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Theranostics 2019; 9(8):2183-2197. doi:10.7150/thno.30834 This issue Cite

Research Paper

High OGT activity is essential for MYC-driven proliferation of prostate cancer cells

Harri M Itkonen^1,2,✉, Alfonso Urbanucci^1,3,4,5, Sara ES Martin^2,6, Aziz Khan¹, Anthony Mathelier^1,7, Bernd Thiede⁸, Suzanne Walker^2,✉, Ian G Mills^1,9,10✉

1. Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, Oslo, Norway.
2. Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA.
3. Department of Core Facilities, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.
4. Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.
5. Institute for Cancer Genetics and Informatics, Oslo University Hospital, Oslo, Norway.
6. Current address: Chemistry Department, The College of Wooster, US.
7. Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway.
8. Department of Biosciences, University of Oslo, Oslo, Norway.
9. PCUK/Movember Centre of Excellence for Prostate Cancer Research, Centre for Cancer Research and Cell Biology (CCRCB), Queen's University Belfast, UK.
10. Nuffield Department of Surgical Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK.

✉ Corresponding authors: Harri M Itkonen; h.m.itkonencom. Department of Microbiology, Harvard Medical School, 4 Blackfan Circle, Boston, MA 02115, Phone: (617) 432-5498. Ian G. Mills; ian.millsox.ac.uk. Suzanne Walker; suzanne_walkerharvard.edu. More

Abstract

O-GlcNAc transferase (OGT) is overexpressed in aggressive prostate cancer. OGT modifies intra-cellular proteins via single sugar conjugation (O-GlcNAcylation) to alter their activity. We recently discovered the first fast-acting OGT inhibitor OSMI-2. Here, we probe the stability and function of the chromatin O-GlcNAc and identify transcription factors that coordinate with OGT to promote proliferation of prostate cancer cells.

Methods: Chromatin immunoprecipitation (ChIP) coupled to sequencing (seq), formaldehyde-assisted isolation of regulatory elements, RNA-seq and reverse-phase protein arrays (RPPA) were used to study the importance of OGT for chromatin structure and transcription. Mass spectrometry, western blot, RT-qPCR, cell cycle analysis and viability assays were used to establish the role of OGT for MYC-related processes. Prostate cancer patient data profiled for both mRNA and protein levels were used to validate findings.

Results: We show for the first time that OGT inhibition leads to a rapid loss of O-GlcNAc chromatin mark. O-GlcNAc ChIP-seq regions overlap with super-enhancers (SE) and MYC binding sites. OGT inhibition leads to down-regulation of SE-dependent genes. We establish the first O-GlcNAc chromatin consensus motif, which we use as a bait for mass spectrometry. By combining the proteomic data from oligonucleotide enrichment with O-GlcNAc and MYC ChIP-mass spectrometry, we identify host cell factor 1 (HCF-1) as an interaction partner of MYC. Inhibition of OGT disrupts this interaction and compromises MYC's ability to confer androgen-independent proliferation to prostate cancer cells. We show that OGT is required for MYC-mediated stabilization of mitotic proteins, including Cyclin B1, and/or the increased translation of their coding transcripts. This implies that increased expression of mRNA is not always required to achieve increased protein expression and confer aggressive phenotype. Indeed, high expression of Cyclin B1 protein has strong predictive value in prostate cancer patients (p=0.000014) while mRNA does not.

Conclusions: OGT promotes SE-dependent gene expression. OGT activity is required for the interaction between MYC and HCF-1 and expression of MYC-regulated mitotic proteins. These features render OGT essential for the androgen-independent, MYC-driven proliferation of prostate cancer cells. Androgen-independency is the major mechanism of prostate cancer progression, and our study identifies OGT as an essential mediator in this process.

Keywords: O-GlcNAc transferase, MYC, prostate cancer, glycosylation, super-enhancer

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