ABRO1 suppresses tumourigenesis and regulates the DNA damage response by stabilizing p53

Abstract

Abraxas brother 1 (ABRO1) has been reported to be a component of the BRISC complex, a multiprotein complex that specifically cleaves 'Lys-63'-linked ubiquitin. However, current knowledge of the functions of ABRO1 is limited. Here we report that ABRO1 is frequently downregulated in human liver, kidney, breast and thyroid gland tumour tissues. Depletion of ABRO1 in cancer cells reduces p53 levels and enhances clone formation and cellular transformation. Conversely, overexpression of ABRO1 suppresses cell proliferation and tumour formation in a p53-dependent manner. We further show that ABRO1 stabilizes p53 by facilitating the interaction of p53 with USP7. DNA-damage induced accumulation of endogenous ABRO1 as well as translocation of ABRO1 to the nucleus, and the induction of p53 by DNA damage is almost completely attenuated by ABRO1 depletion. Our study shows that ABRO1 is a novel p53 regulator that plays an important role in tumour suppression and the DNA damage response.

Figure 1. ABRO1 is frequently downregulated in human liver cancer tissues.

(a) Quantitative RT–PCR analysis of ABRO1 mRNA levels in liver cancer tissues and corresponding adjacent tissue from 22 HCC patients. Data were analysed using Student’s t-test and are shown as the mean±s.d. **P<0.01. (b) ABRO1 expression scores shown as box plots, with the horizontal lines representing the median; the bottoms and tops of the boxes represent the 25th and 75th percentiles, respectively, and the vertical bars represent the range of data. Comparison was performed using the Mann–Whitney U-test. n=310. (c,d) Low ABRO1 expression correlates with poor survival of HCC patients. Kaplan–Meier survival curves of overall survival in Cohort 1 and Cohort 2 (c) and tumour-free survival in Cohort 2 (d) are shown. Comparison was made between groups with high ABRO1 expression (score≥9) and low ABRO1 expression (score<9). The marks on the graph lines represent censored samples. P-value refers to two-sided log-rank tests.

Figure 2. ABRO1 suppresses cell growth in a p53-dependent manner.

(a) Cells were transfected with pCMV-Flag-ABRO1 or pCMV-Flag vectors, and 2 × 10³ cells were plated per 6-cm dish and selected with G418. Clone numbers (>50 cells) were determined after 2 weeks. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. *P<0.05, **P<0.01. (b) HCT116 p53^+/+ and HCT116 p53^−/− cells were transfected with pCMV-Flag-ABRO1 or pCMV-Flag vectors, and the number of G418-resistant clones was determined. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. A representative image of the clones is shown in Supplementary Fig. 3a. (c) HCT116 p53^+/+ and HCT116p53^−/− cells were infected with control lentivirus, with lentivirus encoding shRNA specific to ABRO1 or with lentivirus encoding ABRO1 shRNA and RNAi-immune ABRO1. Clones were counted 2 weeks after initial plating. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. A representative image of the clones is shown in Supplementary Fig. 3b. (d) L02 cells were infected with control lentivirus, with lentivirus encoding shRNA specific to ABRO1 or with lentivirus encoding ABRO1 shRNA and RNAi-immune ABRO1, and plated on soft agar. Clones were counted 2 weeks after initial plating. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. A representative image of the clones is shown in Supplementary Fig. 3c. (e) HCT116 p53^+/+ and HCT116 p53^−/− cells were transfected with pCMV-Flag-ABRO1 or pCMV-Flag vectors, and G418-resistant cells were selected. The cells were pulsed for 1 h with BrdU before being harvested, fixed, stained with anti-BrdU fluorescein isothiocyanate (FITC) and propidium iodide (PI), sorted by flow cytometry and analysed for DNA content. The data shown are the mean±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. (f) Volume of xenograft tumours derived from control or ABRO1-overexpressing HCT116 cells. n=10 in each group. Tumour sizes were measured using a calliper at the indicated time points. The data shown are means±s.d. (g) Excised tumours from control or ABRO1-overexpressing HCT116 cells. Scale bar, 10 mm.

