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Published in final edited form as: Int J Radiat Biol. 2020 Jan 8;96(4):461–468. doi: 10.1080/09553002.2020.1707325

A Potential New Role of ATM Inhibitor in Radiotherapy: Suppressing Ionizing Radiation-Activated EGFR

Siyuan Tang 1, Zhentian Li 2, Lifang Yang 3, Liangfang Shen 1, Ya Wang 2
PMCID: PMC7500639  NIHMSID: NIHMS1623344  PMID: 31859574

Abstract

Purpose:

Although EGFR inhibitor (EGFRi) is used in cancer therapy to suppress tumor growth and resistance to treatment including radiotherapy, EGFRi resistance frequently developed, which significantly reduced treatment outcomes. Therefore, developing alternative approaches for EGFRi is of great importance. Based on our recent observation that ATM inhibitor (ATMi) efficiently inhibited ionizing radiation (IR)-induced EGFR activation in mouse embryo fibroblasts (MEF), the main purpose of this study is to determine whether ATMi could inhibit IR-induced EGFR activation in human tumor cell lines and explore its potential in EGFRi-alternative therapies.

Materials and methods:

We compared the effects of ATMi, EGFRi individually or in combination on IR-induced EGFR phosphorylation, cell growth and radio-sensitization in nine human tumor cell lines including lung adenocarcinoma (A549 and H358), glioblastoma (LN229), cervical cancer (HeLa), colorectal carcinoma (SW480 and HCT116) and nasopharygeal carcinoma (5–8F, 6–10B and HK1) cell lines. In addition, we detected the effects of ATMi, EGFRi alone or both on the efficiency of non-homologous end-joining (NHEJ) and homologous recombination (HR) using I-SceI –GFP based NHEJ or HR reporter cell lines.

Results:

Compared to EGFRi treatment, ATMi treatment decreased IR-induced EGFR phosphorylation, suppressed growth and increased IR sensitization in tested cell lines at a similar or even more efficient level. Combining ATMi and EGFRi did not significantly increased the effects on these phenotypes as ATMi treatment alone. Also, similar to ATMi, EGFRi mainly reduced the efficiency of HR but not NHEJ although combining ATMi and EGFRi further inhibited the HR efficiency.

Conclusion:

Our study demonstrates that ATMi can function like EGFRi in human tumor cells to inhibit tumor cell growth and sensitize the tumor cells to IR, suggesting that ATMi treatment as an alternative approach may exert anticancer effects on EGFRi-resistant tumor cells and facilitate radiotherapy.

Keywords: ATM, EGFR, Ionizing Radiation, DNA Repair, Human Tumor Cell Lines

Introduction

Epidermal growth factor receptor (EGFR) is an important tyrosine kinase that is over expressed or over-activated in a variety of tumors, and is associated with poor prognosis and decreased survival (Wee and Wang 2017). EGFR is a transmembrane glycoprotein that constitutes one of four members of the erbB family of tyrosine kinase receptors. Binding of EGFR to its cognate ligands leads to auto-phosphorylation of receptor tyrosine kinase and subsequent activation of signal transduction pathways that are involved in regulating cellular proliferation, differentiation, and survival, as well as in resistance to chemotherapy and ionizing radiation (IR) treatment in tumor cells (Herbst 2004). Over the past two decades, a number of EGFR inhibitors have been developed and approved by the US Food and Drug Administration (FDA) in combination with cytotoxic treatments, chemotherapy, and radiotherapy (Moore, Goldstein et al. 2007). However, despite the initial remarkable response, patients inevitably developed acquired EGFR inhibitor (EGFRi) resistance within 9–14 months of treatment, resulting in cancer recurrence (Singh, Attri et al. 2016, Blasco, Navas et al. 2019). Therefore, developing alternative approaches to efficiently control EGFR overexpression/overactivation-induced tumor growth or failure in chemo or radiotherapy is of great importance.

