Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation

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.

Efficacy of afatinib, an irreversible ErbB family blocker, in the treatment of intracerebral metastases of non-small cell lung cancer in mice

“…Low albumin concentrations in the CSF contribute to unbound afatinib as the primary form. As such, the PD analysis showed the EC 50 of afatinib was 0.26 nmol/L, a value similar to the in vitro IC 50 (0.28 nmol/L) of PC-9 cells reported in our previous work [20] . EGFR-TKIs are a class of small molecules that permeate across the BBB by two mechanisms: the lipophilic passive pathway and the receptor-mediated active transport pathway [21] .…”

“…WBRT disrupts the BBB and leads to an increased passage of afatinib across the BBB via the passive pathway. Our previous work has shown that afatinib exhibits radiosensitization effects in PC-9 cells and PC-9 gefitinib-resistant cells with sensitization enhancement ratios (SERs) of 1.22 and 1.50, respectively [20] . Afatinib markedly blocked the basal level of EGFR and ERK phosphorylation and caused delays in the radiation-induced phosphorylation of AKT in lung cancer cells.…”

Capilliposide from Lysimachia capillipes inhibits AKT activation and restores gefitinib sensitivity in human non-small cell lung cancer cells with acquired gefitinib resistance

“…Of these cell lines, H1299 expresses wild-type EGFR, PC-9 expresses mutant EGFR with a 15-bp deletion (EGFR19Del), H1975 and PC-9-GR express mutant EGFRs with dual mutations, one of which includes T790M (EGFR-L858R/ T790M for H1975 and EGFR-19Del/T790M for PC-9-GR) [35] , and H460 expresses mutant KRAS (KRAS-Q61H). With IC 50 values determined by the MTS assay, we found that these cells responded to gefitinib treatment with a wide range of IC 50 values, but only PC-9 cells showed an IC 50 value at the nmol/L level (31.00±6.15 nmol/L).…”

“…The human NSCLC cell line PC-9-GR was developed by chronic exposure to gefitinib as we previously reported [35] . All of the cell lines were maintained in RPMI-1640 (Gibco, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Waltham, Massachusetts, USA).…”

“…In this study, we used the IC 30 values as the appropriate dose for LC capilliposide treatment, and we found no obvious inhibitions on cell growth for all of the tested cell lines when they were treated with LC capilliposide alone for 72 h. However, we noticed that the presence of LC capilliposide increased the inhibitory effects of gefitinib in these cells, with decreases of the IC 50 values from 6.80±1.00 to 0.77±0.12 μmol/L for PC-9-GR, 12 Figure 1C). Of interest, the concurrent treatment of LC capilliposide and gefitinib caused the most significant decrease of the IC 50 value of gefitinib in PC-9-GR, a cell line with acquired resistance to gefitinib treatment that was established after longterm exposure to gefitinib [35] . To test whether exposure to LC capilliposide could restore the sensitivity of gefitinib in PC-9-GR cells, we performed median effect analyses.…”