DNA damage alters EGFR signaling and reprograms cellular response via Mre-11

Abstract

To combat the various DNA lesions and their harmful effects, cells have evolved different strategies, collectively referred as DNA damage response (DDR). The DDR largely relies on intranuclear protein networks, which sense DNA lesions, recruit DNA repair enzymes, and coordinates several aspects of the cellular response, including a temporary cell cycle arrest. In addition, external cues mediated by the surface EGF receptor (EGFR) through downstream signaling pathways contribute to the cellular DNA repair capacity. However, cell cycle progression driven by EGFR activation should be reconciled with cell cycle arrest necessary for effective DNA repair. Here, we show that in damaged cells, the expression of Mig-6 (mitogen-inducible gene 6), a known regulator of EGFR signaling, is reduced resulting in heightened EGFR phosphorylation and downstream signaling. These changes in Mig-6 expression and EGFR signaling do not occur in cells deficient of Mre-11, a component of the MRN complex, playing a central role in double-strand break (DSB) repair or when cells are treated with the MRN inhibitor, mirin. RNAseq and functional analysis reveal that DNA damage induces a shift in cell response to EGFR triggering that potentiates DDR-induced p53 pathway and cell cycle arrest. These data demonstrate that the cellular response to EGFR triggering is skewed by components of the DDR, thus providing a plausible explanation for the paradox of the known role played by a growth factor such as EGFR in the DNA damage repair.

Figure 1

DNA damage enhances ligand-induced EGFR phosphorylation and signaling. Normal human dermal fibroblasts (HF) were either left untreated or treated with bleomycin for 30 min, followed by extensive washing and subsequent exposure to HB-EGF (10 ng/ml). After 5 min (A) or at indicated time points (B), cells were lysed. Western blot analyses were performed on cell extracts using antibodies to phosphorylated EGFR (Y1173), phosphorylated AKT, and ERK. Anti-β-actin immunoblotting revealed relative amounts of protein in each lane. (C) HF were treated as above and at the indicated time points cells were collected and immune-stained for cell surface expression of EGFR. MFI: Median fluorescent intensity. (D) Fibroblasts from an ATLD patient (ATLD2) were treated and analyzed as in (A). pEGFR signal intensities were quantified and normalized to actin signal (loading control). The level of EGFR phosphorylation in response to HB-EGF treatment in bleomycin treated cells as compared to that of mock-treated cells (set as 1) are shown (A: n = 9; D: n = 4). Representative results of at least three independent experiments are shown. *p < 0.05; ns: non-significant.

Figure 2

Pharmacological inhibition of Mre-11 interferes with ligand-dependent EGFR signaling in cells suffering from DNA damage. (A) HF were either left untreated or treated with mirin (100 µM) for one hour and then were treated with bleomycin for an additional 30 min followed by HB-EGF (10 ng/ml) treatment for 10 min. (B) ATLD2 cells (Mre-11^−/−) or ATLD2 cells reconstituted with wild type Mre-11 (Mre-11 wt) were treated as in (A). (C) HF were either left untreated or treated with mirin for one hour and then left untreated (mock) and HB-EGF was added for 5 min. Cells were lysed and subjected to Western blot analyses using the indicated antibodies as above. pEGFR signal intensities were quantified and normalized as in Fig. 1 (n = 5). Representative results of at least three independent experiments are shown.

Figure 3

Regulation of Mig-6 expression by DNA damage. HF cells (A), ATLD2 cells (B), or HF cells that were pre-treated with mirin (100 µM) for an hour (C), were either left untreated or treated with bleomycin for 30 min and then RNA was extracted. Mig-6 mRNA expression level was determined by qRT-PCR and normalized to GAPDH. Graphs show an average (± STD) of the indicated number of independent experiments in each group. The expression of Mig-6 in control cells was set as 1. **p < 0.005; ***p < 0.0001; ns: non-significant.

Figure 4

DNA damage alters gene expression profile and cellular response to HB-EGF triggering. (A) HF were treated as in Fig. 1A. After 30 and 90 min RNA was extracted and was subjected to RNAseq analysis. Differentially expressed genes altered with higher than twofold changes were selected. A list of IPA canonical pathways that are enriched in bleomycin treated cells stimulated with HB-EGF compared with those from HB-EGF stimulated mock cells (upper left panel). IPA upstream functional analysis was used to predict the top upstream regulators from differentially expressed genes (lower left panel). Gene set enrichment analysis (GSEA) pathways that are up-regulated (red) or down-regulated (blue) in response to HB-EGF treatment in bleomycin as compared with mock-treated cells (right panel). (B) Mock and peroxide (500 µM) treated cells were pulsed with BrdU for two hours immediately after treatment. After 24 h the cells were fixed and analyzed by flow cytometry. The percentage of BrdU positive cells is shown. Representative results of three independent experiments are shown. (C) Cells were treated as above and after 12 h were collected and stained with Annexin-V. The percentage of Annexin-V positive cells of three independent experiments was normalized to mock (set as 1). *p < 0.05; **p < 0.005; ns: non-significant.

Figure 5

Model effects of DNA damage and repair on EGFR signaling and cellular response. (A) Under homeostatic conditions binding of ligand to EGFR induces canonical signals supporting cell proliferation and survival. (B) Upon induction of DNA damage, the MRN complex is assembled around sites of DSBs leading to a decrease in Mig-6 expression and amplifies ligand-induced EGFR signaling. In turn, amplified EGFR signaling enhances DDR-induced p53 pathway and cell cycle arrest and facilitates DSBs rejoining. Images created using BioRender.