Radiotherapy kills tumor cells mainly through generating DNA double strand breaks (DSBs) that are repaired by two major pathways: non-homologous end joining (NHEJ) and homologous recombination (HR) in mammalian cells. ATM is one of the important effectors and regulators of the DNA damage-activated checkpoint pathway (Bensimon, Aebersold et al. 2011), and induces cell resistance to IR mainly by facilitating HR (Golding, Rosenberg et al. 2004). It was reported that EGFR promoted ATM activation through phosphorylating ATM (Lee, Lan et al. 2015), implicating EGFR as an upstream regulator of ATM. However, it remains unclear whether ATM can affect EGFR function. Recently, when studying the effects of EGFR activation on miR-21 expression, we unexpectedly found that a highly potent, selective ATM inhibitor (ATMi) efficiently inhibited IR-induced EGFR phosphorylation in mouse embryo fibroblasts (MEF) although we did not find that EGFRi affected ATM activation (Tang, Liu et al. 2019). These results suggest that ATMi may efficiently inhibit EGFR activation in human tumors, thus suppressing EGFR-related tumor cell growth and sensitizing tumor cells to IR-induced killing. The main purpose of this study was to test this hypothesis by examining the effects of a regular ATMi on IR-activated EGFR, tumor cell growth and radio-sensitivity using different human tumor cell lines, and combining a reporter assay to determine the effects of EGFR on NHEJ and HR. Our results demonstrate that an ATMi (KU55933) efficiently inhibited IR-induced EGFR activation, tumor cell growth and radio-resistance; an EGFRi (gefitnib) mainly inhibited HR efficiency. ATM inhibitors now are in early clinical development among brain tumor, melanoma, lung and prostate cancer (Chalmers, Ruff et al.2009, Durant, Zheng et al.2018, Shen, Xu et al.2019, Yin, Xu et al.2019). Particular in glioma, ATM inhibitor was found to radiosensitises glioma stem-like cell (Ross, Shafiq et al.2015). Our work show the potential of ATMi to be an alternative approach to overcome tumor cell resistance to EGFRi and thus improving cancer therapy.

Materials and methods

Cell lines and irradiation

A549 and H358 (human lung adenocarcinoma), HeLa (human cervical cancer), LN229 (human glioblastoma), SW480 and HCT116 (human colorectal carcinoma) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, US). These three human nasopharygeal carcinoma cell lines (5–8F, 6–10B and HK1) were obtained from the Cancer Research Institute, School of Basic Medicine Science, Central South University, Changsha, China. Three nasopharygeal carcinoma cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and others were in DMEM supplemented with 10% bovine calf serum, in a humidified incubator at 37°C and 5% CO2. Radiation was performed using an accelerator—Varian 23EX in Xiangya Hospital. The IR dose rate was approximately 600 MU/min, equivalent to 6 Gy/min.

Reagents and antibodies

ATM inhibitor (KU55933) and EGFR inhibitor (gefitnib) were purchased from Selleck Inc. Houston, TX, US. As we previously reported (Tang, Liu et al. 2019), KU55933 (ATMi) was added to cell cultures 1 h before IR, and gefitnib (EGFRi) was added to cell cultures 24 h before IR. An antibody against EGFR (sc-03) was purchased from Santa Cruz Biotechnology Inc CA, US. Antibody against phosphorylated EGFR Y1068 (AP0301) and β-actin were purchased from ABclonal Inc. China.

Cell growth detection

Cell growth was detected by using an MTS assay, which is a colorimetric assay for cell metabolic activity (Stockert, Horobin et al. 2018). NAD(P)H-dependent cellular oxidoreductase enzymes may reflect the number of viable cells present under defined conditions. MTS assay kits (CellTiter 96® Aqueous One Solution Cell Proliferation Assay) were purchased from Promega Corp, USA. Cells (1.0 × 103 per well) were seeded in a 96-well plate with fresh medium and incubated for the specific times with or without ATM or EGFR inhibitor, or both. MTS solution was added into each well at the indicated time points (0, 24, 48, 72 h) following treatment with ATM and/or EGFR inhibitor (the ATMi concentration was 5 μM and EGFRi was 1 nM). Cell viability was evaluated through spectrophotometric reading at 490 nm wavelength following the manufacturer’s instructions. Each cell line has five replicates and the experiments were repeated twice.

