Date Published: January 20, 2016
Publisher: Public Library of Science
Author(s): Takeshi Yoshida, Lanxi Song, Yun Bai, Fumi Kinose, Jiannong Li, Kim C. Ohaegbulam, Teresita Muñoz-Antonia, Xiaotao Qu, Steven Eschrich, Hidetaka Uramoto, Fumihiro Tanaka, Patrick Nasarre, Robert M. Gemmill, Joëlle Roche, Harry A. Drabkin, Eric B. Haura, Pier Giorgio Petronini.
Epithelial-mesenchymal transition (EMT) is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER) by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8), a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin), ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.
Despite the benefit of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) patients with EGFR mutation , acquired resistance to these therapies is a critical clinical problem. Although the T790M secondary EGFR mutation  and MET gene amplification  may together account for 70% of this resistance, mechanisms for the remaining 30% are unclear. The epithelial-mesenchymal transition (EMT) has been negatively associated with EGFR-TKI sensitivity in NSCLC [4–7]. In line with these results, recent studies reported EMT as a possible mechanism of acquired EGFR-TKI resistance in NSCLC cell line models [8,9]. Furthermore, EMT was observed in a subset of NSCLC patients who developed EGFR-TKI resistance [10,11]. However, detailed mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC, as well as the strategies for overcoming it, remain unclear [8,9]. Several signaling pathways, such as FGFR [6,12], TGF-β [8,9], and WNT , as well as transcription factors, such as the Zinc finger E-box-binding homeobox 1 (ZEB1) , have been implicated in the EMT process.
EMT is essential for tumor invasion, metastasis, and drug resistance in many cancers  [16,22,52]. EMT-like changes have been observed in subsets of NSCLC patients with acquired EGFR-TKI resistance [10,11]. Here, we generated HCC4006ER cells that underwent EMT with activation of the TGF-β pathway in the absence of other known mechanisms of EGFR-TKI resistance, such as a T790M gatekeeper mutation or MET amplification. Gene expression microarrays and quantitative RT-PCR showed that the mRNA level of ZEB1 was considerably higher in HCC4006ER cells than in control HCC4006 cells, with the absence of significant changes in other EMT-related transcription factors such as ZEB2, Snail, Slug, or Twist. Also striking was the observation of coordinated regulation of ZEB1-dependent genes indicating the potential reprogramming of HCC4006 cells by ZEB1. Consistent with this idea, knockdown of ZEB1 reversed the EMT phenotype in HCC4006ER cells, and more importantly erlotinib sensitivity in HCC4006ER cells reverted to the same level as parental HCC4006 cells, suggesting that ZEB1 mediates the resistance in these cells. We also demonstrated that ZEB1 upregulation was observed in additional NSCLC cell lines and human samples with EMT-related EGFR-TKI resistance, suggesting that ZEB1 is a possible target to overcome this kind of resistance in clinical lung cancer. Previous reports have pointed out the importance of miR-200c in the EMT process [30–32]. In line with these studies, we observed that miR-200c expression was markedly reduced in HCC4006ER cells. A recent study has shown that EMT process by miR200/ZEB1 axis results in PD-L1 upregulation or CD8(+) TIL immunosuppression in NSCLC, suggesting that anti-PDL1 therapy is a potential strategy against NSCLC with EMT phenotype . Future studies should examine microRNAs and PD-L1 in TKI acquired resistance cell line models as well as in patients with acquired EGFR-TKI resistance. Thus, we conclude that ZEB1 mediates both the EMT process and erlotinib resistance in HCC4006ER cells and that ZEB1 could be regulated by miR-200c in these cells.