Oleg N. Demidov

Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity

Expression of oncogenic Ras in primary human cells activates p53, thereby protecting cells from transformation. We show that in Ras-expressing IMR-90 cells, p53 is phosphorylated at Ser33 and Ser46 by the p38 mitogen-activated protein kinase (MAPK). Activity of p38 MAPK is regulated by the p53-inducible phosphatase PPM1D, creating a potential feedback loop. Expression of oncogenic Ras suppresses PPM1D mRNA induction, leaving p53 phosphorylated at Ser33 and Ser46 and in an active state. Retrovirus-mediated overexpression of PPM1D reduced p53 phosphorylation at these sites, abrogated Ras-induced apoptosis and partially rescued cells from cell-cycle arrest. Inactivation of p38 MAPK (the product of Mapk14) in vivo by gene targeting or by PPM1D overexpression expedited tumor formation after injection of mouse embryo fibroblasts (MEFs) expressing E1A+Ras into nude mice. The gene encoding PPM1D (PPM1D, at 17q22/q23) is amplified in human breast-tumor cell lines and in approximately 11% of primary breast tumors, most of which harbor wildtype p53. These findings suggest that inactivation of the p38 MAPK through PPM1D overexpression resulting from PPM1D amplification contributes to the development of human cancers by suppressing p53 activation.

Mice deficient for the wild-type p53-induced phosphatase gene (Wip1) exhibit defects in reproductive organs, immune function, and cell cycle control

ABSTRACT The Wip1 gene is a serine/threonine phosphatase that is induced in a p53-dependent manner by DNA-damaging agents. We show here that Wip1 message is expressed in moderate levels in all organs, but is present at very high levels in the testes, particularly in the postmeiotic round spermatid compartment of the seminiferous tubules. We have confirmed that Wip1 mRNA is induced by ionizing radiation in mouse tissues in a p53-dependent manner. To further determine the normal biological function of Wip1 in mammalian organisms, we have generated Wip1-deficient mice. Wip1 null mice are viable but show a variety of postnatal abnormalities, including variable male runting, male reproductive organ atrophy, reduced male fertility, and reduced male longevity. Mice lacking Wip1 show increased susceptibility to pathogens and diminished T- and B-cell function. Fibroblasts derived from Wip1 null embryos have decreased proliferation rates and appear to be compromised in entering mitosis. The data are consistent with an important role for Wip1 in spermatogenesis, lymphoid cell function, and cell cycle regulation.

Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by Wip1 phosphatase

The ataxia telangiectasia mutated (ATM) kinase is a key tumor suppressor that regulates numerous cell cycle checkpoints as well as apoptosis. Here, we report that ATM is a critical player in the regulation of apoptosis and lymphomagenesis in the presence of c-myc. In turn, deletion of the inhibitory ATM phosphatase, Wip1, results in ATM up-regulation and suppression of Eμ-myc–induced B cell lymphomas. Using mouse genetic crosses, we show that the onset of myc-induced lymphomas is dramatically delayed in Wip1-null mice in an ATM- and p53-, but not p38 MAPK– or Arf-, dependent manner. We propose that Wip1 phosphatase is critical for regulating the ATM-mediated tumor surveillance network.

ING2 Regulates the Onset of Replicative Senescence by Induction of p300-Dependent p53 Acetylation†

ABSTRACT ING2 is a candidate tumor suppressor gene that can activate p53 by enhancing its acetylation. Here, we demonstrate that ING2 is also involved in p53-mediated replicative senescence. ING2 protein expression increased in late-passage human primary cells, and it colocalizes with serine 15-phosphorylated p53. ING2 and p53 also complexed with the histone acetyltransferase p300. ING2 enhanced the interaction between p53 and p300 and acted as a cofactor for p300-mediated p53 acetylation. The level of ING2 expression directly modulated the onset of replicative senescence. While overexpression of ING2 induced senescence in young fibroblasts in a p53-dependent manner, expression of ING2 small interfering RNA delayed the onset of senescence. Hence, ING2 can act as a cofactor of p300 for p53 acetylation and thereby plays a positive regulatory role during p53-mediated replicative senescence.

Wip1 Phosphatase Regulates p53-Dependent Apoptosis of Stem Cells and Tumorigenesis in the Mouse Intestine

Colorectal cancer is one of the major causes of cancer-related deaths. To gain further insights into the mechanisms underlying its development, we investigated the role of Wip1 phosphatase, which is highly expressed in intestinal stem cells, in the mouse model of APC(Min)-driven polyposis. We found that Wip1 removal increased the life span of APC(Min) mice through a significant suppression of polyp formation. This protection was dependent on the p53 tumor suppressor, which plays a putative role in the regulation of apoptosis of intestinal stem cells. Activation of apoptosis in stem cells of Wip1-deficient mice, but not wild-type APC(Min) mice, increased when the Wnt pathway was constitutively activated. We propose, therefore, that the Wip1 phosphatase regulates homeostasis of intestinal stem cells. In turn, Wip1 loss suppresses APC(Min)-driven polyposis by lowering the threshold for p53-dependent apoptosis of stem cells, thus preventing their conversion into tumor-initiating stem cells.

