The EGF receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent protein kinase (DNA-PKc), a key enzyme in the nonhomologous end joining DNA repair pathway. We previously showed that EGFR-DNA-PKcs interactions are significantly compromised in the context of activating mutations in EGFR in non–small cell lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response. The data reveal that both the kinase activity of DNA-PKcs and radiation-induced phosphorylation of DNA-PKcs by the ataxia telangiectasia–mutated (ATM) kinase are critical prerequisites for EGFR-mediated radioresponse. Alanine substitutions at seven key serine/threonine residues in DNA-PKcs or inhibition of DNA-PKcs by NU7441 completely abrogated EGFR-mediated radioresponse and blocked EGFR binding. ATM deficiency or ATM inhibition with KU55933 produced a similar effect. Importantly, alanine substitution at an ATM-dependent DNA-PKcs phosphorylation site, T2609, was sufficient to block binding or radioresponse of EGFR. However, mutation of a DNA-PKcs autophosphorylation site, S2056 had no such effect indicating that DNA-PKcs autophosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and human bronchial epithelial cells, activating mutations in EGFR specifically abolished the DNA-PKcs phosphorylation at T2609, but not S2056. Our study underscores the critical importance of a reciprocal relationship between DNA-PKcs phosphorylation and EGFR-mediated radiation response and elucidates mechanisms underlying mutant EGFR-associated radiosensitivity in NSCLCs. Mol Cancer Res; 10(10); 1359–68. ©2012 AACR.

This article is featured in Highlights of This Issue, p. 1241

The EGF receptor (EGFR), a 170-kDa-receptor tyrosine kinase, is an important determinant of tumor resistance to ionizing radiation (IR) in a number of cancers including non–small cell lung cancer (NSCLC). In addition to cell proliferation, (1–3) and apoptosis inhibition (4, 5), EGFR has a direct role in the repair of IR-induced double strand breaks (DSB; ref. 6) and [reviewed in refs. (7, 8)].

Evidence from a number of laboratories shows that, in response to IR, EGFR is rapidly internalized and translocates to the nucleus (9–11). Moreover, nuclear EGFR has been shown to interact with the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK) in an IR-dependent manner (6, 9). The precise domains in EGFR and DNA-PK that are involved in this interaction are not known.

DNA-PK plays an important role in the nonhomologous end joining DNA repair pathway. It is composed of the regulatory DNA binding heterodimer, Ku70/80, and a catalytic subunit, DNA-PKcs. Ku70/80 heterodimer binds broken DNA ends of DSBs. Two molecules of DNA-PKcs are then recruited to the DNA break (12). In response to radiation, DNA-PKcs is rapidly activated and phosphorylated at several serine and threonine residues that are organized into distinct clusters (13). These clusters include the 2609 (or ABCDE) cluster (14, 15), the 2056 (or PQR) cluster (16), and a C' terminal site (17, 18). IR-induced serine 2056 (S2056) autophosphorylation (19) is mediated by DNA-PKcs itself, but the phosphorylation of many residues in the 2609 and 2056 clusters, particularly threonine 2609 (T2609), is mediated by the ataxia telangiectasia–mutated kinase (ATM; ref. 20). The autophosphorylation of the 2609 cluster promotes end processing (13), notably through the activity of the Artemis endonuclease (21), whereas the autophosphorylation at the 2056 cluster inhibits end processing and promotes ligation of DNA ends (16). Both DNA-PKcs S2056 autophosphorylation and ATM-mediated T2609 phosphorylation appear to be essential for DNA-PKcs–mediated DSB repair and radioresistance (20). Whether the phosphorylation status of DNA-PKcs affects EGFR-mediated radiation response is not known.

Somatic activating mutations in EGFR have been clinically linked to dramatic responses in NSCLC patients to the EGFR inhibitors, gefitinib and erlotinib (22–25). We previously showed that NSCLCs harboring either an in-frame deletion (ΔE746-E750) in the 19th exon or a leucine to arginine substitution (L858R) in the 21st exon of the EGFR tyrosine kinase domain exhibit dramatic sensitivity to IR (26). Moreover, ectopic expression of L858R or ΔE746-E750 EGFR in different NSCLC cells lines or human bronchial epithelial cell (HBEC) significantly reduced cellular radioresistance in a dominant negative manner (26). We showed that mutant EGFR-associated radiosensitivity manifests as pronounced delays in repair of IR-induced DSBs, inhibition of IR-induced nuclear translocation and absence of IR-induced EGFR-DNA-PKcs interactions (27). However, how mutant EGFR expression affects DNA-PKcs activity and function is not fully understood.

Here, we ectopically express wild type, L858R and ΔE746-E750 forms of EGFR and evaluate their relative contributions to clonogenic survival in the genetic background of various site-specific, phospho-ablating mutations in DNA-PKcs. We interrogate key DNA-PKcs residues for their ability to modulate IR-induced interactions between EGFR and DNA-PKcs and support EGFR-mediated radiation response. Our search reveals that IR-induced phosphorylation of T2609 in DNA-PKcs is a critical requirement for this interaction. Surprisingly, T2609 phosphorylation is also under the influence of EGFR. Our data support a model in which EGFR modulates DNA-PKcs function through stabilization of T2609 phosphorylation.

