Abstract 3513: Ketoconazole-induced O-GlcNAcylated c-Myc expression and inhibition of cell proliferation in cancers
Authors: So Hee Kim, Eun-Sin Du, Aby Joiakim, Sung-Su Park, David Kaplan, David Putt, Hyesook Kim
Journal: Cancer Research (2014)

Abstract Protein N-acetylglucosamine modification (O-GlcNAcylation) plays a critical role in cell-cycle regulation, apoptosis and signal transduction. Thr-58 of c-Myc, a mutational hot spot in lymphomas, is a site for both phosphorylation (primed by Ser-62 phosphorylation) and O-GlcNAcylation. Whereas Thr-58 O-GlcNAcylation induces ubiquitin-dependent c-Myc degradation, Thr-58/Ser-62 phosphorylation increases c-Myc stability and thus induces invasiveness and tumorigenesis of MCF-7 human breast cancer cells. Using form-specific antibodies for Thr-58-O-GlcNAcylated c-Myc, Western blot analysis was carried out and found that Thr-58-O-GlcNAcylated c-Myc proteins were detected primarily in the nuclear fraction as 68 kDa proteins. MCF-7 cells were treated for 4 hr with 1% DMSO or 2 mM ketoconazole [β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, dissolved in DMSO]. Western blot analysis of nuclear lysates of MCF-7 cells revealed that the levels of Thr-58-O-GlcNAcylated c-Myc dramatically increased after ketoconazole treatment. Thr-58-O-GlcNAcylated c-Myc levels also increased in the nuclear lysates of Mia Paca-2 and SW480 cells treated with 50 µg/ml for 48 hours. The ketoconazole treatment increased cell death in MCF-7 cells 84% higher than in MCF10A non-cancerous cells by lactate dehydrogenase (LDH) cytotoxicity assays. Treatment with 50 µg/ml ketoconazole for 72 hr inhibited cell proliferation of colon cancer cells, SW480 and HT29, by 63.3% and 67.4%, respectively, lung cancer cells, H1437 and A549, by 44.9% and 51.3%, respectively, and a pancreatic cancer cell, Mia Paca-2, by 60.2% in a dose-dependent manner. These results demonstrated that treatment of ketoconazole inhibited the proliferation of MCF-7 breast cancer cells as well as colon, lung and pancreatic cancer cells. Wound healing assays were carried out to verify the inhibition of cell migration. Whereas 55.3% of Mia Paca-2 cells migrated toward a scratched field after 24-hr treatment, only 11.6% of the cells migrated after treatment with 100 µg/ml ketoconazole. PANC-1 cells, which failed to increase Thr-58-O-GlcNAcylated c-Myc expression and cell proliferation after ketoconazole treatment, showed no significant difference of cell migration after 24 hr with 100 µg/ml ketoconazole treatment. Surprisingly, colon cancer cells, SW480 and HT29, showed an inhibition of invasiveness as evidenced by the inhibition of cell proliferation and cell migration after treatment with 100 µg/ml ketoconazole. Collectively, our results demonstrated that treatment of ketoconazole, an O-GlcNAcase inhibitor, increased Thr-58-O-GlcNAcylated c-Myc level and inhibited cell proliferation in MCF-7, SW480, HT29, H1437, A549 and Mia Paca-2 cells, which may decrease invasiveness and migration of the breast, colon, lung and pancreatic cancer cells. Supported by NCI SBIR Phase II Contract No. HHSN261201300058C. Citation Format: So Hee Kim, Eun-Sin Du, Aby Joiakim, Sung-Su Park, David Kaplan, David Putt, Hyesook Kim. Ketoconazole-induced O-GlcNAcylated c-Myc expression and inhibition of cell proliferation in cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3513. doi:10.1158/1538-7445.AM2014-3513