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.

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. METHODS AND 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. CONCLUSION AND IMPLICATIONS: 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.

Correction: Multiple Analyses of G-Protein Coupled Receptor (GPCR) Expression in the Development of Gefitinib-Resistance in Transforming Non-Small-Cell Lung Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0044368.].

Multiple analyses of G-protein coupled receptor (GPCR) expression in the development of gefitinib-resistance in transforming non-small-cell lung cancer.

There is increasing evidence that functional crosstalk between GPCRs and EGFR contributes to the progression of colon, lung, breast, ovarian, prostate and head and neck tumors. In this study, we performed multiple analyses of GPCR expression in a gefitinib-resistant non-small cell lung cancer (NSCLC) cell line, H1975, which harbors an L858R/T790M mutation. To determine the expression profile of mRNAs encoding 384 GPCRs in normal human lung fibroblast (NHLF) and H1975 cells, a GPCR-specific microarray analysis was performed. A heat-map of the microarray revealed considerable differences in the expression of GPCRs between NHLF and H1975 cells. From the GPCR expression list, we selected some GPCR agonists/antagonist to investigate whether the respective ligands could affect the growth of H1975 cells. Among them, treatment with either a selective antagonist of adenosine A2a receptors, which were highly expressed in H1975 cell and another gefitinib-resistant NSCLC cells, HCC827GR cells or "small interfering RNA" (siRNA) targeting adenosine A2a receptors produced a significant decrease in cell viability of both H1975 and HCC827GR cells. Among up-regulated GPCRs in H1975 cells, Gs-, Gi- and Gq-coupled GPCRs were expressed almost equally. Among down-regulated GPCRs, Gi-coupled GPCRs were dominantly expressed in H1975 cells. The present results suggest that multilayered crosstalk between GPCRs and EGFR may play an important role in orchestrating downstream signaling molecules that are implicated in the development of gefitinib-resistant NSCLC.