Abstract 1909: Multiple Mdm2 isoforms promote tumorigenesis, disrupt mammary tissue architecture, and are endogenously expressed in cancers.
Authors: Okoro Danielle , Gao Chong , Puente Cindy , Polotskaia Alla , Arva Nicoleta , Rosso Melissa , Bargonetti Jill 
Published: August 2014

Abstract
Mdm2 is a key regulator of the tumor suppressor p53 and over expression of Mdm2 is a negative prognostic marker for many cancers. Mdm2 expression is increased in estrogen-receptor positive breast cancer cells that have the mdm2 G/G single nucleotide polymorphism at position 309 (mdm2 SNP309). In such cells, estrogen activates cell proliferation in an Mdm2 dependent manner. Alternatively spliced mdm2 transcripts have been observed in cancers, however, endogenous protein isoforms have not been detected. The exogenous expression of mdm2 splice variants encodes proteins, transforms cell lines in culture, and in the absence of p53 causes tumors in mice. Therefore we hypothesized that endogenous human Mdm2 protein isoforms participate in p53-independent tumorigenesis. We created a specific human Mdm2-C antibody to the splice junction of exons four and ten (Mdm2 C410) and validated the C410 antibody using in vitro translated Mdm2 compared to Mdm2-C. We detected, for the first time in human cancer cell lines, expression of endogenous Mdm2-C. This was particularly high in G/G mdm2 SNP309 breast cancer cells. In addition, using immunohistochemistry analysis three out of three Liposarcoma tissue samples tested positive for Mdm2-C expression. Using the C410 antibody we detected endogenous Mdm2-C protein in estrogen receptor positive T47D and MCF-7 breast cancer cells. T47D cells (with mutant p53), and MCF-7 cells (with wild-type p53), both showed increased Mdm2-C protein expression after estrogen treatment. We also determined that Mdm2-C did not interact with p53. Interestingly, Mdm2-C protein localized in nucleolar and cytoplasmic speckles. The exogenous expression of Mdm2-C increased colony formation ability indicating that it had transforming capability. However contrary to results with exogenously expressed Mdm2-FL, exogenously expressed Mdm2-C did not influence p53 stability, or p53 transactivation activity. Importantly, siRNA-mediated knockdown of Mdm2-C in T47D cells induced cell death. We engineered miR30-based inducible mdm2 knockdown cell lines of MCF-7 and T47D to evaluate the impact of multiple Mdm2 isoforms. The down-regulation of Mdm2 in MCF-7.shmdm2 and T47D.shmdm2 inhibited cell proliferation in 2D culture and in 3D laminin-rich matrigel. The knockdown of Mdm2 in T47D cells reduced colony size, reverted to cells to more normal mammary architecture, and decreased E2F protein expression. We suggest that survival of estrogen-influenced breast cancer is through a p53-independent, and thus non-canonical, Mdm2 isoform mediated mechanism.
Special thanks to Gerard Evan for help with immunogen peptide design. This work was supported by The Breast Cancer Research Foundation and The NSF (grant MCB-0744316) to J.B. D.O. was partially supported by a CUNY Magnet Award and M.R. was supported by MBRS-RISE Program to Hunter College 3R25-GM060665.
Citation Format: Danielle Okoro, Chong Gao, Cindy Puente, Alla Polotskaia, Nicoleta Arva, Melissa Rosso, Jill Bargonetti. Multiple Mdm2 isoforms promote tumorigenesis, disrupt mammary tissue architecture, and are endogenously expressed in cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1909. doi:10.1158/1538-7445.AM2013-1909