Abstract
The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC50 concentrations of resistant lines exhibited a 4–5 and 11–22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2–4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.
Highlights
EGFR and c-Met are both highly expressed in non-small cell lung cancer (NSCLC) tumors and share common signaling pathways [1,2,3]
Establishment of drug resistant cell lines To identify appropriate concentrations of SU11274, erlotinib and a combination of both tyrosine kinase inhibitors (TKIs) for the development of resistant cell lines, H2170 and H358 cell lines were treated with progressively increasing concentrations of SU11274 (2.5–17 mM) [1], erlotinib (0. 5–14 mM) [39], or both SU11274 (1.25–13.5 mM) and erlotinib (0.25–9 mM) for 96 hours
Cells were treated with increasing concentrations of SU11274 [1], erlotinib [39] or a combination for several weeks after which five individual resistant clones were isolated from single cells, expanded and checked for stable resistance after each serial passage [40]
Summary
EGFR and c-Met are both highly expressed in NSCLC tumors and share common signaling pathways [1,2,3]. Tivantinib, a c-Met TKI which inhibits tumor growth in mice [12], is currently in Phase III clinical trials and has been shown to increase PFS from 9.7 to 16.1 weeks when given in combination with erlotinib [13,14]. In these trials, only certain patient subsets (KRAS mutants, non-squamous histology and EGFR wild-type status) exhibited significantly increased PFS [14], suggesting that new TKIs need to be added to this combination. While the use of combined therapy modalities may limit the ability of tumors to develop resistance [7], understanding the mechanism of resistance is the best approach for improving targeted therapy [16]
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