Abstract Estrogen receptor positive breast cancers often have high levels of Mdm2. We investigated if estrogen signaling in breast cancers occurred through an Mdm2 mediated pathway. We examined the effect of long-term 17β-estradiol (E2) treatment in ERα positive breast cancer cell lines that contain wild-type p53 (MCF-7) and mutant p53 (T-47D). These cell lines also contain the mdm2 SNP at position 309, which increases the expression of Mdm2 by Sp1 driven transcription. In both these cell lines, we observed that E2 treatment dramatically increased the amount of Mdm2 without decreasing the corresponding wild-type or mutant p53. Surprising the amount of p53 protein in both cell lines increased with E2 treatment. We engineered miR30-based inducible mdm2 knockdown cell lines of MCF-7 and T-47D to evaluate to impact of Mdm2 on their cancer cell phenotypes. The down-regulation of Mdm2 in MCF-7.shmdm2 and T-47D.shmdm2 inhibited cell proliferation in 2D culture and in 3D laminin-rich matri-gel. Knockdown of Mdm2 did not cause an increase in p53 protein. These data suggest a p53-independent signal transduction pathway for Mdm2-mediated activation of proliferation. Alternative spliced mdm2 transcripts have been observed in many cancers including breast, but no endogenous protein isoforms from the spliced isoforms have been described. We asked if the spliced variant Mdm2-C transcript (that lacks most of the N-terminal p53 binding domain) expressed endogenous protein in these breast cancer cell lines. We created an Mdm2-C specific antibody using a synthetic peptide to the splice junction of exons four and ten (C4-10) to detect the protein. We validated the C4-10 antibody using in vitro translated Mdm2 compared to Mdm2-C. High expression of Mdm2-C occurred in the G/G mdm2 SNP309 ER+ breast cancer cell line T-47D, but only a slight increase was detected in the presence of E2. Additionally, Mdm2-C was found in nucleolar and cytoplasmic foci before and after estrogen treatment, while the mutant p53 was predominantly nuclear. We report the first identification of endogenous tumor-associated Mdm2-C protein in breast cancer. We suggest that Mdm2 and its isoforms work through non-canonical pathways to transform estrogen influenced breast cancers through molecular pathways that remain to be elucidated. Acknowledgements: This work was supported by Breast Cancer Research Foundation to J.B. and NSF grant to JB MCB-0744316. D.O. was also partially supported by a CUNY Graduate Center Magnet Award. Special thanks to Gerard Evan for help with immunogen peptide design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1169. doi:1538-7445.AM2012-1169