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Cancer Research 67, 5267, June 1, 2007. doi: 10.1158/0008-5472.CAN-07-0242
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Somatic Mutations in the Tyrosine Kinase Domain of Epidermal Growth Factor Receptor (EGFR) Abrogate EGFR-Mediated Radioprotection in Non–Small Cell Lung Carcinoma

Amit K. Das1, Benjamin P. Chen1, Michael D. Story1, Mitsuo Sato5, John D. Minna2,3,4,5, David J. Chen1 and Chaitanya S. Nirodi1

Departments of 1 Radiation Oncology, 2 Internal Medicine, 3 Pathology, and 4 Pharmacology and 5 Hamon Cancer Center for Therapeutic Oncology and Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas

Requests for reprints: Chaitanya S. Nirodi, Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC 7.208, Mail Code 9187, Dallas, TX 75390. Phone: 214-648-7318; Fax: 214-648-5995; E-mail: chaitanya.nirodi{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The epidermal growth factor receptor (EGFR) is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in several cancers, including non–small cell lung carcinoma (NSCLC). We reported recently that NSCLC cell lines harboring somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR exhibit significant delays in the repair of DNA double-strand breaks (DSB) and poor clonogenic survival in response to radiation. Here, we explore the mechanisms underlying mutant EGFR-associated radiosensitivity. In three representative NSCLC cell lines, we show that, unlike wild-type (WT) EGFR, receptors with common oncogenic TKD mutations, L858R or {Delta}E746-E750, are defective in radiation-induced translocation to the nucleus and fail to bind the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK), a key enzyme in the nonhomologous end-joining repair pathway. Moreover, despite the presence of WT EGFR, stable exogenous expression of either the L858R or the {Delta}E746-E750 mutant forms of EGFR in human bronchial epithelial cells significantly delays repair of ionizing radiation (IR)–induced DSBs, blocks the resolution of frank or microhomologous DNA ends, and abrogates IR-induced nuclear EGFR translocation or binding to DNA-PK catalytic subunit. Our study has identified a subset of naturally occurring EGFR mutations that lack a critical radioprotective function of EGFR, providing valuable insights on how the EGFR mediates cell survival in response to radiation in NSCLC cell lines. [Cancer Res 2007;67(11):5267–74]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In several cancers, including non–small cell lung carcinoma (NSCLC), the epidermal growth factor receptor (EGFR) is an important determinant of radioresponse (1–5) Thus, it is of great importance to learn how EGFR mediates tumor responses to ionizing radiation (IR). There is evidence that IR-induced activation of EGFR increases tumor cell proliferation through the activation of the EGFR/RAS/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase pathway (6, 7), which is thought to result in rapid repopulation after radiation exposure (8–10). EGFR may also promote survival through the activation of the phosphatidylinositol 3-kinase and protein kinase B/AKT kinase (11, 12). A third mechanism implicates a direct role of EGFR as a mediator in the repair of IR-induced DNA damage. Recently, Dittmann et al. (13) showed that when A549 NSCLC cells are exposed to IR, the wild-type (WT) EGFR rapidly translocates to the nucleus and binds both the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK). DNA-PK is a critical component of the nonhomologous end-joining (NHEJ) repair pathway that plays a dominant role in repair of IR-induced DNA damage in higher eukaryotes. DNA-PK comprises three subunits, the 465-kDa catalytic subunit, DNA-PKcs, and two regulatory subunits, Ku70 (70-kDa) and Ku80 (80-kDa), which associate with XRCC4, DNA ligase IV, and Artemis at sites of double-strand breaks (DSB) to bring about the physical rejoining of DSB ends. Thus, interactions with DNA-PKcs may constitute an integral component of EGFR-mediated radioprotection. However, the physiologic significance of EGFR-mediated DNA damage response is uncertain for primarily two reasons: (a) no naturally occurring mutations that abrogate interactions between EGFR and DNA damage response pathways have thus far been identified and (b) radioresponses in appropriate knockout or transgenic mutant animal models are not known.

We reported recently that NSCLC cell lines that harbored somatic, activating mutations in the tyrosine kinase domain (TKD) of EGFR exhibit a marked sensitivity to IR and pronounced delays in the repair of IR-induced DNA DSBs (14). These EGFR mutations were linked previously to dramatic tumor sensitivity in NSCLC patients to EGFR tyrosine kinase inhibitors, gefitinib and erlotinib (15–18). The TKD mutations frequently occur as {Delta}E746-E750 deletion in the 19th exon and an L858R replacement in the 21st exon in adenocarcinomas of NSCLC patients having East Asian origin, female gender, and nonsmoking status (reviewed in ref. 19). However, the exact mechanism underlying mutant EGFR-associated radiosensitivity is not known.

