Document Type

Thesis

College

College of Pharmacy and Health Sciences

Department

Pharmaceutical and Administrative Sciences

Degree

MS in Pharmaceutical Sciences

Date Completed

5-2025

First Committee Member

Kinney, Shannon

Second Committee Member

Lipkens, Bart

Third Committee Member

English, Anthony

Abstract

Despite available treatments, prostate cancer is a leading cause of cancer related morbidity and mortality in the United States. Thus, there is a need for more targeted and efficacious therapies. Epigenetic DNA modifications, such as methylation (5mC) and hydroxymethylation (5hmC), whose normal patterns are disrupted in cancer, can influence gene regulation. Although the role of loss or gain of 5mC in cancer is better established, the function of 5hmC in cancer progression is much less clear. The use of epigenetic modifying agents, particularly Ten Eleven Translocation (TET) enzyme inhibitors, may prove useful in preventing the creation of 5hmC and restoring tumor suppressors, or downregulating oncogenic pathways. In the current study we treated three cell lines (PNT2 – normal prostate, LNCaP – earlier stage prostate cancer, and PC3 – later stage more aggressive prostate cancer) with a TET inhibitor (TETi76) and assessed its effects on TET1, TET2, and TET3 mRNA expression, global genomic levels of 5hmC, and cell viability. We also examined transcriptome and locus specific genome-wide 5mC and 5hmC patterns in PC3 cells, with and without TETi76 treatment. Finally, we treated PC3 cells with a combination of a DNA methylation inhibitor (DAC) and TETi76 to examine potential additive or synergistic effects. From these experiments we observed no effects on TET expression, but significant global genomic hypo-hydroxymethylation (approximately 50-80% decrease) and a dose dependent decrease in cell viability with TETi76 treatment, in each of the cell lines tested. The IC50s were calculated to be 23.16mM, 10.68mM, and 17.35mM for PNT2, LNCaP, and PC3 cells, respectively. Although cell cycle and apoptosis analysis results were not significant, there was an increase in the percentage of G0/G1 phase cells with TETi76 treatment (approximately 3% increase in PNT2 and LNCaP). PC3 cells treated with 25 mM TETi76 displayed decreased expression of MYT1 and DOCK2, as well as increased expression of an antisense RNA to GRM5, which are all involved in mitogenic signaling pathways. Interestingly, combination of DAC with low doses of TETi76 caused additional decreases in cell viability, while higher doses of TETi76 in combination with DAC were not as effective as TETi76 alone. Combination of DAC and TETi76 resulted in higher G0/G1 phase cells (from 43.3±2.4% DAC alone to 48.7±2.5% DAC and TETi76) and fewer G2/M phase cells (from 39.6±3.4% DAC alone to 34.9±2.9% DAC and TETi76) than with DAC alone. Additional experiments are needed to delve deeper into the mechanism of TETi76 in the model used here, but our results thus far suggest that TETs and 5hmC have oncogenic function in prostate cancer and TET inhibitors may be an option for the treatment of this disease.

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