title: Endogenous human MDM2-C is highly expressed in human cancers and functions as a p53-independent growth activator.
abstract: Human cancers over-expressing mdm2, through a T to G variation at a single nucleotide polymorphism at position 309 (mdm2 SNP309), have functionally inactivated p53 that is not effectively degraded. They also have high expression of the alternatively spliced transcript, mdm2-C. Alternatively spliced mdm2 transcripts are expressed in many forms of human cancer and when they are exogenously expressed they transform human cells. However no study to date has detected endogenous MDM2 protein isoforms. Studies with exogenous expression of splice variants have been carried out with mdm2-A and mdm2-B, but the mdm2-C isoform has remained virtually unexplored. We addressed the cellular influence of exogenously expressed MDM2-C, and asked if endogenous MDM2-C protein was present in human cancers. To detect endogenous MDM2-C protein, we created a human MDM2-C antibody to the splice junction epitope of exons four and ten (MDM2 C410) and validated the antibody with in vitro translated full length MDM2 compared to MDM2-C. Interestingly, we discovered that MDM2-C co-migrates with MDM2-FL at approximately 98 kDa. Using the validated C410 antibody, we detected high expression of endogenous MDM2-C in human cancer cell lines and human cancer tissues. In the estrogen receptor positive (ER+) mdm2 G/G SNP309 breast cancer cell line, T47D, we observed an increase in endogenous MDM2-C protein with estrogen treatment. MDM2-C localized to the nucleus and the cytoplasm. We examined the biological activity of MDM2-C by exogenously expressing the protein and observed that MDM2-C did not efficiently target p53 for degradation or reduce p53 transcriptional activity. Exogenous expression of MDM2-C in p53-null human cancer cells increased colony formation, indicating p53-independent tumorigenic properties. Our data indicate a role for MDM2-C that does not require the inhibition of p53 for increasing cancer cell proliferation and survival.
authors.affiliation: Department of Biological Sciences Hunter College and The Graduate Center Departments of Biology and Biochemistry, City University of New York, New York City, United States of America.
authors.forename: Danielle R
authors.initials: DR
authors.lastname: Okoro
authors.forename: Nicoleta
authors.initials: N
authors.lastname: Arva
authors.forename: Chong
authors.initials: C
authors.lastname: Gao
authors.forename: Alla
authors.initials: A
authors.lastname: Polotskaia
authors.forename: Cindy
authors.initials: C
authors.lastname: Puente
authors.forename: Melissa
authors.initials: M
authors.lastname: Rosso
authors.forename: Jill
authors.initials: J
authors.lastname: Bargonetti
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2