Gain-of-function mutations of PPM1D/Wip1 impair the p53-dependent G1 checkpoint

Abstract

The DNA damage response (DDR) pathway and its core component tumor suppressor p53 block cell cycle progression after genotoxic stress and represent an intrinsic barrier preventing cancer development. The serine/threonine phosphatase PPM1D/Wip1 inactivates p53 and promotes termination of the DDR pathway. Wip1 has been suggested to act as an oncogene in a subset of tumors that retain wild-type p53. In this paper, we have identified novel gain-of-function mutations in exon 6 of PPM1D that result in expression of C-terminally truncated Wip1. Remarkably, mutations in PPM1D are present not only in the tumors but also in other tissues of breast and colorectal cancer patients, indicating that they arise early in development or affect the germline. We show that mutations in PPM1D affect the DDR pathway and propose that they could predispose to cancer.

Figure 1.

The PPM1D gene is mutated in selected cancer cell lines. (A) Whole-cell lysates from indicated cell lines were probed with anti-Wip1 (Bethyl Laboratories, Inc.), anti-Wip1 (Santa Cruz Biotechnology, Inc. [SC]), and anti–14-3-3 (loading control) antibodies. Note the additional Wip1-reactive band around 60 kD in U2OS and HCT116 cells. PC3 cells are p53 negative and served as a control. (B) Wip1 was depleted in U2OS and HCT116 cells by siRNA, and lysates were probed with the indicated antibodies. (C) Sequencing chromatograms of PPM1D from genomic DNA isolated from U2OS and HCT116 cells. Numbering is based on NCBI GenBank reference sequence NT_010783.15. Mutations are indicated by arrowheads and underlined. WT, wild-type PPM1D; Mut, mutated PPM1D; c., nucleotide sequence; p., Wip1 peptide sequence.

Figure 2.

Truncated Wip1 mutants are enzymatically active. (A) Soluble and chromatin fractions from cells expressing FL or truncated FLAG-Wip1 were probed with the indicated antibodies. (B) Cells expressing EGFP-Wip1-FL (FL), EGFP-Wip1-D314A (phosphatase dead), or truncated EGFP-Wip1 were fixed 3 h after irradiation (3 Gy). 53BP1 nuclear foci were counted by automated image analysis (1,000 cells per condition). Average number of 53BP1 foci per nucleus in transfected (GFP+) and neighboring control cells (GFP−) is shown. (C) Expression of Wip1-FL, Wip1-D314A, or truncated FLAG-Wip1 was induced by tetracycline 12 h before irradiation (3 Gy). Whole-cell lysates were probed with the indicated antibodies. (D) Phosphatase activity of immunopurified FLAG-Wip1-FL, -L450X, -R458X, or -D314A (phosphatase dead) was measured in vitro using a synthetic phosphopeptide corresponding to pSer15-p53 (bottom), and the precipitated material was probed with anti-Wip1 antibody as a control of equal loading (top). n = 4. (E) Quantification of the signal intensity corresponding to the endogenous levels of FL and truncated Wip1 in HCT116 and U2OS cells. siRNA of Wip1 was used as a control of the signal specificity. Error bars indicate standard deviations. mut, mutated; WT, wild type.

Figure 3.

Truncated Wip1 mutants show increased protein stability. (A) HCT116 and U2OS cells were treated for the indicated times with cycloheximide. Normalized cell lysates were probed using the monoclonal anti-Wip1 antibody (Santa Cruz Biotechnology, Inc. [SC]). (B) Signal intensity corresponding to the FL-Wip1 and truncated Wip1 from A was quantified using ImageJ software. The relative change in signal intensity is shown. Statistical significance was determined by unpaired two-tailed t test (n = 3; *, P < 0.05). (C) U2OS cells were treated with cycloheximide, proteasomal inhibitor MG132 or a combination of both for the indicated times and analyzed as in A. (D) U2OS cells were transfected with plasmid DNA coding for EGFP or EGFP fused to the C-terminal region of Wip1 (aa 380–605). Cells were treated for the indicated times with cycloheximide. Normalized cell lysates were probed with an anti-GFP antibody, and the signal intensity was quantified using ImageJ software. n = 3. Error bars indicate standard deviations. exp., exposure.

Figure 4.

Mutated Wip1 impairs the p53-dependent G1 checkpoint. (A) U2OS cells were transfected by siRNA targeting luciferase, Wip1 and/or p53 and grown for 48 h. Cells were treated with BrdU and STLC, irradiated or not irradiated with 4 Gy, and grown for a further 16 h. Cells were analyzed by immunoblotting (top) or by flow cytometry (bottom) to determine the fraction of G1 cells (2n; BrdU negative). (B) U2OS cells were transfected by siRNA targeting various isoforms of Wip1, irradiated, and probed with the indicated antibodies (left) or analyzed by cytometry (right). The fraction of BrdU-negative 2n cells corresponds to cells arrested in G1. (C, left) FUCCI-expressing HCT116 cells were transfected with Wip1 or luciferase siRNA (siLuc), irradiated (4 Gy), and followed by live-cell imaging. Cumulative progression into S phase was determined based on the loss of FUCCI-G1 marker (red) and appearance of the FUCCI-S/G2 marker (green) Numbers of analyzed cells are indicated in parentheses. (right) Representative images of four individual cells transfected with Wip1 or Luciferase siRNA are shown. Bars, 10 µm. (D) Retinal pigment epithelium (RPE1), U2OS, and HCT116 cells were transfected with Wip1 or luciferase siRNA and grown for 48 h before irradiation (5 Gy). Cells were fixed 4 h after IR and probed for total p53 and p21. Bars, 50 µm. (E) U2OS cells were treated as in D, collected 6 h after IR, and probed with the indicated antibodies. SC, Santa Cruz Biotechnology, Inc.

Figure 5.

Truncation mutations of Wip1 are present in cancer patients. (A) Chromatograms of four truncating mutations identified by screening of the PPM1D gene in cancer patients. Mutations are indicated by arrowheads and underlined. WT, wild-type PPM1D; Mut, mutated PPM1D; c., nucleotide sequence; p., Wip1 peptide sequence. (B) Cells expressing EGFP–Wip1-FL, -R458X, -L484X, -F534X, or -K535X mutants were irradiated, and the number of 53BP1 foci was analyzed as in Fig. 2 B. (C) FLAG–Wip1-FL, -R458X, -L484X, -F534X, or -K535X mutants were immunoprecipitated, and phosphatase activity was determined as in Fig. 2 D. (D) Wip1 expression in leukocytes from a healthy control or the #BRCA1855 patient was analyzed by immunoblotting. The asterisk indicates a cross-reacting band in the blood sample. Note the increased expression level of the truncated Wip1 in leukocytes from cancer patient. (E) Mutation of PPM1D was analyzed in microdissected mammary noncancer tissue and in cancer tissue from the #BRCA1855 patient. Error bars indicate standard deviations.