DNA damage response (DDR) pathway protects cells from genome instability and prevents cancer development. Tumor suppressor p53 is a key molecule that interconnects DDR, cell cycle checkpoints, and cell fate decisions in the presence of genotoxic stress. Inactivating mutations in TP53 and other genes implicated in DDR potentiate cancer development and also influence the sensitivity of cancer cells to treatment. Protein phosphatase 2C delta (referred to as WIP1) is a negative regulator of DDR and has been proposed as potential pharmaceutical target. Until recently, exploitation of WIP1 inhibition for suppression of cancer cell growth was compromised by the lack of selective small-molecule inhibitors effective at cellular and organismal levels. Here, we review recent advances in development of WIP1 inhibitors and discuss their potential use in cancer treatment.
- MeSH
- Protein Conformation MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- Neoplasms drug therapy metabolism MeSH
- Oncogenes MeSH
- DNA Damage MeSH
- Protein Phosphatase 2C antagonists & inhibitors chemistry immunology metabolism MeSH
- Antineoplastic Agents pharmacology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Genotoxic stress triggers a combined action of DNA repair and cell cycle checkpoint pathways. Protein phosphatase 2C delta (referred to as WIP1) is involved in timely inactivation of DNA damage response by suppressing function of p53 and other targets at chromatin. Here we show that WIP1 promotes DNA repair through homologous recombination. Loss or inhibition of WIP1 delayed disappearance of the ionizing radiation-induced 53BP1 foci in S/G2 cells and promoted cell death. We identify breast cancer associated protein 1 (BRCA1) as interactor and substrate of WIP1 and demonstrate that WIP1 activity is needed for correct dynamics of BRCA1 recruitment to chromatin flanking the DNA lesion. In addition, WIP1 dephosphorylates 53BP1 at Threonine 543 that was previously implicated in mediating interaction with RIF1. Finally, we report that inhibition of WIP1 allowed accumulation of DNA damage in S/G2 cells and increased sensitivity of cancer cells to a poly-(ADP-ribose) polymerase inhibitor olaparib. We propose that inhibition of WIP1 may increase sensitivity of BRCA1-proficient cancer cells to olaparib.
- MeSH
- Tumor Suppressor p53-Binding Protein 1 metabolism MeSH
- Apoptosis drug effects MeSH
- Drug Resistance, Neoplasm drug effects MeSH
- Chromatin metabolism MeSH
- Phthalazines pharmacology MeSH
- HEK293 Cells MeSH
- Homologous Recombination genetics MeSH
- G2 Phase Cell Cycle Checkpoints MeSH
- S Phase Cell Cycle Checkpoints MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Breast Neoplasms metabolism MeSH
- DNA Repair genetics physiology MeSH
- Poly(ADP-ribose) Polymerase Inhibitors pharmacology MeSH
- Piperazines pharmacology MeSH
- DNA Damage genetics physiology MeSH
- Cell Proliferation drug effects MeSH
- BRCA1 Protein metabolism MeSH
- Protein Phosphatase 2C antagonists & inhibitors genetics metabolism MeSH
- Antineoplastic Agents pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.
- MeSH
- Acetylation MeSH
- Adaptor Proteins, Signal Transducing genetics metabolism MeSH
- Biological Assay methods MeSH
- Cell Nucleus genetics metabolism MeSH
- Phosphorylation MeSH
- Protein Interaction Domains and Motifs MeSH
- Humans MeSH
- Tumor Cells, Cultured MeSH
- Tumor Suppressor Protein p53 genetics metabolism MeSH
- Bone Neoplasms genetics metabolism pathology MeSH
- DNA Repair MeSH
- Osteosarcoma genetics metabolism pathology MeSH
- DNA Damage MeSH
- Protein Phosphatase 2C genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
PP2C family serine/threonine phosphatase WIP1 acts as a negative regulator of the tumor suppressor p53 and is implicated in silencing of cellular responses to genotoxic stress. Chromosomal locus 17q23 carrying the PPM1D (coding for WIP1) is commonly amplified in breast carcinomas and WIP1 was proposed as potential pharmacological target. Here we employed a cellular model with knocked out PPM1D to validate the specificity and efficiency of GSK2830371, novel small molecule inhibitor of WIP1. We have found that GSK2830371 increased activation of the DNA damage response pathway to a comparable level as the loss of PPM1D. In addition, GSK2830371 did not affect proliferation of cells lacking PPM1D but significantly supressed proliferation of breast cancer cells with amplified PPM1D. Over time cells treated with GSK2830371 accumulated in G1 and G2 phases of the cell cycle in a p21-dependent manner and were prone to induction of senescence by a low dose of MDM2 antagonist nutlin-3. In addition, combined treatment with GSK2830371 and doxorubicin or nutlin-3 potentiated cell death through a strong induction of p53 pathway and activation of caspase 9. We conclude that efficient inhibition of WIP1 by GSK2830371 sensitizes breast cancer cells with amplified PPM1D and wild type p53 to chemotherapy.
- MeSH
- Aminopyridines pharmacology MeSH
- Apoptosis drug effects MeSH
- Cell Cycle drug effects MeSH
- Drug Resistance, Neoplasm * MeSH
- Dipeptides pharmacology MeSH
- Imidazoles pharmacology MeSH
- Humans MeSH
- Tumor Cells, Cultured MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- Breast Neoplasms drug therapy enzymology pathology MeSH
- Piperazines pharmacology MeSH
- DNA Damage drug effects MeSH
- Cell Proliferation drug effects MeSH
- Protein Phosphatase 2C antagonists & inhibitors genetics metabolism MeSH
- Proto-Oncogene Proteins c-mdm2 antagonists & inhibitors genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G(1) phase to G(2) and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G(1) cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.
- MeSH
- DNA Primers genetics MeSH
- Fluorescent Antibody Technique MeSH
- Phosphorylation MeSH
- Mass Spectrometry MeSH
- M Phase Cell Cycle Checkpoints physiology MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Humans MeSH
- RNA, Small Interfering genetics MeSH
- Mitosis physiology MeSH
- Cell Line, Tumor MeSH
- DNA Damage * MeSH
- Phosphoprotein Phosphatases metabolism MeSH
- Gene Expression Regulation physiology MeSH
- Signal Transduction physiology MeSH
- Transfection MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- MeSH
- Cell Cycle MeSH
- G1 Phase * MeSH
- Genetic Predisposition to Disease MeSH
- HeLa Cells MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Mutation * MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 genetics MeSH
- Neoplasms metabolism MeSH
- DNA Damage MeSH
- Phosphoprotein Phosphatases genetics physiology MeSH
- Gene Expression Regulation, Neoplastic * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.
- MeSH
- Ataxia Telangiectasia Mutated Proteins metabolism MeSH
- Models, Biological MeSH
- Cell Line MeSH
- Chromatin metabolism MeSH
- Phosphorylation MeSH
- G2 Phase Cell Cycle Checkpoints * MeSH
- Humans MeSH
- Protein Interaction Mapping MeSH
- Protein Processing, Post-Translational MeSH
- Protein Serine-Threonine Kinases metabolism MeSH
- Protein Phosphatase 2C metabolism MeSH
- Cell Cycle Proteins metabolism MeSH
- Proto-Oncogene Proteins metabolism MeSH
- Repressor Proteins metabolism MeSH
- Fluorescence Resonance Energy Transfer MeSH
- Models, Theoretical MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