In this study, we test the hypothesis that somatic activating mutations in the TKD of EGFR interfere with a critical radioprotective function of EGFR that involves IR-induced nuclear import of EGFR and binding to DNA-PK. The consequences of this interference on IR-induced DSB resolution kinetics, DNA end-joining capability, and clonogenic survival are examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. The NSCLC cell lines, NCI-A549, NCI-H820, NCI-HCC827, and NCI-H1975, were obtained from the Hamon Cancer Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center. NSCLC cell lines were cultured at 37°C and 5% CO2 in RPMI 1640 with glutamine (Hyclone) containing 5% fetal bovine serum (FBS; Gemini Biosciences). AA8 and V3 Chinese hamster ovarian (CHO) fibroblast cell lines, from the laboratory of David J. Chen (University of Texas Southwestern Medical Center, Dallas, TX), were maintained at 37°C and 5% CO2 in HyQ MEM Alpha Modification (Hyclone) medium supplemented with 10% FBS. Immortalized human bronchial epithelial (HBEC) lines (20) and AA8 or V3 cell lines were stably transfected with either an empty vector, a cytomegalovirus promoter-driven LacZ construct, the WT EGFR, a mutant EGFR construct with an L858R replacement in the exon 21, or an EGFR construct with a deletion in the exon 19 as described previously (14). HBECs were grown at 37°C and 5% CO2 in keratinocyte serum-free medium (Invitrogen/Life Technologies) supplemented with 0.2 ng/mL recombinant human EGF and 30 µg/mL bovine pituitary extract.

Clonogenic cell survival assay. Parental strains or V3 and AA8 transfectants of WT, L858R, and {Delta}E746-E750 EGFR were seeded in triplicate 60-mm dishes at various densities commensurate with dose of radiation. Cells were irradiated at various doses using the 137Cs irradiator (model Mark I-68, J.L. Shepherd Associates). Colonies containing >50 cells were stained with crystal violet and manually counted using a microscope. Surviving fraction values were plotted as a function of radiation dose. Cell survival curves were generated using the multitarget, single-hit cell survival equation S = 1 – (1– e–D/D0)n, where S is the surviving fraction at dose D, D0 is the dose required to reduce the surviving fraction to 37%, and n is the total number of targets at 0 Gy.

Preparation of cytosolic and nuclear extracts from NSCLC and HBEC. Cells were plated at 70% confluency in 100-mm dish. Twenty-four hours later, cells were irradiated at 4 Gy and harvested at various time points. Nuclear and cytosolic fractions were prepared according to a procedure from Dignam et al. (21). For Western blot analysis, 80 to 100 µg of cytosolic or nuclear extract were loaded on an 8% SDS-PAGE gel. Western blot was done and membranes were probed with anti-EGFR antibody (Santa Cruz Biotechnology, Inc.). Calnexin (Santa Cruz Biotechnology) and lamin B (Santa Cruz Biotechnology) antibodies were used as cytosolic and nuclear fraction markers, respectively. NSCLC and HBEC cells expressing L858R were also probed with a mutant EGFR-specific antibody (Cell Signaling).

Coimmunoprecipitation of DNA-PKcs with EGFR. NSCLC and HBEC cells were plated at 70% confluency. Twenty-four hours later, cells were irradiated with 4 Gy IR and harvested at various time points. Cells were lysed in a buffer containing 50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 1% Tween 20, 1 mmol/L DTT, and protease/phosphatase inhibitors, incubated on ice for 30 min, and lysates were clarified by centrifugation at 5,000 rpm for 5 min. EGFR was immunoprecipitated from 200 to 300 µg of whole-cell lysate using an anti-EGFR antibody (clone R19/48, Biosource International, Inc.). Immune complexes were resolved by electrophoresis on 8% low bis-acrylamide SDS gel transferred to polyvinylidene difluoride membrane and blots were probed with antibodies against DNA-PKcs (Lab Vision), EGFR (Santa Cruz Biotechnology), and Ku70 (Santa Cruz Biotechnology). Blots were stripped and reprobed with anti-phosphotyrosine-pY-20 (BD Transduction Laboratories).

