Functional effects of EpCAM N-glycosylation in MDA-MB-468 breast cancer cells.

EpCAM, the epithelial cell adhesion molecule, is a cancer cell marker whose expression is associated with worse prognosis in several epithelial cancers, including breast cancer. Despite being an attractive therapeutic target, it is unclear whether EpCAM plays a causative role in cancer progression and metastasis, and the mechanisms of this possible role remain unknown. To investigate EpCAM, we generated stable human MDA-MB-468 breast cancer cell lines with EpCAM knockout, EpCAM overexpression, or mutant unglycosylated EpCAM expression, in which all three normally N-glycosylated asparagine residues had been mutated to glutamines to abrogate EpCAM N-glycosylation. Our data establish that the lack of N-glycosylation in mutant unglycosylated EpCAM decreased EpCAM protein stability. EpCAM colocalization with cell membrane markers decreased in unglycosylated EpCAM, and colocalization with endoplasmic reticulum (ER) markers increased. Despite these clear effects on EpCAM protein stability and its subcellular localization, we did not observe any effects of unglycosylated EpCAM on the expression of downstream EpCAM targets including EGFR, E-cadherin, β-catenin, cyclin E, cyclin A, and c-myc. Similarly, there was no effect of unglycosylated EpCAM on cell viability, migration, invasion, or homotypic adhesion, although we did observe slight increases in homotypic cell-cell adhesion in EpCAM overexpressing cells. These findings show that N-glycosylation has a significant impact on stability and subcellular localization of EpCAM, but not on several critical breast cancer cell properties important for cancer progression and metastasis in human triple-negative MDA-MB-468 breast cancer cells.