Tristetraprolin functions as a tumor suppressor in metastatic breast cancer cells by a novel mechanism independent of mRNA binding

Abstract

The RNA-binding protein tristetraprolin (TTP) targets and initiates decay of many mRNAs containing AU-rich elements. TTP expression is repressed across a wide range of human tumors and cancer cell lines relative to non-transformed tissues suggesting that TTP may function as a tumor suppressor in many cancers, and low TTP expression is a negative prognostic indicator in breast cancer. The range of pro-tumorigenic factors encoded by TTP-targeted mRNAs and the variety of neoplastic phenotypes that TTP has been reported to modulate in different cell types prompted the hypothesis that TTP may coordinately attenuate multiple tumor suppressive pathways in a single cell background. Here we show that TTP slows proliferation of the triple negative MDA-MB-231 breast cancer cell model by restricting passage through the G1/S checkpoint while concomitantly inhibiting stemness, cell migration, expression of select regulatory proteins including cyclin D1 and c-Myc, and tumor growth in murine xenograft models. However, suppression of cellular tumorigenic phenotypes and cell cycle markers was independent of TTP's canonical mRNA-destabilizing function, which is inactivated by constitutive ERK but not p38 MAP kinase signaling in MDA-MB-231 cells. Consistent with this model, a non-RNA-binding TTP mutant similarly suppressed MDA-MB-231 proliferation, stemness, and migration together with cyclin D1 and c-Myc levels. Protein turnover assays revealed that TTP expression accelerates the decay of cyclin D1 and c-Myc by a proteasome-dependent mechanism. Microarray analysis of MDA-MB-231 cells expressing TTP also revealed attenuated regulation of c-Myc target genes, however shRNA studies showed c-Myc knockdown in MDA-MB-231 cells does not result in the same attenuation of gene regulation, nor does loss of c-Myc phenocopy the effects of TTP expression. However, despite being dispensable for suppressing tumorigenic characteristics of MDA-MB-231 cells, the RNA-binding activity of TTP did induce a variety of specific gene expression changes despite limitations on its mRNA-destabilizing ability. Collectively our data establish a novel mechanism by which TTP can suppress several tumorigenic phenotypes and gene expression programs in a single cell model independently of its mRNA-destabilizing function. Given the diverse range of cancers where MAP kinase pathways are activated, these non-canonical TTP functions may be applicable to many tumor types.