Western blotting

Cells were treated with EGFR (1 nM) 24 h or ATM (5 μM) inhibitor 1 h before irradiation. After 10 Gy exposure, cells were incubated at 37°C for 3 h and then were collected to detect protein levels. Cells were lysed at 4°C for 30 min in RIPA buffer, and the protein concentration was determined by BCA protein assay (Bio-Rad Laboratories, Hercules, CA, US) according to the manufacturer’s instructions. Proteins were then separated by SDS PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Piscataway, NJ, US). Primary antibodies were mused at the following dilutions: anti-phospho-EGFR (dilution 1: 1000), anti-EGFR (dilution 1: 1000), and anti-β-actin as an internal control (dilution 1: 3000). Relative protein levels were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, US).

Cell radio-sensitivity assay

Cell radio-sensitivity was detected using a clonogenic survival assay as described previously (Hu, Wang et al. 2017). Briefly, cells were seeded in 6-well plates (1–2 ×103 per well), and then were treated by EGFRi (1 nM) for 24 h and ATMi (5 μM) 1 h before different doses of radiation. Medium was replaced with 3 ml fresh medium 24 h after radiation, and cells were incubated for 8–10 days. Colonies were stained by crystal violet and scanned for colony counting. Colony with ≥ 30 cells was counted and surviving fractions were calibrated with non-irradiated cell colonies. Final results were obtained by analyzing the data from three independent experiments.

Reporter assay

U2OS-DR-GFP and HEK293/PC222 reporter cell lines were described previously (Gunn, Bennardo et al. 2011, Hu, Wang et al. 2017). Briefly, the U2OS-DR-GFP line contains a chromosomal reporter cassette with a promoter and an inactive GFP cDNA that is disrupted by a meganuclease I-SceI target site, GFP can be restored by HR using a downstream donor template. The HEK293/PC222 line contains a chromosomal reporter cassette with a promoter and an intact RFP cDNA that is separated by a GFP cDNA flanking by two I-SceI target sites; I-SceI challenge induces removal of the intervening GFP sequence and joining of the RFP cDNA to promoter by NHEJ, which activates RFP expression. The reporter cells were treated with 1 nM EGFRi, or 5 μM ATMi, alone or both. I-SceI expressing plasmid pRRL sEF1a HA.NLS.Sce(opt). T2A.IFP (purchased from Addgene, (#31484), Watertown, MA, US) was transfected using the jetPRIME Transfection Reagent (Polyplus-transfection® SA). GFP positive cells were analyzed using a BD LSR II flow cytometer (BD Biosciences), data was processed using FlowJo (BD Biosciences).

Statistical analysis

Differences in cell growth and radio-sensitivities between two groups (comparing the values from one cell line treated with or without inhibitor treatment; or treated with different inhibitors) were evaluated using Student’s t-test. P values < 0.05 were regarded as significant.

Results

ATMi suppressed IR-stimulated EGFR phosphorylation in tumor cell lines

To examine whether ATMi could suppress IR-activated EGFR in human tumor cells as we observed in MEFs (Tang, Liu et al. 2019), we compared the effects of an ATMi (KU55933) and an EGFRi (gefitnib) on IR-induced EGFR phosphorylation in nine different human tumor cell lines. These include lung adenocarcinoma (A549 and H358), glioblastoma (LN229), cervical cancer (HeLa), colorectal carcinoma (SW480 and HCT116) and nasopharygeal carcinoma (5–8F, 6–10B and HK1) cell lines. To enhance the IR-stimulated EGFR phosphorylation signal, we examined EGFR status 3 hours after 10 Gy IR exposure as reported previously (Tang, Liu et al. 2019). As EGFR-mediated cell growth and response to DNA damage are fully dependent on EGFR activity, we mainly detected phosphorylated and total EGFR level. All cell lines showed significantly increased EGFR phosphorylation after IR (Figure 1A, B), and treatment with EGFRi dramatically decreased IR-stimulated EGFR phosphorylation (Figure 1A, B). Notably, ATMi treatment also inhibited EGFR phosphorylation although the degree of inhibition was less than that with EGFRi (Figure 1A, B). These data suggest that not merely in MEF cells, inhibition of ATM could reduce IR-induced EGFR activation in human cells.

