Zoledronic Acid Produces Combinatory Anti-Tumor Effects with Cisplatin on Mesothelioma by Increasing p53 Expression Levels

We examined anti-tumor effects of zoledronic acid (ZOL), one of the bisphosphonates agents clinically used for preventing loss of bone mass, on human mesothelioma cells bearing the wild-type p53 gene. ZOL-treated cells showed activation of caspase-3/7, -8 and -9, and increased sub-G1 phase fractions. A combinatory use of ZOL and cisplatin (CDDP), one of the first-line anti-cancer agents for mesothelioma, synergistically or additively produced the cytotoxicity on mesothelioma cells. Moreover, the combination achieved greater anti-tumor effects on mesothelioma developed in the pleural cavity than administration of either ZOL or CDDP alone. ZOL-treated cells as well as CDDP-treated cells induced p53 phosphorylation at Ser 15, a marker of p53 activation, and up-regulated p53 protein expression levels. Down-regulation of p53 levels with siRNA however did not influence the ZOL-mediated cytotoxicity but negated the combinatory effects by ZOL and CDDP. In addition, ZOL treatments augmented cytotoxicity of adenoviruses expressing the p53 gene on mesothelioma. These data demonstrated that ZOL-mediated augmentation of p53, which was not linked with ZOL-induced cytotoxicity, played a role in the combinatory effects with a p53 up-regulating agent, and suggests a possible clinical use of ZOL to mesothelioma with anti-cancer agents.     

MDM4 Overexpressed in Acute Myeloid Leukemia Patients with Complex Karyotype and Wild-Type TP53

Acute myeloid leukemia patients with complex karyotype (CK-AML) account for approximately 10–15% of adult AML cases, and are often associated with a poor prognosis. Except for about 70% of CK-AML patients with biallelic inactivation of TP53, the leukemogenic mechanism in the nearly 30% of CK-AML patients with wild-type TP53 has remained elusive. In this study, 15 cases with complex karyotype and wild-type TP53 were screened out of 140 de novo AML patients and the expression levels of MDM4, a main negative regulator of p53-signaling pathway, were detected. We ruled out mutations in genes associated with a poor prognosis of CK-AML, including RUNX1 or FLT3-ITD. The mRNA expression levels of the full-length of MDM4 (MDM4FL) and short isoform MDM4 (MDM4S) were elevated in CK-AML relative to normal karyotype AML (NK-AML) patients. We also explored the impact of MDM4 overexpression on the cell cycle, cell proliferation and the spindle checkpoint of HepG2 cells, which is a human cancer cell line with normal MDM4 and TP53 expression. The mitotic index and the expression of p21, BubR1 and Securin were all reduced following Nocodazole treatment. Moreover, karyotype analysis showed that MDM4 overexpression might lead to aneuploidy or polyploidy. These results suggest that MDM4 overexpression is related to CK-AML with wild-type TP53 and might play a pathogenic role by inhibiting p53-signal pathway.     

Inhibition of the MDM2 E3 Ligase Induces Apoptosis and Autophagy in Wild-Type and Mutant p53 Models of Multiple Myeloma,and Acts Synergistically with ABT-737

Intracellular proteolytic pathways have been validated as rational targets in multiple myeloma with the approval of two proteasome inhibitors in this disease, and with the finding that immunomodulatory agents work through an E3 ubiquitin ligase containing Cereblon. Another E3 ligase that could be a rational target is the murine double minute (MDM) 2 protein, which plays a role in p53 turnover. A novel inhibitor of this complex, MI-63, was found to induce apoptosis in p53 wild-type myeloma models in association with activation of a p53-mediated cell death program. MI-63 overcame adhesion-mediated drug resistance, showed anti-tumor activity in vivo, enhanced the activity of bortezomib and lenalidomide, and also overcame lenalidomide resistance. In mutant p53 models, inhibition of MDM2 with MI-63 also activated apoptosis, albeit at higher concentrations, and this was associated with activation of autophagy. When MI-63 was combined with the BH3 mimetic ABT-737, enhanced activity was seen in both wild-type and mutant p53 models. Finally, this regimen showed efficacy against primary plasma cells from patients with newly diagnosed and relapsed/refractory myeloma. These findings support the translation of novel MDM2 inhibitors both alone, and in combination with other novel agents, to the clinic for patients with multiple myeloma.     

Disruption of the MDM2�Cp53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2–p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.     

A novel Aurora-A-mediated phosphorylation of p53 inhibits its interaction with MDM2

Purpose: Crosstalk between Aurora-A kinase and p53 has been proposed. While the genetic amplification of Aurora-A has been observed in many human cancers, how p53 is regulated by Aurora-A remains ambiguous. In this study, Aurora-A-mediated phosphorylation of p53 was analyzed by mass spectrometry in order to identify a new phosphorylation site. Subsequently, the functional consequences of such phosphorylation were examined. Experimental design: In vitro phosphorylation of p53 by Aurora-A was performed and the phosphorylated protein was then digested with trypsin and enriched for phosphopeptides by immobilized metal affinity chromatography. Subsequently, a combination of β-elimination and Michael addition was applied to the phosphopeptides in order to facilitate the identification of phosphorylation sites by MS. The functional consequences of the novel phosphorylation of p53 on the protein–protein interactions, protein stability and transactivation activity were then examined using co-immunoprecipitation, Western blotting and reporter assays. Results: Ser-106 of p53 was identified as a novel site phosphorylated by Aurora-A. A serine-to-alanine mutation at this site was found to attenuate Aurora-A-mediated phosphorylation in vitro. In addition, phosphate-sensitive Phos-tag SDS-PAGE was used to confirm that the Ser-106 of p53 is in vivo phosphorylated by Aurora-A. Finally, co-immunoprecipitation studies suggested that Ser-106 phosphorylation of p53 decreases its interaction with MDM2 and prolongs the half-life of p53. Conclusions: The inhibition of the interaction between p53 and MDM2 by a novel Aurora-A-mediated p53 phosphorylation was identified in this study and this provides important information for further investigations into the interaction between p53 and Aurora-A in terms of cancer biology.     