Immunostaining for confocal microscopy. HBEC cells overexpressing WT, L858R, and {Delta}E746-E750 mutant EGFR were plated in chambered slides. At the various time points following 4 Gy IR, slides were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% bovine serum albumin in PBS. Cells were then incubated sequentially with anti-EGFR antibody (Santa Cruz Biotechnology), rhodamine red–conjugated goat antirabbit IgG (Molecular Probes), and Alexa Fluor 488 phalloidin (Molecular Probes) and finally mounted in Vectashield with 4',6-diamidino-2-phenylindole (Vector Laboratories). Images were acquired at x63 using the Zeiss LSM510 Meta system.

NHEJ and microhomologous end-joining assay. p-MND-luciferase construct was digested with SmaI for use in a frank DNA end-joining assay and with BclI for a microhomologous end-joining (MHEJ) assay. V3, AA8, or V3+DNA-PKcs cells or HBEC cells overexpressing WT, L858R, and {Delta}E746-E750 mutant EGFR were cotransfected with RSV-Renilla luciferase and either uncut p-MND-luciferase or SmaI-digested or BclI-digested p-MND-luciferase. Twenty-four hours after transfection, cells were lysed and firefly and beetle (Renilla) luciferase activities were sequentially measured using the PolarStar Optima fluorescence plate reader (BMG Laboratories) using the Dual-Luciferase Assay System (Promega).

Phospho-{gamma}H2AX foci determination. IR-induced phospho-{gamma}H2AX foci were measured in HBEC cells overexpressing WT, L858R, and {Delta}E746-E750L mutant EGFR as described previously (14) except that images were acquired at x40 magnification using the Olympus BX5 fluorescence microscope. At each time point, foci were visually counted in at least 100 nuclei.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DSB end joining and repair is compromised in cells ectopically expressing TKD-mutated forms of EGFR. Previously, we showed that ectopic expression of L858R or {Delta}E746-E750 mutant forms of EGFR compromised survival in the isogenic backgrounds of immortalized HBEC cells as well as A549 and H1299 NSCLCs (14). To test whether ectopic expression of TKD-mutated forms of EGFR has any effect on repair of IR-induced DNA damage, we measured IR-induced phospho-{gamma}H2AX foci as a surrogate for DSBs in HBEC cells that were stably transfected with WT, L858R, or {Delta}E746-E750 mutant form of EGFR. HBEC cells stably expressing the WT EGFR showed a ~90% decrease in the number of IR-induced phospho-{gamma}H2AX foci within 4 h of IR treatment (Fig. 1A ). The number of phospho-{gamma}H2AX foci returned to basal levels by 8 h following IR. By contrast, the rate of IR-induced DSB resolution was significantly lower in cells ectopically expressing either the L858R or the {Delta}E746-E750 mutant form of EGFR (Fig. 1A). At 4 h following IR, mutant EGFR-expressing HBEC transfectants retained ~70% of IR-induced phospho-{gamma}H2AX foci at 4 h and ~50% foci at 24 h following IR. Thus, ectopic expression of TKD-mutated EGFR significantly inhibits repair of IR-induced DSBs.


Figure 1
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  		Figure 1. Ectopic expression of TKD-mutated EGFR blocks repair of IR-induced DSBs. A, kinetics of IR-induced DSB resolution. Stable HBEC transfectants of LacZ ({blacktriangleup}), WT ({square}), L858R ({blacksquare}), or {Delta}E746-E750 EGFR ({circ}), were irradiated at 1 Gy, and {gamma}H2AX foci were measured at different time points as described in Materials and Methods. Points, mean of 100 nuclear events; bars, SD. Inset, levels of EGFR expression confirmed by Western blotting. B, left, schematic diagram of the principle of NHEJ assays; right, relative ability of AA8 (gray column), V3 (gray checkered column), and V3+DNA-PKcs (gray horizontal lines column) cell lines or HBEC cells stably transfected with LacZ (white column), WT EGFR (black column), L858R (black checkered column), or {Delta}E746-E750 (black horizontal lines column) to rejoin frank DNA ends generated by SmaI digestion of MnD promoter. C, left, schematic diagram of the principle of MHEJ assay; right, relative ability of error-free filling and rejoining of 5' overhangs generated by BclI digestion at a unique site of the luciferase ORF in AA8 (gray column), V3 (gray checkered column), and V3+DNA-PKcs (gray horizontal lines column) cell lines or HBEC cells stably transfected with LacZ (white column), WT EGFR (black column), L858R (black checkered column), or {Delta}E746-E750 (black horizontal lines column). Columns, luciferase activity recovered from cells transfected with SmaI- or BclI-digested p-MnD-luciferase relative to transfectants of uncut construct, respectively (B and C). *, P values obtained from an ANOVA test done between WT EGFR and L858R transfectants (black columns) and between WT EGFR and DE746-E750 transfectants (gray columns).

