A small molecule inhibitor of p53-inducible protein phosphatase PPM1D

PPM1D is a p53-inducible Ser/Thr protein phosphatase. PPM1D gene amplification and overexpression have been reported in a variety of human tumors, including breast cancer and neuroblastoma. Because the phosphatase activity of PPM1D is essential for its oncogenic role, PPM1D inhibitors should be viable anti-cancer agents. In our current study, we showed that SPI-001 was a potent and specific PPM1D inhibitor. SPI-001 inhibited PPM1D phosphatase activity in PPM1D-overexpressing human breast cancer cells and increased phosphorylation of p53. Furthermore, SPI-001 suppressed cell proliferation by inducing apoptosis. Our present study suggested that SPI-001 was a potential lead compound in developing anti-cancer drugs.     

PPM1D regulates p21 expression via dephoshporylation at serine 123

The cyclin-dependent kinase inhibitor p21 plays a critical role in regulating cell cycle and cell proliferation. We previously cloned the dog p21 gene and found that unlike human p21, dog p21 is expressed as 2 isoforms due to the proline-directed phosphorylation at serine 123 (S123). Here, we identified that PPM1D, also called Wip1 and a Mg²⁺-dependent phosphatase, dephosphorylates dog p21 protein at serine 123. Specifically, we showed that the level of S123-phosphorylated dog p21 is increased by a PPM1D inhibitor in a dose-dependent manner. We also showed that over-expression of PPM1D decreases, whereas knockdown of PPM1D increases, the level of S123-phosphorylated dog p21 regardless of p53. Additionally, in vitro phosphatase assay was performed and showed that phosphorylated S123 in dog p21 is dephosphorylated by recombinant rPPM1D, which contains the catalytic domain of human PPM1D (residue 1–420), but not by the phosphatase dead rPPM1D (D314A). Furthermore, dephosphorylation of S123 by rPPM1D can be abrogated by PPM1D inhibitor or by withdrawal of Mg²⁺. Finally, we showed that upon PPM1D inhibition, the level of S123-phosphorylated dog p21 was increased, concomitantly with decreased expression of cyclin A, cyclin B, Rb, and PCNA. Together, our results indicate that PPM1D functions as a phosphatase of dog p21 at serine 123 and plays a role in cell cycle control via p21.     

PPM1D regulates p21 expression via dephoshporylation at serine 123

The cyclin-dependent kinase inhibitor p21 plays a critical role in regulating cell cycle and cell proliferation. We previously cloned the dog p21 gene and found that unlike human p21, dog p21 is expressed as 2 isoforms due to the proline-directed phosphorylation at serine 123 (S123). Here, we identified that PPM1D, also called Wip1 and a Mg²⁺-dependent phosphatase, dephosphorylates dog p21 protein at serine 123. Specifically, we showed that the level of S123-phosphorylated dog p21 is increased by a PPM1D inhibitor in a dose-dependent manner. We also showed that over-expression of PPM1D decreases, whereas knockdown of PPM1D increases, the level of S123-phosphorylated dog p21 regardless of p53. Additionally, in vitro phosphatase assay was performed and showed that phosphorylated S123 in dog p21 is dephosphorylated by recombinant rPPM1D, which contains the catalytic domain of human PPM1D (residue 1–420), but not by the phosphatase dead rPPM1D (D314A). Furthermore, dephosphorylation of S123 by rPPM1D can be abrogated by PPM1D inhibitor or by withdrawal of Mg²⁺. Finally, we showed that upon PPM1D inhibition, the level of S123-phosphorylated dog p21 was increased, concomitantly with decreased expression of cyclin A, cyclin B, Rb, and PCNA. Together, our results indicate that PPM1D functions as a phosphatase of dog p21 at serine 123 and plays a role in cell cycle control via p21.     

