Amplification and Over-Expression of the MDM2 Gene in Human Soft Tissue Tumours

Purpose. Amplification of genetic sequences on chromosome 12q13 is frequently found in soft tissue tumours. However, for the MDM2 gene, over-expression of the MDM2 protein has not always been shown to accompany gene amplification, raising the possibility that amplification of genetic sequences targets alternative genes on chromosome 12q13 for over-expression. To investigate this discrepancy, we have examined 129 soft tissue tumours for amplification of the MDM2 gene using Southern analysis, and 39 of these tumours were also examined by immunohistochemical staining for MDM2 over-expression.Results. Gene amplification was identified in 14/114 (12.3%) of the malignant tumours, but was not identified in any of the benign tumours; 21/39 (54%) of the malignant tumours also demonstrated MDM2 over-expression. Within this group the MDM2 gene was over-expressed in every tumour in which the gene amplification was found, and over-expression in the absence of gene amplification was also found in an additional 10 tumours.Discussion. These data demonstrate a clear correlation between the presence of MDM2 amplification and MDM2 over-expression, and provide persuasive evidence therefore that the amplification of genetic sequences on chromosome 12q13 in soft tissue sarcomas targets the MDM2 gene for over-expression. These data also indicate that alternative mechanisms may contribute to MDM2 over-expression within some tumours.     

P53 mutations in Ewing's sarcoma

The p53 tumor suppressor gene is one of the most frequently altered genes in human malignancies. To explore the implication of p53 alteration in Ewing's sarcoma, we analyzed the deletion and sequence alterations of p53 and abnormal amplification of MDM2, which acts as a functional inhibitor of p53, in 35 tissue specimens. Quantitative genomic PCR analysis showed that 2 of 35 tumors have extremely low levels of the p53 gene, indicating a homozygous deletion of the gene. Mutational analysis of exons 4 to 9 of p53 by PCR-SSCP revealed that 3 of 35 tumors carry sequence alterations in exons 5 or 8, and DNA sequencing analysis identified missense point mutations at codon 132 (AAG-->ATG, lysine-->methionine) and codon 135 (TGC-->TCC, cystein-->serine) in exon 5, and codon 287 (GAG-->GTG, glutamic acid-->valine) in exon 8 from these tumors. No abnormal amplification of the MDM2 gene was recognized. Taken together, our data demonstrate that p53 is genetically altered in a small fraction of Ewing's sarcoma.     

Activation of p53 by roscovitine-mediated suppression of MDM2 expression.

The p53 tumor suppressor is regulated by the MDM2 oncoprotein. Overexpression of MDM2 maintains p53 at low levels and contributes to the functional inactivation of p53 in a subset of tumors. We found that treatment with roscovitine and olomoucin, which were originally developed as cyclin-dependent kinase (CDK) inhibitors, can efficiently stabilize and activate nuclear p53 in tumor cells with MDM2 amplification or cytoplasmic p53. These inhibitors block the degradation of p53 without affecting p53-MDM2 binding and the nuclear shuttling function of p53 and MDM2. Roscovitine also induces stabilization of the p53 Ala-315 mutant, indicating that it does not act by regulating the CDK phosphorylation of serine 315. Roscovitine induces down-regulation of MDM2 expression at both protein and mRNA levels. Ectopic expression of MDM2 can abrogate the ability of roscovitine to induce p53 stabilization. Low concentrations of roscovitine cooperate with the DNA-damaging agent camptothecin to activate p53 in a synergistic fashion. These results show that the small molecule CDK inhibitors can be used to activate p53 through their potent inhibitory effect on MDM2 expression and may be useful as sensitizing agents for other DNA-damaging drugs.     

Abnormal expression of MDM-2 in breast carcinomas

MDM-2 is a cellular oncoprotein that binds to the p53 protein and abrogates its growth-suppressing function. At least seven MDM-2 mRNAs and five proteins (p90, p85, p76, p74, and p57) have been reported in tissue culture. MDM-2 gene amplification occurs in human sarcomas and high-grade gliomas. MDM-2 overexpression without gene amplification has been reported in leukemias and lymphomas. Here we report MDM-2 mRNA overexpression in 24 (73%) out of 33 cases of human breast carcinoma as compared with normal breast tissue. The MDM-2 overexpression was seen in the absence of MDM-2 gene amplification. MDM-2 protein expression was studied by western blot analysis in 21 of these cases of carcinoma. We found complete concordance between MDM-2 mRNA overexpression and MDM-2 protein levels. MDM-2 proteins were overexpressed in 15 of 21 breast carcinoma tissue samples but not in normal breast tissue controls. Ten of these fifteen cases overexpressed MDM-2 p57 protein, two cases overexpressed both p57 and p90, and three cases overexpressed only p90. MDM-2 overexpression was confirmed by immunohistochemistry. p53 overexpression was also studied by immunohistochemistry; 69% of breast carcinomas that overexpressed the MDM-2 mRNA had detectable nuclear p53 protein. These findings demonstrate that MDM-2 oncoprotein expression is altered in primary human breast carcinomas at both mRNA and protein levels. In addition, our results suggest that MDM-2 p57 protein represents the main MDM-2 protein altered in breast carcinomas.     

