Abstract
Afatinib is a second-generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor and has shown a significant clinical benefit in non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. However, the potential therapeutic effects of afatinib combining with other modalities, including ionizing radiation (IR), are not well understood. In this study, we developed a gefitinib-resistant cell subline (PC-9-GR) with a secondary EGFR mutation (T790M) from NSCLC PC-9 cells after chronic exposures to increasing doses of gefitinib. The presence of afatinib significantly increases the cell killing effect of radiation in PC-9-GR cells harboring acquired T790M, but not in H1975 cells with de novo T790M or in H460 cells that express wild-type EGFR. In PC-9-GR cells, afatinib remarkable blocks baseline of EGFR and ERK phosphorylations, and causes delay of IR-induced AKT phosphorylation. Afatinib treatment also leads to increased apoptosis and suppressed DNA damage repair in irradiated PC-9-GR cells, and enhanced tumor growth inhibition when combined with IR in PC-9-GR xenografts. Our findings suggest a potential therapeutic impact of afatinib as a radiation sensitizer in lung cancer cells harboring acquired T790M mutation, providing a rationale for a clinical trial with combination of afatinib and radiation in NSCLCs with EGFR T790M mutation.
Highlights
Lung cancer is the leading cause of cancer-related mortality in the world [1]
PC-9 cells were first treated with 8 nM of gefitinib, a dose equivalent to IC30 in parental PC-9, and were exposed to increased doses of gefitinib until 8.0μM
amplification refractory mutation system (ARMS) assays were used to determine the T790M mutation and the 15 bp deletion of epidermal growth factor receptor (EGFR) in cell samples with DNA extracted from PC-9-GR cells. ddH2O was used as negative control, and a DNA template with T790M mutation or 15bp deletion was included as positive control in these assays
Summary
Lung cancer is the leading cause of cancer-related mortality in the world [1]. Despite the advances in the diagnosis and treatment, the overall survival of this disease remains disappointing, and 5-year survival rate is approximately 15% [2]. Acquired TKI resistance remains to be a major problem for therapeutic failure, which develops in most of the patients treated with reversible first-generation of TKI for 10 to 16 months, and almost 50% of cases are caused by acquired or de novo T790M mutation [4,5,6]. For these patients with re-progressed tumors with developed TKIs-resistance, treatments with second-line chemotherapeutic agents such as XL-647 [7], dasatinib [8], and neratinib [9] failed to show obvious improvement of clinical outcomes
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