Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4.
TL;DR: This work presents a novel and scalable approach to gene expression engineering that allows for real-time annotation of gene expression changes in response to cancerigenicity and shows promise in finding novel and efficient treatments for cancer.
Abstract: 1 Laboratory For Molecular Cancer Biology, Flanders Interuniversity Institute for Biotechnology (VIB), University of Ghent, Technologiepark, 927, Ghent B9052, Belgium 2 Salk Institute for Biological Studies, Gene Expression Laboratory, La Jolla, CA 92037, USA 3 Gene Expression and Diseases Unit, Institut Pasteur, Paris, France 4 The University of Texas Graduate School of Biomedical Sciences and department of Molecular Genetics, Section of Cancer Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA * Corresponding author: J-C Marine, Laboratory For Molecular Cancer Biology, VIB, Technologiepark, 927, Ghent B-9052, Belgium. Tel: þ 32-93-313-640; Fax: þ 32-93-313-516; E-mail: chris.marine@dmbr.ugent.be
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Dissertation•
22 Jan 2016
TL;DR: A P53-independent and stemness maintaining function of MDM2 which supported the de-differentiation process of induced pluripotent stem (iPS) cells and inhibited differentiation of mesenchymal stem cells into osteoblasts and accelerated clonogenic cancer cell survival in the absence of P53.
Abstract: The E3 ubiquitin ligase MDM2 is the most well-known physiological antagonist of the tumor suppressor P53. P53 induces cell cycle arrest or apoptosis in the case of DNA damage, whereas MDM2 targets it for proteasomal degradation during unstressed conditions. Loss of MDM2 in the murine organism is embryonically lethal but can be rescued by a concomitant loss of P53, which led to the assumption that MDM2’s only function is based on P53 regulation. Still, several tumor species have been identified which are supported by high levels of MDM2 even in the absence of P53.
In this project, we have analyzed a P53-independent and stemness maintaining function of MDM2 which supported the de-differentiation process of induced pluripotent stem (iPS) cells. It furthermore inhibited differentiation of mesenchymal stem cells (MSCs) into osteoblasts and accelerated clonogenic cancer cell survival in the absence of P53. In each system, loss of MDM2 resulted in the deregulation of Polycomb group (PcG) family target genes. The PcG consists of the two protein complexes, PRC1 and PRC2, and silences gene expression through methylation of histone H3K27 (PRC2) and ubiquitination of H2AK119 (PRC1), which is often essential for stemness maintenance and cancer cell survival. MDM2 directly interacted with the PRC2 proteins EZH2 and SUZ12 and was recruited to PcG target gene promoters through EZH2. On the chromatin, MDM2 enhanced H3K27me3 and H2AK119ub1 which correlated to its gene regulatory function. H2AK119 is mainly ubiquitinated by the PRC1 protein RING1B. Loss of both E3 ligases, MDM2 and RING1B, decreased H2AK119ub1 levels and induced target gene expression further than the loss of each factor alone. Moreover, loss of RING1B and MDM2 was synthetically lethal in primary mouse- and cancer cells.
A close homolog of the MDM2 protein is MDM4. MDM2 and MDM4 support each other in the regulation of P53 but they cannot compensate each other according to in vivo loss of function studies. Preliminary data in our setting indicated a similar gene regulatory and H3K27me3 stabilizing function of MDM4 as detected for MDM2.
Taken together, MDM proteins enhanced the repression of lineage specific genes in cooperation with the PcG family, in the absence of P53. Thus, the oncogenic function of MDMs is not limited to controlling P53, but extended to chromatin modification and a stem-like cell phenotype.
3 citations
Cites background from "Keeping p53 in check: essential and..."
...As most embryonic and adult cells do not produce high wt P53 protein levels, proteasomal degradation explained the considerably high levels of P53 mRNA (Marine et al, 2006)....
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Dissertation•
01 Jan 2008
TL;DR: The role for the chaperone-associated ubiquitin ligase CHIP in ubiquitination of mutant p53, although other unidentified ubiquitIn ligases appear to contribute is suggested, suggesting p73 can play a role in regulation of stability of mutants p53.
