Abstract
Introduction
Approximately 70-80% of diagnosed breast cancer (BC) cases present with the estrogen receptor positive (ER+) subtype. Although targeted endocrine therapies such as tamoxifen and fulvestrant are effective, a significant portion of patients experience disease recurrence due to de novo or acquired endocrine resistance. Alternations in biomolecular glycosylation (proteins and lipids) are well-known drivers of malignant progression, epithelial to mesenchymal transition (EMT), stemness, and metastasis. Recently cell-surface glycoRNAs were identified as a new class of glycosylated biomolecules with emerging roles in cell biology, including neutrophil recruitment and malignant transformation. However, the expression profile and biological significance of glycoRNAs in BC endocrine resistance remains unexplored. Here, we map the molecular landscape of glycoRNAs remodeled during the transition to endocrine resistance in ER+ BC.
Methods
To model progressive endocrine resistance, a panel of breast cell lines including benign (MCF10A), endocrine-sensitive (MCF7), tamoxifen-resistant (MCF7-TAM1), and multi-endocrine resistant (MCF7-LCC9) were used. Cell-surface or total sialoglycoRNAs are labeled either with metabolic copper-free chemistry, or via direct periodate oxidation followed by aldehyde ligation (rPAL). Purified RNAs were isolated by TriZol or RNAzol combined with silica columns and rigorous DNA/protein digestion. The purity was validated by NanoDrop spectrophotometry and fluorometric assays. GlycoRNA size distributions, glycan structures, and enzymatic requirements were characterized using size fractionation (>200 vs 200 nts), targeted glycosidase/nuclease digestions, and Northern Blot analysis with SYBR Gold and streptavidin-conjugated fluorophores.
Results
RNA with high purity (A260/280 ≥ 2.0; RIN > 9.0) was routinely isolated from all breast cell lines. SialoglycoRNAs were readily detected in benign MCF10A and endocrine-sensitive MCF7 cells but were markedly reduced or undetectable in endocrine-resistant variants (MCF7-TAM1 and MCF7-LCC9), revealing an inverse correlation between cell-surface glycoRNA abundance and endocrine resistance/malignancy. Notably, while tamoxifen-resistant MCF7-TAM1 cells retained intracellular (total) glycoRNA signals, their cell-surface display was substantially lost. Signal sensitive to RNase but not Proteinase K confirms glycoRNA identity. Size fractionation and enzymatic digestion demonstrated that glycoRNA signal is predominantly carried by small RNAs (200 nt) bearing N-linked glycans, with distinct shifts in both N-glycan and O-glycan alterations are seen across our resistance model.
Conclusion
Cell-surface glycoRNAs are displayed on non-malignant and endocrine-sensitive breast cells, and their surface expression is inversely associated with malignancy and endocrine resistance. The loss of cell-surface glycoRNA display in resistant models points to a dramatic remodeling of the surface glyco-landscape. Future studies will identify specific glycoRNA transcripts lost during disease progression and elucidate their fundamental roles in maintaining breast tissue homeostasis and endocrine sensitivity.