Doctoral Dissertations

Date of Award

12-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Seung J. Baek

Committee Members

Michael M. Fry, Karla J. Matteson, Jay Whelan

Abstract

This dissertation explores the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on human colorectal cancer (CRC) cell proteins related to cellular structure and adhesion. NSAIDs are extensively used not only to treat inflammatory diseases but also to prevent cancer among high-risk groups. Their mechanisms are not fully understood, but both cyclooxygenase (COX) dependent and independent pathways play a role in NSAID-induced anti-tumorigenesis. Our lab previously reported that NSAIDs induce other anti-tumorigenic genes in a COX-independent manner (Chapter 1). Human CRC cells treated with the NSAID sulindac sulfide (SS) showed dramatic morphological changes under differential interference contrast and fluorescent microscopy, as well as weakened cellular adhesion and loss of membrane permeability as measured by micro-impedance. To elucidate a molecular mechanisms involved, two independent microarray analysis were performed using HCT-116 cells, and the gene Nesprin-2 was selected for further study based on its novelty in relation to cancer and its role in cell organization and structure (Chapter 2). SS-treated cells diminished Nesprin-2 mRNA expression compared to vehicle-treated cells. Other NSAIDs were also tested and demonstrated that inhibition of Nesprin-2 was not unique to SS. Additionally, immunohistochemistry showed higher levels of Nesprin-2 in the tumors of many tissue types in comparison with normal tissue. Further micro-impedance experiments on cells with reduced Nesprin-2 expression showed a proportional decrease in cellular adhesion. Next, we examined the effects of SS on another potentially oncogenic protein, epithelial cell adhesion molecule (EpCAM), which is over-expressed in many cancers including CRC, breast, pancreas, and prostate. We found EpCAM to be regulated by SS in a manner that is independent of: COX activity, transcription, de novo protein synthesis, and proteasomal degradation. Our findings demonstrate that SS drives an uncharacterized cleavage of EpCAM between arginine residues at positions 80 and 81 which is blocked by mutation to alanine residues as well as deletions (Chapter 3). Findings presented in this dissertation (1) our data suggests that Nesprin-2 maybe a potential novel cancer-associated protein and NSAIDs decrease its expression (2) SS facilitates proteolysis of EpCAM at amino acids 80/81 and provides a novel COX-independent mechanism of NSAID anti-tumorigenesis at the post-translational level.

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