Date of Award

1-1-2009

Document Type

Campus Access Dissertation

Department

Biological Sciences

First Advisor

Dan A. Dixon

Abstract

Colorectal cancer is a leading cause of cancer mortality among adults and the risk of developing colon cancer is increased in those individuals suffering from chronic inflammatory syndromes such as inflammatory bowel disease (IBD). Multiple pro-oncogenic and inflammation associated immediate early-response genes demonstrate elevated levels of expression in both colon cancer cells and tumors. One well-established gene that is overexpressed in colorectal cancer is cyclooxygenase-2 (COX-2). This enzyme catalyzes the rate-limiting step of increased prostaglandin formation in inflammatory states. Normally COX-2 expression levels are tightly regulated through transcriptional and post-transcriptional mechanisms, allowing for rapid COX-2 expression in response to pro-inflammatory and growth signaling events. However, uncontrolled expression of COX-2 is observed in a variety of cancers and chronic inflammatory diseases, indicating that COX-2 may play a role carcinogenesis. To identify the molecular mechanisms that contribute to COX-2 overexpression in disease states, this work examines the post-transcriptional regulation of COX-2 mediated through its 3' untranslated region (3'UTR). AU-rich sequence elements (AREs) that exist within the 3' untranslated region (3'UTR) of tightly regulated mRNAs like COX-2 allow for highly controlled mRNA expression and help signal for rapid mRNA decay when the cell no longer requires it. This is facilitated through cellular RNA-binding proteins that function as mediators of mRNA decay, stability, or control of translation. Altered expression of the RNA-binding protein HuR has recently been associated with colorectal cancer progression.

The purpose of the findings described in this work was to further elucidate the underlying post-transcriptional mechanisms associated with altered regulation of COX-2 mRNA stability in the context of an ARE-binding protein HuR. The studies presented in chapter one demonstrate that increased expression and cytoplasmic localization of HuR can have a dramatic effect on COX-2 expression within colon cancer cells and in intestinal tumors due to decreased mRNA and protein expression levels of the mRNA decay factor TTP colon cancer cells, adenomas, and adenocarcinomas as compared to normal tissue. Furthermore, re-introduction of TTP into colon cancer cells significantly diminished COX-2 mRNA and protein expression, in addition to drastically altering cell growth. These results suggest that loss of TTP expression in tumor cells may allow for COX-2 and other ARE-containing mRNA stabilization in concert with HuR overexpression.

In chapter two the role of miR-16 in ARE-specific microRNA-mediated regulation of COX-2 in colon cancer were examined using HeLa cells stimulated to express COX-2 as well as HCA7 and Moser colon cancer cell lines that constitutively express COX-2. Expression of miR-16 specifically inhibited COX-2 expression at both the mRNA and protein level, by binding to 2 target sites within the COX-2 3'UTR. Moreover, miR-16 expression was observed to be decreased in intestinal tumors isolated from human and murine tissue, in addition to colon cancer cell lines. It was next established that HuR and miR-16 share overlapping ARE-binding specificity, and overexpression of HuR can supersede the ability of miR-16 to target the COX-2 3'UTR to stabilize COX-2 mRNA with a half-life to more than 4 hours that may occur due to the ability of HuR to associate with endogenous miR-16 when HuR is overexpressed and present in the cytoplasm. These findings imply that alterations in the function of miR-16 as a result of cytosolic expression of HuR may allow for elevated COX-2 expression.

The third chapter determined physiological impact overexpression of HuR had specifically in the gastrointestinal epithelium in vivo. By way of the GI-specific A33 promoter, a transgenic mouse that expresses the HuR/GFP bicistronic transgene exclusively in the epithelial tissue of the large and small intestine was created. When HuR-transgenic mice were crossed with the APCMIN/+ mouse to yield HuR-Tg/APCMIN/+ mice, overexpression of HuR acted to enhance intestinal tumor burden 2-3 fold by 15 weeks. Overexpression of HuR in the APCMIN/+ mouse promoted an increase in COX-2 mRNA and protein in colonic tumors and treatment of <APCMIN/+ and HuR-Tg/APCMIN/+ mice with the COX-2-selective inhibitor celecoxib resulted in a significant decrease in tumor burden for mice with or without elevated expression of HuR. In a colitis-associated model of colorectal tumorigenesis using the carcinogen AOM in combination with the colitis promoter DSS, similar results were observed. These findings indicate that HuR functions to enhance COX-2 expression in a manner that facilitates tumor growth and/or promotion downstream of a tumor-initiating event. Overall, the work presented here identifies cellular processes, at both the molecular and physiological level, that contribute to the role of HuR in colorectal carcinogenesis.

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