Cell culture

The NSCLC cell lines, NCI-A549, NCI-H820, NCI-HCC827, and NCI-H1975 were from the American Type Culture Collection. The immortalized HBEC line was originally obtained from John D. Minna (UT Southwestern Medical center; ref. 28). All cell lines were maintained as previously described (27). Chinese hamster ovary (CHO) cell lines, V3-7A, V3-WT DNA-PKCS, V3-S2056A, and V3-T2609A, and fibroblast cell lines, 1BR3, and AT5, were a generous gift from Dr. David J. Chen and were maintained as previously described (20, 29). The wild-type EGFR, L858R, and ΔE746-E750 forms of EGFR were tagged with V5 epitope in lentiviral vectors through recombinational cloning using the Gateway system (Invitrogen/Gibco-BRL). Immortalized HBEC or CHO cell lines, were genetically modified by lentivirus infection of V5-tagged EGFR forms or an unrelated LacZ construct and maintained as previously described (28, 30).

Clonogenic cell survival assay

Clonogenic survival was measured as described before (26, 27). Where inhibitors were used, cells received a 2-hour pretreatment with vehicle, 10 μmol/L NU7441, or KU55933 before irradiation and were plated at various densities 8 hours later (delayed plating). Mean SF was plotted as a function of radiation dose from 3 or more independent experiments, each conducted in triplicate samples per dose. Curves were fitted to the linear quadratic equation.

Coimmunoprecipitation and Western blot assays

EGFR was immunoprecipitated using an anti-EGFR antibody (Clone R19/48, Biosource,44-796G) and DNA-PKcs and EGFR were detected by Western blot (WB) using anti-DNA-PKcs, and EGFR antibodies as described previously (27). Phosphorylated ATM was detected by WB assay using p-ATM antibody (p-S1981, 200–301-400, Rockland Inc,) and blots were stripped and reprobed with ATM antibody (5C2, GeneTex).

Proximity ligation assay for protein–protein interactions

Stable complexes of EGFR and DNA-PKcs or PP2A and DNA-PKcs were detected using the Duolink proximity ligation assay (PLA) kit according to the manufacturer's instructions (Olink Bioscience). NSCLC or HBEC expressing wild type, L858R and ΔE746-E750 mutant EGFR were exposed to 4 Gy IR. For inhibition experiments, cells were pretreated for 2 hours before IR with 10 μmol/L of NU7441 or KU55933. At various time points, cells were fixed for immunofluorescence staining as described before (27) and simultaneously incubated with mouse DNA-PKcs antibody (Clone 25–4, Lab Vision, dilution 1:150) and rabbit anti-EGFR antibody (Santa Cruz, sc-03, 1:1000) or rabbit anti-PP2A antibody (Clone 81G5, Cell Signaling, S2041, 1:100 dilution). Cells were incubated with complementary oligonucleotide-conjugated anti-rabbit and anti-mouse secondary antibodies followed by ligation and rolling circle amplification in the presence of a Texas Red conjugated nucleotide. The fluorescent amplicons manifest as red fluorescent dots, with each dot representing a specific and stable interaction between the 2 interacting proteins. Cells were costained with 4′,6-Diamidino-2-phenylindole and images were acquired using Zeiss Axiophot fluorescence microscope with a ×40 objective. After correcting for illumination, integrated fluorescence intensity of foci in 600 to 800 nuclei per experiment was measured using the Cell profiler image analysis software (31). Mean integrated fluorescence intensity per nucleus and standard error of means from 3 or more independent experiments was used to quantify EGFR-DNA-PKcs or PP2A-DNA-PKcs binding at various time points following IR.

Quantification of phosphorylated forms of DNA-PKcs by immunocytochemical staining

DNA-PKcs phosphorylation in response to 4 Gy IR was measured by staining formalin fixed, Triton-X-100-permeabilized cells with antibodies against pT2609 or S2056 (20) which were detected by Alexa-488-conjugated secondary antibodies. Images were acquired using a ×40 objective of the Zeiss Axiophot fluorescence microscope. After correcting for illumination, integrated fluorescence intensity of phospho DNA-PKcs per nucleus in 450 to 600 nuclei per experiment was measured using the Cell profiler image analysis software (31). Mean integrated fluorescence intensity per nucleus and standard error of means from 2 or more independent experiments were reported.

Mutations in the 2609 and 2056 clusters of DNA-PKcs abrogate EGFR-mediated radiation response

To examine the effect of DNA-PKcs phosphorylation on wild type or mutant EGFR-mediated radiation responses, we stably expressed the wild type, L858R, or ΔE746-E750 forms of EGFR in 3 different DNA-PKcs backgrounds: DNA-PKcs-deficient V3 CHO cells, V3 cells stably expressing wild type human DNA-PKcs (V3-WT) or V3 cells stably expressing a catalytically active, but DSB-repair defective, mutant form of human DNA-PKcs (V3-7A) in which 7 alanine replacements in the 2609 and 2056 clusters (Fig. 1A and B). Consistent with previous observations (27), in V3-WT DNA-PKCS cells, ectopic expression of wild-type EGFR significantly increased clonogenic survival, whereas expression of the L858R and ΔE746-E750 activating mutant forms of EGFR had a pronounced dominant-negative radiosensitizing effect relative to untransfected cells (Fig. 1C, middle). This contrasting pattern of radiation responses associated with wild type and mutant EGFR expression was completely abrogated in V3 cells (Fig. 1C, left) or V3–7A cells (Fig. 1C, right). The data indicate EGFR-mediated radiation response is not only DNA-PKcs dependent but also requires phosphorylation of residues in the 2056 and 2609 clusters of DNA-PKcs.

