The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells

Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair, and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i.e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC.

Scientific RepoRts | 5:11305 | DOi: 10.1038/srep11305 ten facilities in the world; this situation may not be substantially improved in the next few decades because of high costs. Therefore, the selection of locally-advanced NSCLC cases in which CIRT is beneficial is of great importance.
Recent genome-wide mutation analyses revealed that NSCLCs possess genetic alterations, called " driver gene mutations", that play significant roles in carcinogenesis by abnormally activating oncogenes [10][11][12] . In most cases, these driver gene mutations are mutually exclusive 13,14 ; in other words, NSCLCs can be classified based on driver gene mutation status. Drugs that target activated oncogene products have begun to replace conventional cytotoxic chemotherapy, even for first-line use 10 . However, little is known about the association between driver gene mutation status and relative biological effectiveness (RBE) of carbon-ion beams in NSCLC. If it affects RBE, the driver gene mutation status may be a useful predictor of response to CIRT. To investigate this issue, we analyzed the sensitivity of 15 NSCLC lines with known mutations in EGFR and KRAS, genes frequently mutated in NSCLC 13,14 , to X-rays and carbon-ion beams.

Results and Discussion
We first examined the sensitivity of 15 NSCLC lines with different EGFR and KRAS mutational statuses to X-rays or carbon-ion beams by clonogenic survival assay (Fig. 1a, Supplementary Fig. 1). The mutation status of these lines is listed in Supplementary Table 1 [15][16][17][18][19][20] . The X-ray dose producing 10% survival (D 10 ) varied widely among the cell lines (3.8-10.9). The D 10 of EGFR-mutant lines was significantly lower than that of EGFR wild-type lines (Fig. 1c, upper left panel). These data indicate that NSCLC cells show varying degrees of sensitivity to X-rays and that EGFR-mutant cells are more sensitive to X-rays than EGFR wild-type cells. The D 10 achieved by carbon-ion beams was lower than that achieved by X-rays in all of the cell lines and was scored within a relatively narrow range (1.5-4.0) (Fig. 1a). No significant difference was observed between the carbon-ion beam D 10 in EGFR-mutant lines and that in EGFR wild-type lines (Fig. 1c, upper middle panel). These data indicate that carbon-ion beams have a higher cell-killing effect than X-rays regardless of EGFR mutation status. Carbon-ion beam RBEs calculated from the D 10 values obtained for X-rays and carbon-ion beams ranged from 1.5 to 3.8 (Fig. 1b). The RBEs in EGFR-mutant lines were significantly lower than those in wild-type lines (Fig. 1c, upper right panel). In contrast to EGFR, there was no significant difference in X-ray D 10 , carbon-ion beam D 10 , and the RBE between the 4 KRAS-mutant and the 11 KRAS wild-type lines (Fig. 1a,b,c, lower panels).
To confirm the greater X-ray sensitivity and RBE of EGFR-mutant cells compared to EGFR wild-type cells, we compared the sensitivity to X-rays and carbon-ion beams using isogenic A549 cells stably expressing wild-type or mutant EGFR proteins. A549-WT, A549-Δ E746-A750, and A549-L858R cells, which express, respectively, wild-type EGFR, a Δ E746-A750 deletion mutant, and a L858R point mutant, were used. Among the various EGFR mutations identified in human NSCLC, Δ E746-A750 and L858R are the most frequent (39.4%) and second most frequent (37.5%), respectively 21 . A549-Δ E746-A750 and A549-L858R cells showed higher sensitivity to X-rays than A549-WT cells (Fig. 2), but all three cell lines showed similar sensitivity to carbon-ion beams. Thus, the RBE observed in A549-Δ E746-A750 and A549-L858R cells was lower than that observed in A549-WT cells (Supplementary Table 2). Taken together, these data suggest that EGFR-mutant NCSLC cells show a low RBE due to their high sensitivity to X-rays and to the strong EGFR mutation-independent cell-killing effect of carbon-ion beams.