Figure 3. ABRO1 translocates to the nucleus after DNA damage and regulates the p53-dependent DNA damage response.

(a) HCT116 p53^+/+ cells were treated with DOX (2 μg ml⁻¹), VP16 (20 μM), or irradiation (10 Gy) for the indicated times. Proteins were extracted and subjected to western blotting. (b) HCT116 p53^+/+ cells were transfected with siABRO1-1 or siNC. After 48 h, cells were treated with DOX (2 μg ml⁻¹) for the indicated times. Proteins were extracted and subjected to western blotting. (c) HCT116 cells were transfected with siABRO1-1 or siNC. Forty-eight hours after transfection, the cells were treated with DOX (2 μg ml⁻¹) for 12 h, and cell cycle profiles were determined by FACS as described in the legend to Fig. 2. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. (d) HCT116 cells were infected with control lentivirus or with lentivirus encoding shRNA specific to ABRO1 and treated with DOX (2 μg ml⁻¹) for 12 h. Cell apoptosis was examined by FACS. The values shown are the mean±s.d. of three independent experiments; data were analysed using Student’s t-test. **P<0.01. (e) HCT116 p53^+/+ and A549 cells were treated with DOX (2 μg ml⁻¹), VP16 (20 μM), IR (10 Gy) or CPT (2 μM). The subcellular localization of ABRO1 was detected by immunochemistry with ABRO1 antibody, and 4',6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. Leptomycin B (LMB) was used as a positive control. Scale bar, 10 μm. (f) HCT116 p53^+/+ cells were treated as indicated. After harvesting and fractionation of the cells, cellular fractions were blotted with the indicated antibodies. (C, cytoplasmic; N, nuclear).

Figure 4. ABRO1 increases the protein stability of p53.

(a). HCT116 p53^+/+, HepG2 and A549 cells were transfected with increasing concentrations of plasmid encoding Flag–ABRO1. After 36 h, proteins were extracted and subjected to western blotting. (b) HCT116 p53^+/+ cells were transfected with different ABRO1 siRNAs and siNC. After 36 h, proteins were extracted and subjected to western blotting. (c) HCT116 p53^+/+ cells were co-transfected with plasmids encoding Flag-ABRO1 and Myc-MDM2. Proteins were extracted and subjected to western blotting. (d) HCT116 p53^+/+ cells were treated with cycloheximide (CHX) (1 μg μl⁻¹) at the indicated times after transfection with pCMVFlag-ABRO1. Proteins were extracted and subjected to western blotting. (e) HCT116 p53^+/+ cells were treated with CHX (1 μg μl⁻¹) at the indicated times after transfection with siABRO1-2 or siNC. Proteins were extracted and subjected to western blotting. (f) HCT116 p53^+/+ cells were co-transfected with the indicated constructs, and reporter activity was determined as described in Experimental Procedures. Data were generated from three independent experiments, each performed in triplicate (The data shown are the mean±s.d.). Data were analysed using Student’s t-test.*P<0.05, **P<0.01.

Figure 5. ABRO1 regulates p53 stability by inhibiting p53 ubiquitination.

(a) HCT 116 p53^+/+ cells were co-transfected with the indicated constructs. Twenty-four hours later, the cells were treated with 20 μM MG-132 for 4 h. Proteins were extracted, immunoprecipitated with p53 (FL-393) antibody and immunoblotted with anti-Ub antibody (left) or p53 antibody (DO-1) (right). (b) HCT 116 p53^+/+ cells were transfected with various ABRO1 siRNAs or with siNC. After 48 h, the cells were treated with 20 μM MG-132 for 4 h before harvesting and lysis for immunoprecipitation with p53 (FL-393) antibody and immunoblotting with anti-Ub antibody. (c) HCT 116 p53^+/+ cells were seeded onto small slide glass coverslips in 24-well culture plates and transfected with ABRO1 siRNAs or siNC. Forty-eight hours later, the cells were treated with 20 μM MG-132 for 4 h and the subcellular localization of p53 was detected by immunochemistry with p53 antibody. Scale bar, 10 μm. (d) HCT 116 p53^+/+ cells were treated as in c. The nuclear protein fractionation assay was performed, and the samples were immunoblotted with the indicated antibodies. (e) MEF mdm2^−/−/p53^−/− cells were co-transfected with Flag-ABRO1 and HA-p53 plasmids. Proteins were extracted and subjected to western blotting.