Figure 1.

Mutations in the 2609 and 2056 cluster of DNA-PKcs abrogate EGFR-mediated radiation response. A, schematic illustration of 7A-mutated DNA-PKcs with alanine substitutions in the 2056 and 2609 clusters. B, WB: relative expression of DNA-PKcs (top) or V5 epitope-tagged EGFR (bottom) in V3, V3-WT, and V3-7A CHO cells. C, clonogenic survival in V3 (left), V3-WT(middle), and V3-7A (right) CHO cells mock-transfected or stably expressing LacZ, (closed circle) wild-type EGFR (square), L858R (triangle up), and ΔE746-E750 (triangle down). Symbols: mean surviving fraction (SF) and error bars: standard deviation (SD) from 3 independent experiments.

Figure 1.

Mutations in the 2609 and 2056 cluster of DNA-PKcs abrogate EGFR-mediated radiation response. A, schematic illustration of 7A-mutated DNA-PKcs with alanine substitutions in the 2056 and 2609 clusters. B, WB: relative expression of DNA-PKcs (top) or V5 epitope-tagged EGFR (bottom) in V3, V3-WT, and V3-7A CHO cells. C, clonogenic survival in V3 (left), V3-WT(middle), and V3-7A (right) CHO cells mock-transfected or stably expressing LacZ, (closed circle) wild-type EGFR (square), L858R (triangle up), and ΔE746-E750 (triangle down). Symbols: mean surviving fraction (SF) and error bars: standard deviation (SD) from 3 independent experiments.

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ATM deficiency abrogates EGFR-mediated radiation responses

Previous reports have shown that IR-induced phosphorylation of many of the residues in the 2056 and 2609 cluster of DNA-PKcs is ATM dependent (20). To test whether ATM deficiency has any effect on EGFR-mediated effects on cellular radiosensitivity, we overexpressed wild type, L858R, or ΔE746-E750 forms of EGFR in ATM-proficient, 1BR3, or ATM-deficient, AT5, backgrounds (Fig. 2A). The data in Fig. 2B show that both wild-type EGFR-mediated radioprotection and mutant EGFR-mediated radiosensitization are intact in 1BR3 cells. In AT5 cells, on the other hand, expression of either wild-type or mutant EGFR did not significantly affect clonogenic survival relative to mock- or LacZ-transfected cells. The data indicate that IR-induced ATM-driven DNA-PKcs phosphorylation is an essential requirement for EGFR-mediated radiation response.

Figure 2.

ATM deficiency abrogates EGFR-mediated radiation responses. A, WB: relative levels of V5-tagged EGFR, DNA-PKcs, and ATM in ATM-proficient 1BR3 and AT5 fibroblast cells. B, clonogenic survival in 1BR3 (left) and AT5 (right) cells mock transfected (open circle) or stably expressing LacZ (closed circle), wild-type EGFR (square), L858R (triangle up), and ΔE746-E750 (triangle down). Symbols: mean SF and error bars: SD from 3 independent experiments.

Figure 2.

ATM deficiency abrogates EGFR-mediated radiation responses. A, WB: relative levels of V5-tagged EGFR, DNA-PKcs, and ATM in ATM-proficient 1BR3 and AT5 fibroblast cells. B, clonogenic survival in 1BR3 (left) and AT5 (right) cells mock transfected (open circle) or stably expressing LacZ (closed circle), wild-type EGFR (square), L858R (triangle up), and ΔE746-E750 (triangle down). Symbols: mean SF and error bars: SD from 3 independent experiments.

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DNA-PKcs phosphorylation of 2609 and 2056 cluster is critical for IR-induced binding to EGFR

A key step in EGFR-mediated radiation response requires binding of EGFR and DNA-PKcs that is abrogated by activating mutations, L858R or ΔE746-E750 in EGFR (27) and Supplementary Fig. S1C. We first tested whether phospho-ablating mutations in 7A-DNA-Pkcs affected EGFR binding. Figure 3A shows that in V3-WT cells, interactions between wild type DNA-PKcs and wild-type EGFR occurred as early as 5 minutes following 4 Gy IR, persisted until 90 minutes and diminished shortly thereafter. As expected, WT DNA-PKcs failed to coprecipitate with L858R or the ΔE746-E750 mutant form of EGFR (27). Interestingly, the 7A mutant form of DNA-PKcs was undetectable in immune complexes of wild-type or mutant EGFR. The data indicate that alanine substitution in 7 of the 11 phosphorylation sites in DNA-PKcs completely abolished radiation-induced binding of EGFR. We reasoned that the reported abrogation of DNA-PKcs phosphorylation in the context of ATM deficiency (20) should similarly affect EGFR-DNA-PKcs binding. As expected, DNA-PKcs coprecipitated with wild-type EGFR (Fig. 3B) in ATM-proficient 1BR3 fibroblast cells but such interaction was undetectable in ATM-deficient AT5 cells even at 90 minutes following IR. The data indicate that radiation-induced interactions between EGFR and DNA-PKcs are dependent, in part, on ATM-driven phosphorylation of DNA-PKcs.

Figure 3.

DNA-PKcs phosphorylation of 2609 and 2056 cluster is critical for IR-induced binding to EGFR. Co-IP/WB assay: EGFR and DNA-PKcs in complexes with EGFR immunoprecipitated from A, V3-WT and V3-7A transfectants of wild-type L858R and ΔE7460-E750 EGFR or B, 1BR3 and AT5 transfectants of wild-type EGFR at indicated time points following 4 Gy IR.