 
To test whether ectopic expression of TKD-mutated EGFR has any effect on nonhomologous DNA end joining of unirradiated HBEC cells, we used two plasmid-based assays modified from a protocol originally developed by Zhuang et al. (22). The DNA NHEJ assay (Fig. 1B, left) tests the intrinsic ability of cells to recover luciferase reporter activity by rejoining frank DNA ends generated by SmaI in the MnD promoter that drives reporter gene expression. Similarly, the MHEJ assay (Fig. 1C, left) measures luciferase activity recovered by error-free rejoining and repair of 5' overhangs generated by BclI in the open reading frame (ORF) of the luciferase reporter gene. To test the validity of the assay, we used AA8 CHO fibroblast cell line that contains functional DNA-PKcs and the DNA-PKcs–deficient V3 CHO cells as positive and negative controls, respectively (23–26). The results in Fig. 1B (right) show that, relative to the activity of the intact reporter, reporter activity was efficiently restored through rejoining of the cut promoter in AA8 DNA-PKcs–proficient cells. However, in V3 DNA-PKcs–deficient cells, only 20% of reporter activity was recovered through rejoining of DNA ends. Ectopic expression of DNA-PKcs in V3 cells (V3+DNA-PKcs) restored the DNA end-rejoining ability of these cells to 100% relative to the intact promoter. We examined the effect of ectopic WT or TKD-mutated EGFR expression on the NHEJ efficiency in unirradiated HBEC cells. HBEC cells, endogenously (LacZ transfected) or ectopically expressing the WT EGFR, showed ~100% end-rejoining capabilities. In contrast, cells expressing L858R or the {Delta}E746-E750 mutant form of EGFR restored only 65% of reporter activity, indicating that DNA end-joining capability in these transfectants was compromised.

We used the MHEJ assay to examine whether ectopic expression of WT and TKD-mutated EGFR has any effect on the efficiency of error-free repair of microhomologous DNA ends in unirradiated HBEC cells. Reporter activity in AA8 (DNA-PKcs proficient) cells transfected with BclI-linearized plasmid was restored to ~28% relative to cells transfected with the intact plasmid (Fig. 1C, right). The low efficiency of MHEJ activity is consistent with previous estimates (22) and reflects on the low frequency of error-free repair associated with the intrinsically error-prone NHEJ repair pathway. By contrast, repair-incompetent, DNA-PKcs–deficient V3 cells transfected with BclI-linearized plasmid restored <5% of reporter activity, whereas V3 cells ectopically expressing DNA-PKcs fully restored MHEJ activity. WT EGFR-expressing HBEC cells were able to recover 30% to 40% of luciferase activity, whereas, in cells expressing either the L858R or the {Delta}E746-E750 forms of EGFR, the recovery was <15%. Together, the data show that ectopic expression of TKD-mutated forms of EGFR compromises IR-induced DNA repair as well as the rejoining of nonhomologous or microhomologous DNA ends.

