri, Nagasawa, Atsumi, Aiuchi, Toshiki, Suda, Yukari, Hamada, Yusuke, Ikegami, Daigo, Iwasawa, Chizuru, Arakawa, Kazuhiko, Igarashi, Katsuhide, Kuzumaki, Naoko, Yoshioka, Yusuke, Ochiya, Takahiro, Takeshima, Hideyuki, Ushijima, Toshikazu, Narita, Minoru
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 22-02-2017 Public Library of Science (PLoS)
Subjects:	Antineoplastic Agents - pharmacology Biology and life sciences Cancer Cancer cells Carcinoma, Non-Small-Cell Lung - genetics Carcinoma, Non-Small-Cell Lung - metabolism Carcinoma, Non-Small-Cell Lung - pathology Care and treatment Cell Line, Tumor Clonal deletion Disease control Disease resistance Dosage and administration Drug Resistance, Neoplasm - genetics E-cadherin Enzyme inhibitors Epidermal growth factor Epidermal growth factor receptors Epigenesis, Genetic - drug effects Epigenetics Epithelial-Mesenchymal Transition - drug effects Gefitinib Gene deletion Gene Expression Regulation, Neoplastic - drug effects Humans Inhibition Inhibitor drugs Kinases Life sciences Lung cancer Lung diseases Lung Neoplasms - genetics Lung Neoplasms - metabolism Lung Neoplasms - pathology Medicine and Health Sciences Mesenchyme Metastasis MicroRNA MicroRNAs MicroRNAs - genetics MicroRNAs - metabolism miRNA Mutation Non-small cell lung cancer Non-small cell lung carcinoma Pharmaceutical sciences Pharmacology Pharmacy Protein-tyrosine kinase Quinazolines - pharmacology Ribonucleic acid RNA RNA polymerase Sensitivity Smad2 protein Targeted cancer therapy Tyrosine Vimentin
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Summary:	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. 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. 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing Interests: The authors have declared that no competing interests exist. Conceptualization: Minoru Narita Michiko Narita.Formal analysis: Michiko Narita YH DI KI NK.Funding acquisition: Minoru Narita.Investigation: Michiko Narita ES AN TA YS DI CI KA YY NK.Methodology: Michiko Narita KI YY HT.Project administration: Minoru Narita.Resources: Michiko Narita YS YH CI.Supervision: TO TU.Validation: Michiko Narita.Visualization: YH.Writing – original draft: Michiko Narita ES.Writing – review & editing: Minoru Narita.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0172115