Regulation and Function of WIP1 Phosphatase and its Role in Tumor Cells

Division Of Basic Sciences - Nci

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Abstract

The wild-type p53-induced phosphatase Wip1 (PP2Cdelta or PPM1D) is a member of the serine/threonine protein phosphatase 2C (PP2C) family. Although Wip1 is expressed at low levels in most normal cells, its transcription is induced by p53 after exposure of cells to DNA damage-inducing agents, such as ionizing radiation (IR) or ultraviolet (UV) light. The Wip1 protein is frequently overexpressed or the PPM1D gene is amplified in several human cancers, and this increased expression is associated with worse outcomes. Studies on human cells have shown that overexpression of Wip1 compromises tumor suppressor functions, and studies of mice that lack Wip1 show that they are resistant to tumorigenesis. The current research on Wip1 is focused on understanding its regulation and functions, identifying its functional targets and performing high-throughput screens (HTS) of small molecule libraries to identify specific modulators of Wip1 phosphatase activity. High-resolution structure of the Wip1 catalytic domain is crucial for understanding its activity and for development of specific inhibitors of Wip1 phosphatase activity. To that end, we have recently reported the first crystal structure of the PPM1D catalytic domain to 1.8 A resolution in Journal of Biological Chemistry. The structure reveals the active site with two Mg2+ ions bound. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short beta-strands that are followed by an irregular loop. A nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided mechanistic insights into the regulation of PPM1D catalytic activity. In addition, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. As Wip1 is amplified or overexpressed in numerous human cancers developing inhibitors of Wip1 activity may be beneficial in the treatment of several human cancers. Wip1, though, can function as a tumor suppressor in cancer cells bearing inactive mutated p53. Therefore, developing activators of Wip1 is as important as characterizing inhibitors of this phosphatase. We have used two validated, orthogonal HTS assays to identify potent and specific modulators of Wip1, with minimal cytotoxicity. The combined use of both assays has led us to the discovery of both inhibitors and activators. Currently, starting with the crystal structure that we newly determined, we are using virtual screening to identify binding sites of both inhibitors and activators, and search for new candidates from databases of millions of in-stock compounds. Further biophysical, biochemical, and cell-based studies of confirmed hits will be carried out and, additionally, the best compounds will be tested using an animal-based model. To investigate the effects of ablating Wip1 in the immune system on tumor progression, we used a mouse with a specific ablation of Wip1 in neutrophils by crossbreeding Ppm1d floxed mice (tm1c) with mice expressing the cre recombinase under the control of the mrp8 promoter, a protein specifically expressed in neutrophils (tm1c/mrp8cre). LLC1 lung tumor cells injected subcutaneously showed decreased tumor growth when injected into tm1c/mrp8cre mice compared to tm1c mice. This decrease in growth was accompanied by an increased neutrophil infiltration, and a switch from N2 pro-tumoral neutrophils to N1 anti-tumor neutrophils. To study the impact of Wip1 deletion in neutrophils, tumor-associated neutrophils (TANs) from tm1c and tm1c/mrp8cre mice injected with LLC1 lung cancer cells were isolated and their differential gene expression analyzed by RNAseq. We observed that Wip1 deletion led to profound gene expression changes, most notably in pathways linked to phagocytosis, interaction with symbiont, secretary vesicle and immune cell activation. Moreover, we observed a strong increase in genes involved in antigen presentation, notably members of the MHCI and MHCII complex, and CD48, a co-activator protein. Neutrophils with antigen-presenting capabilities (nAPCs) have been previously. We have further performed cell biological and omics methods to characterize the role of Wip1 in nAPCs. PPM1D gain-of-function variants linked to cancer and neurodevelopmental disorders have been reported. With Drs. Ana Esteve-Sole and Sergio D. Rosenweig, Clinical Center, NIH we showed that PPM1D is important for a balanced p53/PPM1D axis for normal B- and NK-cell development, maturation and localization in both in humans and mice. It proved essential for neurogenesis in Ppm1d-deficient mice, with decreased embryonic viability and disrupted neurogenesis. In patient-derived unguided neural organoids (UNOs), PPM1D loss- and gain-of-function showed dysregulated neurodevelopmental pathways. Further, patient-derived UNOs show that PPM1D-gene dosage is key for proper brain development. Overall, these data highlight the importance of PPM1D gene dosage and regulation in health and disease and established a new role for targeted treatment.