Abstract: Tumour suppressor p53 is frequently mutated in cancers While wild type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilised and accumulate to high levels in tumour cells Several studies have shown that mutant p53 acquires oncogenic properties and actively contributes to tumourigenesis It is therefore important to understand how the stability of mutant p53 is regulated This thesis shows that mutant and wild type p53 are ubiquitinated and degraded through overlapping but distinct pathways While Mdm2 can drive the degradation of both mutant and wild type p53, this study suggests that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner The contribution of Mdm2 to the degradation of mutant p53 may reflect an ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome Ubiquitination does not efficiently target mutant p53 for the proteasomal degradation, however ubiquitinated p53 mutants localize to the cytoplasm This thesis suggests the role for the chaperone-associated ubiquitin ligase CHIP in ubiquitination of mutant p53, although other unidentified ubiquitin ligases appear to contribute Interaction of mutant p53 with its family member p73 decreases ubiquitination, suggesting p73 can play a role in regulation of stability of mutant p53
3 citations
Cites background from "Keeping p53 in check: essential and..."
...The recent study of roles of Mdm2 and Mdmx done with mice with conditional p53 expression suggests that Mdmx inhibits the transcriptional activity of p53 independently of Mdm2, whereas Mdm2 is required for regulation p53 protein levels (351, 352)....
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...Loss of Mdmx does no lead to accumulation of p53, suggesting that Mdmx does not regulate p53 stability (351, 352)....
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01 Jan 2010
TL;DR: This chapter examines current models of how the dynamic equilibrium between p53 and its two negative regulators is maintained in proliferating cells, and is targeted by multiple signaling pathways to control the magnitude and duration of the p53-dependent transcriptional response to genotoxic stress.
Abstract: Murine double minute-2 (MDM2) was first described as a p53-associated protein and potential oncogene in the early 1990s.1,2 Its paralogue MDMX was subsequently identified in a screen for p53-binding proteins.3 Extensive evidence now confirms both proteins to be oncogenic in both mice and humans, largely through their ability to negatively regulate the tumor-suppressor activities of p53. It is now clear that the two proteins form heterodimers, and act in concert to regulate p53 activity in proliferating and stressed cells. In this chapter I firstly review the several mechanisms whereby MDM2 and MDMX are potentially able to regulate p53 function independently of each other. I then examine how heterodimerization between the two molecules influences how they regulate the abundance and activity of both p53, and each other. I conclude by examining current models of how the dynamic equilibrium between p53 and its two negative regulators is maintained in proliferating cells, and is targeted by multiple signaling pathways to control the magnitude and duration of the p53-dependent transcriptional response to genotoxic stress.
3 citations
TL;DR: The results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm 2 RING homodimers and MDM2-MdmX heterodimering may not be entirely structurally equivalent, despite their apparent similarity.
Abstract: Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules in vivo, suggesting that one conserved amino acid residue in the RINGs of Mdm2 and MdmX could serve as the determinant of the differential ability of these domains to form dimers and their E3 activity. In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity.
3 citations
Cites background from "Keeping p53 in check: essential and..."
...…which work together to control the levels and activity of the tumor suppressor p53 during embryonic development and in unstressed healthy cells (Jones et al., 1995; Montes de Oca Luna et al., 1995; Parant et al., 2001; Migliorini et al., 2002; Marine et al., 2006; Ringshausen et al., 2006)....
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TL;DR: In this article, a quantitative proteomic approach was used to investigate rete testis invasion (RTI)-associated proteins, including 14-3-3γ, ezrin, filamin A, Parkinsonism-associated deglycase 7 (PARK7), vimentin and vinculin.
