Crucial roles of GALNT6-mediated O-glycosylation of GRP78/Bip in proliferation of breast cancer cells.

Increased expression of the polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), an O-glycosyltransferase, has been reported to play a crucial role in mammary carcinogenesis. Here, we demonstrate that GALNT6 O-glycosylates Glucose-regulated protein 78/Binding immunoglobulin protein (GRP78/Bip), a key regulator of the unfolded protein response (UPR), by adding N-acetylgalactosamine (GalNAc), thereby modulating its stability in breast cancer cells. Functional inhibition of either GALNT6 or GRP78/Bip suppressed the proliferation of the luminal-type breast cancer cell line, ZR-75-1, understanding their importance in tumor cell growth. We further found that GRP78/Bip, which is primarily localized in the endoplasmic reticulum (ER), is transported to the Golgi apparatus under the ER stress conditions, where it undergoes O-glycosylation at Thr203 by the Golgi-resident GALNT6. Substitution of Thr203 with alanine inhibited the binding of GRP78/Bip to IRE1, an ER stress sensor, suggesting that the O-glycosylation at Thr203 in GRP78/Bip facilitates sustained activation of the UPR. These findings define the GALNT6-GRP78/Bip axis as a novel mechanism driving persistent UPR activation and tumor cell adaptation to ER stress, offering a potential new therapeutic target for luminal-type breast cancers.