Figure 3.

DNA-PKcs phosphorylation of 2609 and 2056 cluster is critical for IR-induced binding to EGFR. Co-IP/WB assay: EGFR and DNA-PKcs in complexes with EGFR immunoprecipitated from A, V3-WT and V3-7A transfectants of wild-type L858R and ΔE7460-E750 EGFR or B, 1BR3 and AT5 transfectants of wild-type EGFR at indicated time points following 4 Gy IR.

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Inhibition of DNA-PKcs or ATM abrogates EGFR DNA-PKcs interactions and EGFR-mediated radioresponse

We next examined whether EGFR-mediated radioresponses and radiation-induced EGFR-DNA-PKcs binding are dependent on the kinase activity of DNA-PKcs or ATM. NU7441 (2-N-morpholino-8-dibenzothiophenyl-chromen-4-one) and KU55933 (2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one) are highly potent, selective inhibitors of DNA-PKcs and ATM respectively (32, 33). In HBEC, a 2-hour pretreatment with either NU7441 or KU55933 completely abolished the survival responses associated with both wild-type and mutant EGFR (Fig. 4A). Moreover, NU7441 or KU55933 pretreatment completely eliminated IR-induced associations between DNA-PKcs and EGFR in coimmunoprecipitation (Co-IP) assays (Fig. 4B). To quantify the effects of NU7441 or KU55933 on EGFR-DNA-PKcs binding in NSCLCs and HBEC, we used the PLA. PLA relies on in situ detection of protein–protein interactions through a fluorescent signal that is generated only when 2 interacting proteins are physically associated with each other. PLA not only allows the quantitative assessment of protein–protein interactions but also reveals the subcellular location where they predominate. Fig. 4C shows that in wild type EGFR expressing A549 cells, relative to untreated cells, at 1 hour following 4 Gy IR, there was a significant (12.5-fold) increase in EGFR-DNA-PKcs complexes, occurring predominantly in the nuclear region of the cells. Both basal and radiation-induced PLA fluorescence was undetectable in the ΔE746-E750 expressing H820 cells. More importantly, NU7441 or KU55933 treatment completely abrogated both basal and radiation-induced EGFR-DNA-PKcs complexes in A549 cells and showed no appreciable change over baseline in H820 cells. In the isogenic settings of HBEC (Fig. 4C, bottom), NU7441 or KU55933 pretreatment similarly eliminated the approximately 200-fold IR-induced increase in EGFR-DNA-PKcs associations in wild type EGFR expressing HBEC. In contrast, cells expressing the ΔE746-E750 EGFR showed no such increase and were not further affected by treatment with either inhibitor. The data indicate that kinase activities of both, DNA-PKcs and ATM, are critical for the radiation-induced binding of DNA-PKcs and EGFR in NSCLCs and HBEC.

Figure 4.

Inhibition of DNA-PKcs or ATM abrogates EGFR DNA-PKcs interactions and EGFR-mediated radioresponse. A, clonogenic survival in HBEC-3KT, mock transfected (dashed gray line), stably expressing LacZ (green), wild-type EGFR (black), L858R (red), or ΔE746-E750 (blue) following a 2-hour pretreatment with either 10 μmol/L NU74421 or 10 μmol/L KU55933. Mean SF (symbols) and SD (error bars). B, Co-IP/WB assay: WB of EGFR and DNA-PKcs in complexes with EGFR immunoprecipitated, pretreated for 2 hours with either 10 μmol/L NU74421 or 10 μmol/L KU55933 at indicated times following 4 Gy IR. C, PLA: NSCLCs or HBEC-3KT expressing wild type or ΔE746-E750 forms of EGFR following a 2-hour pretreatment with vehicle, 10 μmol/L NU7441 or 10 μmol/L KU55933 were mock irradiated or exposed to 4 Gy IR and fixed at 60 minutes. Left: representative images of nuclei (blue) showing EGFR-DNA-PKcs complexes (intense red dots). Original images in Supplementary Fig. S2. Right: mean integrated fluorescence intensity (columns) and SEM (error bars) from 2 independent experiments.

Figure 4.

Inhibition of DNA-PKcs or ATM abrogates EGFR DNA-PKcs interactions and EGFR-mediated radioresponse. A, clonogenic survival in HBEC-3KT, mock transfected (dashed gray line), stably expressing LacZ (green), wild-type EGFR (black), L858R (red), or ΔE746-E750 (blue) following a 2-hour pretreatment with either 10 μmol/L NU74421 or 10 μmol/L KU55933. Mean SF (symbols) and SD (error bars). B, Co-IP/WB assay: WB of EGFR and DNA-PKcs in complexes with EGFR immunoprecipitated, pretreated for 2 hours with either 10 μmol/L NU74421 or 10 μmol/L KU55933 at indicated times following 4 Gy IR. C, PLA: NSCLCs or HBEC-3KT expressing wild type or ΔE746-E750 forms of EGFR following a 2-hour pretreatment with vehicle, 10 μmol/L NU7441 or 10 μmol/L KU55933 were mock irradiated or exposed to 4 Gy IR and fixed at 60 minutes. Left: representative images of nuclei (blue) showing EGFR-DNA-PKcs complexes (intense red dots). Original images in Supplementary Fig. S2. Right: mean integrated fluorescence intensity (columns) and SEM (error bars) from 2 independent experiments.