Figure 6. ABRO1 forms a complex with USP7 and p53.

(a) Lysates of HCT116 p53^+/+ cells with or without exposure to IR (10 Gy) were subjected to immunoprecipitation with control IgG or anti-p53 antibody. The immunoprecipitates were then blotted with ABRO1 antibody. (b) 293 cells were co-transfected with the indicated constructs, and lysates were subjected to immunoprecipitation with anti-myc. The immunoprecipitates were then blotted with ABRO1 antibody and anti-myc antibody. (c) 293 cells were co-transfected with the indicated constructs, and lysates of the cells were subjected to immunoprecipitation with anti-myc. The immunoprecipitates were then blotted with anti-Flag antibody and anti-myc antibody. (d) Lysates of HCT116 p53^+/+ cells with or without exposure to IR (10 Gy) were subjected to immunoprecipitation with control IgG and anti-USP7. The immunoprecipitates were then blotted with ABRO1 antibody. (e,f) 293 cells were co-transfected with the indicated constructs, and cell lysates were subjected to immunoprecipitation with anti-myc. The immunoprecipitates were then blotted with anti-Flag antibody and anti-myc antibody. (g) HCT116 p53^+/+ cell lysates were subjected to immunoprecipitation with control IgG or anti-ABRO1 antibody. The immunoprecipitates were washed with high-salt elution buffer and sequential immunoprecipitation was performed with the indicated antibodies. (h) HCT 116 p53^+/+ cells were seeded on glass coverslips in 24-well cell culture plates and then treated with or without IR. The subcellular localization of ABRO1, USP7 and p53 was detected by immunochemistry using the indicated antibodies. Scale bars, 10 μm.

Figure 7. ABRO1 facilitates p53 deubiquitination in a herpes-associated ubiquitin-specific protease (HAUSP)-dependent manner, and its level in primary RCC correlates with expression of p53.

HCT116 p53^+/+ cells transfected with the indicated constructs (a) or with siNC or siABRO1 (b) were treated with MG132 for 4 h before harvest. Lysates were immunoprecipitated with control IgG or anti-p53 (FL-393) antibody and analysed by immunoblotting with the indicated antibodies. (c) HCT116 p53^+/+ cells co-transfected with the indicated constructs were treated with MG132 for 4 h before harvest. Cell lysates were immunoprecipitated with anti-p53 (FL-393) antibody and analysed by immunoblotting with anti-Ub antibody. HCT116 p53^+/+ cells transfected with the indicated constructs and siABRO1 (d) or siUSP7 (e) were treated with MG132 for 4 h before harvest. Cell lysates were immunoprecipitated with anti-p53 (FL-393) antibody and analysed by immunoblotting with the indicated antibodies. (f) HCT116 p53^+/+ cells were co-transfected with Flag-ABRO1 and siBRE or siNC as indicated. Cell lysates were analysed by immunoblotting with the indicated antibodies. (g) Representative images showing immunohistochemical staining of ABRO1 and p53 expression in two serial sections of the same RCC tumour from three cases of different histological grades (1–3). Scale bars, 50 μm. (h) Box plot of p53 expression in RCC from 136 subjects. The subjects were divided into three groups based on ABRO1 expression scores in the tumours. The samples shown represent low (score 1–4), medium (score 5–8) and high (score 9–12) expression of ABRO1. Outliers are marked with circles and extreme cases are marked with asterisks. The data were analysed using the one-way analysis of variance test with Games–Howell’s correction. Horizontal lines represent the median; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively; and the vertical bars represent the range of data. (i) The percentage of p53-positive tumours in the three groups of subjects described in h. The data were analysed using Pearson’s χ²-test. (j) Working model of p53 regulation by ABRO1.