Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function

As a major negative regulator of p53, the MDM2 oncogene plays an important role in carcinogenesis and tumor progression. MDM2 promotes p53 proteasomal degradation and negatively regulates p53 function. The mechanisms by which the MDM2-p53 interaction is regulated are not fully understood, although several MDM2-interacting molecules have recently been identified. To search for novel MDM2-binding partners, we screened a human prostate cDNA library by the yeast two-hybrid assay using full-length MDM2 protein as the bait. Among the candidate proteins, ribosomal protein S7 was identified and confirmed as a novel MDM2-interacting protein. Herein, we demonstrate that S7 binds to MDM2, in vitro and in vivo, and that the interaction between MDM2 and S7 leads to modulation of MDM2-p53 binding by forming a ternary complex among MDM2, p53 and S7. This results in the stabilization of p53 protein through abrogation of MDM2-mediated p53 ubiquitination. Consequently, S7 overexpression increases p53 transactivational activities, induces apoptosis, and inhibits cell proliferation. The identification of S7 as a novel MDM2-interacting partner contributes to elucidation of the complex regulation of the MDM2-p53 interaction and has implications in cancer prevention and therapy.     

Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. A majority of these mutations are missense mutations in the DNA-binding domain. As a result, the mutated p53 gene encodes a full-length protein incapable of transactivating its target genes. In addition to this loss of function, mutant p53 can have a dominant negative effect over wild-type p53 and/or gain of function activity independently of the wild-type protein. To better understand the nature of the tumorigenic activity of mutant p53, we have investigated the mechanism by which mutant p53 can exert a dominant negative effect. We have established several stable cell lines capable of inducibly expressing a p53 mutant alone, wild-type p53 alone, or both proteins concurrently. In this context, we have used chromatin immunoprecipitation to determine the ability of wild-type p53 to bind to its endogenous target genes in the presence of various p53 mutants. We have found that p53 missense mutants markedly reduce the binding of wild-type p53 to the p53 responsive element in the target genes of p21, MDM2, and PIG3. These findings correlate with the reduced ability of wild-type p53 in inducing these and other endogenous target genes and growth suppression in the presence of mutant p53. We also showed that mutant p53 suppresses the ability of wild-type p53 in inducing cell cycle arrest. This highlights the sensitivity and utility of the dual inducible expression system because in previous studies, p53-mediated cell cycle arrest is not affected by transiently overexpressed p53 mutants. Together, our data showed that mutant p53 exerts its dominant negative activity by abrogating the DNA binding, and subsequently the growth suppression, functions of wild-type p53.     

MDM2/p53 co-expression in oral premalignant and malignant lesions: potential prognostic implications.

The expression of MDM2 protein in betel and tobacco related oral malignancies in Indian population, its relationship to clinicopathological parameters and p53 protein expression was investigated. Sixty five oral squamous cell carcinomas (SCCs), 33 premalignant lesions (leukoplakia) and 30 normal oral tissues were assessed by immunohistochemical analysis. MDM2 protein was overexpressed in 51/65 (78%) oral SCCs and 17/33 (52%) premalignant lesions; 11/23 hyperplastic lesions and 6/10 dysplastic lesions. mdm2 gene amplification is an infrequent event in oral tumorigenesis. Elevation in the level of MDM2 protein not only in oral SCCs but also in premalignant lesions suggests that altered MDM2 expression is an early even in the pathogenesis of oral neoplasia. The hallmark of the study was the significant association of MDM2 expression with the p53 protein accumulation in 16/33 (49%) oral premalignant lesions (p = 0.001) and 39/65 (60%) malignant lesions (p = 0.021), suggesting an active role for MDM2 in binding and inactivating p53 in oral tumorigenesis. Further, significant association of MDM2/p53 co-expression was observed with advanced tumour stage (p = 0.0009), as well as lymph node metastasis (p = 0.0325) features associated with aggressive tumour behaviour and poor prognosis. Discordant MDM2+/p53-phenotype was observed in 12/65 (18%) oral SCCs suggesting a p53-independent role for MDM2 in the pathogenesis of a subset of oral carcinomas. In conclusion, alterations in MDM2 and p53 expression are early events likely to be involved in preinvasive stages in oral tumorigenesis and may be indicative of a 'gain of function' phenotype with more aggressive characteristics.     