Optimization of a cyclic peptide inhibitor of Ser/Thr phosphatase PPM1D (Wip1)

PPM1D (PP2Cδ or Wip1) was identified as a wild-type p53-induced Ser/Thr phosphatase that accumulates after DNA damage and classified into the PP2C family. It dephosphorylates and inactivates several proteins critical for cellular stress responses, including p38 MAPK, p53, and ATM. Furthermore, PPM1D is amplified and/or overexpressed in a number of human cancers. Thus, inhibition of its activity could constitute an important new strategy for therapeutic intervention to halt the progression of several different cancers. Previously, we reported the development of a cyclic thioether peptide with low micromolar inhibitory activity toward PPM1D. Here, we describe important improvements in the inhibitory activity of this class of cyclic peptides and also present a binding model based upon the results. We found that specific interaction of an aromatic ring at the X1 position and negative charge at the X5 and X6 positions significantly increased the inhibitory activity of the cyclic peptide, with the optimized molecule having a K(i) of 110 nM. To the best of our knowledge, this represents the highest inhibitory activity reported for an inhibitor of PPM1D. We further developed an inhibitor selective for PPM1D over PPM1A with a K(i) of 2.9 μM. Optimization of the cyclic peptide and mutagenesis experiments suggest that a highly basic loop unique to PPM1D is related to substrate specificity. We propose a new model for the catalytic site of PPM1D and inhibition by the cyclic peptides that will be useful both for the subsequent design of PPM1D inhibitors and for identification of new substrates.     

Development of a substrate-based cyclic phosphopeptide inhibitor of protein phosphatase 2Cdelta, Wip1

The wild-type p53-induced phosphatase, Wip1 (PP2Cdelta or PPM1D) is a member of the protein phosphatase 2C (PP2C) family and functions as a negative regulator of the p38 MAP kinase-p53 signaling pathway. PPM1D is amplified or Wip1 is overexpressed in several human cancers, and it acts as a weak oncogene. Although inhibition of Wip1 may have therapeutic value, no specific inhibitors are available. In this study, we designed phosphopeptide inhibitors for Wip1 on the basis of its optimal substrate sequence. We found that phosphoserine-containing diphosphorylated peptides with the sequence pSXpY inhibited Wip1 phosphatase activity, whereas phosphothreonine-containing peptides with the sequence pTXpY were physiological substrates. Moreover, the X residue in the pSXpY sequence modulated inhibitor activity, and beta-branched amino acid-substituted (Ile or Val) phosphopeptides showed high inhibitory potencies. A thioether cyclic phosphopeptide c(MpSIpYVA) had a K(i) <1.0 microM. Two serine/threonine phosphatases, PP2Calpha and PP2A, were not significantly inhibited by the cyclic phosphopeptide with a nonhydrolyzable phosphoserine mimetic. A homology model of Wip1 bound to a cyclic phosphopeptide and site-directed mutagenesis helped to identify residues important for Wip1 inhibitor selectivity among the PP2C family. These results provide the first proof of concept of a specific inhibitor of the catalytic site of Wip1 and should be useful for developing potential anti-cancer drugs.     

PPM1D exerts its oncogenic properties in human pancreatic cancer through multiple mechanisms

Protein phosphatase, Mg²⁺/Mn²⁺ dependent, 1D (PPM1D) is emerging as an oncogene by virtue of its negative control on several tumor suppressor pathways. However, the clinical significance of PPM1D in pancreatic cancer (PC) has not been defined. In this study, we determined PPM1D expression in human PC tissues and cell lines and their irrespective noncancerous controls. We subsequently investigated the functional role of PPM1D in the migration, invasion, and apoptosis of MIA PaCa-2 and PANC-1 PC cells in vitro and explored the signaling pathways involved. Furthermore, we examined the role of PPM1D in PC tumorigenesis in vivo. Our results showed that PPM1D is overexpressed in human PC tissues and cell lines and significantly correlated with tumor growth and metastasis. PPM1D promotes PC cell migration and invasion via potentiation of the Wnt/β-catenin pathway through downregulation of apoptosis-stimulating of p53 protein 2 (ASPP2). In contrast to PPM1D, our results showed that ASPP2 is downregulated in PC tissues. Additionally, PPM1D suppresses PC cell apoptosis via inhibition of the p38 MAPK/p53 pathway through both dephosphorylation of p38 MAPK and downregulation of ASPP2. Furthermore, PPM1D promotes PC tumor growth in vivo. Our results demonstrated that PPM1D is an oncogene in PC.     

