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

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.

O-GlcNAc regulates gene expression by controlling detained intron splicing

“…In the case of OGT , low O-GlcNAc results in more functional mRNA by promoting the skipping of the poison exon, while in the case of OGA , low O-GlcNAc enhances splicing of the frame-shifted NMD substrate (Figure 3E). The rapid, reciprocal, O-GlcNAc-controlled changes we observed in OGT and OGA splicing pathways provide an explanation for a well-known observation, namely, that OGT and OGA levels change in opposite directions when either protein is blocked (18,19,50,66). These O-GlcNAc-regulated splicing changes provide an elegant mechanism for how a single signal can be read out to produce opposing gene expression outcomes through the structural configuration of exons and introns, enabling OGT and OGA levels to be precisely tuned to buffer nutrient fluctuations.…”

“…OSMI-2 is a cell-permeable OGT inhibitor with a submicromolar K d and a well-defined binding mode characterized by X-ray crystallography (Figure 1A) (19). This compound was shown previously to reduce global O-GlcNAc levels by 4 h of treatment in a number of cell lines, including HEK-293T cells (19,50). Because shorter treatment times were not examined, we first asked whether OSMI-2 reduced O-GlcNAc at earlier time points.…”

“…We asked whether the global decrease in DIs could be due to decreases in total transcript levels for the affected genes because several findings have suggested that O-GlcNAc levels can affect transcription in a variety of ways. Numerous transcription factors as well as the C-terminus of RNA Pol II are known to be O-GlcNAc modified (50, 67), and OGT has been observed to be enriched at promoters (68). We analyzed changes at the total gene transcript level using RNA-seq data and found that 635 genes showed at least a 2-fold decrease in transcription, while 350 genes showed at least a 2-fold increase, with >0.95 posterior probability of differential expression being considered significant (Supplementary Figure S8).…”

“…There are two important findings from this study (Figure 5). First, we found that OGT and OGA levels, which are known to change reciprocally when either enzyme is blocked (50,66), are regulated by detained intron splicing in an O-GlcNAc-dependent manner. These rapid splicing changes serve to maintain O-GlcNAc homeostasis by tuning levels of the O-GlcNAc cycling enzymes as cellular conditions change.…”

CRISPR-Cas9-mediated depletion ofO-GlcNAc hydrolase and transferase for functional dissection ofO-GlcNAcylation in human cells

“…For instance, it has been shown that OGT inhibition leads to a markedly decreased expression of OGA [9][10][11][12][13][14][15] . Since OGA expression is also modulated by the total levels of O-GlcNAc modification, treatment of cells with an OGA inhibitor increases O-GlcNAc levels, concomitantly elevating the expression of OGA and decreasing OGT expression as a compensatory mechanism 15 .…”