Abstract Metformin and γ-tocotrienol (GT3) are promising chemopreventative agents with studies showing improved patient outcomes with metformin use in several cancer types, including prostate cancer. However, the cellular mechanisms underlying the improved survival rates in cancer patients treated with these drugs are incompletely understood. Inhibition of mammalian target of rapamycin (mTOR), alteration of p-c-JUN and p-ERK signaling pathways and inhibition of mitochondrial complex 1 have all been implicated as part of metformin's anti-tumorigenesis activities. More recently, metformin was shown to influence the activity of epigenetic modifying enzymes resulting in genome-wide alterations in DNA methylation patterns in breast cancer cell lines. In this study, we investigated whether GT3 and metformin treatment would alter methylation pathways in prostate cancer cells.The prostate cancer cell line PC-3 (androgen independent) and normal prostate control cell line RWPE-1 were treated with varying concentrations of either GT3, metformin, the methylation inhibitor azacitidine, or metformin plus azacitidine for 6 hours. Genomic DNA was isolated, and methylation assessed using the Illumina Infinium methylation EPIC BeadChip array. Analysis revealed treatment of PC-3 cells with metformin promoted hypermethylation of CC2D2B, GHDC, LINC01377, PLPP2, and PTK2 and decreased methylation of TSPY4 as compared to normal prostate controls. qRT-PCR experiments confirmed decreased expression of PTK2 mRNA, consistent with hypermethylation of PTK2. In contrast, GT3 did not affect methylation of any identifiable recognizable genes. This study suggests metformin, but not GT3, may inhibit prostate cancer proliferation partially through epigenetic changes which disrupt downstream focal adhesion kinase (FAK) signaling pathways. Metformin is a promising chemopreventive agent; studies investigating the impact of metformin on cellular adhesion and FAK pathways are ongoing in our laboratory. Citation Format: Christine Ann Moore, Janet Lightner, Michelle Duffourc, Koyamangalath Krishnan. Metformin, not γ-tocotrienol, increases gene methylation and decreases expression of genes responsible for cell signal transduction in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1274.