Abstract 1970: The mRNA-destabilizing protein tristetraprolin suppresses tumorigenic phenotypes in a triple-negative breast cancer cell model via a non-canonical mechanism

Abstract

Abstract The purpose of this study was to define mechanisms by which the mRNA-destabilizing factor tristetraprolin (TTP) impacts tumorigenic phenotypes in an aggressive model of metastatic breast cancer. Widespread repression of TTP levels in human tumors and cancer cell lines relative to non-transformed tissues suggests that TTP may function as a tumor suppressor in diverse neoplastic contexts, and low TTP expression is a negative prognostic indicator in breast cancer. TTP is a tandem zinc finger protein that binds to AU-rich elements (AREs) and targets their associated mRNAs for degradation. AREs are potent cis-acting determinants of cytoplasmic mRNA turnover in mammalian cells, and are essential for limiting cellular production of many clinically important gene products including regulators of inflammation, cell proliferation, and apoptosis. In this study, we show that restoration of TTP levels attenuates several tumorigenic phenotypes in the aggressively metastatic breast cancer line MDA-MB-231. Notably, TTP-expressing cell lines replicate approximately 70% slower than non-transfected controls. Inhibition of cell proliferation did not result from apoptosis but rather by a delay at the G1/S checkpoint. TTP expression also significantly reduced the formation of cultured mammospheres based on both sphere-forming efficiency frequency and extreme limiting dilution analyses, indicating that this protein reduces stemness and non-adherent growth potential. Finally, cell motility was suppressed by TTP, demonstrated using wound healing assays. These findings are consistent with a tumor suppressor role for TTP. To identify potential mechanisms linking TTP to diminution of tumorigenic phenotypes, we surveyed the expression of genes encoding select pro-tumorigenic factors, including several known to encode TTP-targeted mRNAs. Expression of cyclin D1, cyclin E and c-Myc proteins were significantly reduced in TTP-expressing MDA-MB-231 cells, however, this was not mediated by accelerated mRNA decay. Interestingly, we observed that the mRNA-destabilizing function of TTP is abrogated by the constitutively active ERK signaling pathway in MDA-MB-231 cells, suggesting that TTP suppresses tumorigenic properties in this cell model independently of its canonical mRNA-destabilizing function. Consistent with this model, expressing a non-RNA-binding mutant form of TTP (C147R) robustly attenuated the same tumorigenic properties suppressed by the wild type protein. Together, these findings show that the mRNA-destabilizing activity of TTP is dispensable for its tumor suppressive properties in MDA-MB-231 cells, and by extension that TTP must limit diverse tumorigenic properties in these cells via an as yet uncharacterized non-canonical mechanism. Citation Format: Christina R. Ross, Gerald M. Wilson. The mRNA-destabilizing protein tristetraprolin suppresses tumorigenic phenotypes in a triple-negative breast cancer cell model via a non-canonical mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1970. doi:10.1158/1538-7445.AM2015-1970