Date on Master's Thesis/Doctoral Dissertation

5-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Degree Program

Physiology and Biophysics, PhD

Committee Chair

Galandiuk, Susan

Committee Co-Chair (if applicable)

Joshua, Irving

Committee Member

Maldonado, Claudio

Committee Member

Schuschke, Dale

Committee Member

Bhatnagar, Aruni

Author's Keywords

miRNA; colorectal cancer; cancer cell lines; mTOR

Abstract

Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth most common cause of death. These are staggering statistics for a disease that can essentially be cured if caught early and the pathology is favorable to therapeutic intervention. There is currently a drastic decrease in five year survival as the cancer stage increases from locally confined disease to metastatic disease. These statistics suggest that although some strides have been made with colon cancer screening and early intervention, there is still much room for improvement in both screening and treatment of CRC. One of the pathways that have been linked to CRC is the mammalian target of rapamycin (mTOR) pathway. Recently this has been the subject of intense study particularly with a view to better targeted drug therapy for colorectal cancer. Much is known about this pathway; however, multiple regulatory elements have yet to be elucidated. MicroRNAs (miRNAs) have been shown to influence some aspects of the mTOR pathway in other cancers, however their role in the mTOR pathway in CRC has yet to be fully explored. The microRNA-99 (miR-99) family of miRNAs has recently been implicated in regulation of mTOR signaling in other diseases. Studies were conducted using normal colon epithelial and CRC cell lines provided the following key findings: 1. Transfection of miRNA-99a mimic into CCD-841 (normal epithelium) and CRC cell lines, HT-29 (Dukes C), HCT-116 (Dukes D), results in a decrease in the amount of phosphorylated mTOR protein in all 3 cell. 2. miRNA-99a transfection also affected expression of downstream protein products, with a decrease phosphorylated S6K1 in HT-29 and CCD-841, while this same transfection showed an increase in this phosphorylated protein in cell line HCT-116. The opposite was observed for protein 4EBP1 after transfection, which showed an increase in phosphorylated 4EBP1 in cell lines HT-29 and CCD-841, but a decrease in phosphorylated protein the HCT-116 cell line. 1. Transfection of miRNA-99a mimic resulted in decreased motility and invasion in cell lines HT-29 and HCT-116 as compared to cell lines transfected with the negative control. Transfection with the miRNA-99a antagomir resulted in increased motility in both CRC cells lines but minimal changes or decrease in invasion. The effects of miRNA-99a on mTOR protein, S6K1 and 4EBP1 were elucidated by this project providing further evidence as to the importance of mTOR inhibition for possible regulation of CRC metastasis. This supports new avenues of study to further understand CRC metastasis as well as possible targets for therapeutic intervention.