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DNA-PKcs phosphorylation at T2609 but not S2056 is critical for radiation-induced binding to EGFR

Previous studies show that radiation-induced phosphorylation of DNA-PKcs at T2609 in the 2609 cluster is ATM-dependent, whereas S2056 is autophosphorylated by DNA-PKcs in an ATM-independent manner (20). Our next objective was to examine whether alanine substitution at either of these residues would affect EGFR-DNA-PKcs binding and in turn alter EGFR-mediated survival responses. Toward this end, we stably expressed wild-type and mutant EGFR in V3 cells expressing either a S2056A-mutated (V3-S2056A) or T2609A-mutated (V3-T2609A) forms of DNA-PKcs (Fig. 5A). V3-S2056A cells were more radiosensitive compared with V3-T2609A cells (Fig. 5B). However, only V3-S2056A cells exhibited the characteristic decrease in radiosensitivity associated with wild-type EGFR and increase in radiosensitivity typical of L858R or ΔE746-E750 EGFR. In contrast, survival of V3-T2609A cells was strikingly unresponsive to wild type or mutant EGFR expression, indicating that T2609 was crucial in supporting EGFR-mediated survival response to radiation. We compared IR-induced binding of EGFR with S2056A or T2609A mutant forms of DNA-PKcs. In V3-S2056A cells, we observed a robust 40-fold IR-induced increase in nuclear complexes of EGFR and the S2056A mutant DNA-PKcs (Fig. 5C). In V3-T2609A cells, however, IR-induced complexes between EGFR and the T2609A mutant DNA-PKcs were virtually undetectable. Coimmunoprecipitiation assay essentially confirmed these findings in Fig. 5D. The data in Fig. 5 offer compelling evidence that DNA-PKcs phosphorylation at T2609, but not S2056 is critical for radiation-induced EGFR-DNA-PKcs binding and EGFR-mediated radioresponses.

Figure 5.

DNA-PKcs phosphorylation at T2609 is critical for EGFR-mediated radioresponse. A, WB analysis showing relative levels of DNA-PKcs (left) and wild type, L858R and ΔE746-E750 EGFR in V3-S2056A and V3-T2609A CHO cells. B, clonogenic survival in V3-S2056A and V3-T2609A CHO cells either mock transfected (open circle), or stably expressing LacZ (closed circle), wild-type EGFR (square), L858R (triangle up), or ΔE746-E750 (triangle down). Symbols: mean SF and error bars SD. C, PLA to detect EGFR-DNA-PKcs complexes in V3-WT DNA-PKCS, V3-S2056A, and V3-T2609A CHO cells expressing wild-type EGFR which were either mock irradiated or exposed to 4 Gy IR. Left: representative images of nuclei (blue) and EGFR-DNA-PKcs complexes (red PLA fluorescence dots) after processing for PLA. Original images in Supplementary Fig. S3. Right: fold increase in fluorescence (columns) relative to untreated cells at 1 hour following IR, and SEM (error bars) from 2 independent experiments each with 350 nuclei per sample or more. D, Co-IP/WB assay: WB analysis of EGFR and DNA-PKcs in complexes with EGFR was immunoprecipitated from V3-S2056A and V3-T2609A transfectants of wild-type L858R and ΔE7460-E750 EGFR at indicated time points following 4 Gy IR.

Figure 5.

DNA-PKcs phosphorylation at T2609 is critical for EGFR-mediated radioresponse. A, WB analysis showing relative levels of DNA-PKcs (left) and wild type, L858R and ΔE746-E750 EGFR in V3-S2056A and V3-T2609A CHO cells. B, clonogenic survival in V3-S2056A and V3-T2609A CHO cells either mock transfected (open circle), or stably expressing LacZ (closed circle), wild-type EGFR (square), L858R (triangle up), or ΔE746-E750 (triangle down). Symbols: mean SF and error bars SD. C, PLA to detect EGFR-DNA-PKcs complexes in V3-WT DNA-PKCS, V3-S2056A, and V3-T2609A CHO cells expressing wild-type EGFR which were either mock irradiated or exposed to 4 Gy IR. Left: representative images of nuclei (blue) and EGFR-DNA-PKcs complexes (red PLA fluorescence dots) after processing for PLA. Original images in Supplementary Fig. S3. Right: fold increase in fluorescence (columns) relative to untreated cells at 1 hour following IR, and SEM (error bars) from 2 independent experiments each with 350 nuclei per sample or more. D, Co-IP/WB assay: WB analysis of EGFR and DNA-PKcs in complexes with EGFR was immunoprecipitated from V3-S2056A and V3-T2609A transfectants of wild-type L858R and ΔE7460-E750 EGFR at indicated time points following 4 Gy IR.

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DNA-PKcs phosphorylation at T2609 but not S2056 is abrogated in mutant EGFR expressing NSCLCs and HBEC

Radiation-induced EGFR-DNA-PKcs interactions are absent in cells expressing the L858R or ΔE746-E750 mutant forms of EGFR [Fig. 3A and (27)]. We investigated whether this had any effect on the phosphorylation status of DNA-PKcs. We used an immune-fluorescence microscopy approach to quantify the extent and subcellular location of DNA-PKcs phosphorylation. We focused on 2 specific residues. Phosphorylation of T2609 is ATM dependent, whereas S2056 is autophosphorylated by DNA-PKcs in an ATM-independent manner (20). Figure 6A shows that, in wild-type EGFR expressing HBEC, DNA-PKcs phosphorylation at both, T2609 and S2056 rapidly increased in response to 4 Gy. Cells expressing the L858R or ΔE746-E750 forms of EGFR, showed a similar IR-induced increase in S2056 phosphorylation. Surprisingly, IR-induced DNA-PKcs phosphorylation at T2609 was virtually undetectable even at 60 minutes following IR. The data indicate that mutant EGFR expression specifically abrogates radiation-induced phosphorylation of DNA-PKcs at T2609 but not S2056.