EGFR-mediated radioresponse requires DNA-PKcs. Previous studies have linked EGFR-DNA-PKcs interactions to a radiotolerant phenotype in A549 NSCLC cells (13). We hypothesized that if DNA-PKcs is indeed critical for EGFR-mediated radioresponses, then the radiomodulating effects of WT and mutant EGFR expression would be abrogated in a DNA-PKcs–deficient genetic background. To test this hypothesis, AA8 CHO cells containing the WT DNA-PKcs or V3 CHO cells that were genetically deficient in DNA-PKcs activity were stably transfected with either the WT, the L858R mutant, or the {Delta}E746-E750 mutant forms of EGFR, and clonogenic survival in response to various doses of IR was examined (Fig. 2 ). Ectopic expression of the WT EGFR in the DNA-PKcs–proficient background of AA8 cells (Fig. 2A) significantly enhanced clonogen survival, which was at least 3-fold higher at 5 Gy IR compared with mock-transfected or LacZ-transfected controls. Moreover, ectopic expression of either the {Delta}E746-E750 mutant or the L858R mutant forms of EGFR produced a significant radiosensitizing effect and decreased clonogen survival 3-fold relative to mock-transfected or LacZ-transfected controls. The radiomodulating effects of the WT and TKD-mutated forms of EGFR were consistent with those observed previously in other NSCLCs and HBEC isogenic cell lines (14). In V3 cells, which lack DNA-PKcs, clonogenic survival in mock-transfected cells was reduced ~100-fold at 5 Gy IR relative to similarly treated AA8 cells (Fig. 2B). In striking contrast to their radioresponse modulating effects in the AA8 DNA-PKcs–proficient background, in the V3 DNA-PKcs–deficient background, expression of neither the WT nor the mutant forms of EGFR had any effect. Thus, the absence of functional DNA-PKcs abrogates the radiomodulating effects of both WT and TKD-mutated forms of EGFR, suggesting a critical requirement of DNA-PKcs in EGFR-mediated radioresponse.


Figure 2
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  		Figure 2. DNA-PKcs is required from EGFR-mediated radioresponses. Clonogenic cell survival assay in (A) AA8 (DNA-PKcs proficient) and (B) V3 (DNA-PKcs deficient) CHO fibroblast cell lines that were stably transfected with either LacZ vector ({circ}), or WT ({blacktriangledown}), L858R ({Delta}), or {Delta}E746-E750 ({blacksquare}) forms of EGFR. EGFR-mediated radioresponses are lost in a DNA-PKcs background. Points, mean of triplicate measurements of one of three independent experiments with similar result; bars, SD. C, levels of EGFR expression confirmed by Western blot.

 
EGFRs with somatic, activating mutations in the TKD fail to bind DNA-PKcs. Huang et al. (27) and Dittmann et al. (13) have shown that IR induces the binding of the WT EGFR in A549 NSCLC to the catalytic and regulatory subunits of DNA-PK. To account for the radiosensitive phenotype associated with TKD-mutated forms of EGFR, we tested the hypothesis that mutant forms of EGFR may be defective in binding to DNA-PKcs. We examined EGFR-DNA-PKcs interactions in three representative NSCLC cell lines that had either the WT (A549), L858R mutant (H1975), or the {Delta}E746-E750 deleted forms of EGFR (HCC827). Figure 3A shows a result of an immunoprecipitation and Western blot assay with whole-cell lysates from irradiated NSCLC cell lines. Unirradiated A549 cells showed no EGFR-DNA-PKcs complexes. However, within 15 min of exposure to 4 Gy IR, DNA-PKcs was detected in immune complexes precipitated with an anti-EGFR antibody in A549 cells. The level of DNA-PKcs-EGFR interactions peaked within 60 min after IR, gradually decreased over the next 12 h, and returned to basal levels by the 24-h time point. In striking contrast to WT EGFR-expressing A549 NSCLC cells, although both the {Delta}E746-E750-expressing HCC827 and the L858R-expressing H1975 contained high levels of endogenous EGFR, DNA-PKcs was undetectable in immunoprecipitates of the EGFR in these cell lines. To confirm that TKD-mutated forms are indeed defective in binding to DNA-PKcs, we examined EGFR-DNA-PKcs interactions in HBEC cells stably transfected with either the WT, L858R, or the {Delta}E746-E750 EGFR. In WT EGFR-transfected cells, binding of DNA-PKcs and Ku70 to EGFR occurred within 5 min following IR, persisted until 4 h, and returned to basal levels, thereafter. The mobility of the EGFR band appears slightly distorted in WT EGFR lysates (Fig. 3B, top) relative to the bands in the mutant EGFR-expressing cell lines. This distortion is most likely due to a "smiling" effect caused by electrophoretic conditions because it was not reproduced in other experiments with this cell line. The kinetics of EGFR-DNA-PKcs association in the HBEC transfectants closely paralleled the kinetics observed in A549 NSCLCs cells. In striking contrast, binding to DNA-PKcs or Ku70 could not be detected in HBEC cells stably transfected with either the L858R or the {Delta}E746-E750 mutant forms of EGFR.


