Abstract A26: Effects of O-GlcNAcase inhibitors on O-GlcNAcylated c-myc expression in MCF-7 cells
Authors: Kim Hyesook , Kim So Hee , Joiakim Aby , Kaplan David , Putt David 
Published: October 2015

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, which are conserved among human rat and mouse. Whereas Thr-58O-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.
c-Myc antibodies specific for (a) Thr-58-O-GlcNAcylation, (b) Thr-58-unmodification and (c) Thr-58/Ser-62 phosphorylation were produced and specificities of the antibodies have been characterized by Western blot analyses using BSA conjugated with synthetic peptides containing the O-GlcNAcylated, unmodified and phosphorylated sites of c-myc. Western blot analysis of MCF-7 cells revealed that, whereas ~68 kDa Thr-58-O-GlcNAcylated c-myc proteins were primarily detected in the nuclear fraction, the 65-68 kDa Thr-58/Ser-62 phosphorylated and 65 kDa and 40 kDa Thr-58 unmodified c-myc proteins were expressed in both nuclear and cytosolic fractions.
When MCF-7 cells were treated with 1% DMSO, 2 mM ketoconazole, a medicine-like human and bacterial β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, or 2 mM streptozotocin (STZ), an irreversible inhibitor of O-GlcNAcase, dissolved in DMSO (final concentration, 1%) for 4 hr, Thr-58-O-GlcNAcylated c-myc protein levels in nuclear and total cell lysates increased after 2 mM ketoconazole treatment but not with STZ treatment. Thr-58/Ser-62 phosphorylated protein levels did not change after either ketoconazole or STZ treatments. Treatment of the cells with 1% DMSO, 2 mM ketoconazole, buspirone or acetazolamide dissolved in DMSO (final concentration, 1%) or N6-methyladenosine 5'-monophosphate (dissolved in media), a medicine-like O-GlcNAcase inhibitor, for 4 hr revealed that Thr-58-O-GlcNAcylated c-myc protein levels in nuclear lysates increased only when the cells were treated with 2 mM ketoconazole. None of the O-GlcNAcase inhibitors including ketoconazole treatments changed levels of c-myc proteins unmodified at the Thr-58 site or phosphorylated at the Thr-58/Ser-62 sites. Whereas treatment of the MCF-7 cells with 2 mM STZ and N6-methyladenosine 5'-monophosphate failed to induce cell death and treatment with 2 mM buspirone and acetazolamide induced minimal cell death, 2 mM ketoconazole treatment, which dramatically increased Thr-58-O-GlcNAcylated c-myc protein levels, induced severe cell death.
Subsequent lactate dehydrogenase (LDH) cytotoxicity assays demonstrated that the ketoconazole treatment increased cell death in MCF-7 cells 84% higher than in MCF10A non-cancerous cells by lactate dehydrogenase (LDH) cytotoxicity assays. Dose-dependent cell proliferation assays were carried out by treatment of the cells with and without 10, 20, 50 or 100 µM ketoconazole dissolved in DMSO for 72 hr, staining the cells with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). The ketoconazole treatment inhibited cell proliferation of the MCF-7 cells by 60% and 90% at 50 and 100 µM, respectively (p &lt; 0.05).
These results suggest that, in MCF-7 cells, ketoconazole, a medicine-like O-GlcNAcase inhibitor, dramatically increased Thr-58-O-GlcNAcylated c-myc proteins, which coincided with increased cell death and inhibited cell proliferation. Supported by NCI SBIR Phases I and II Contracts N261201100073C and N261201300058C.
Citation Format: Hyesook Kim, So Hee Kim, Aby Joiakim, David Kaplan, David Putt. Effects of O-GlcNAcase inhibitors on O-GlcNAcylated c-myc expression in MCF-7 cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A26.