MET amplification and epithelial-to-mesenchymal transition exist as parallel resistance mechanisms in erlotinib-resistant, EGFR-mutated, NSCLC HCC827 cells

K R Jakobsen,D Hussmann,B S Sorensen,A L Nielsen,A T Madsen,C Demuth,J Vad-Nielsen

doi:10.1038/oncsis.2017.17

K R Jakobsen, D Hussmann + Show 5 more

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https://doi.org/10.1038/oncsis.2017.17

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Journal: Oncogenesis	Publication Date: Apr 1, 2017
Citations: 42	License type: open-access

Affiliation: Aarhus University Hospital, Aarhus University

Abstract

Although many epidermal growth factor receptor (EGFR)-mutated lung cancer patients initially benefit from the EGFR-inhibitor erlotinib, all acquire resistance. So far, several mechanisms implicated in resistance have been identified, but the existence of multiple resistance mechanisms in parallel have only been sparsely investigated. In this study, we investigated parallel resistance mechanisms acquired by HCC827, an EGFR-mutated adenocarcinoma cell line dependent on EGFR activity and sensitive to erlotinib. The cell line was treated with erlotinib by stepwise escalation of the drug-concentration and erlotinib-resistant (HCC827ER) cells created. HCC827ER cells depicted a mixed epithelial and mesenchymal phenotype. To clarify potential parallel resistance mechanisms, 14 resistant subclones were established by limited dilution. Interestingly, all HCC827ER subclones harbored either a MET-amplification (6/14) or underwent EMT (8/14), mechanisms both found in previous studies, but not in co-occurrence. Both subclone-types were resistant to erlotinib, but only MET-subclones responded to the MET-inhibitors crizotinib and capmatinib. EMT-subclones on the other hand had markedly increased FGFR1 expression and responded to the FGFR-inhibitor AZD4547, whereas MET-subclones did not. Monitoring gene expression through the development of HCC827ER revealed upregulation of FGFR1 expression as an early response to erlotinib. In addition, FGFR1 expression increased upon short-term erlotinib treatment (48 h) identifying a physiological role immediately after erlotinib exposure. The high FGFR1 expression seen in EMT-subclones was stable even after five passages without erlotinib. Here we show, that parallel resistance mechanisms appear during erlotinib-resistance development in EGFR-mutated NSCLC cells and highlight a role for FGFR1 expression changes as an early response to erlotinib as well as a bypass-signaling mechanism.

Highlights

Non-small cell lung cancer (NSCLC) is the leading cause of cancerrelated death in many developed countries
It was soon discovered that gefitinib (another EGFRdirected tyrosine kinase inhibitor (TKI)) and erlotinib were especially effective in patients with an activating mutation in epidermal growth factor receptor (EGFR).[2,3,4]
Based on earlier studies reporting MET amplification as an EGFR-inhibitor resistance mechanism in HCC827, we investigated the copy number variation of MET throughout the course of resistance development

Summary

Introduction

Non-small cell lung cancer (NSCLC) is the leading cause of cancerrelated death in many developed countries. The poor outcome depicts the advanced disease stage and degree of metastasis at diagnosis, and the fact that most patients develop resistance to the given treatment and quickly experience progression of their disease. A kinase inhibitor targeting EGFR, initially was used successfully as a second-line treatment in NSCLC patients that progressed on standard chemotherapy.[1]. It was soon discovered that gefitinib (another EGFRdirected tyrosine kinase inhibitor (TKI)) and erlotinib were especially effective in patients with an activating mutation in EGFR.[2,3,4] These patients are offered EGFR-TKIs as first-line treatment.[5,6] Many EGFR-mutated patients experience a pronounced initial effect of this treatment, but all acquire resistance over time. Some resistance mechanisms have been discovered including the T790M secondary mutation in EGFR,[7]

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