Laboratory of Experimental Molecular Pathology, Department of Pathology, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy.
Lab Invest. 2013 Nov;93(11):1232-40. doi: 10.1038/labinvest.2013.107. Epub 2013 Sep 9.
The molecular marker of well-differentiated/de-differentiated liposarcomas is MDM2 gene amplification coupled with protein overexpression and wild-type TP53. MDMX is a recently identified MDM2 homolog and its presence in this tumor is unexplored. Our aim was to investigate the role of full-length MDM2 and MDMX proteins and their isoforms in surgical specimens of well-differentiated/de-differentiated liposarcomas in view of Nutlin-3A (a MDM2 inhibitor) treatment. Frozen and matched formalin-fixed, paraffin-embedded material from surgical specimens was examined by means of: (1) fluorescence in situ hybridization to determine MDM2 and MDMX gene copy numbers; (2) RT-PCR and densitometry to analyze alternative splicing forms of mdm2 and mdmx; (3) immunoblotting and immunohistochemistry to assess the corresponding translated proteins; and (4) in vitro and in silico assays to determine their affinity for Nutlin-3A. All these cases showed MDM2 gene amplification with an MDMX disomic pattern. In all cases, the full-length mdm2 transcript was associated with the mdm2-b transcript, with ratios ranging from 0.07 to 5.6, and both were translated into protein; mdmx and mdmx-s were co-transcripted, with ratios ranging from 0.1 to 5.6. MDMX-S was frequently more upregulated than MDMX at both transcriptional and protein level. Each case showed different amounts of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and the corresponding proteins. In vitro assays showed that Nutlin-3A was ineffective against MDM2-B and was unable to disrupt the MDMX/TP53 and MSMX-S/TP53 complexes. Molecular simulations confirmed these in vitro findings by showing that MDM2 has high Nutlin-3A affinity, followed by MDMX-S, MDMX, and MDM2-B. Nutlin-3A is predicted to be a good therapeutic option for well-differentiated/de-differentiated liposarcomas. However, our findings predict heterogeneous responses depending on the relative expression of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and proteins.
高分化/去分化脂肪肉瘤的分子标志物是 MDM2 基因扩增，伴有蛋白过表达和野生型 TP53。MDMX 是最近鉴定的 MDM2 同源物，其在这种肿瘤中的存在尚未被探索。我们的目的是研究全长 MDM2 和 MDMX 蛋白及其同工型在高分化/去分化脂肪肉瘤手术标本中的作用，因为 Nutlin-3A（一种 MDM2 抑制剂）的治疗作用。通过以下方法检查手术标本的冷冻和匹配的福尔马林固定、石蜡包埋材料：（1）荧光原位杂交以确定 MDM2 和 MDMX 基因拷贝数；（2）RT-PCR 和密度测定法分析 mdm2 和 mdmx 的选择性剪接形式；（3）免疫印迹和免疫组化评估相应的翻译蛋白；（4）体外和计算机模拟测定其对 Nutlin-3A 的亲和力。所有这些病例均显示 MDM2 基因扩增，MDMX 为二倍体模式。在所有情况下，全长 mdm2 转录物与 mdm2-b 转录物相关，比值范围为 0.07 至 5.6，两者均翻译为蛋白质；mdmx 和 mdmx-s 共同转录，比值范围为 0.1 至 5.6。MDMX-S 在转录和蛋白水平上通常比 MDMX 上调更多。每个病例显示出不同数量的 mdm2、mdm2-b、mdmx 和 mdmx-s 转录物和相应的蛋白质。体外试验表明，Nutlin-3A 对 MDM2-B 无效，无法破坏 MDMX/TP53 和 MSMX-S/TP53 复合物。分子模拟通过显示 MDM2 对 Nutlin-3A 具有高亲和力，其次是 MDMX-S、MDMX 和 MDM2-B，证实了这些体外发现。预测 Nutlin-3A 是治疗高分化/去分化脂肪肉瘤的良好选择。然而，我们的研究结果预测根据 mdm2、mdm2-b、mdmx 和 mdmx-s 转录物和蛋白质的相对表达，会产生不同的反应。