Nuclear exclusion of p53 in a subset of tumors requires MDM2 function

Wild type p53 accumulates in the cytoplasm in a subset of tumors such as neuroblastomas and breast carcinomas through an unknown mechanism. Exclusion of p53 from the nucleus may lead to inactivation of p53 during tumor development. We present evidence that MDM2 plays a significant role in promoting the degradation of nuclear p53 in tumor cells with a cytoplasmic p53 phenotype. Inhibition of MDM2 expression using antisense oligonucleotide, inhibition of MDM2 function by the tumor suppressor ARF or a MDM2 deletion mutant result in the accumulation of nuclear p53. p53 point mutants deficient in MDM2 binding have increased nuclear localization. Inhibition of nuclear export by leptomycin B also results in retention of nascent p53 in the nucleus, suggesting that cytoplasmic distribution of p53 results from efficient export of nuclear p53 in combination with MDM2-mediated degradation. These results suggest that MDM2 is an important determinant of p53 subcellular distribution and may contribute to p53 inactivation without overexpression.     

Wild-type p53 overexpression and its correlation with MDM2 and p14ARF alterations: an alternative pathway to non-small-cell lung cancer.

PURPOSE: We found a relatively reduced frequency of p53 mutation with a much greater frequency of p53 protein overexpression, which reflected stabilization of p53 protein in the absence of p53 gene mutation. Therefore, we investigated the possibility of alternative mechanisms leading to p53 protein stabilization. PATIENTS AND METHODS: We performed gene and protein alteration studies on p53 and its upstream effectors, MDM2 and p14ARF, in tumors from 94 non-small-cell lung cancer (NSCLC) patients. RESULTS: Immunohistochemical and sequencing analyses indicated that 37 tumors showed overexpression of wild-type p53. An absence of nuclear staining of MDM2 protein was found in 95% of these tumors (35 of 37; P < .001). The tumors with negative MDM2 staining showed a significantly high concordance of loss of Akt activity and low MDM2 mRNA expression (P < .001). Sequencing analysis revealed five distinct MDM2 splicing variants disrupting the conserved p53 binding domain. Corresponding variant proteins were detected in three lung cancer cell lines using the Western blot analysis. Our results also indicated that among the tumors with overexpression of the wild-type p53, 92% (34 of 37) showed immunoreactivity to p14ARF (P = .001). In addition, the deregulation of p53 and MDM2 genes was significantly associated with squamous lung cancer (P < .05) and was correlated with advanced stages (P < .05) and poor prognosis (P < .05). CONCLUSION: Our data suggest that immunopositivity of p14ARF together with a low expression of MDM2 contributes to accumulation of the wild-type p53, and that deregulation of the p53-MDM2-p14ARF pathway is important in the pathogenesis and outcome of a subset of NSCLC.     

Dysfunction of p53 pathway in human colorectal cancer: analysis of p53 gene mutation and the expression of the p53-associated factors p14ARF, p33ING1, p21WAF1 and MDM2

Mutations of p53 tumor suppressor gene increase with tumor progression in colorectal cancers. In this study, we examined the expressions of p33ING1, p14ARF, MDM2 and p21WAF1 mRNA in 25 advanced colorectal cancers by quantitative RT-PCR method, and compared the expression levels of p33ING1, p14ARF, p21WAF1 and MDM2 in relation to p53 status in the tumors. Fifteen of 25 colorectal cancers (60%) showed abnormal accumulation of p53 protein in the nucleus, and the remaining 10 colorectal cancers (40%) were negative for p53 immunostaining. We found a G --> T transition (nonsense mutation) at the first nucleotide of codon 298 (exon 8) in one p53-negative case, and a frame shift mutation on exon 7 in another p53-negative case. In remaining eight p53-negative cases, there was no mutation in the entire open reading frame of p53 cDNA. Interestingly, in eight cases with p53 wild-type gene, 6 cases (75%) showed a marked down-regulation of p14ARF mRNA, and three cases (37.5%) over-expressed MDM2 mRNA. Only one case with wild-type p53 gene showed normal level expression of p53 regulatory-factors (p33ING1, p14ARF, and MDM2). Thus, p53 tumor suppressor pathway was disrupted in 24 of 25 colorectal cancers (96%).     

HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN,irrespective of p53 mutational status

The MDM2-p53 pathway plays a prominent role in well-differentiated liposarcoma (LPS) pathogenesis. Here, we explore the importance of MDM2 amplification and p53 mutation in LPS independently, to determine whether HDACi are therapeutically useful in LPS. We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone. HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS. In control mesothelioma cell lines, HDACi treatment resulted in down-regulation of p53 in the p53 mutant cell line JMN1B, but resulted in no changes of MDM2 and p53 in two mesothelioma lines with normal MDM2 and wild-type p53. HDACi treatment substantially decreased LPS and mesothelioma proliferation and survival, and was associated with upregulation of PTEN and p21, and inactivation of AKT. Our findings indicate that wild-type p53 depletion by HDACi is MDM2 amplification-dependent. These findings underscore the importance of targeting both MDM2 and p53 in LPS and other cancers harboring p53 mutations. Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.     

Amplification of the MDM2 gene in human breast cancer and its association with MDM2 and p53 protein status.

The present study reports on the frequency of MDM2 gene amplification and MDM2 protein expression in a series of 100 breast carcinomas and its association with accumulation of the p53 protein. Of the 100 cases, frozen samples for 82 cases were available for Southern blotting. Three of the 82 (4%) demonstrated MDM2 gene amplification of up to 6-fold. Immunohistochemical analysis of the formalin-fixed, paraffin-embedded tumours demonstrated that 7/97 (7%) had nuclear expression for MDM2 in 10-50% of the tumour cells (type 2 staining) and were denoted MDM2+. Two of the MDM2-amplified samples were MDM2+ with one of the two tumours also displaying type 2 p53 nuclear staining. Finally at the protein level, MDM2+ tumours were significantly associated with tumours having low levels of p53 staining (0-10% cells positive) (P = 0.03). We conclude that MDM2 gene amplification occurs at a lower frequency in breast cancer than in non-epithelial tumours. Alterations in MDM2 and p53 may represent alternative pathways in tumorigenesis, but they are not mutually exclusive in all cases.     

Pharmacological activation of the p53 pathway by nutlin-3 exerts anti-tumoral effects in medulloblastomas

Medulloblastomas account for 20% of pediatric brain tumors. With an overall survival of 40%-70%, their treatment is still a challenge. The majority of medulloblastomas lack p53 mutations, but even in cancers retaining wild-type p53, the tumor surveillance function of p53 is inhibited by the oncoprotein MDM2. Deregulation of the MDM2/p53 balance leads to malignant transformation. Here, we analyzed MDM2 mRNA and protein expression in primary medulloblastomas and normal cerebellum and assessed the mutational status of p53 and MDM2 expression in 6 medulloblastoma cell lines. MDM2 expression was elevated in medulloblastomas, compared with cerebellum. Four of 6 medulloblastoma cell lines expressed wild-type p53 and high levels of MDM2. The tumor-promoting p53-MDM2 interaction can be inhibited by the small molecule, nutlin-3, restoring p53 function. Targeting the p53-MDM2 axis using nutlin-3 significantly reduced cell viability and induced either cell cycle arrest or apoptosis and expression of the p53 target gene p21 in these 4 cell lines. In contrast, DAOY and UW-228 cells harboring TP53 mutations were almost unaffected by nutlin-3 treatment. MDM2 knockdown in medulloblastoma cells by siRNA mimicked nutlin-3 treatment, whereas expression of dominant negative p53 abrogated nutlin-3 effects. Oral nutlin-3 treatment of mice with established medulloblastoma xenografts inhibited tumor growth and significantly increased survival. Thus, nutlin-3 reduced medulloblastoma cell viability in vitro and in vivo by re-activating p53 function. We suggest that inhibition of the MDM2-p53 interaction with nutlin-3 is a promising therapeutic option for medulloblastomas with functional p53 that should be further evaluated in clinical trials.     