Figure 1.

Figure 1.

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ATMi reduced IR-activated EGFR phosphorylation but had no significant effect on total EGFR protein level in different tumor cell lines. (A) Tumor cells (A549, H358, HeLa, HK1, 5–8F, 6–10B, SW480, HCT116 and LN229) were treated with 1 nM EGFR inhibitor for 24 h and 5 μM ATM inhibitor for 1 h and were irradiated (10 Gy). Then cells were collected 3 h after IR and whole cell lysates were prepared. The levels of total EGFR and phosphorylated EGFR were detected by western blotting. Actin was used as an internal loading control for each blot. (B) Quantified representation of the data with mean and error bar obtained by repeated western blot analysis in (A).

The ATMi and EGFRi suppressed tumor cell growth

The inhibitory effects of ATMi on IR-stimulated EGFR activity suggest that the ATMi may suppress EGFR-promoted tumor cell growth. To verify this hypothesis, we compared the effects of ATMi and EGFRi treatment on the growth of these tumor cell lines for 72 hours. All of the tumor cells grew well in the absence of inhibitor, but cell growth was inhibited by treatment with either ATMi or EGFRi alone (Figure 2). Notably, compared with the EGFRi, the ATMi showed even greater growth inhibitory effects in A549, H358, 5–8F, SW480 and HCT116 cells (Figure 2). These results indicate that similar to EGFRi, ATMi can efficiently inhibit tumor cell growth, which may involve inhibition of EGFR activity.

Figure 2.

Figure 2.

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ATMi and EGFRi suppressed tumor cell growth. A549 (A), H358 (B), HeLa (C), HK1 (D), 5–8F (E), 6–10B (F), SW480 (G), HCT116 (H) and LN229 (I) cells were treated with 5 μM ATM inhibitor and 1 nM EGFR inhibitor. At 0, 24, 48, 72 h after inhibitor treatment, cells were collected by MTS assay at the wavelength of 490 nm. Experiments were repeated twice.

ATMi sensitized tumor cells to IR, combining with EGFRi did not significantly increase Sensitization

Previously, it was reported that EGFR decreased radio-sensitivity involved in facilitating NHEJ by promoting expression of DNA-PK catalytic subunit (DNA-PKcs) (a major NHEJ factor) (Kang, Zhu et al. 2012) or DNA-PKcs activity through IR-induced nuclear translocation of EGFR and interactions of EGFR with DNA-PKcs (Dittmann, Mayer et al. 2005, Friedmann, Caplin et al. 2006, Chen and Nirodi 2007). However, as we discovered in this study (Figure 1), inhibition of ATM efficiently inhibited IR-activated EGFR, indicating that EGFR as an target of ATM, may involve in HR pathway since ATM mainly promotes HR to protect cells from IR-induced cell death and has fewer effects on NHEJ (Golding, Rosenberg et al. 2004). We compared the effects of treatment with the ATMi, and EGFRi alone or in combination on radio-sensitization of these tumor cells to determine EGFR facilitates which DSB repair pathway. Treatment with ATMi alone potently sensitized all of the tested tumor cell lines to IR, more significantly than that seen with EGFRi alone (Figure 3A–J). Combining ATMi and EGFRi did not significantly increase the sensitization levels of these tumor cells to IR compared with treatment with the ATMi alone (Figure 3A–J). These results support that ATM function as a regulator of EGFR plays a major role in promoting HR to repair IR-induced DSBs. In addition, these results reveal that EGFR plays a minor role in NHEJ since any inhibition of NHEJ in an HR-deficient cell (such as ATMi-treated cells) should significantly increase the sensitization level of the cells to IR.

Figure 3.

Figure 3.

