In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas

• Published in: Laboratory investigation 2013-11, Vol.93 (11), p.1232-1240
• Main Authors: Bozzi, Fabio, Conca, Elena, Laurini, Erik, Posocco, Paola, Lo Sardo, Alessandra, Jocollè, Genny, Sanfilippo, Roberta, Gronchi, Alessandro, Perrone, Federica, Tamborini, Elena, Pelosi, Giuseppe, Pierotti, Marco A, Maestro, Roberta, Pricl, Sabrina, Pilotti, Silvana
• Format: Article
• Language: English
• Subjects: 631/1647/2258/1267; 692/699/67/1798; Adult; Aged; Aged, 80 and over; Alternative Splicing; Antineoplastic Agents - metabolism; Antineoplastic Agents - pharmacology; Biomarkers, Tumor - genetics; Biomarkers, Tumor - metabolism; Cell Cycle Proteins; Cell Differentiation; Computer Simulation; Drug Resistance, Neoplasm - genetics; Drug Resistance, Neoplasm - physiology; Female; Gene Dosage; Humans; Imidazoles - metabolism; Imidazoles - pharmacology; In Situ Hybridization, Fluorescence; Laboratory Medicine; Liposarcoma - drug therapy; Liposarcoma - metabolism; Liposarcoma - pathology; Male; Medicine; Medicine & Public Health; Middle Aged; Models, Molecular; Molecular Dynamics Simulation; Nuclear Proteins - antagonists & inhibitors; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Pathology; Piperazines - metabolism; Piperazines - pharmacology; Polymorphism, Single Nucleotide; Protein Conformation; Protein Isoforms - antagonists & inhibitors; Protein Isoforms - genetics; Protein Isoforms - metabolism; Proto-Oncogene Proteins - antagonists & inhibitors; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-mdm2 - antagonists & inhibitors; Proto-Oncogene Proteins c-mdm2 - genetics; Proto-Oncogene Proteins c-mdm2 - metabolism; research-article; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism
• ISSN: 0023-6837; 1530-0307
• DOI: 10.1038/labinvest.2013.107

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.