WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response

Abstract

Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells.

Keywords: ITCH; WW domain-containing oxidoreductase; ataxia telangiectasia-mutated; common fragile sites; genomic instability.

Fig. 1.

Induction of WWOX expression early after DNA damage stimuli. (A) Real-time PCR analysis of WWOX, FHIT, and MAF in MCF7 after ionizing radiation treatment for the indicated time points. (B–E) Immunoblot blot analyses of WWOX levels in (B and C) MCF7 and (D and E) early-passage MEFs after treatment with ionizing radiation (10 Gy) or NCS (200 ng/mL) for different time points. Equal loading was confirmed by blotting with α-GAPDH–specific antibody. Fold change of WWOX expression upon DNA damage relative to untreated cells is shown below each blot.

Fig. 2.

Depletion of WWOX renders cells more susceptible to DSBs and compromises DNA damage-induced ATM checkpoint activation. (A) Comet assay. Control- (EV-Sh) and WWOX-depleted MCF7 (WWOX-Sh) cells were treated or not treated with NCS (200 ng/mL) for 1 h. Single cells were then electrophoresed in agarose gel on a slide and stained with SyberGreen. Labeled DNA was visualized under a fluorescence microscope using 60× magnification. Representative images are shown. (B) Quantification of the comet assay in A. Bars show the comet tail as measured using ImageJ 1.47g software. Results show that MCF7-sh (WWOX-depleted) treated with NCS accumulates more DSBs relative to EV-control cells. (C) Control- and WWOX-depleted MCF7 cells were untreated or treated with ionizing radiation (10 Gy). Whole-cell lysates were prepared at the indicated time points, and immunoblot analysis was performed using specific antibodies against ATM, p-ATM (Ser1981), KAP1, p-KAP1 (pThr824), total H2A.X, γ-H2AX (pSer139), WWOX, and GAPDH. (D) WWOX-depleted MCF7 cells (MCF7-sh) were transduced with lentiviral vectors expressing scramble (EV), WT WWOX, or WT WWOX-WFPA mutant and selected with neomycin. The different cells were untreated or treated with NCS (200 ng/mL) for the indicated time points. Whole-cell lysates were prepared at the indicated time points, and immunoblot analysis was performed using specific antibodies against ATM, p-ATM, KAP1, p-KAP1, WWOX, and GAPDH. IR, ionizing radiation.

Fig. 3.

Impaired γ-H2AX signals and DNA repair in WWOX-manipulated cells after DNA damage. (A) WT or KO MEFs were untreated or treated with ionizing radiation (10 Gy) and fixed at the indicated time points, and immunofluorescence analysis for γ-H2AX (red) was carried out. DAPI staining was performed to indicate the positions of nuclei. Cells were examined by confocal microscopy under 60× magnification. Close-up views of one cell are shown in column 5 (zoom = 5×). Quantification of γ-H2AX foci is shown under the panels as the average foci number per cell ± SEM; P < 0.002 in treatment of 30 min between WT and KO MEFs. (B) KO and WT MEFs were transduced with Ad-WWOX or Ad-GFP, treated with ionizing radiation (10 Gy), and fixed after 15 min, and immunofluorescence analysis for γ-H2AX (red) was carried out. A representative experiment is shown. Quantification of γ-H2AX foci is shown on the right side as the average foci number per cell ± SEM (P = 0.155 comparing KO-AdWWOX with WT-AdGFP; P = 0.118 comparing KO-AdWWOX with WT-AdWWOX). (C) Quantification of γ-H2AX foci in MCF7 control or WWOX-depleted cells after treatment with ionizing radiation (5 Gy) for different times. Representative figures are shown in Fig. S3B. The average of γ-H2AX foci in 10 fields is shown in the different treatments. UNT, untreated. (D and E) WWOX is important for HR. (D) WWOX-depleted MCF7 cells exhibit impaired micro-HR as revealed in cells transfected with pGL2-Luc vector that was linearized with either HindIII or EcoRI. Luciferase activity after EcoRI treatment was normalized to that of HindIII. (E) U2OS-DR-GFP cells were transduced with indicated Lenti-WWOX constructs (WWOX, WWOX-WFPA, or WWOX-K274R) (Fig. S3C). The newly generated cells were then transfected with I-SecI. Forty-eight hours later, cells were harvested and subjected to flow cytometric analysis to analyze the HR efficiency. The percentage of GFP-positive cells is shown. Data represent averages from three independent experiments.

Fig. 4.

