Wip1, the product of the PPM1D gene, is a conserved PP2C phosphatase that was first identified in my laboratory as a gene whose induction following DNA damage required wild-type p53. In humans and mice, Wip1 is generally expressed at low levels in most tissues, but its expression is higher in tissues with greater proliferative potential. Wip1 overexpression, either reflecting gene amplification or other mechanisms, is found in a substantial fraction of cases of several human cancers, including breast cancers, ovarian clear cell carcinomas and neuroblastoma, and is generally associated with a worse prognosis. To better understand the connections between Wip1 activity and the control of cellular proliferation, we are identifying targets of Wip1 phosphatase activity, investigating the regulation of Wip1 expression and activity, and developing inhibitors of Wip1 phosphatase activity. The substrates of Wip1 that have been characterized to date include p38MAPK, UNG2, Chk1, Chk2 and ATM. Wip1 dephosphorylates serine and threonine residues within pTXpY and pTQ/pSQ motifs, and we have recently characterized its substrate specificity using biochemical studies. Many of the known pTQ/pSQ substrates of Wip1 are phosphorylated by ATM. To identify potential novel substrates, we searched for motifs compatible with Wip1 specificity among known ATM targets. Among these, we identified Ser112 of 4E-BP1, an inhibitor of EIF4E-dependent mRNA export and cap-dependent translation. Wip1 is able to dephosphorylate this residue in vitro using either a purified phospho-peptide or a recombinant protein phosphorylated in vitro as a substrate. In addition, co-immunoprecipitation experiments showed that Wip1 interacted with 4E-BP1. Modulation of Wip1 levels resulted in changes in EIF4E activity consistent with dephosphorylation of Ser112 of 4E-BP1. The use of specific kinase inhibitors showed that the effect of decreasing Wip1 level on EIF4E activity was independent of mTOR and p38MAPK activity, but required ATM activity. Therefore, the modulation of EIF4E activity by Wip1 may be occurring at two levels: by inhibiting ATM activity through dephosphorylation of Ser1981 and by dephosphorylation of Ser112 of 4E-BP1. In cells containing wild-type p53, the levels of Wip1 mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter. Transient transfection and chromatin immunoprecipitation experiments in HCT116 cells were used to characterize a conserved p53 response element located in the 5'UTR of the PPM1D gene that is required for the p53-dependent induction of transcription from the human PPM1D promoter. CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or ionizing radiation, the abundance of transcripts with short 5'UTRs increased in cells containing wild-type p53, indicating increased utilization of downstream transcription initiation sites. In these cells exposure to UV resulted in increased Wip1 protein levels even when the mRNA levels remained constant, indicating post-transcriptional regulation of Wip1 protein levels. Thus the location of the p53 RE within the 5′UTR provides a mechanism for transcriptional and post-transcriptional regulation of the PPM1D gene by p53. In addition, we have initiated studies into the regulation of Wip1 protein levels by determining the protein half-life under stressed and non-stressed conditions and investigating the mechanism of Wip1 protein degradation in unstressed cells and after cellular stress. These studies will provide important insights into how the Wip1 mRNA and protein levels are regulated and how this regulation may be altered in tumors. The PPM1D promoter also contains several binding sites for cell cycle-related transcription factors, suggesting that in non-stressed cells, PPM1D mRNA and protein levels may be regulated in a cell cycle-dependent manner. To explore the importance of these response elements in the PPM1D promoter, we have begun to analyze the changes in PPM1D mRNA and protein levels in different stages of the cell cycle. We have determined that PPM1D mRNA and protein levels do vary at different stages in the cell cycle in two different cell types. Many human tumors in which the PPM1D gene is amplified or overexpressed contain wt p53. Our results indicate that Wip1 phosphatase is a candidate proto-oncogene that promotes tumorigenesis through inactivation of wld-type p53. Through a combination of rational design and a screening assay using recombinant Wip1, we have developed a series of small molecules that mimic the three-dimensional arrangement of the polar and hydrophobic functional groups of our previously identified cyclic-peptide inhibitor of Wip1. Our inhibitors are selective for the Wip1 phosphatase over related enzymes in vitro. We have used solution-based synthesis of the best inhibitor to produce quantities sufficient for crystallographic analysis, which will facilitate further optimization of the binding selectivity and affinity. We hypothesize that treatment of cancers that over-express Wip1 with a Wip1 inhibitor will lead to increased activation of p53 and subsequent cell killing. Although the Wip1 inhibitors would induce cell death via activation of p53, almost half of all tumors contain mutated p53. In those tumors, it is crucial, then, to activate apoptosis via p53-independent mechanisms. In a screen for small molecules that would induce apoptosis, we have identified a novel inhibitor of the Bcl-2 family member Bcl-x(L). One mechanism that cancer cells use to evade death is to over-produce specific proteins that inhibit apoptosis. One such group of proteins is the Bcl-2 family, of which Bcl-x(L) is an important member. We identified a small molecule, designated as, Bang52, that induces apoptosis in a lymphoblast-derived cell line by decreasing the level of Bcl-x(L). The apoptotic response is independent of p53, as similar results were observed regardless of the p53 status in the cells. Since Bang52 bears no resemblance to the known inhibitors of Bcl-x(L), we believe that degradation of the protein is stimulated by a new type of pathway. These findings highlight a novel approach to the development of small molecules that promote apoptosis. By combining inhibitors such as Bang52 with Wip1 inhibitors, a broad spectrum of tumors could be treated, and since tumors are heterogeneous, use of inhibitor combinations such as these may be a critical part of the future treatment of cancer.
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