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

Fabio Bozzi, Elena Conca, Erik Laurini, Paola Posocco, Alessandra Lo Sardo, Genny Jocollè, Roberta Sanfilippo, Alessandro Gronchi, Federica Perrone, Elena Tamborini, Giuseppe Pelosi, Marco A. Pierotti, Roberta Maestro, Sabrina Pricl, Silvana Pilotti

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)1232-1240
Number of pages9
JournalLaboratory Investigation
Volume93
Issue number11
DOIs
Publication statusPublished - 2013

Keywords

  • MDM2
  • MDMX
  • molecular modeling
  • Nutlin-3A
  • TP53

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Fingerprint Dive into the research topics of 'In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas'. Together they form a unique fingerprint.

Cite this