AKT-dependent phosphorylation of Niban regulates nucleophosmin- and MDM2-mediated p53 stability and cell apoptosis

Although Niban is highly expressed in human cancer cells, the cellular functions of Niban remain largely unknown. We demonstrate here that ultraviolet irradiation induces phosphorylation of Niban at S602 by AKT, which increases the association of Niban with nucleophosmin and disassociation of nucleophosmin from the MDM2 complex. This leads to the promotion of MDM2-p53 interaction and subsequent p53 degradation, thereby providing an antiapoptotic effect. Conversely, depletion of or deficiency in Niban expression promotes stabilization of p53 with increased cell apoptosis. Our findings illustrate a pivotal role for AKT-mediated phosphorylation of Niban in protecting cells from genotoxic stress-induced cell apoptosis.     

羽扇豆醇介导MDM2-p53通路对胃癌细胞生物学行为的影响

目的:探讨羽扇豆醇介导鼠双微基因2(Mouse double microgene 2,MDM2)-p53通路对胃癌细胞生物学行为的影响及相关机制。方法:对数生长期的胃癌小鼠MFC细胞株随机分为三组。实验1组与实验2组给予10 mg/L和20 mg/L的羽扇豆醇处理,对照组以等体积的1×磷酸盐缓冲液处理。对比三组MFC细胞细胞增殖、凋亡、迁移与侵袭,及MDM2-p53通路蛋白表达。结果:细胞处理后6 h与12 h,实验1组与实验2组的细胞增殖指数、细胞迁移与侵袭指数、MDM2蛋白相对表达水平显著低对于对照组,实验2组也低于实验1组,对比差异都有统计学意义(P<0.05)。细胞处理后6 h与12 h,实验1组与实验2组的细胞凋亡指数、p53蛋白相对表达水平显著高于对照组,实验2组也高于实验1组,对比差异都有统计学意义(P<0.05)。结论:羽扇豆醇能促进胃癌细胞p53蛋白的表达,抑制MDM2蛋白的表达,从而促进细胞凋亡,抑制胃癌的增殖、侵袭与转移,且具有剂量依赖性。     

DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner

Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function.     

Association of p53 and MDM2 polymorphisms with risk of human papillomavirus (HPV)-related esophageal squamous cell carcinoma (ESCC)

PurposeThough polymorphisms of the tumor suppressor gene p53 have been extensively investigated in numerous tumors, particularly tumors associated with human papillomavirus (HPV) infection. However, the results remain controversial. Our previous study showed that HPV serostatus is not an independent risk factor for esophageal squamous cell carcinoma (ESCC) in nonsmokers and nondrinkers. Given the roles of p53 and HPV E6 as well as MDM2 oncoproteins in p53 degradation, we validated the association of p53 and MDM2 polymorphisms with ESCC risk stratified by HPV16 sero-status.MethodsSingle nucleotide polymorphisms of p53 Arg72Pro (rs1042522) and MDM2 (rs937283) in 307 ESCC patients and 311 healthy controls were genotyped. The presence or absence of HPV16 in serum was measured by enzyme-linked immunosorbent assay. Multivariable logistic regression analysis was used to evaluate the possible associations of p53 and MDM2 polymorphisms with ESCC risk stratified by HPV16 sero-status.ResultsPatients carrying p53 Arg/Arg or Arg/Pro had a higher risk of esophageal SCC (P < 0.001, Odds ratio [OR] 4.98, 95% confidential interval [CI] 3.46–7.17), however, not found in MDM2 rs937283. The risk of esophageal SCC increased significantly among patients carrying p53 Arg/Arg, or Arg/Pro and HPV16-seropositivity (P < 0.001, OR 9.33, 95% CI 5.44–16.0), but not for MDM2 rs937283. The risk of esophageal SCC was further elevated among patients carrying Arg/Arg or Arg/Pro and HPV16-seropositivity who were smokers (P < 0.001, OR 27.05, 95% CI 11.06–66.16) or drinkers (P < 0.001, OR 13.20, 95% CI 5.74–30.38).ConclusionHPV16 seropositivity synergized with p53 Arg/Arg or Arg/Pro and increased ESCC risk, especially in smokers or drinkers.     