Figure 6.

Activating mutations in EGFR specifically block IR-induced DNA-PKcs phosphorylation at T2609. A, immuno-fluorescence detection of DNA-PKcs phosphorylation in HBEC stably expressing wild type, L858R or ΔE746-E750 forms of EGFR at various time points following 4 Gy IR. Top: representative images with anti-p-T2609 and anti-p-S2056 DNA-PKcs antibodies showing nuclei (blue) and phosphorylated DNA-PKcs (green). Original images in Supplementary Fig. S4. Bottom: fluorescence intensity from p-T2609 and p-S2056 signals as a function of time. Columns: mean fluorescence intensity relative to 100% fluorescence at 60 minutes. Error bars: SEM from 3 experiments, each with 300 to 450 nuclei per sample. B, representative images of p-T2609 immunofluorescence (green) in nuclei (blue) in indicated NSCLCs (left). Original images in Supplementary Fig. S5. Fluorescence intensity levels (right) in indicated NSCLCs. C, p-T2609 immunofluorescence intensity levels (right) in A549 and HCC827 cells, from images (left) captured 1-hour postirradiation following a 2-hour pretreatment with 10 μmol/L NU7441 or 10 μmol/L KU55933. Original images in Supplementary Fig. S6. For (B) and (C), columns: mean p-T2609 fluorescence per nucleus and error bars: SEM from 2 independent experiments, each with 450 to 600 nuclei per sample.

Figure 6.

Activating mutations in EGFR specifically block IR-induced DNA-PKcs phosphorylation at T2609. A, immuno-fluorescence detection of DNA-PKcs phosphorylation in HBEC stably expressing wild type, L858R or ΔE746-E750 forms of EGFR at various time points following 4 Gy IR. Top: representative images with anti-p-T2609 and anti-p-S2056 DNA-PKcs antibodies showing nuclei (blue) and phosphorylated DNA-PKcs (green). Original images in Supplementary Fig. S4. Bottom: fluorescence intensity from p-T2609 and p-S2056 signals as a function of time. Columns: mean fluorescence intensity relative to 100% fluorescence at 60 minutes. Error bars: SEM from 3 experiments, each with 300 to 450 nuclei per sample. B, representative images of p-T2609 immunofluorescence (green) in nuclei (blue) in indicated NSCLCs (left). Original images in Supplementary Fig. S5. Fluorescence intensity levels (right) in indicated NSCLCs. C, p-T2609 immunofluorescence intensity levels (right) in A549 and HCC827 cells, from images (left) captured 1-hour postirradiation following a 2-hour pretreatment with 10 μmol/L NU7441 or 10 μmol/L KU55933. Original images in Supplementary Fig. S6. For (B) and (C), columns: mean p-T2609 fluorescence per nucleus and error bars: SEM from 2 independent experiments, each with 450 to 600 nuclei per sample.

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We next verified the relationship between EGFR mutation status and T2609 phosphorylation in NSCLC cell lines. Exposure of wild type expressing A549 NSCLC to 4 Gy resulted in an approximately 1,000-fold increase in DNA-PKcs phosphorylation at T2609 (Fig. 6C). Both basal and radiation-induced DNA-PKcs phosphorylation was undetectable in the L858R expressing H1975 cells or the ΔE746-E750 expressing H820 cells. However, HCC827, which also harbors the ΔE746-E750 mutant, exhibited an approximately 75-fold increase in T2609 phosphorylation with radiation, although this was dramatically lower than the approximately 1,000-fold increase observed in A549 NSCLCs. Moreover, while IR-induced T2609 phosphorylation in A549 cells was NU7441-insensitive, but KU55933-sensitive, IR-induced T2609 phosphorylation in HCC827 NSCLC cells was unaffected by either ATM- or DNA-PKcs-inhibition, indicating a mechanism unrelated to ATM or DNA-PKcs.

Mutant EGFR expression reverses IR-induced decrease in DNA-PKcs-PP2A binding

The data in Fig. 6 indicate that activating mutations in EGFR adversely influence radiation-induced phosphorylation of DNA-PKcs at T2609. We considered 2 possibilities. First, we examined whether mutant EGFR directly influences DNA-PKcs p-T2609 although an ATM-dependent mechanism. Results in Fig. 7A and B indicate that in both NSCLCs and HBEC, L858R or ΔE746-E750 expression had no effect on overall ATM levels or ATM phosphorylation at S1981. Second, we examined whether wild type or mutant EGFR expression had any effect on interactions between DNA-PKcs and the protein phosphatase, PP2A. There is evidence that radiation-induced DNA-PKcs phosphorylation is regulated by PP2A that dephosphorylates and inactivates DNA-PKcs (34). Data in Fig. 7C and D show that in unirradiated A549 and wild-type EGFR expressing HBEC, levels of PP2A-DNA-PKcs interactions predominate but are significantly reduced (∼3-fold) on exposure to 4 Gy IR. In striking contrast, ΔE746-E750 mutant EGFR expressing H820 and HBEC have higher basal levels of PP2A-DNA-PKcs complexes and exposure to IR resulted in a further (≥2-fold) increase in PP2A-DNA-PKcs binding. The data provide compelling evidence that activating mutations in EGFR significantly augment PP2A-DNA-PKcs interactions and suggest a possible mechanism underlying mutant EGFR-associated abrogation of T2609 phosphorylation.