Low grade amplification of MDM2 gene in a subset of human breast cancers without p53 alterations

MDM2 protein is thought to bind to p53 tumor suppressor protein leading to inhibition of p53-mediated transactivation. Amplification of the MDM2 gene has been frequently observed in human sarcoma, and relevant overexpression of the MDM2 protein is assumed to contribute to tumorigenesis through inactivation of the p53 function. In order to determine whether MDM2 amplification plays a role in the development of human breast cancer without genetic alteration of p53, we analyzed, MDM2 gene amplification by quantitative hybridization and genetic alteration of p53, in 32 primary tumors and 26 metastatic lymph nodes. Low grade amplification of the MDM2 gene (2-6 fold) was observed in four cases, none of which showed even subtle genetic alterations of p53 or loss of alleles on 17p. Moreover, in three of the four cases with MDM2 gene amplification, the level of gene amplification in the metastatic lymph nodes was slightly higher than that in the primary tumors. These results, taken together with previous findings, suggest that a subset of breast cancers without genetic alteration of p53 may also arise by inactivation of the p53 function through interaction with the overexpressed MDM2 protein induced by gene amplification.     

p53基因突变和STK15基因过表达与喉癌发生的关系

目的　探讨p5 3基因突变和STK15基因表达与喉癌发生发展的关系。方法　自 5 5例术前未经化疗和放疗的喉鳞状细胞癌患者的新鲜手术标本中 ,分别取配对癌组织和癌旁正常组织进行以下检测 :(1)提取DNA ,采用聚合酶链反应 单链构象多态性 (PCR SSCP)银染结合DNA直接测序 ,检测喉鳞癌组织中p5 3基因第 7外显子 (p5 3E7)和第 8外显子 (p5 3E8)的突变情况 ;(2 )提取总RNA ,反转录合成cDNA ,以 β actin为内对照进行PCR扩增 ,分析STK15基因表达的水平。结果　 5 5例喉癌组织中 ,17例 (30 .9% )发生p5 3E7突变 ,未发现p5 3E8突变 ;癌组织STK15基因表达与 β actin表达平均密度比值 (ADV)为 1.2 2± 0 .4 9。癌旁正常组织发生p5 3E7突变 1例 (1.8% ) ,p5 3E8突变 1例 (1.8% ) ;癌旁正常组织ADV为 0 .99± 0 .5 4。喉癌组织p5 3E7突变率显著高于癌旁正常组织 (χ2 =8.6 6 ,P <0 .0 1)。癌组织STK15基因表达高于癌旁正常组织 (t=4 .5 39,P <0 .0 1)。 17例p5 3基因突变的患者中 ,14例 (82 .4 % )癌组织STK15基因表达高于癌旁正常组织 ;38例癌组织STK15基因表达高于癌旁正常组织的患者中 ,14例 (36 .8% )存在p5 3E7突变。 2 5 .5 %的喉癌患者同时发生p5 3E7突变与STK15基因表达增高。结论　同时发生p5 3基因突变和STK     

Prognostic and therapeutic value of the Hippo pathway,RABL6A,and p53-MDM2 axes in sarcomas

Additional prognostic and therapeutic biomarkers effective across different histological types of sarcoma are needed. Herein we evaluate expression of TAZ and YAP, the p53-MDM2 axis, and RABL6A, a novel oncoprotein with potential ties to both pathways, in sarcomas of different histological types. Immunohistochemical staining of a tissue microarray including 163 sarcomas and correlation with clinical data showed that elevated YAP and TAZ independently predict worse overall and progression-free survival, respectively. In the absence of p53 expression, combined TAZ and YAP expression adversely affect overall, progression free, and metastasis free survival more than TAZ or YAP activation alone. RABL6A independently predicted shorter time to metastasis and was positively correlated with p53, MDM2 and YAP expression, supporting a possible functional relationship between the biomarkers. Network analysis further showed that TAZ is positively correlated with MDM2 expression. The data implicate all five proteins as clinically relevant downstream players in the Hippo pathway. Finally, a novel inhibitor of MDM2 (MA242), effectively suppressed the survival of sarcoma cell lines from different histological types regardless of p53 status. These findings suggest both independent and cooperative roles for all five biomarkers across different histological types of sarcoma in predicting patient outcomes and potentially guiding future therapeutic approaches.