Abstract 2130: The RNA-binding protein tristetraprolin controls intestinal cell differentiation and tumorigenesis through the Notch signaling pathway
Authors: Zhuang Shufei , Hu Liangyan , Sanduja Sandhya , Umar Shahid , Anant Shrikant , Dixon Dan A. 
Published: August 2015

Colorectal cancer (CRC) is the 2nd leading cause of cancer death in U.S. With lifetime incidence rates as high as 1 in 20, it is critical to better define the molecular events controlling intestinal homeostasis and tumor initiation/progression. In normal intestinal homeostasis, the Notch pathway plays a fundamental role in controlling epithelial cell fate through juxtacrine signaling between membrane-bound ligands to the Notch receptor present on contacting cells. This complex promotes intestinal epithelial cell fate through transcription factors Hes1 and Atoh1/Math1, allowing for differentiation of absorptive enterocyte lineage by Hes1 or secretory (goblet, enteroendocrine and Paneth) cell lineage controlled by Atoh1. In CRC however, constitutive Notch activation is observed allowing for enhanced Hes1 expression associated with tumor progression and self-renewal of tumor-initiating cells. Yet, a clear mechanism for the constitutive activation of this pathway in CRC is not known. Here, we investigated the regulation of Notch signaling by the mRNA decay factor Tristetraprolin (TTP) in CRC and TTP knockout mice. TTP is a member of the TIS11 family of RNA-binding proteins, and the major factor promoting decay of mRNAs bearing AU-rich element (ARE) motifs in their 3′UTR. TTP plays an essential role in controlling pro-inflammatory and oncogenic gene expression in the GI tract, however during CRC progression, TTP expression is silenced in tumors. In this study, TTP-inducible CRC cells were used to show that expression of TTP suppresses proliferation and anchorage-independent growth, impacts cell cycle, and reduces xenograft tumor growth. Interestingly, TTP expression altered CRC cells such that cells now displayed a secretory cell-like morphology, consistent with studies showing effects of Notch signaling attenuation. Alcian blue staining of acidic mucins was significantly increased in TTP-expressing CRC cells and in xenograft tumors, suggesting a role for TTP in directing secretory cell differentiation. Furthermore, gene expression arrays and reporter gene assays showed that levels of Notch signaling genes were significantly impacted by TTP and TTP preferentially promoted a 3-fold downregulation of Notch1 receptor as compared to Notch2-4. The ability of TTP to target Notch1 mRNA for rapid decay occurred through its ARE-containing 3′UTR as observed using 3′UTR reporter and RNA-binding assays. To determine if these effects were specific to tumor cells, intestinal cell lineage was examined in TTP knockout mice with a remarkable reduction in goblet cell number observed as compared to wild-type mice. These findings demonstrate a novel role for TTP in controlling intestinal cell differentiation via post-transcriptional regulation of Notch1 and provide mechanistic insights into constitutive Notch activation observed in CRC occurring through loss of TTP expression.

Citation Format: Shufei Zhuang, Liangyan Hu, Sandhya Sanduja, Shahid Umar, Shrikant Anant, Dan A. Dixon. The RNA-binding protein tristetraprolin controls intestinal cell differentiation and tumorigenesis through the Notch signaling pathway. [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 2130. doi:10.1158/1538-7445.AM2015-2130