Figure 3
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  		Figure 3. Immune complexes with EGFR contain DNA-PKcs in irradiated WT but not TKD-mutated EGFR-expressing NSCLCs and HBEC transfectants. Whole-cell extracts at different time points from NSCLC cell lines (A) or HBEC transfectants of WT, L858R, and {Delta}E746-E750 EGFR (B) were immunoprecipitated (IP) with total EGFR antibody and blots (WB) were probed with antibodies against DNA-PKcs or EGFR. TKD-mutated forms of EGFR do not bind DNA-PKcs.

 
EGFRs with somatic, activating mutations in the TKD are defective in IR-induced nuclear translocation. Dittmann et al. (13) showed recently that, in A549 NSCLC cells, IR induced the translocation of the EGFR into the nucleus (27). We considered the possibility that our inability to detect binding of DNA-PKcs with TKD-mutated forms of EGFR in NSCLCs and stably transfected HBEC cells could be due to an inability of the mutant forms of EGFRs to translocate to the nucleus in an IR-dependent manner. To test this possibility, we examined levels of the WT and TKD-mutated forms of receptor in cytosolic and nuclear fractions of three representative NSCLC cell lines, A549, H820, and H1975, that endogenously expressed the WT, the {Delta}E746-E750, and the L858R forms of EGFR, respectively. To verify the efficiency of subcellular fractionation, we probed gels for calnexin, a cytoplasm-specific marker, and lamin B, a nucleus-specific marker. Results in Fig. 4A show that in response to 4 Gy IR, cytoplasmic levels of the WT EGFR in A549 cell decreased within 30 min with a concomitant increase in nuclear levels of EGFR. In striking contrast to WT EGFR NSCLC, in both mutant NSCLCs, H820 and H1975, the TKD-mutated forms of EGFR failed to appear in the nucleus even after 60 min following IR exposure. Moreover, the cytoplasmic levels of EGFR remained either unchanged (H820) or increased at least 2.5-fold (H1975) over untreated samples. Almost identical results were obtained in an experiment conducted with HBEC cells that ectopically expressed the WT L858R and {Delta}E746-E750 mutant forms of EGFR. IR induced the nuclear import of the ectopically expressed WT but not the L858R or {Delta}E746-E750 mutant EGFRs. We reprobed the blots using an antibody specific to the L858R mutant form of EGFR and confirmed that the L858R mutant form of EGFR is defective in IR-induced nuclear translocation. Interestingly, a pan-EGFR antibody failed to detect even the endogenously expressed WT EGFR in nuclei of irradiated HBEC cells stably expressing the L858R or the {Delta}E746-E750 mutant form of receptor. The bands corresponding to lamin B appeared slightly different in HBEC cells compared with lamin B bands in NSCLC cells probably due to differences in the batch of antibody used in the two separate experiments. We suspect that the earlier lots, used in the NSCLC experiments, reacted with both forms of lamin, B1 and B2, whereas the more recent lots, used in the HBEC experiments, are more specific for lamin B1. The data suggest that the TKD-mutated forms of EGFR may exert a dominant-negative effect on IR-induced nuclear translocation of the WT EGFR.


Figure 4
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  		Figure 4. IR induces nuclear translocation of WT but not TKD-mutated EGFR in NSCLCs and stably transfected HBEC cells. Cytosolic and nuclear fractions of NSCLC cell lines (A) or HBEC transfectants of WT, L858R, or {Delta}E746-E750 EGFR (B) were resolved by electrophoresis, and Western blots were probed with pan-EGFR or L858R EGFR-specific antibody. Blots were probed for calnexin and lamin B to identify cytosolic and nuclear fractions, respectively.

 
EGFRs with somatic, activating mutations in the TKD do not appear in nuclei of IR-treated transfected HBEC. To further confirm that TKD-mutated EGFRs are defective in IR-induced nuclear translocation, we examined subcellular localization of EGFR in HBEC cells stably expressing either the WT L858R or the {Delta}E746-E750 mutant form of receptor at various time points after exposure to 4 Gy IR by laser scanning confocal microscopy (Fig. 5 ). Consistent with earlier observations in A549 NSCLC cells (13), in unirradiated HBEC cells, ectopically expressed WT EGFR was seen in the perinuclear space of the cell. However, on exposure to IR, the WT EGFR was observed entering the nucleus within 15 min. At 60 min, the EGFR was observed almost entirely in the nucleus with virtually no EGFR staining in the perinuclear space. In contrast, in HBEC cells ectopically expressing either the L858R or the {Delta}E746-E750 mutant form of receptor, there was a complete absence of EGFR in the nucleus even at 60 min following IR.


