KLF8 and OGT/O‐GlcNAcylation regulate breast cancer stem‐like cells

One of the main challenges in treating breast cancer is the intra-tumor heterogeneity, which, partly, is maintained and promoted by a sub-population of breast cancer cells called breast cancer stem-like cells (BCSCs). BCSCs shares traits of mammary stem cells, capable of self-renewing and differentiating, while promotes invasion, metastasis, drug resistance and relapse. BCSCs are highly adaptive, capable of reprogramming metabolism and signaling activity in response to tumor microenvironment. One key component of cell nutrient sensing mechanism, linking alteration in metabolism to cell signaling, is O-GlcNAc transferase (OGT). OGT is responsible to adding O-GlcNAc moieties to target nuclear, cytoplasmic and mitochondrial proteins from UDP-GlcNAc - final product of the hexosamine biosynthetic pathway. This modification is termed O-GlcNAcylation and both OGT and O-GlcNAcylation are upregulated in many cancers. Recently, we showed that OGT/O-GlcNAcylation is enriched in BCSCs, promoting the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. RNA-seq analysis of BCSC-enriched mammospheres with OGT overexpression revealed Kruppel-like-factor 8 (KLF8) as a downstream target of OGT in promoting BCSCs. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE) and NANOG expression and ALDH activity in breast cancer cells, while overexpressing KLF8 increased MFE, NANOG+ and ALDH+BCSC population. Knockdown of KLF8 also reduced breast tumor growth in vivo. Importantly, KLF8 knockdown abolished the effect of elevated OGT and O-GlcNAcylation in breast cancer cell MFE and BCSC population, while KLF8 overexpression rescued OGT inhibition in MFE and BCSC population. suggesting that KLF8 is required in promoting BCSCs. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O-GlcNAcylation, suggesting a possible feed-forward loop between OGT and KLF8 in promoting BCSCs. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple-negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem-like cells population.