DNA double-strand breaks (DSBs) are most critical lesions contributing to the cell-killing effect of ionizing irradiation. Therefore, we investigated the association between the EGFR mutation status and the capacity for repair of X-ray-or carbon-ion beam-induced DSBs. DSB repair was assessed by scoring the number of Ser139-phosphorylated histone H2AX (γ H2AX) foci, which are markers for DSBs, 24 h post-irradiation using immunofluorescence staining 22 . H1299, H1703, and A549 cells, which showed the three highest D 10 values for X-rays, and HCC827, H1650 and Ma-24 cells, which showed the three lowest, were used. In EGFR wild-type lines, the numbers of γ H2AX foci after X-ray irradiation were slightly higher than those in non-treated controls, indicating that a major proportion of X-ray-induced DSBs were repaired 24 h post-irradiation (Fig. 3a). In EGFR-mutant lines, the numbers of γ H2AX foci after X-ray irradiation were significantly higher than those in X-ray-irradiated EGFR wild-type lines (Fig. 3a, Supplementary Table 3). These data suggest that the capacity of EGFR-mutant cells for repair of X-ray-induced DSBs is lower than that of EGFR wild-type cells.
Previous studies showed that the EGFR Δ E746-A750 deletion mutant and L858R point mutant are defective in translocation to the nucleus and in binding to the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in response to ionizing irradiation [23][24][25] . DNA-PKcs plays central roles in non-homologous end joining (NHEJ), a major DSB repair pathway 26 . After ionizing irradiation, DNA-PKcs is recruited to DSB sites and autophosphorylated. Then DNA-PKcs contributes DNA end ligation through the recruitment of x-ray cross-complementing gene 4 (XRCC4) and DNA ligase IV (LIG4) 27 . Together, we investigated NHEJ in the EGFR-mutant lines using NU7441, which inhibits DNA-PKcs activity 28 . In the presence of NU7441, the number of X-ray-induced γ H2AX foci was comparable regardless of the EGFR mutation status, i.e., the additive effect of NU7441 on the increase in X-ray-induced γ H2AX foci number was smaller in EGFR-mutant lines than in wild-type lines (Fig. 3a,  Supplementary Table 3). If the mutant EGFRs function in homologous recombination, an alternative to NHEJ in DSB repair, the additive effect of NU7441 should not depend on the EGFR mutation status. Therefore, these data indicate that EGFR-mutant cells are defective in NHEJ. Finally, we examined the repair of carbon-ion beam-induced DSB in EGFR-mutant and wild-type cells. No significant difference in the number of γ H2AX foci was observed after irradiation between EGFR-mutant and wild-type lines (Fig. 3b, Supplementary Table 3). Meanwhile, in EGFR wild-type cells, the box plots of irradiation-alone and that of irradiation plus NU7441 were closer in carbon-ion beams than in X-rays. Furthermore, when the cells were irradiated with X-rays or carbon-ion beams for the same dose of 2 Gy, the number of γ H2AX foci was significantly smaller in X-rays than in carbon-ion beams, in all the EGFR-mutant and the EGFR wild-type cells examined (Supplementary Fig. 2). Taken together, these data indicate that the repair efficacy for carbon-ion beam-induced DSBs is lower than that of X-ray-induced DSBs regardless of the EGFR mutation status. The low repair efficacy of carbon-ion beam-induced DSBs may be attributable in part to the structural complexity of the DSB ends 29,30 .
The results of the present study highlight the potential value of the EGFR mutation status as a predictor of CIRT RBE. To the best of our knowledge, this study is the first to report the association of driver gene mutation status with the RBE of carbon-ion beams. The results suggest that NSCLCs driven by mutations in oncogenes other than EGFR, including KRAS, show high RBE and thus should be selected as candidates for CIRT. Validation in animal models should be conducted. Nevertheless, previous research demonstrates an excellent agreement between the radiosensitivity of cancer cells assessed by clonogenic survival assay and the clinical response to radiotherapy [31][32][33] . Therefore, our results provide a valuable biological basis for selecting NSCLC patients for CIRT.
