Epidermal growth factor receptor dependence of radiation-induced transcription factor activation in human breast carcinoma cells

Abstract

Ionizing radiation (1-5 Gy) activates the epidermal growth factor receptor (EGFR), a major effector of the p42/44 mitogen-activated protein kinase (MAPK) pathway. MAPK and its downstream effector, p90 ribosomal S6 kinase (p90RSK), phosphorylate transcription factors involved in cell proliferation. To establish the role of the EGFR/MAPK pathway in radiation-induced transcription factor activation, MDA-MB-231 human breast carcinoma cells were examined using specific inhibitors of signaling pathways. Gel-shift analysis revealed three different profile groups: 1) transcription factors that responded to both radiation (2 Gy) and epidermal growth factor (EGF) (CREB, Egr, Ets, and Stat3); 2) factors that responded to radiation, but not EGF (C/EBP and Stat1); and 3) those that did not respond significantly to either radiation or EGF (AP-1 and Myc). Within groups 1 and 2, a two- to fivefold maximum stimulation of binding activity was observed at 30-60 min after irradiation. Interestingly, only transcription factors that responded to EGF had radiation responses significantly inhibited by the EGFR tyrosine kinase inhibitor, AG1478; these responses were also abrogated by farnesyltransferase inhibitor (FTI) or PD98059, inhibitors of Ras and MEK1/2, respectively. Moreover, radiation-induced increases in CREB and p90RSK phosphorylation and activation of Stat3 and Egr-1 reporter constructs by radiation were all abolished by AG1478. These data demonstrate a distinct radiation response profile at the transcriptional level that is dependent on enhanced EGFR/Ras/MAPK signaling.

Figure 1

Time-dependent increases in the radiation-induced binding of MDA-MB-231 nuclear extracts to transcription factor consensus sequences. The experimental protocol is described in the legend of Figure 2, except AG1478 treatment was not used in these experiments. Fold-changes represent the mean of three independent experiments.

Figure 2

Inhibition of nuclear extract binding to transcription factor consensus sequences by AG1478 (AG). Cells were preincubated with 5 μM AG1478 for 1 h, irradiated with 2 Gy, and nuclear extracts were isolated at various times. Extracts were incubated with ³²P-labeled consensus oligonucleotides, run on TBE-PAGE gels, and autoradiography was performed. The free oligo (FO) samples were incubated without nuclear extract. A 20-fold excess of nonradioactive oligonucleotides were added to the 60 min sample as a “cold” competitor (CC). Fold-changes represent the mean of three independent experiments.

Figure 3

Effect of epidermal growth factor (EGF) treatment on the binding of nuclear extracts to transcription factor consensus sequences. EGF treatments (10 ng/ml) were for 30 min at 37°C before nuclei extraction. For irradiated samples, nuclei were extracted 60 min after a 2-Gy dose. The rest of the protocol is the same as described in the legend of Figure 2. 0, untreated control; R, treatment with radiation alone; E, treatment with EGF alone.

Figure 4

Effect of farnesyltransferase inhibitor I (FTI) on the radiation-induced maximum increase in binding of nuclear extracts to transcription factor consensus sequences. Cells were treated with 50 μM FTI for 18 h before irradiation, and drug was present until protein extraction. Otherwise, the protocol is the same as described in the legend of Figure 2. Fold-changes represent the mean of three independent experiments.

Figure 5

Effect of PD98059 (PD) on the radiation-induced maximum increase in binding of nuclear extracts to transcription factor consensus sequences. Cells were treated with 10 μM PD98059 for 1 h before irradiation, and drug was present until protein extraction. Otherwise, the protocol is the same as described in the legend of Figure 2. Fold-changes represent the mean of three independent experiments.

Figure 6

Gel-shift supershift and cold-competitor controls for transcription factors dependent on the EGFR (CREB, Egr, Ets, and Stat3) or MAPK (C/EBP). Control nuclear extracts (0) were from untreated cells. Supershift antibodies against CREB (all forms), Stat3, Egr-1, Ets-2, C/EBP-α, and C/EBP-β were added to reactions after nuclear extract incubation with the CREB, Stat3, Egr, Ets, and C/EBP oligonucleotides, respectively (SS). A 20-fold excess of nonradioactive oligonucleotides were added to nuclear extracts as a “cold” competitor (CC).

Figure 7

Time-dependent radiation-induced increase in phospho-CREB phosphorylation and inhibition of this effect by AG1478 (1 h pretreatment). Cells were irradiated with 2 Gy, incubated for various times, and extracts were analyzed by Western blotting using phospho-specific antibodies. The bottom panel shows the same blot probed for nonphosphorylated CREB. Fold-changes represent the mean of three independent experiments; maximum fold-increases and inhibition by AG1478 were statistically significant (p < 0.05).

Figure 8

(A) Time-dependent radiation-induced increase in p90RSK phosphorylation. Cells were irradiated with 2 Gy and incubated for various times, and extracts were analyzed by Western blotting. The top panel shows phospho-p90RSK, and the bottom panel shows the same blot probed for β-actin. (B) Inhibition of p90RSK phosphorylation at 30 min after irradiation by either AG1478 or PD98059. Fold-changes represent the mean of three independent experiments. The fold-change at 30 min and inhibition by either compound were significant (p < 0.05).

Figure 9

Radiation-induced increase in reporter construct activation. Cells were cotransfected with reporter constructs containing transcription factor binding sites, and β-galactosidase reporter constructs (to obtain transfection efficiencies values for normalization). Cells were irradiated or EGF-treated and incubated at 37°C for 3 or 6 h. AG1478 was present for 1 h before and after irradiation. Error bars, ±SEM of three independent experiments. Maximum fold-changes and inhibition by AG1478 were statistically significant (p < 0.05). (A) Egr-1 reporter construct; (B) Stat3 reporter construct.

Figure 10

A schematic representation of the effects of EGFR, MAPK, and Ras inhibition on radiation-induced transcription factor activation and proliferation control. The events that are shown downstream of transcription factors in this model are referenced in the DISCUSSION; each transcription factor regulates one or more of the indicated proteins that are involved in proliferation control.