Abstract 3201: PTBP1 stability is increased in ovarian and breast cancer cell lines compared to matched controls.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC PTBP1 (polypyrimidine tract-binding) protein is a splicing factor that is frequently upregulated in ovarian and breast tumors. Knockdown (KD) of PTBP1 expression by shRNA impairs ovarian tumor cell growth in vivo and in vitro, as well as colony formation and invasiveness in vitro (He et al, Oncogene 26:4961-8, 2007). We have also found that PTBP1 is progressively upregulated in immortalized human mammary epithelial cells (HMECs) and human ovarian surface epithelial (HOSE) cells, correlating with their transformation state. These results prompted us to determine the factors that play a role in the upregulation of PTBP1 protein expression. We first asked whether the increased PTBP1 protein in human ovarian and breast cell lines is accompanied by proportional increases of PTBP1 mRNA. We found that there was a discordance between PTBP1 mRNA and protein levels, as measured by RT-PCR and western blot, in these cancer cell lines in relation to the control cells (finite lifespan HMEC or HOSE, respectively). Accordingly, we attempted to understand the basis for this discrepancy, and measured PTBP1 protein half-life. We treated A2780 (ovarian cancer), MCF7 (breast cancer) cells, and “finite lifespan” HMEC (184A1) with cycloheximide to terminate protein synthesis and followed the fate of PTBP1 protein over time by western blot. We found that the half-life of PTBP1 protein is ∼3-5 h in the HMECs with finite lifespan, whereas it ranges from ∼9-12h in human epithelial ovarian and breast cancer cells. These results demonstrate that PTBP1 expression is regulated in part by events that control the stability of the protein and suggest that the normal regulation of PTBP1 protein turnover is altered in cancer cell lines. Bioinformatics analysis identified one possible SUMO-binding motif site within the PTBP1 protein (SUMOsp 2.0 - SUMOylation Site Prediction, http://sumosp.biocuckoo.org/), suggesting that SUMOylation may play a role in PTBP1 stability; we are currently examining this possibility. Findings presented herein will enhance our understanding of the regulation of PTBP1 expression and the mechanisms underlying its variation in protein and mRNA levels in ovarian and breast tumors. (Support in part by NCI grants R01 CA40570 and R01 CA138762 [to WTB], by OCRF [to XH], and by UIC.) Citation Format: Ahmet Dirim Arslan, Szilard Asztalos, Martha Stampfer, Debra Tonetti, Xiaolong He, William T. Beck. PTBP1 stability is increased in ovarian and breast cancer cell lines compared to matched controls. [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 3201. doi:10.1158/1538-7445.AM2013-3201