The present study focused on EGFR and KRAS, however, alterations in other genes were identified as drivers of NSCLC 13,14 . For example, H1299 cells carry a Q61K mutation in the oncogene NRAS 18 . Moreover, recent genome-wide analyses and functional validation demonstrated that genes that have not been recognized as classical oncogenic drivers, including genes involved in chromatin remodeling and DNA damage responses, are frequently mutated in human cancers, underscoring the pathogenic significance of these mutations 34,35 . Studies assessing the association between the mutation status of a wide panel of cancer-related genes and the sensitivity of cancer cells to X-rays and carbon-ion beams will further elucidate genetic profiles that affect radiosensitivity and RBE, and will provide biological basis for the establishment of useful predictors for personalized radiotherapy. To this end, the mutational analysis of 409 known cancer-related genes in the 15 NSCLC lines used in the present study is ongoing.
In summary, EGFR mutation-negative NSCLCs show a high RBE compared to EGFR-mutant NSCLCs and may, therefore, benefit from CIRT.  Irradiation. X-ray irradiation was performed using a Faxitron RX-650 (100 kVp, 1.14 Gy/min; Faxitron Bioptics, Tucson, AZ, USA). Carbon-ion beam irradiation was performed at the Gunma University Heavy Ion Medical Center using the same beam specifications used in clinical settings (290 MeV/nucleon and an average linear energy transfer at the center of a 6 cm spread-out Bragg peak of approximately 50 keV/μ m). Carbon-ion beams were delivered in a vertical direction so that cells on culture plates could receive the dose evenly.

Methods
Clonogenic survival assay. Cells were seeded into 6-well plates and exposed to X-rays or carbon-ion beams. After incubation for 10 days, the cells were fixed with methanol and stained with crystal violet. Colonies of at least 50 cells were counted. The surviving fraction was normalized to the corresponding controls. The D 10 was calculated using the linear-quadratic model as described previously 37 . Immunofluorescence staining. Cells were seeded on glass coverslips in 35 mm dishes and incubated overnight. The culture medium was changed to that containing 10 μ M of the DNA-PKcs inhibitor NU7441 (R&D Systems, Minneapolis, MN, USA). After incubation for 1 h, the cells were exposed Figure 3. Repair of X-ray-or carbon-ion beam-induced DSBs in EGFR-mutant or wild-type NSCLC lines assessed by immunofluorescence staining of γH2AX. Cells were exposed to X-rays (6 Gy) or carbonion beams (2 Gy) in the presence or absence of the DNA-PKcs inhibitor NU7441 (10 μM) and stained with an antibody to γH2AX 24 h post-irradiation. The number of γ H2AX foci per nucleus was scored in 30-50 cells for each experimental condition using a fluorescence microscope at ×100 magnification. The results of a representative experiment are shown as box plots. (a) X-rays. (b) Carbon-ion beams (C-ion). to X-rays or carbon-ion beams. After incubation for 24 h, the coverslips were stained with antibodies against γ H2AX (Merck Millipore, Billerica, MA, USA) as described previously 38 . The number of γ H2AX foci per nucleus was scored in sequential 2D images captured from multiple focal planes using a fluorescence microscope (Eclipse Ni, Nikon, Tokyo, Japan) at ×100 magnification. Statistical analysis. Statistical analysis was performed using SigmaPlot 12.0 (Hulinks, Tokyo, Japan) 28 . Normality was tested by Shapiro-Wilk test. For the data sets that followed a normal distribution, variance was further tested by Levene Median test; if the variance was equal, significance was then tested by unpaired Student's t-test. For the data sets that did not follow a normal distribution, significance was tested by Man-Whitney U test. P < 0.05 was considered significant.