title: In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas.
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.
authors.affiliation: Laboratory of Experimental Molecular Pathology, Department of Pathology, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Bozzi
authors.forename: Elena
authors.initials: E
authors.lastname: Conca
authors.forename: Erik
authors.initials: E
authors.lastname: Laurini
authors.forename: Paola
authors.initials: P
authors.lastname: Posocco
authors.forename: Alessandra
authors.initials: A
authors.lastname: Lo Sardo
authors.forename: Genny
authors.initials: G
authors.lastname: Jocollè
authors.forename: Roberta
authors.initials: R
authors.lastname: Sanfilippo
authors.forename: Alessandro
authors.initials: A
authors.lastname: Gronchi
authors.forename: Federica
authors.initials: F
authors.lastname: Perrone
authors.forename: Elena
authors.initials: E
authors.lastname: Tamborini
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pelosi
authors.forename: Marco A
authors.initials: MA
authors.lastname: Pierotti
authors.forename: Roberta
authors.initials: R
authors.lastname: Maestro
authors.forename: Sabrina
authors.initials: S
authors.lastname: Pricl
authors.forename: Silvana
authors.initials: S
authors.lastname: Pilotti
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017398:Alternative Splicing; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D003198:Computer Simulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics / Q000502:physiology; D005260:Female; D018628:Gene Dosage; D006801:Humans; D007093:Imidazoles / Q000378:metabolism / Q000494:pharmacology*; D017404:In Situ Hybridization, Fluorescence; D008080:Liposarcoma / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010879:Piperazines / Q000378:metabolism / Q000494:pharmacology*; D020641:Polymorphism, Single Nucleotide; D011487:Protein Conformation; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D018797:Cell Cycle Proteins; D007093:Imidazoles; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D010879:Piperazines; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2