WWOX association with ATM. (A and B) WWOX–ATM physical association. (A) MCF7 cells were untreated or treated with NCS (200 ng/mL) for 1 h. Lysates were immunoprecipitated with anti-WWOX mAb. Anti-IgG was used as the negative control. The immunoprecipitates were analyzed by immunoblotting using antibodies against p-ATM, p-KAP1, and WWOX. (B) HEK293 cells were transfected with Myc-WWOX expression vector. Twenty-four hours later, cells were irradiated (10 Gy), and 30 min later, cells were lysed. Lysates were immunoprecipitated with anti-Myc antibody and blotted with anti–p-ATM, anti–p-KAP1, and Myc (WWOX). (C and D) WWOX–ATM functional association. (C) MEF-WT and MEF-KO cells were treated with ionizing radiation (10 Gy), and 2 h later, cells were fixed and stained for p-ATM (green) and p-H2AX (red). Nuclei were counterstained with DAPI (blue). Cells were visualized with confocal microscopy under 60× magnification. (D) WWOX depletion is associated with reduced ATM monomerization. HEK293-Sh-EV and HEK293-Sh-WWOX cells were cotransfected with Flag-ATM and YFP-ATM. At 48 h, cells were untreated or treated with NCS (200 ng/mL) for an additional 1 h. Whole-cell lysates were prepared and precleared with mouse anti-IgG followed by immunoprecipitation with anti-IgG (as negative control; lanes 1 and 4) or anti-Flag antibody overnight at 4 °C. Complexes were then washed, separated on SDS/PAGE, and analyzed by immunoblotting using antibodies against FLAG (self-immunoprecipitation) and YFP (coimmunoprecipitation). (Left) Whole-cell lysates were analyzed by immunoblotting using the indicated antibodies. GAPDH was used for normalization. IP, immunoprecipitation.

Fig. 5.

WWOX nuclear accumulation and ubiquitination. (A) MCF7 cells were untreated or treated with NCS (200 ng/mL). At 24 h after NCS treatment, the cells were fractionated into cytosolic and nuclear extracts. The fractionated cytosol and nuclear extracts were immunoblotted using antibodies against WWOX, HSP90, or Lamin. (B) WWOX ubiquitination after DNA damage. HEK293 cells transfected with HA-Ub and pEBG-WWOX or pEBG-WWOX-WFPA plasmids. At 24 h, cells were treated with NCS (200 ng/mL) for an additional 1 h. Cells were then subfractionated into nuclear and cytoplasmic fractions, and GST pull down was performed. Lysates were blotted against ATM, p-ATM, GST (WWOX), Lamin (nuclear fraction), GAPDH (cytoplasmic fraction), and anti-HA (Ub) antibodies. Arrows indicate ubiquitinated WWOX.

Fig. 6.

Ubiquitination of WWOX at Lys274 after DNA damage. (A) HEK293 cells were transfected with HA-Ub and GST-WWOX or GST-WWOX-K274R plasmids. At 24 h, cells were treated with NCS (200 ng/mL) for an additional 1 h. Cell lysates were blotted against p-ATM, GST (WWOX), and GAPDH. Pulled down complexes were blotted with anti-HA (Ub), anti-GST (WWOX), and anti–p-ATM antibodies. (B) HeLa cells were transfected with HA-Ub and GST-WWOX or GST-WWOX-K274R plasmids. At 24 h, cells were treated with NCS (200 ng/mL) for 15 min. Immunostaining was performed using anti-GST (red) and anti–p-H2AX (green). GST is also observed in control untreated cells, likely because anti-GST stains endogenous GST. DAPI was used as a marker for nuclei. Cells were examined by confocal microscopy under 60× magnification. Quantification of γ-H2AX foci is shown on the right side as the average foci number per cell ± SEM (P < 0.001).

Fig. 7.

ITCH ubiquitinates WWOX upon DNA damage. (A) HEK293 cells were transfected with HA-Ub, GST-WWOX, and FLAG-ITCH or FLAG-ITCHC830A plasmids. At 24 h, cells were mock or KU55933 treated overnight followed by NCS (200 ng/mL) treatment of 1 h. Cell lysates were blotted against the indicated antibodies. Pulled down complexes were blotted with anti-HA (Ub), anti-GST (WWOX), and anti-ITCH antibodies. (B) Control- and ITCH-depleted HEK293 cells were transfected with GST-WWOX and HA-Ub. At 24 h, cells were incubated with NCS (200 ng/mL) for an additional 1 h. Cell lysates and pulled down complexes were treated as in A. (C) Itch-KO and WT MEFs were treated with NCS (200 ng/mL) for the indicated times. Lysates were blotted using the indicated antibodies. (D) Hypothetical model of WWOX action in DDR. WWOX deficiency leads to an increased number of DNA strand breaks after DNA damage. After DNA damage, ATM positively enhances ITCH-mediated ubiquitination and translocation of WWOX into the nucleus. Nuclear WWOX physically interacts with ATM and mediates ATM monomerization and activation in a positive feed-forward loop manner. When WWOX is lost, ATM function is hampered.