Abstract: Rete testis invasion (RTI) is an unfavourable prognostic factor for the risk of relapse in clinical stage I (CS I) seminoma patients. Notably, no evidence of difference in the proteome of RTI-positive vs. -negative CS I seminomas has been reported yet. Here, a quantitative proteomic approach was used to investigate RTI-associated proteins. 64 proteins were differentially expressed in RTI-positive compared to -negative CS I seminomas. Of them, 14-3-3γ, ezrin, filamin A, Parkinsonism-associated deglycase 7 (PARK7), vimentin and vinculin, were validated in CS I seminoma patient cohort. As shown by multivariate analysis controlling for clinical confounders, PARK7 and filamin A expression lowered the risk of RTI, while 14-3-3γ expression increased it. Therefore, we suggest that in real clinical biopsy specimens, the expression level of these proteins may reflect prognosis in CS I seminoma patients.
3 citations
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TL;DR: A gene is identified, named WAF1, whose induction was associated with wild-type but not mutant p53 gene expression in a human brain tumor cell line and that could be an important mediator of p53-dependent tumor growth suppression.
8,339 citations
TL;DR: In this article, the authors identify potent and selective small-molecule antagonists of MDM2 and confirm their mode of action through the crystal structures of complexes, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts.
Abstract: MDM2 binds the p53 tumor suppressor protein with high affinity and negatively modulates its transcriptional activity and stability. Overexpression of MDM2, found in many human tumors, effectively impairs p53 function. Inhibition of MDM2-p53 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. Here, we identify potent and selective small-molecule antagonists of MDM2 and confirm their mode of action through the crystal structures of complexes. These compounds bind MDM2 in the p53-binding pocket and activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice.
4,397 citations
TL;DR: It is proposed that the Mdm2-promoted degradation of p53 provides a new mechanism to ensure effective termination of the p53 signal.
Abstract: The p53 tumour-suppressor protein exerts antiproliferative effects, including growth arrest and apoptosis, in response to various types of stress. The activity of p53 is abrogated by mutations that occur frequently in tumours, as well as by several viral and cellular proteins. The Mdm2 oncoprotein is a potent inhibitor of p53. Mdm2 binds the transcriptional activation domain of p53 and blocks its ability to regulate target genes and to exert antiproliferative effects. On the other hand, p53 activates the expression of the mdm2 gene in an autoregulatory feedback loop. The interval between p53 activation and consequent Mdm2 accumulation defines a time window during which p53 exerts its effects. We now report that Mdm2 also promotes the rapid degradation of p53 under conditions in which p53 is otherwise stabilized. This effect of Mdm2 requires binding of p53; moreover, a small domain of p53, encompassing the Mdm2-binding site, confers Mdm2-dependent detstabilization upon heterologous proteins. Raised amounts of Mdm2 strongly repress mutant p53 accumulation in tumour-derived cells. During recovery from DNA damage, maximal Mdm2 induction coincides with rapid p53 loss. We propose that the Mdm2-promoted degradation of p53 provides a new mechanism to ensure effective termination of the p53 signal.
4,311 citations
TL;DR: It is shown that interaction with Mdm2 can also result in a large reduction in p53 protein levels through enhanced proteasome-dependent degradation, which may contribute to the maintenance of low p53 concentrations in normal cells.
Abstract: The tumour-suppressor p53 is a short-lived protein that is maintained at low, often undetectable, levels in normal cells. Stabilization of the protein in response to an activating signal, such as DNA damage, results in a rapid rise in p53 levels and subsequent inhibition of cell growth. Tight regulation of p53 function is critical for normal cell growth and development, and one mechanism by which p53 function is controlled is through interaction with the Mdm2 protein. Mdm2 inhibits p53 cell-cycle arrest and apoptic functions and we show here that interaction with Mdm2 can also result in a large reduction in p53 protein levels through enhanced proteasome-dependent degradation. Endogenous levels of Mdm2 are sufficient to regulate p53 stability, and overexpression of Mdm2 can reduce the amount of endogenous p53. Because mdm2 is transcriptionally activated by p53, this degradative pathway may contribute to the maintenance of low p53 concentrations in normal cells. Furthermore, mechanisms regulating the Mdm2-induced degradation of p53 may play a role in controlling the extent and duration of the p53 response.
3,298 citations
TL;DR: The data suggest that the MDM2 protein, which is induced by p53, functions as a ubiquitin ligase, E3, in human papillomavirus‐uninfected cells which do not have E6 protein.
1,962 citations