The role of the MKK6/p38 MAPK pathway in Wip1-dependent regulation of ErbB2 -driven mammary gland tumorigenesis

There is increasing evidence for the role of wild-type p53 induced phosphatase 1 (Wip1) phosphatase in the regulation of tumorigenesis. To evaluate Wip1 as a breast cancer oncogene, we generated a mouse strain with targeted expression of Wip1 to the breast epithelium. We found that these mice are prone to cancer when intercrossed with transgenics expressing the ErbB2 oncogene but not conditional knockouts for Brca2. This tumor-prone phenotype of Wip1 is fully eliminated through attenuation of proliferation by activating the MKK6/p38 mitogen-activated protein kinases (MAPK) cascade in mice bearing a constitutively active form of MKK6. We propose that Wip1 phosphatase operates within the MKK6/p38 MAPK signaling pathway to promote ErbB2-driven mammary gland tumorigenesis.

p38MAPK Controls Expression of Multiple Cell Cycle Inhibitors and Islet Proliferation with Advancing Age

Aging is a complex organismal process that is controlled by genetic, environmental, and behavioral factors. Accumulating evidence supports a role for different cell cycle inhibitors in mammalian aging. Little is known, however, about the upstream signals that induce their expression. Here, we explore the role of p38MAPK by generating a dominant-negative allele (p38(AF)) in which activating phosphorylation sites Thr180 and Tyr182 are mutated. Heterozygous p38(AF) mice show a marked attenuation of p38-dependent signaling and age-induced expression of multiple cell cycle inhibitors in different organs, including pancreatic islets. As a result, aged p38(AF/+) mice show enhanced proliferation and regeneration of islets when compared to wild-type littermates. We further find an age-related reduction in expression of the p38-specific phosphatase Wip1. Wip1-deficient mice demonstrate decreased islet proliferation, while Wip1 overexpression rescues aging-related decline in proliferation and regenerative capacity. We propose that modulation of p38MAPK activity may provide new avenues for treating certain age-related degenerative diseases.

Chemical inhibition of Wip1 phosphatase contributes to suppression of tumorigenesis

Wip1 is an amplified oncogene whose deletion causes a tumor resistant phenotype in mice. These observations provide justification for a search for Wip1 chemical inhibitors as potential anticancer drugs. Here we report a group of Wip1 inhibitors with anticancer properties both in vitro and in vivo. In vitro, inactivation of Wip1 reduces the proliferation rate of breast cancer cell lines and enhances growth inhibition caused by doxorubicin. In vivo, administration of Wip1 inhibitors decreases proliferation of xenograph tumors and tumors developed in MMTV-c-Neu transgenic mice. We propose that these agents may serve as lead compounds for the development of anticancer drugs targeting Wip1 phosphatase.

Wip1 Phosphatase-Deficient Mice Exhibit Defective T Cell Maturation Due To Sustained p53 Activation

The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1−/− and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1−/− DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal αβ T cell development.

Wip1 Regulates the Generation of New Neural Cells in the Adult Olfactory Bulb through p53-Dependent Cell Cycle Control†‡

Continual generation of new neural cells from adult neural stem/progenitor cells (NPCs) is an important component of life‐long brain plasticity. However, the intrinsic regulation of this process remains poorly defined. Here we report that Wip1 phosphatase, previously studied in oncogenesis, functions as a crucial physiological regulator in adult neural cell generation. Wip1 deficiency resulted in a 90% decrease in new cell formation in adult olfactory bulb, accompanied by aberrantly decreased NPC amplification, stem cell frequency, and self‐renewal. At a cellular level, Wip1 knockout NPCs exhibit a prolonged cell cycle, an accumulation at G2 to M phase transition, and enhanced p53 activity. Interestingly, the impaired M‐phase entry and NPC amplification of Wip1‐null mice can be reversed in Wip1/p53 double‐null mice. Importantly, there is no difference in NPC amplification between p53‐null and Wip1/p53 double‐null mice. Our data demonstrate that Wip1 regulates the generation of new neural cells in adult olfactory bulb specifically through p53‐dependent M‐phase entry of the NPC cell cycle. STEM CELLS 2009;27:1433–1442