Polymorphisms of p53 codon 72 and MDM2 promoter 309 and the risk of endometrial cancer

Genetic polymorphisms of p53 and its negative regulator murine double minute 2 homolog (MDM2) have been shown to be closely associated with tumorigenesis in a variety of human cancers. In the present study, single nucleotide polymorphism (SNP) at p53 codon 72 and MDM2 promoter 309 was examined for germline DNA samples from 102 endometrial cancer cases and 95 controls using polymerase chain reaction-based fragment analysis. There were no significant differences in the genotype and allele prevalence between control subjects and endometrial cancer patients for p53 codon 72. The GG genotype frequency of MDM2-SNP309 was statistically higher in endometrial cancer patients than that in normal healthy women when compared with the TG genotype ( P = 0.0088). However, no statistically significant differences were found between the TT and TG or GG genotype frequencies and allele prevalence. Interestingly, the combination of the homozygous Arg/Arg genotype of p53 codon 72 and homozygous GG genotype of MDM2 SNP309 polymorphisms was significantly associated with the risk of endometrial cancer (odds ratio = 3.28, 95% confidence interval = 1.13 to 9.53, P = 0.0212). The homozygous variants of wild p53 codon 72 and mutant MDM2 promoter 309 may cooperatively increase the risk of endometrial cancer in a Japanese population.     

Linc-ROR regulates apoptosis in esophageal squamous cell carcinoma via modulation of p53 ubiquitination by targeting miR-204-5p/MDM2

The long intergenic noncoding RNA, regulator of reprogramming (linc-ROR) has been reported to participate in tumorigenesis, while its functions and fundamental mechanisms in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, gain-of-function assays showed that linc-ROR upregulation enhanced cell viability, promoted cell proliferation, and inhibited apoptosis. Mechanistically, the regulatory network of linc-ROR/miR-204-5p/MDM2 was established with bioinformatics analysis and online databases, then validated via dual-luciferase reporter assays, RNA immunoprecipitation assays in ESCC cells. Linc-ROR positively regulates the expression of MDM2 as a molecular sponge of miR-204-5p. Moreover, results of western blot and coimmunoprecipitation indicated that linc-ROR overexpression enhanced the ubiquitination level of p53, and its downstream apoptosis-related genes have showed higher bcl-2 expression, lower bax, and cleaved caspase-3 expressions, while miR-204-5p could counteract with this effect. Finally, small interfering RNAs tailored to linc-ROR were established to further evaluate its effects on ESCC comprehensively. In summary, this study revealed that linc-ROR modulated cell apoptosis and regulated p53 ubiquitination via targeting miR-204-5p/MDM2 axis, which provides a novel therapeutic insight into treatments for ESCC.     

CREB represses p53-dependent transactivation of MDM2 through the complex formation with p53 and contributes to p53-mediated apoptosis in response to glucose deprivation

Recently, we have described that CREB (cAMP-responsive element-binding protein) has the ability to transactivate tumor suppressor p53 gene in response to glucose deprivation. In this study, we have found that CREB forms a complex with p53 and represses p53-mediated transactivation of MDM2 but not of p21^WAF1. Immunoprecipitation analysis revealed that CREB interacts with p53 in response to glucose deprivation. Forced expression of CREB significantly attenuated the up-regulation of the endogenous MDM2 in response to p53. By contrast, the mutant form of CREB lacking DNA-binding domain (CREBΔ) had an undetectable effect on the expression level of the endogenous MDM2. During the glucose deprivation-mediated apoptosis, there existed an inverse relationship between the expression levels of MDM2 and p53/CREB. Additionally, p53/CREB complex was dissociated from MDM2 promoter in response to glucose deprivation. Collectively, our present results suggest that CREB preferentially down-regulates MDM2 and thereby contributing to p53-mediated apoptosis in response to glucose deprivation.     