Bim directly antagonizes Bcl-xl in doxorubicin-induced prostate cancer cell apoptosis independently of p53

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.     

Mutant p53 exhibits trivial effects on mitochondrial functions which can be reactivated by ellipticine in lymphoma cells

Increasing evidence has shown that a fraction of the wild-type (wt) form of the tumor suppressor p53, can translocate to mitochondria due to genotoxic stress. The mitochondrial targets of wt p53 have also been studied. However, whether mutant p53, which exists in 50% of human cancers, translocates to mitochondria and affects mitochondrial functions is unclear. In this study, we used doxorubicin, a chemotherapeutic drug, to treat five human lymphoma cell lines with wt, mutant or deficient in p53, to induce p53 activation and mitochondrial translocation. Our results demonstrated that mutant p53, like wt p53, was induced upon doxorubicin treatment. Similarly, a fraction of mutant p53 also translocated to mitochondria. However, Complex I and II activities in the mitochondria were compromised only in wt p53-bearing cells after doxorubicin treatment, but not in mutant p53-bearing cells. Similarly, doxorubicin treatment caused greater cell death only in wt p53-bearing cells, but not in mutant p53-bearing cells. When p53 deficient Ramos cells were transfected with mutant p53 (249S), the cells showed resistance to doxorubicin-induced cell death and decreases in complex activities. To reactivate mutant p53 and reverse chemoresistance, ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) was used to treat mutant p53 cells. Ellipticine enhanced p53 mitochondrial translocation, decreased Complex I activity, and sensitized p53 mutant cells to doxorubicin-induced apoptosis. In summary, our studies suggest that mutations in p53 may not hinder p53’s mitochondrial translocation, but impair its effects on mitochondrial functions. Therefore, restoring mutant p53 by ellipticine may sensitize these cells to chemotherapy.     

Mechanisms of cell death induced by histone deacetylase inhibitors in androgen receptor-positive prostate cancer cells

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.     

Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner

Constitutive activation of the PI3 kinase/Akt pathway is associated with the neoplastic phenotype of a large number of human tumor cells. As the anti-apoptotic role of the PI3 kinase/Akt pathway has been established, we have examined whether specific blockade of this pathway sensitizes tumor cells to DNA-damaging agent-induced cytotoxicity by enhancing apoptotic cell death. Although a PI3 kinase inhibitor, LY294002, by itself does not induce apoptotic cell death, LY294002 selectively and markedly enhances the apoptosis-inducing efficacy of doxorubicin: such an enhanced cell death is only detected in tumor cells in which the PI3 kinase/Akt pathway is constitutively activated, and it is totally dependent on the functional p53 pathway. These results suggest that the combination of a PI3 kinase/Akt pathway inhibitor and doxorubicin provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the PI3 kinase/Akt pathway is constitutively activated and the p53 pathway is functional.     

Combining p53 stabilizers with metformin induces synergistic apoptosis through regulation of energy metabolism in castration-resistant prostate cancer

Since altered energy metabolism is a hallmark of cancer, many drugs targeting metabolic pathways are in active clinical trials. The tumor suppressor p53 is often inactivated in cancer, either through downregulation of protein or loss-of-function mutations. As such, stabilization of p53 is considered as one promising approach to treat those cancers carrying wild type (WT) p53. Herein, SIRT1 inhibitor Tenovin-1 and polo-like kinase 1 (Plk1) inhibitor BI2536 were used to stabilize p53. We found that both Tennovin-1 and BI2536 increased the anti-neoplastic activity of metformin, an inhibitor of oxidative phosphorylation, in a p53 dependent manner. Since p53 has also been shown to regulate metabolic pathways, we further analyzed glycolysis and oxidative phosphorylation upon drug treatments. We showed that both Tennovin-1 and BI2536 rescued metformin-induced glycolysis and that both Tennovin-1 and BI2536 potentiated metformin-associated inhibition of oxidative phosphorylation. Of significance, castration-resistant prostate cancer (CRPC) C4-2 cells show a much more robust response to the combination treatment than the parental androgen-dependent prostate cancer LNCaP cells, indicating that targeting energy metabolism with metformin plus p53 stabilizers might be a valid approach to treat CRPC carrying WT p53.     