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ATMi or EGFRi sensitized tumor cells to IR, but combined ATMi and EGFRi treatment did not enhance the effects. Cells were seeded in plates and incubated overnight, then treated with EGFRi for 24 h and ATM inhibitor for 1 h before different doses of IR (0, 1, 2, 4 Gy). At 24 hours after radiation, medium was replaced and cells were incubated for 8–10 days. Colonies were stained with p-iodonitrotetrazolium violet and colonies containing >30 cells were counted to calculate surviving fractions. Survival curve for A549 (A), H358 (B), HeLa (C), HK1 (D), 5–8F (E), 6–10B (F), SW480 (G), HCT116 (H) and LN229 (I) cells showed increased sensitivity to different degrees. The graph depicts the mean and standard deviation for each cell line from three independent experiments. (J) Images of cell colony formation of ATMi and/or EGFRi treated cells following 4 Gy of IR exposure.

EGFRi mainly inhibited the efficiency of HR but not NHEJ

To verify our survival results shown in Figure 3, and determine whether EGFR mainly affects HR but not NHEJ, we compared the effects of treatment with the EGFRi and ATMi respectively or in combination on the efficiency of HR or NHEJ in U2OS-DR-GFP (for HR) and U2OS-EJ5 (for NHEJ) reporter cell lines. Consistently, treatment with ATMi potently inhibited HR efficiency (Figure 4A, B), but had no effect on NHEJ efficiency (Figure 4C, D), which confirmed the role of ATM in HR to repair DSBs. Similarly, treatment with EGFRi also efficiently inhibited HR efficiency (Figure 4A, B), but had no significant effects on NHEJ efficiency (Figure 4C, D). Combining the two inhibitors did not affect the efficiency of NHEJ but led to greater inhibition of HR efficiency (Figure 4). These results indicate that EGFR mainly promotes DSB repair through facilitating HR but not NHEJ, which may be involved in both ATM dependent and independent pathways.

Figure 4.

Figure 4.

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Frequencies of HR and NHEJ in reporter cells at 72 h post I-SceI challenge. (A) U2OS-DR-GFP reporter cells were treated as indicated (EGFRi treatment started 24 h and, ATMi treatment started 1 h before transfection). The florescence signals were measured by a flow cytometer. (B) HR frequency was obtained by calculating the signal ratio of GFP to IFP that represents I-SceI transfection efficiency, which was further normalized to non-inhibitor treated cells. (C) HEK293/PC222 reporter cells were treated similarly as in (A). (D) NHEJ frequency was obtained by calculating the signal ratio of RFP to GFP then to IFP, which was further normalized to non-inhibitor treated cells. Experiments were performed in 3 independent replicates, statistical analysis was performed by one-way ANOVA (*. P<0.05, **, P<0.01). Error bars denote standard deviation, and “ns” represents no significant difference.

Taken together, the data shown in this study reveal a novel finding that ATMi suppresses IR-activated EGFR, inhibits tumor cell growth and sensitizes tumor cells to IR, and EGFR mainly promotes DSB repair through HR, not NHEJ, which shows the potential of an ATMi may be applied to overcome the resistance of tumor cells to EGFRi, thus improving cancer therapy.

Discussion

Our work in this study suggest that an ATMi can be applied to suppress EGFR function in tumor cells. It is well known that EGFR overactivation in many tumors is not only caused by EGFR overexpression but also by EGFR mutations (Erdem-Eraslan, Gao et al. 2015, Graham, Treece et al. 2018). Although different EGFR mutants may function in tumor cells through different partners and show different phenotypes, EGFR overactivation occurs exclusively through EGFR auto-phosphorylation that mainly reflects the EGFR activation status. For instance, EGFRvIII, a frequently occurring mutant in primary glioblastoma, results in a protein product that cannot bind ligand but is constitutively activated. EGFRvIII functions alone or in heterodimers with wild type EGFR; however, EGFRvIII is a substrate of EGFR (Fan, Cheng et al. 2013). Inhibiting wild type (WT) EGFR using an EGFRi could efficiently inhibit the activation of the mutant EGFR. These results strongly support that ATMi treatment may be an alternative approach to different tumors with over-activated EGFR caused by either over-expressed WT EGFR or mutant EGFR.