Figure 5
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  		Figure 5. Confocal microscopy reveals IR-induced nuclear translocation of WT but not TKD-mutated EGFR in HBEC transfectants. HBEC cells stably transfected with WT, L858R, or {Delta}E746-E750 EGFR were fixed and sequentially stained for EGFR (red), actin (green), and nucleus (blue).

 
Translocation-defective TKD-mutated forms of EGFR exhibit high levels of basal tyrosine phosphorylation that are not altered by exposure to IR. It is known that IR induces tyrosine phosphorylation of EGFR (10, 13), which might be necessary for IR-induced EGFR nuclear import or binding of EGFR to DNA-PKcs. We considered the possibility that defective IR-induced nuclear translocation and/or DNA-PKcs binding TKD-mutated forms of EGFR is associated in some way to altered kinetics of IR-induced phosphorylation. We examined EGFR tyrosine phosphorylation at various time points following IR in A549, H820, and H1975 NSCLC cell lines by Western blot analysis of the total EGFR that was immunoprecipitated by a pan-EGFR antibody. The results in Fig. 6A reveal that levels of tyrosine-phosphorylated WT EGFR in A549 cells dramatically increased within 5 min following IR, peaked at 60 min, decreased within 4 h of IR treatment, and returned to basal levels thereafter. By contrast, TKD-mutated forms of EGFR in both, H820 and H1975, NSCLCs showed high levels of basal phosphorylation that were not altered with IR treatment. Thus, despite the high basal phosphorylation, TKD-mutated forms of EGFR failed to bind DNA-PKcs. These differences in IR-induced tyrosine phosphorylation were exactly reproduced when the WT and TKD-mutated forms of EGFR were ectopically expressed in the isogenic system of HBEC cells (Fig. 6B), in which tyrosine phosphorylation of the EGFR, immunoprecipitated by a pan-EGFR antibody in Fig. 3B, was examined. The mobility differences in the total EGFR in the WT EGFR panel are likely due to electrophoretic distortion. It is unlikely that these differences in mobility (Fig. 3B, WT EGFR and 6B, WT EGFR) are due to changes in phosphorylation status of the receptor because, although the WT EGFR in A549 cells (Fig. 6A, A549) and other NSCLC cell lines (data not shown) exhibited dramatic increases in tyrosine phosphorylation following IR, no such differences in EGFR mobility were observed in those cell lines.


Figure 6
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  		Figure 6. IR induces tyrosine phosphorylation of WT but not TKD-mutated EGFR. EGFR was immunoprecipitated in whole-cell lysates from NSCLCs. A, Western blots of immune complexes were probed with antibodies against phosphorylated tyrosine residues (pY-20). Blots were stripped and reprobed with a pan-EGFR antibody to confirm pull-down efficiency. B, Western blots from Fig. 3B containing EGFR-immunoprecipitated form WT, L858R, or {Delta}E746-E750 HBEC transfectants were probed with antibodies against phosphorylated tyrosine residues (pY-20). Pull-down efficiency was confirmed by probing with pan-EGFR antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Evidence presented in this study supports a model for EGFR-mediated radioprotection in NSCLCs. In its simplest form, the model predicts that, during an immediate early response to IR, EGFR translocates to the nucleus and binds to DNA-PKcs, a key enzyme in the NHEJ repair pathway. Our study underscores a critical role for DNA-PKcs in EGFR-mediated radioresponse because in a DNA-PKcs–deficient genetic background, the radioprotective effect of the EGFR is lost. Thus, interactions with DNA-PKcs form a critical component of EGFR-mediated radioprotection.

The data in Supplementary Fig. S2 suggest that at least in A549 NSCLC cells, but not in HBEC cells, the WT EGFR binds two proteins of different sizes reactive to the anti-DNA-PKcs antibody. Binding to the larger protein increases with time following IR, whereas binding of EGFR to the smaller polypeptide correspondingly decrease. The identity of the two EGFR-associated DNA-PKcs polypeptides is not known at this time. It is likely that the two bands represent two spliced forms of DNA-PKcs. Spliced variants of DNA-PKcs with predicted molecular masses 469, 465, and 441 kDa have been reported recently (28). Of these, only the full-length (469 kDa) product seems to be fully functional, and ubiquitously distributed, and is therefore the focus of our study. In contrast to A549 cells, immune complexes of WT EGFR in HBEC cells showed only a single band of DNA-PKcs, suggesting that HBEC cells may lack other spliced variants of DNA-PKcs and contain a single, possibly full-length, isoform.

