Epigenetic Regulation of Sialyltransferase ST6GAL1 Drives Breast Tumor Heterogeneity

Background/Objectives: Tumor progression is often accompanied by sialylation of cell-surface N-glycan glycoproteins with α2-6-linked sialic acids by the sialyltransferase ST6GAL1. Moreover, a hallmark of human cancers is heterogeneity in ST6GAL1 expression among cancer cells, yet the mechanisms and significance of this variability remain poorly understood. Here, we investigate whether epigenetic changes in ST6GAL1 DNA methylation, which affect intracellular ST6GAL1 and cell-surface α2,6-sialylation of glycoproteins, are associated with the biology of human breast cancer (BCa) cells. Methods: We analyzed CpG island methylation across spatially distinct regions of the human ST6GAL1 promoter (P2 and P3) and the associated loss of ST6GAL1 expression in primary human breast tumors compared with adjacent normal tissue controls. Epigenetic changes in ST6GAL1 DNA methylation and patient progression-free survival (PFS) were evaluated. Small-molecule epigenetic inhibitors (5-aza-dC and MS-275) were used to assess ST6GAL1 transcriptional reactivation and its effects on intracellular ST6GAL1, cell-surface α2,6-sialylation, and cell adhesion to collagen I in cultured BCa cell models. Results: In estrogen receptor (ER)+ and human epidermal growth factor receptor 2 (HER2)+ primary BCa tissues, ST6GAL1 P3-promoter hypermethylation, but not P2-promoter methylation, significantly correlated with loss of ST6GAL1 expression compared with adjacent normal controls. Higher promoter methylation was significantly associated with improved PFS in ER+ and HER2+ BCa subtypes compared with triple-negative breast cancer (TNBC). ST6GAL1 protein expression and relative transcript levels are higher in adjacent normal controls or human normal breast epithelial cells than in human BCa tissue or cultured cells. In BCa cells, native ST6GAL1 is sensitive to degradation by cycloheximide or MG-132 and even to sialidase treatment. Pharmacological reactivation with 5-aza-dC or MS-275 restored intracellular ST6GAL1 expression and functional cell-surface α2,6-sialylation, thereby enhancing BCa cell adhesion to collagen I. Since ST6GAL1 is also released into the extracellular milieu by cancer cells, and extracellular ST6GAL1 (exoST6) may contribute to cancer cell-surface protein α2,6-sialylation, we also found that 5-aza-dC increased exoST6 levels released in the form of exosome vesicles. Conclusions: Our results suggest that ST6GAL1 promoter methylation, together with copy-number alterations, drives loss of expression and phenotypic heterogeneity in human BCa, thereby reducing α2,6-sialylation and correlating with improved PFS in the ER+ and HER2+ subtypes. Demethylating agents restore both endogenous ST6GAL1 expression and exosomal exoST6 release in BCa cells, highlighting this targetable epigenetic axis as a promising prognostic biomarker and therapeutic strategy to improve patient survival in progressive diseases. Future studies will focus on evaluating the functional outcomes of targeting cell-native ST6GAL1 versus the exoST6/sialylation pathways and on defining ST6GAL1's role in driving disease progression in vivo.