Targeting the MDM2-p53 interaction for cancer therapy

p53 is a powerful tumor suppressor and is an attractive cancer therapeutic target because it can be functionally activated to eradicate tumors. The gene encoding p53 protein is mutated or deleted in half of human cancers, which inactivates its tumor suppressor activity. In the remaining cancers with wild-type p53 status, its function is effectively inhibited through direct interaction with the human murine double minute 2 (MDM2) oncoprotein. Blocking the MDM2-p53 interaction to reactivate the p53 function is a promising cancer therapeutic strategy. This review will highlight the advances in the design and development of small-molecule inhibitors of the MDM2-p53 interaction as a cancer therapeutic approach.     

p53, p21, Rb, and MDM2 proteins in tongue carcinoma from patients < 35 versus > 75 years

Relatively rare squamous cell carcinomas of the tongue in young patients may be associated with different etiologic factors and pathogenetic mechanisms than carcinomas from the same site in older patients. Alterations in cell cycle proteins likely contribute to the biologic behavior of these neoplasms. The purpose of this investigation was to evaluate cell cycle proteins (p53, p21, Rb, MDM2) in lateral tongue cancers from patients at the two ends of the age spectrum. All available archived lateral tongue carcinomas from patients < 35 years (n = 36, 23 males and 13 females) were sectioned, immunohistochemically stained, and evaluated. Protein expression was scored as percent positive nuclei. An equal number of sequentially accessioned lateral tongue specimens from patients > 75 years (23 males and 13 females) were stained and compared. Positive p53 staining was seen in 18/36 of the < 35-year group versus 24/36 of the > 75-year group (p = 0.149). Increased p21 staining (both percent of positive cells and intensity) was evident in 25/32 of the < 35-year group versus 24/32 of the > 75-year group (p = 1.0). Increased p21 expression was seen in both p53-positive and -negative cases in both age groups. Rb protein was increased in 16/29 of the < 35-year group versus 17/26 of the > 75-year group (p = 0.58). Fourteen cases (4/35 vs 10/36, p = 0.135) showed positive MDM2 staining; MDM2-positive cases were also p53 positive in 4/4 younger and 8/10 older patients. We conclude that p53, p21, Rb, and MDM2 are over-expressed in lateral tongue cancers, and that immunohistochemical profiles are heterogeneous. A p53-independent pathway of p21 induction is supported by the results; p53 suppression may be associated with MDM2 protein expression in a subset of cancers. Significant differences in the expression of p53, p21, Rb, and MDM2 proteins are not evident in lateral tongue carcinomas from patients < 35 years as compared to patients > 75 years.     

Retinoblastoma protein modulates gankyrin-MDM2 in regulation of p53 stability and chemosensitivity in cancer cells

MDM2 is a key ubiquitin E3 ligase for p53 and its activity is critically regulated by a set of modulators, including ARF, p300, YY1 and recently by gankyrin, an oncoprotein frequently overexpressed in human heptocellular carcinomas. We have previously shown that MDM2 binds to and promotes retinoblastoma protein (Rb) degradation. Here we show that Rb inhibits MDM2 E3 ligase activity resulting in stabilization of p53. In addition, we demonstrated that Rb inhibits MDM2-mediated p53 ubiquitination in a gankyrin-dependent manner and the Rb-gankyrin interaction is critical for Rb-induced p53 stabilization. Furthermore, acute ablation of Rb facilitates gankyrin-mediated p53 destabilization, and desensitizes cancer cells for chemotherapy-induced apoptosis. These results indicate that Rb antagonizes gankyrin to inhibit MDM2-mediate p53 ubiquitination in cancer cells and suggest that the status of both p53 and Rb is important for efficacy of cancer chemotherapy.     

The expression of MDM2 and p53 protein breast carcinoma

We studied the expression of MDM2 protein and p53 protein in 67 cases of the breast carcinoma. The result demonstrated no significant relationship between the MDM2 protein expression and any of the clinicopathological parameters except for the estrogen receptor status (p=0.033). Furthermore, in p53 protein negative cases, there was a significant relationship between MDM2 protein expression and the ER status (p=0.027). This study showed the expression of ER to be associated with MDM2 protein in p53 protein negative breast carcinoma. These results thus suggested that an investigation of both p53 and MDM2 gene alteration revealed precise information regarding breast carcinoma.