Date of Award

January 2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Seema Somji

Abstract

Bladder cancer constitutes one of the most prevalent cancers and is among the leading causes of cancer-related deaths in the United States. This work sought to better characterize and understand the role of the proto-oncogene anterior gradient 2 (AGR2) in bladder cancers. Immunohistochemical analysis of AGR2 expression in a range of bladder cancer specimens indicated that the expression of AGR2 decreases with an increase in the stage and grade of bladder cancers. The environmental carcinogens arsenic and cadmium (Cd2+) have been implicated in various cancers. This laboratory has demonstrated that arsenite (As3+) and Cd2+ can malignantly transform the bladder epithelium cell line UROtsa. This study therefore sought to determine AGR2 expression in six As3+ and seven Cd2+-transformed UROtsa cell lines in which real-time PCR and western blotting data indicated that AGR2 expression was increased in several of the transformed cell lines. Next AGR2 expression was evaluated in mouse heterotransplant tumors arising from the injection of the previously transformed UROtsa cell lines in athymic nude (Foxn1nu) mice. Results from real-time PCR analysis and immunohistochemistry on tumor samples demonstrated that AGR2 expression remained increased in most of the Cd2+ heterotransplants but was significantly increased in only a couple of the As3+ heterotransplants. UROtsa cells were further exposed to 2, 4, or 6 μM As3+ or to 1, 2, or 4 μM Cd2+ for up to 72 hours and real-time PCR analysis was performed to evaluate AGR2 expression. While AGR2 exhibited increased expression in

response to 48 hours As3+ exposure, expression was not induced to the same extent in Cd2+ exposed UROtsa cells. Spheroids containing cancer initiating cells (CICs) were generated from the transformed and the parent UROtsa cells and the expression of AGR2 was determined. The results obtained suggested that there was an increase in the expression of AGR2 in all of the spheroids isolated from the As3+-transformed cells whereas some of the spheroids isolated from the Cd2+-transformed cells expressed high levels of AGR2 when compared to the spheroids isolated from the parent UROtsa cells.

This laboratory has also shown that As3+ and Cd2+ can cause malignant transformation of a breast epithelial cell line, MCF-10A. Previous studies have shown that the expression of AGR2 promotes breast tumorigenesis in mice. This gene is known to play a role in promoting cellular transformation, tumor growth, and metastasis in various cancers. This study was interested in determining the expression level of AGR2 in As3+ and Cd2+-transformed MCF-10A cells. Real-time PCR and Western analysis indicated that the expression of AGR2 was significantly increased in the MCF-10A cells transformed with As3+ when compared to the Cd2+-transformed cells. Exposure of the parent MCF-10A cells to 4, 8, and 16 μM As3+ for 48 hours resulted in a significant increase in the expression of AGR2 whereas exposure to Cd2+ did not increase the expression of AGR2, suggesting that As3+ has the potential to induce AGR2 in MCF-10A cells. In order to further investigate the effects of AGR2 expression on breast epithelial cells, MCF-10A cells were transfected with the AGR2 gene. Overexpression of AGR2 in the MCF-10A cells increased the ability of the cells to migrate faster in the wound scratch assay when compared to the blank vector transfected cells. In addition, MTT and scratch assays revealed increased growth and migration in the As3+-transformed cells when compared to the parent MCF-10A cells and the Cd2+-transformed MCF-10A cells further implicating AGR2 in enhancing cell growth and migration. Treatment of the MCF-10A cells with the histone deacetylase inhibitor MS-275 and the demethylating agent, 5-Aza-2'-deoxycytidine (5-AZC) increased the expression of AGR2 suggesting that epigenetic modifications may be involved in regulating the expression of AGR2 in MCF-10A cells. These data also suggest that the expression of AGR2 in the MCF-10A cells may be regulated independently of the estrogen receptor status of the cells. Seeking to gain a better understanding of the relationship between AGR2 expression and breast cancer grade and stage, immunohistochemical staining was performed on a range of breast cancer specimens. Results showed elevated AGR2 expression with increasing grade of breast carcinoma. In conclusion, this study demonstrated the usefulness of AGR2 expression as a biomarker for bladder and breast cancers, further characterized the effect of AGR2 on the growth and migration of MCF-10A cells, and began to evaluate possible mechanisms by which AGR2 expression may be induced.

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