Date of Award

1-1-2012

Document Type

Campus Access Dissertation

Department

Biomedical Science

First Advisor

Dan A Dixon

Abstract

Colorectal cancer (CRC) is the third most common cancer among American adults and a leading cause of cancer-related mortality. The initiation and progression of colorectal tumorigenesis is characterized by genetic alterations that account for uncontrolled expression of many growth- and inflammation-associated genes. In the normal, non-transformed intestinal epithelium, oncogenic gene expression is tightly regulated by cis-acting AU-rich elements (ARE) present in the 3' untranslated region (3'UTR) of their mRNA, thereby targeting them for rapid decay in a process known as ARE-mediated mRNA decay. It has been previously shown that ARE-mediated mRNA decay occurs in cytoplasmic RNA decay centers known as processing bodies (P-bodies). However, the mechanism that drives P-body assembly is largely understudied. The mRNA decay factor tristetraprolin (TTP), by virtue of its ability to bind AREs, promotes ARE-mediated mRNA decay by sequestering its target transcripts to P-bodies. Whereas TTP levels are low-to-undetectable in non-transformed cells, its expression is robustly induced by the growth-inhibitory cytokine TGF-β. Here, we demonstrate that treatment of non-transformed intestinal epithelial cells with TGF-β inhibited ARE-mRNA expression. This effect of TGF-β occurred through transcriptional induction of TTP expression and led to a 2-fold increase in cytoplasmic P-body assembly. Additionally, fluorescent-based RNA visualization of ARE reporter genes indicated that TGF-β treatment selectively localized ARE-mRNAs to P-bodies. These findings implicate TTP as a central regulator of ARE-mediated mRNA decay by virtue of its ability to promote P-body formation. However, ARE-mediated mRNA decay is compromised during colorectal tumorigenesis, as gene expression profiling revealed a 3- to 4-fold enrichment in ARE-containing genes in colorectal adenocarcinomas. Based on the fact that TTP expression is lost in >75% of colorectal adenocarcinomas, we evaluated the functional consequence of loss of TTP expression during the process of colon tumorigenesis with respect to P-body formation. To this extent, loss of TTP expression resulted in loss of TTP-mediated sequestration of ARE-containing mRNAs to P-bodies. Furthermore, P-body assembly was severely compromised in TTP-deficient colorectal tumors, thereby implicating TTP as a physiological driver of P-body formation in the colonic epithelium. Although loss of TTP expression is a key contributing factor to loss of ARE-mediated mRNA decay during colorectal tumorigenesis, previous work has demonstrated that elevated expression of the mRNA stability factor Hu antigen R (HuR) occurs in >94% of colorectal adenocarcinomas. The concomitant changes in the expression of HuR and TTP account for a concerted mechanism that promotes loss of ARE-mediated mRNA decay and pathogenic gene expression in colon cancer. To this extent, elevated HuR expression is correlated with poor clinical prognosis and rapid disease progression. Therefore, HuR has emerged as a novel pharmacological target with potential selectivity for cancer cells. This was examined by treating HuR-overexpressing CRC cell lines with the novel small molecule inhibitor of HuR MS-444. Growth inhibition was observed in all CRC lines with IC50 values of 7-12 µM and cancer cell selectivity was observed with 4- to 5-fold increased MS-444 IC50 in non-transformed intestinal and colonic epithelial cells. This occurred by virtue of MS-444's ability to prevent HuR cytoplasmic localization, where it is known to exert its mRNA stabilizing function. Additionally, MS-444 was effective in promoting ARE-mediated mRNA decay both in vitro and in vivo, as MS-444 treatment of mice bearing tumor xenografts resulted in tumor growth inhibition and attenuation of the ARE-containing gene COX-2, a key contributor to CRC progression. Taken together, loss of TTP expression occurs concomitantly with HuR overexpression, thereby promoting loss of ARE-mediated mRNA decay. Through their ability to modulate expression of a majority of oncogenic factors, these findings define a role for HuR and TTP as novel tumor-promoting and tumor-suppressing factors in the context of colorectal cancer.

Rights

© 2012, Fernando Filippini Blanco

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