Figure 7.

Mutant EGFR expression reverses IR-induced decrease in DNA-PKcs-PP2A binding. WB analysis with antibodies against phospho-serine 1981 ATM or total ATM in A, NSCLC or B, HBEC expressing wild type, L858R or ΔE746-E750 forms of EGFR at time points following 4 Gy IR. C, PLA to detect PP2A-DNA-PKcs interactions in NSCLCs and HBEC expressing WT or ΔE746-E750 EGFR fixed 1 hour following 0 or 4 Gy IR. Left: representative images of nuclei (blue) and PP2A-DNA-PKcs complexes (red: PLA fluorescence). Original images in Supplementary Fig. S7. Right: mean integrated fluorescence intensity per nucleus (columns) and SEM from 4 independent experiments, each with 500 to 750 nuclei per sample. Single asterisk (*): statistically significant decrease in EGFR-PP2A interactions determined by Student's t test between 0 and 4 Gy samples of WT EGFR expressing NSCLCs or HBEC (P < 0.01). Double asterisk (**): statistically significant increase in EGFR-PP2A interactions between 0 and 4 Gy samples of ΔE746-E750 EGFR expressing NSCLCs or HBEC (P < 0.01). E, Co-IP/WB assay: top: EGFR and of PP2A (subunits a and c) in complexes with EGFR immunoprecipitated from A549 and H820 cell lines. Bottom: WB assay to detect levels of total PP2A and Y307 phosphorylated PP2A in NSCLCs and HBEC cells.

Figure 7.

Mutant EGFR expression reverses IR-induced decrease in DNA-PKcs-PP2A binding. WB analysis with antibodies against phospho-serine 1981 ATM or total ATM in A, NSCLC or B, HBEC expressing wild type, L858R or ΔE746-E750 forms of EGFR at time points following 4 Gy IR. C, PLA to detect PP2A-DNA-PKcs interactions in NSCLCs and HBEC expressing WT or ΔE746-E750 EGFR fixed 1 hour following 0 or 4 Gy IR. Left: representative images of nuclei (blue) and PP2A-DNA-PKcs complexes (red: PLA fluorescence). Original images in Supplementary Fig. S7. Right: mean integrated fluorescence intensity per nucleus (columns) and SEM from 4 independent experiments, each with 500 to 750 nuclei per sample. Single asterisk (*): statistically significant decrease in EGFR-PP2A interactions determined by Student's t test between 0 and 4 Gy samples of WT EGFR expressing NSCLCs or HBEC (P < 0.01). Double asterisk (**): statistically significant increase in EGFR-PP2A interactions between 0 and 4 Gy samples of ΔE746-E750 EGFR expressing NSCLCs or HBEC (P < 0.01). E, Co-IP/WB assay: top: EGFR and of PP2A (subunits a and c) in complexes with EGFR immunoprecipitated from A549 and H820 cell lines. Bottom: WB assay to detect levels of total PP2A and Y307 phosphorylated PP2A in NSCLCs and HBEC cells.

Close modal

The data so far indicate that EGFR mediates survival response to radiation through IR-induced interactions with DNA-PKcs that are abrogated in a dominant negative manner in the context of radiosensitizing L858R or ΔE746-E750 EGFR mutations. Our study shows for the first time that DNA-PKcs phosphorylation is an essential prerequisite for EGFR-DNA-PKcs interaction. Alanine substitutions in DNA-PKcs that prevent its radiation-induced phosphorylation abrogate EGFR-DNA-PKcs binding (Fig. 3A, 5C and D). Of these, the T2609A and S2056A mutations are particularly relevant. Evidence shows that p-T2609 is an ATM-dependent phosphorylation event, whereas p-S2056 is an ATM-independent DNA-PKcs autophosphorylation event (20). Our data indicate that p-T2609 is a site of interaction between DNA-PKcs and EGFR because T2609A, but not S2056A, substitution abrogated EGFR binding (Fig. 5C and D). Moreover, ATM deficiency (Fig. 2) or inhibition (Fig. 4), which abrogates T2609 (Fig. 6C), but not S2056 phosphorylation (20), also blocked EGFR-DNA-PKcs binding. DNA-PKcs or ATM alterations had no effect on IR-induced EGFR nuclear translocation (Supplementary Fig. S1A–C) suggesting a direct impact on nuclear EGFR-DNA-PKcs interaction.

Both, DNA-PKcs-7A and T2609A, mutants are catalytically active but do not bind EGFR (Fig. 3A and 5D). Moreover, alanine substitution at S2056, a substrate of DNA-PKcs kinase activity, did not affect EGFR-DNA-PKcs binding (Fig. 5C and D). However, DNA-PKcs inhibition by NU7441 completely inhibited EGFR-DNA-PKcs binding (Fig. 4B) raising the possibility that other DNA-PKcs autophosphorylation sites could be involved.