Our findings indicate that somatic mutations in the TKD represent naturally occurring mutations that affect a critical radioprotective function of EGFR. We have shown previously that expression of TKD-mutated EGFR correlates with compromised repair of IR-induced DNA damage and clonogenic survival in NSCLCs (14). In the present study, we further show that somatic mutations in the TKD prevent EGFR nuclear import and/or association with DNA-PKcs in two mutant EGFR NSCLC cell lines. Stable ectopic expression of TKD-mutated EGFRs in the isogenic HBEC system perfectly recapitulates the deficiencies in IR-induced nuclear translocation, DNA-PKcs binding, and DNA repair and survival. Thus, the data suggest a causal link between TKD mutations in EGFR, failure of DNA repair, and poor survival in response to IR. These findings further underscore the importance of EGFR-DNA-PKcs interactions in DNA repair and survival in response to IR.

The evidence indicates that TKD-mutated forms of EGFR not only lack a radioprotective function but also exert a radiosensitizing effect. First, in patient-derived NSCLC cell lines, TKD-mutated EGFRs are heterozygous and, despite the presence of a WT EGFR allele, these cell lines exhibit a marked sensitivity to IR. Second, ectopic expression of TKD-mutated EGFR overcomes the radioprotective functions of the endogenous WT EGFR and produces a significant radiosensitizing effect. A similar radiosensitizing effect is seen when WT EGFR containing A549 NSCLC cells but not H820 NSCLC cells that contain the {Delta}E746-E750 mutant EGFR are treated with the anti-EGFR monoclonal antibody, C225 (cetuximab; Supplementary Fig. S1). C225 has been shown previously to radiosensitize NSCLC cells by cytosolic sequestration of EGFR (29). Third, in both, NSCLCs and transfected cell lines expressing the TKD-mutated forms of EGFR, there is no evidence of IR-induced nuclear translocation of the endogenous WT EGFR or its binding to DNA-PKcs. It is likely that the translocation-defective TKD-mutated EGFRs may sequester the WT EGFR in the cytosol and prevent nuclear entry.

In both NSCLCs and HBEC transfectants, we observed striking IR-induced tyrosine phosphorylation of WT EGFR that correlates with the kinetics of nuclear import, DNA-PKcs binding, and repair of IR-induced DSBs. Furthermore, consistent with previous reports (30–36), TKD-mutated forms of EGFR showed high basal constitutive tyrosine phosphorylation that was unaltered by IR. This suggests two possibilities: either IR-induced tyrosine phosphorylation is not a prerequisite for nuclear translocation and EGFR-DNA-PKcs interactions or that IR-induced phosphorylation occurs at different tyrosine residues in WT and TKD-mutated EGFRs. The role of EGFR tyrosine phosphorylation in IR-induced nuclear translocation or DNA-PKcs is not clear. It is uncertain whether the phosphorylation precedes translocation and DNA-PKcs binding or is after these events.

How mutations in TKD abrogate IR-induced nuclear translocation and/or binding to DNA-PKcs remains to be determined. Recent X-ray crystallography studies suggest that, at least for the L858R form of EGFR, the mutation is likely to generate profound changes in the conformation and position of critical domains of the EGFR (37). These changes may adversely affect binding to importins {alpha} and ß1, which have been shown to bind the WT EGFR in A549 NSCLC cells (13).

A thorough understanding of how EGFR modulates radioresponses in NSCLCs will require determining the temporal sequence of events leading to IR-induced EGFR-DNA-PKcs interactions, the consequence of these interactions on DNA-PKcs activity and NHEJ repair, and how these events contribute to clonogenic survival in response to IR.

Our study shows that EGFR-DNA-PKcs interactions constitute an important component of EGFR-mediated radioprotection. Strategies that specifically target this component and simulate the radiosensitizing effect of TKD-mutated EGFR could potentially be used to radiosensitize NSCLC tumors in patients.


   Acknowledgments
 
Grant support: American Cancer Society, Institutional Research grant ACS-IRG-196-02, NIH grant CA50519, and National Cancer Institute, Lung Cancer Specialized Programs in Research Excellence grant P5070907.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 1/19/07. Revised 3/ 2/07. Accepted 3/23/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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