Identification of novel therapeutic targets associated with aberrant glycosylation in stem cells of triple-negative breast cancer

Abstract

[eng] Despite the generally high cure rates for breast cancer, a subset of patients still experiences metastasis and relapse, with triple-negative breast tumors (TNBC) being the most aggressive and associated with a poorer prognosis. A small population of cells within tumors, known as cancer stem- like cells (CSCs), are poorly differentiated and highly plastic, driving processes like migration, metastasis, and recurrence. Targeting these cells remains challenging due to their similarity to normal stem cells. However, critical distinctions between normal breast stem cells and breast cancer stem cells (BCSCs) have been identified. One notable difference is the presence of posttranslational modifications in BCSCs, particularly aberrant glycosylation, which often occurs on stemness markers.

Using our novel GlycoCRISPR library, which targets protein glycosylation genes, we performed the first comprehensive interrogation of the genes essential for maintaining stemness in TNBC cell lines. MDA-MB-231 cells (malignant) and MCF10A cells (non-transformed) were used for these screenings. Bioinformatic analysis revealed that ten glycosylation-related genes are specifically essential for maintaining the stem phenotype in cancer cells. Based on public patient data and extensive literature review, three key genes—EXT1, ST3GAL1, and DHDDS—were selected for further study. Clinical analysis using the METABRIC database showed that overexpression of these genes is significantly associated with worse prognosis, including lower overall survival and relapse-free survival rates.

In vitro experiments demonstrated that EXT1, ST3GAL1, and DHDDS are overexpressed in TNBC cells cultured in suspension, a condition used to enrich the stem-like cell population. Knockdown studies of EXT1 and DHDDS in triple negative breast cancer cell lines validated their role in stemness. A tumorsphere assay confirmed a significant reduction in sphere formation capacity following gene silencing in three different TNBC cell lines. Although no significant differences were observed in the CD44+/CD24-/low population or stem-related gene expression between knockdown and control cells, the ALDH+ population was notably reduced when EXT1 and DHDDS were silenced, supporting their role in maintaining stemness.

In mouse tumor formation experiments, tumors with EXT1 and DHDDS knockdown showed significantly reduced growth compared to controls, with smaller tumor masses observed upon extraction. This underscores the role of these genes in sustaining the stem-like phenotype in TNBC.

Finally, by enzymatically degrading heparan sulfate in 231 cells using heparinase III and inhibiting N- glycosylation with tunicamycin to mimic the effects of EXT1 and DHDDS downregulation, respectively, we demonstrate that abnormal glycosylation can influence and sustain stem cell potential.