S1. OGT and O-GlcNAc not regulated by loss of cell adhesion in cancer cells. S2. OGT inhibition attenuates mammosphere formation and CD44HCD24L TIC population. S3. OGT inhibition attenuates mammosphere formation and CD44HCD24L TIC population. S4. Elevated OGT and O-GlcNAc ameliorate mammosphere formation and CD44HCD24L TIC population. S5. OGT/O-GlcNAc modulation affects NANOG-GFP+ and ALDH+ TIC population. S6. OGT/O-GlcNAc modulation affects NANOG-GFP+ TIC population. S7. OGT regulates EMT and stem cell markers. S8. OGT regulates EMT and stem cell markers. S9. KLF8 is a critical regulator of mammosphere formation and required for OGT-mediated mammosphere growth.
ARTICLE ABSTRACT
Breast tumors are heterogeneous and composed of different subpopulation of cells, each with dynamic roles that can change with stage, site, and microenvironment. Cellular heterogeneity is, in part, due to cancer stem–like cells (CSC) that share properties with stem cells and are associated with treatment resistance. CSCs rewire metabolism to meet energy demands of increased growth and biosynthesis. O-GlcNAc transferase enzyme (OGT) uses UDP-GlcNAc as a substrate for adding O-GlcNAc moieties to nuclear and cytoplasmic proteins. OGT/O-GlcNAc levels are elevated in multiple cancers and reducing OGT in cancer cells blocks tumor growth. Here, we report that breast CSCs enriched in mammosphere cultures contain elevated OGT/O-GlcNAcylation. Inhibition of OGT genetically or pharmacologically reduced mammosphere forming efficiency, the CD44H/CD24L, NANOG+, and ALDH+ CSC population in breast cancer cells. Conversely, breast cancer cells overexpressing OGT increased mammosphere formation, CSC populations in vitro, and also increased tumor initiation and CSC frequency in vivo. Furthermore, OGT regulates expression of a number of epithelial-to-mesenchymal transition and CSC markers including CD44, NANOG, and c-Myc. In addition, we identify Krüppel-like factor 8 (KLF8) as a novel regulator of breast cancer mammosphere formation and a critical target of OGT in regulating CSCs.
These findings demonstrate that OGT plays a key role in the regulation of breast CSCs in vitro and tumor initiation in vivo, in part, via regulation of KLF8, and thus inhibition of OGT may serve as a therapeutic strategy to regulate tumor-initiating activity.