Surprisingly, in addition to its pivotal role in EGFR-DNA-PKcs binding, T2609 phosphorylation is also influenced by EGFR. Our study shows for the first time, that activating mutations in EGFR specifically inhibit IR-induced phosphorylation of DNA-PKcs at T2609, but not S2056, a pattern that closely resembles EGFR blockade by anti-EGFR antibody, C225 (9, 35) or ATM inhibition by KU55933 (Fig. 6 and (20)). At least with mutant EGFRs, the mechanism is likely ATM independent, because IR-induced ATM S1981 phosphorylation (Fig. 7A and B) was not affected.

Our data reveal an inverse relationship between EGFR-DNA-PKcs binding and DNA-PKcs association with protein phosphatase, PP2A. Wild-type EGFR expression was associated with dramatic IR-induced increases in EGFR-DNA-PKcs binding, corresponding reductions in PP2A-DNA-PKcs complexes and robust IR-induced DNA-PKcs-T2609 phosphorylation. In contrast, in both basal and IR-induced settings, mutant EGFR expression was associated with absence of EGFR-DNA-PKcs complexes (Fig. 3A), significantly elevated levels of PP2A-DNA-PKcs complexes (Fig. 7C and D) and abrogation of IR-induced DNA-PKcs-T2609 phosphorylation (Fig. 6). However, PP2A phosphorylation at Y307 (Fig 7E, bottom) or EGFR-PP2A binding (Fig. 7E, top) was unaffected by radiation or EGFR mutation status. Thus, EGFR modulates radiation response predominantly through interactions with DNA-PKcs that likely stabilizes DNA-PKcs phosphorylation.

Mutant EGFR-associated abrogation of T2609 phosphorylation has important implications on DNA-PKcs function. Studies show that DNA-PKcs phosphorylation at different sites govern either the stability of the DSB-DNA-PKcs complex required for end processing, or the timely dissociation of DNA-PKcs, which appears critical for DNA end ligation (14, 16, 37, 38). Uematsu and colleagues observed that wild type and T2609A-mutated DNA-PKcs had similar kinetics of association or dissociation at DSBs (29). We observed no change in the dissociation kinetics of wild-type DNA-PKcs with L858R or ΔE746-E750 mutant EGFR expression (data not shown). Two groups have shown that phosphorylation of the 2609 cluster in DNA-PKcs plays a critical role in regulating the intrastrand endonuclease activity of the Artemis nuclease (21, 39). It is, therefore, conceivable that mutant EGFR-mediated inhibition of p-T2609 adversely influences Artemis activity and DNA end processing.

Our study proffers important insights on EGFR's contribution to cellular radiosensitivity. In V3-WT DNA-PKcs cell, effects of L858R or ΔE746-E750 mutant EGFR expression (Fig. 1C, middle) on radiosensitivity were not as dramatic compared with DNA-PKcs ablation (Fig. 1C, left) or NU7441 inhibition (Fig. 4A, middle). This is logical because DNA-PKcs has multiple roles in DSB repair and cellular radioresistance, only some of which may be EGFR-dependent. Nagasawa and colleagues recently showed that, relative to wild-type DNA-PKcs, single T2609A or S2056A mutations exhibit modest radiosensitivity, whereas a T2609A/S2056A double mutation has a synergistic effect on radiosensitivity (36). The synergism of the T2609A/S2056A double mutation suggests that p-T2609 and p-S2056 govern distinct, nonoverlapping functions of DNA-PKcs. Our study reveals that at least one of these nonoverlapping DNA-PKcs functions, pT2609, is modulated by EGFR. Wild-type or mutant EGFR effects on radiosensitivity were evident in the S2056A, but not T2609A, genetic background. Moreover, L858R or ΔE746-E750 expression had a strikingly similar radiosensitizing effect on V3-S2056A cells (Fig. 5B, left) as the T2609A/S2056A double mutation (36).

Our study underscores a modulatory role for EGFR in DSB repair and radiation resistance. In its simplest form, our model suggests that initial IR-induced, ATM-dependent DNA-PKcs phosphorylation at T2609 is a prerequisite for the binding of nuclear EGFR to DNA-PKcs. This association adversely affects PP2A-DNA-PKcs interactions and stabilizes DNA-PKcs-T2609 phosphorylation, which is critical for Artemis-mediated DSB end possessing. Activating mutations in EGFR prevent EGFR-DNA-PKcs interaction, favor DNA-PKcs-PP2A association and compromise p-T2609 stability that adversely affects DSB processing. Our study has important implications on how radiotherapy in combination with EGFR blockade may benefit NSCLC patients, especially those that harbor radioresistant tumors with wild-type EGFR.

No potential conflicts of interest were disclosed.

Conception and design: P. Javvadi, D.J. Chen, B.P. Chen, C.S. Nirodi

Development of methodology: P. Javvadi, H. Makino, A.K. Das, D.J. Chen, C.S. Nirodi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P. Javvadi, H. Makino, D.J. Chen, C.S. Nirodi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P. Javvadi, H. Makino, B.P. Chen, C.S. Nirodi

Writing, review, and/or revision of the manuscript: P. Javvadi, A.K. Das, B.P. Chen, C.S. Nirodi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P. Javvadi, Y.-F. Lin,

Study supervision: C.S. Nirodi

This study was funded by NIH/NCI grant CA129364 (C.S. Nirodi) and the Simmons Comprehensive Cancer Center Bridge funds (C.S. Nirodi).

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.

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Supplementary data