Our reporter cell line data showed that combining ATMi and EGFRi more effectively inhibited HR efficiency than either inhibitor alone, indicating that in addition to the shared pathway by which ATM and EGFR promote HR, ATM and EGFR may also promote HR via affecting different factors. This is supported by the fact that excision repair cross complementation group 1 (ERCC1) functions in 3’flap removal in mammalian HR pathway (Verma and Greenberg 2016), which might be independent of ATM activity in HR. EGFR was shown to interact with ERCC1 in the repair of IR-induced DNA damage (Liccardi, Hartley et al. 2014), implicating that EGFR facilitates HR through both ATM-dependent and ATM-independent pathways. Combination of ATMi and EGFRi did not render the tested tumor cells more sensitive to IR than either inhibitor alone, which might be due to the heterogeneous characteristics of overactivation of a variety of pathways in different tumors overcoming the combined inhibition. Previously it was reported that EGFR facilitates NHEJ, supported by the presence of DNA-PKcs in cytosolic lipid rafts (Lucero, Gae et al. 2003), which involves nuclear EGFR binding to the catalytic subunit DNA-PKcs, and the regulatory subunit Ku70 of DNA-PK. However, the subcellular compartment in which EGFR-DNA-PKcs interactions occurs remains uncertain (Chen and Nirodi 2007). Our radiation sensitivity data and reporter cell line results do not support that EGFR plays an important role in promoting NHEJ. Compared with the HR pathway, the NHEJ pathway includes fewer proteins that expressed at a higher level, and is a relatively simple and repaid process. Therefore, it is reasonable to speculate that most tumors with relatively consistent NHEJ efficiency showed a significant difference in mutations in various proteins involved in the HR pathway, which resulted in HR efficiency being more readily affected by different inhibitors than NHEJ efficiency in most tumor cells. However, this prediction requires more studies to verify in the future.

Our study reveals that ATMi suppresses IR-activated EGFR and EGFR mainly promotes DSB repair through HR, which may provide new insights for the strategy of cancer therapy using different inhibitors. In the near future, it is necessary to further elucidate the mechanism how ATMi inhibits IR-induced EGFR’s activation.

Funding

This work was supported by Emory University provided start-up fund (YW) and Central South University provided fellowship (ST).

Abbreviations

ATMi

ATM inhibitor

EGFR

epidermal growth factor receptor

EGFRi

EGFR inhibitor

ERCC1

excision repair cross complementation group 1

DNA-PKcs

DNA protein kinase catalytic subunit

DSB

double strand break

FDA

Food and Drug Administration

HR

homologous recombination repair

IR

ionizing radiation

MEFs

mouse embryonic fibroblasts

NHEJ

non-homologous end-joining

SSB

single strand break

WT

wild-type

Notes on contributors

Siyuan Tang, MD, received her doctor degree from Central South University, Changsha, China. She has a joint training at Emory University, Atlanta, USA and Xiangya Hospital, Central South University, Changsha, China and now she is a postdoctoral fellow in Xiangya Hospital, Central South University, China.

Zhentian Li, MD/PhD, received his doctor degree from Wuhan University, Wuhan, China. He currently works at the Department of Radiation Oncology, School of Medicine, Emory University, Atlanta, USA.

Lifang Yang, PhD, received his doctor degree from Central South University, Changsha, China. He currently as a professor works at Cancer Research Institute, School of Basic Medicine Science, Central South University, Changsha, China.

Liangfang Shen, MD, received his doctor degree from Norman Bethune Health Science Center of Jilin University, Changchun, China. He currently as a professor and director works at the Department of Radiation Oncology, Central South University, Changsha, China.

Ya Wang, PhD. is a professor and director of the Division of Experimental Radiation Oncology in the Department of Radiation Oncology, School of Medicine, Emory University, Atlanta, USA.

Footnotes

Disclosure statement

The authors declare no conflict interest. The authors are responsible for the content of the paper.

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