Cooperation among Numb, MDM2 and p53 in the development and progression of pancreatic cancer

We study the expression of Numb, MDM2 and p53 for clinical significance in pancreatic cancer (PC) and their functional relationship in regulating biological behaviors of PC cells. IHC, IB and qRT-PCR were used to detect Numb, MDM2 and p53 expression in PC. Transfection and drug intervention were used to investigate their functional relationship in PC cells. IHC showed that Numb expression was negatively associated with tumor size, differentiation and UICC stage, while expression of MDM2 and p53 was positively associated with tumor T and UICC stages, respectively (P?<?0.05). Numb was an independent prognostic indicator in PC (P?<?0.05). Patients with Numb-positive expression or combined with MDM2-negative expression had a significantly better overall survival (P?<?0.05). Altered expression of Numb can regulate wild-type but not mutant p53 expression, while MDM2 knockdown increased Numb but not mutant p53 protein level. Meanwhile, Numb knockdown increased chemoresistance but decreased activated p53 and cleaved-caspase-3 protein expression in gemcitabine-treated Capan-2 cells. Moreover, Numb co-immunoprecipitated with p53 to prevent p53 ubiquitin-dependent protein degradation and this ubiquitin-dependent regulation plays an important role in the coordinate function of these three proteins on cell invasion and migration in PC cells. Our study is the first to demonstrate the clinical significance and functional cooperation among Numb, MDM2 and p53 involved in the development and progression of PC.     

XI-011 enhances cisplatin-induced apoptosis by functional restoration of p53 in head and neck cancer

Head and neck cancer (HNC), one of the most common cancers worldwide, frequently involves mutation of the TP53 gene and dysregulation of the p53 pathway. Overexpression of MDM2 or MDM4 inactivates the tumor-suppressive function of p53. Restoration of p53 function that counteracts these p53 repressors can lead to in vivo tumor regression. Therefore, the present study assessed the ability of the small molecule p53 activator XI-011 (NSC146109) to induce apoptosis in HNC by restoring p53 function. We tested the effects of XI-011 treatment in HNC cell lines, either individually or in combination with cisplatin and assessed growth suppression, cell cycle arrest, and apoptosis. The drug effects on in vivo growth of HNC cells were examined in mice xenograft model. XI-011 exerted the highest growth suppression in tumor cells that overexpress MDM4, in which p53 is degraded. XI-011 treatment downregulated MDM4 mRNA and protein levels, and upregulated expression of proapoptotic genes and promoted apoptosis, in a dose-dependent manner. The apoptotic response was blocked by inhibition of p53 or expression of MDM4, demonstrating that the effects of XI-011 depend on p53 and MDM4. In combination treatments, XI-011 acted synergistically with cisplatin to inhibit growth of HNC cells in vitro and in vivo. MDM4 inhibition and functional restoration of p53 by XI-011 effectively enhanced cisplatin-induced cytotoxicity in HNC cells, an activity that suggests a promising strategy for treating HNC.     

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.     

The Replicative Capacities of Large E1B-Null Group A and Group C Adenoviruses Are Independent of Host Cell p53 Status

Recent reports suggest that an early region 1B (E1B) 55,000-molecular-weight polypeptide (55K)-null adenovirus type 5 (Ad5) mutant (dl1520) can replicate to the same extent as wild-type (wt) Ad5 in cells either deficient or mutated in p53, implicating p53 in limiting viral replication in vivo. In contrast, we show here that the replicative capacity of Ad5 dl1520 is wholly independent of host cell p53 status, as is the replicative capacity of comparable Ad12 E1B 54K-null adenoviruses (Ad12 dl620 and Ad12 hr703). Furthermore, we show that there is no requirement for complex formation between p53 and Ad5 E1B 55K or Ad12 E1B 54K for a productive infection, such that wt Ad5 and wt Ad12 will both replicate in cells which are null for p53. In addition, we find that these Ad5 and Ad12 mutant viruses induce S phase irrespective of the p53 status of the cell and that, therefore, S-phase induction does not correlate with the replicative capacity of the virus. Interestingly, the replicative capacities of the large E1B-null adenoviruses correlated positively with the ability to express E1B 19K and were related to the ability to repress premature adenovirus-induced apoptosis. Infection of primary human cells indicated that Ad5 dl1520, wt Ad5, and wt Ad12 replicated better in cycling normal human skin fibroblasts (HSFs) than in quiescent HSFs. Thus, the cell cycle status of the host cell, upon infection, also influences viral yield.     

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC₅₀ values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.     

Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.