The C terminus of p53 family proteins is a cell fate determinant

The p53 tumor suppressor is the most commonly mutated gene in human cancers. The ability of p53 to induce cell cycle arrest, apoptosis, DNA repair, and other p53-dependent activities is well known; however, the mechanism by which p53 induces a specific activity over another is unclear. Here, we showed that stringent regulation of and by p53 family isoforms facilitates differential target gene expression and thus determines cell fate. Through the use of engineered deletion mutants, we found that activation domain 2 is required for induction of the proapoptotic target gene insulin-like growth factor binding protein 3 (IGFBP3) by p53 and that the basic domain inhibits induction of this gene by p53. Thus, for the first time we provide evidence that the basic domain of p53 is inhibitory in vivo as has been determined in vitro. We also showed that the in vivo inhibitory activity of the basic domain depends upon activation domain 1, such that combined deletion of activation domain 1 and the basic domain was required to alleviate the inhibition by the basic domain. Importantly, deletion of the inhibitory functional domains, namely N-terminal activation domain 1 and the C-terminal basic domain, is paralleled in nature. We found that the IGFBP3 promoter was activated by p53(DeltaNDeltaBD), which mimics a naturally occurring N- and C-terminally truncated human p53 isoform, and by p53AS, a C-terminally truncated murine p53 isoform generated through alternative splicing, but not by full-length human or murine p53. In addition, we found that the C termini of p63 and p73 inhibit the induction of IGFBP3, such that C-terminally truncated p63 and p73 isoforms induce the expression of IGFBP3, whereas full-length ones cannot. We also demonstrated that IGFBP3 is an important effector of the apoptosis induced by N- and C-terminally truncated p53, such that knockdown of IGFBP3 by using an IGFBP3 neutralizing antibody or IGFBP3 small interfering RNA partially rescues the cell death induced by N- and C-terminally truncated p53. In addition, we identified that histone deacetylase activity, not p53 DNA binding ability, governs the regulation of IGFBP3 by full-length p53 family proteins, as inhibition of histone deacetylases restores the induction of IGFBP3 by exogenous full-length p53, p63, and p73 proteins. Furthermore, we found that activation of p53 or inhibition of histone deacetylases alone was not sufficient to induce IGFBP3; however, combined treatment endowed endogenous p53 with this activity. To better understand the significance of this regulation, we performed a microarray study and identified several target genes differentially regulated by full-length p53 and p53 lacking the N-terminal activation domain 1 and the C-terminal basic domain. Taken together, our data suggest a novel mechanism by which p53 family proteins differentially regulate gene expression and provide an insight for designing a combined therapy for cancer treatment.     

Wip1 contributes to cell homeostasis maintained by the steady-state level of Wtp53

Wip1, a human protein Ser/Thr phosphatase also called PPM1D, stands for wild type p53 induced phosphatase 1. Emerging evidences indicate that Wip1 can act as an oncogene largely by turning off DNA damage checkpoint responses. Here we report an unrecognized role of Wipl in normally growing cells. Wip1 can be induced by wild type p53 under not only stressed but also non-stressed conditions. It can trigger G₂/M arrest in wild type p53 containing cells, which was attributed to the decreased Cdc2 kinase activity resulting at least partly from a high level of inhibitory tyrosine phosphorylation on Cdc2 protein at Tyr-15. Furthermore, we also found that Wip1 not only causes G₂/M arrest but also decreases cell death triggered by microtubule assembly inhibitor in mouse fibroblasts when wild type p53 function was restored. These results indicate that Wip1 can provide ample time for wild type p53-containing cells to prepare entry into mitosis and avoid encountering mitotic catastrophe. Therefore, Wipl may play important roles in cell/tissue homeostasis maintained by wild type p53 under normal conditions, enhancing our understanding of how p53 makes cell-fate decisions.     

Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage

Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.