Targeting the conformational transitions of MDM2 and MDMX: Insights into key residues affecting p53 recognition

The oncogenic proteins MDM2 and MDMX have distinct and critical roles in the control of the activity of the p53 tumor suppressor protein. Recently, we have used spatial coarse graining simulations to analyze the conformational transitions manifest in the p53 recognition of MDM2 and MDMX. These conformational movements are different between MDM2 and MDMX and unveil the presence of conserved and nonconserved interactions in the p53 binding cleft that may be exploited in the design of selective and dual modulators of the oncogenic proteins. In this study, we investigate the conformational profiles of apo‐ and p53‐bound states of MDM2 and MDMX using molecular dynamic simulations along a time scale of 60 ns. The analysis of the trajectories is instrumental to discuss energetical and conformational aspects of p53 recognition and to point out specific key residues whose conformational shifts have crucial roles in affecting the apo‐ and p53‐bound states of MDM2 and MDMX. Among these, in particular, linear discriminant analyses identify diverse conformations of Y99/Y100 (MDMX/MDM2) as markers of the apo‐ and p53‐bound states of the oncogenic proteins. The results of this study shed further light on different p53 recognition in MDM2 and MDMX and may prove useful for the design and identification of new potent and selective synthetic modulators of p53‐MDM2/MDMX interactions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

Differential response between the p53 ubiquitin-protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells

Pirh2, a recently identified ubiquitin-protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after gamma irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.     

5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 M 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 M 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 M 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.     

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.     

Involvement of Annexin A2 in p53 induced apoptosis in lung cancer

Tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis and DNA repair in different cell types including lung cancer. There are different p53 apoptotic pathways in high and low metastatic ability lung cancer cells. However, the exactly mechanism in the pathway is still unclear. Here we found that Annexin A2, a Ca²⁺-dependent phospholipid-binding protein, is involved in p53-mediated apoptosis. First, by using mRNA differential display technique, down-regulated Annexin A2 expression was found in all cell lines transfected of Ad-p53 (adenoviral expression construct encoding wild type p53 gene) especially in highly metastatic Anip973 lung cancer cells. Then, decreased expression of Annexin A2 was further confirmed by Northern blot and Western blot analysis. At last, knock down of Annexin A2 by siRNA inhibited cellular proliferation in BE1 cell line with highly metastatic ability. Taken together, our results suggested that Annexin A2 may play roles in p53 induced apoptosis and it is also involved in regulation of cell proliferation. The authors Yun Huang, Yan Jin and Cheng-hui Yan contributed equally to this work.     

Radiosensitization of wildtype p53 cancer cells by the MDM2-inhibitor PXN727 is associated with altered heat shock protein 70 (Hsp70) levels

The oncoprotein MDM2 (murine double minute 2) is often overexpressed in human tumors and thereby attenuates the function of the tumor suppressor p53. In this study, we investigated the effects of the novel MDM2-inhibitor PXN727 on p53 activation, cell proliferation, cell cycle distribution and radiosensitivity. Since the localization of heat shock protein 70 (Hsp70) exerts different effects on radioresistance of tumor cells, we investigated the impact of PXN727 on intracellular, membrane, and secreted Hsp70 levels. We could show that PXN727 exerts its effects on wildtype p53 (HCT116 p53^+/+, A549) but not p53 depleted (HCT116 p53^−/−) or mutated (FaDu) tumor cells. PXN727 activates p53, induces the expression of p21, reduces the proportion of cells in the radioresistant S-phase and induces senescence. Radiosensitivity was significantly increased by PXN727 in HCT116 p53^+/+ tumor cells. Furthermore, PXN727 causes a downregulation of Hsp70 membrane expression and an upregulated secretion of Hsp70 in wildtype p53 tumor cells. Our data suggest that re-activation of p53 by MDM2-inhibition modulates Hsp70 membrane expression and secretion which might contribute to the radiosensitizing effect of the MDM2-inhibitor PXN727.