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.

Cancer cells often have high expression of Mdm2. However, in many cancers mdm2 is alternatively spliced, with more than 40 mRNA variants identified. Many of the alternative spliced mdm2 mRNAs have the potential to encode truncated Mdm2 isoforms. These putative Mdm2 isoforms can theoretically increase the diversity of the cancer proteome. The 3 best characterized are Mdm2-A, Mdm2-B, and Mdm2-C. As described in this review, the exogenous expression of these isoforms results in paradoxical phenotypes of transformation-associated growth as well as the inhibition of growth. Interestingly, these Mdm2 isoforms contribute tumor-promoting capacity in p53-null backgrounds. Herein we describe how alternative splicing of mdm2 may result in Mdm2 protein products that alter signal transduction to promote tumorigenesis. The tumor promoting capacity of Mdm2 isoforms is discussed in the context of functions that do not require the inhibition of p53. When N-terminal portions of Mdm2 are missing, the biochemical functions encoded by exon 12 are proposed to become more important. This may result in growth promoting functions when wild-type p53 is absent or compromised. The p53-independent tumor promoting activity of Mdm2 is proposed to result from C-terminal biochemical contributions of DNA binding, RNA binding, nucleolar localization, and nucleotide binding.