Critical Role of Estrogen Receptor Alpha O-Glycosylation by N-Acetylgalactosaminyltransferase 6 (GALNT6) in Its Nuclear Localization in Breast Cancer Cells

Abstract

Alteration of protein O-glycosylation in various human cancers including breast cancer is well known, but molecular roles of their aberrant glycosylations on cancer have not been fully understood. We previously reported critical roles of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6 or GalNAc-T6) that was upregulated in a great majority of breast cancer tissues. Here we further report O-glycosylation of estrogen receptor alpha (ER-α) by GALNT6 and the significant role of its nuclear localization in breast cancer cells. Knockdown of GALNT6 expression in two breast cancer cell lines, T47D and MCF7, in which both ER-α and GALNT6 were highly expressed, by small interfering RNA could significantly attenuate expression of ER-α. Immunocytochemical analysis clearly demonstrated the drastic decrease of ER-α protein in the nucleus of these cancer cells. Accordingly, the downstream genes of the ER-α pathway such as MYC, CCND1, and CTSD were significantly downregulated. We confirmed GALNT6-dependent ER-α O-glycosylation and identified O-glycosylation of S573 in an F domain of ER-α by GALNT6 through LC-MS/MS analysis. We also obtained evidences showing that the glycosylation of ER-α at S573 by GALNT6 is essential for protein stability and nuclear localization of ER-α in breast cancer cells. Furthermore, we designed cell membrane–permeable peptides including the O-glycosylation site and found a significant decrease of the cell viability of breast cancer cells by treatment of these peptides in a GALNT6 expression–dependent manner. Our study suggests that targeting the GALNT6 enzymatic activity as well as the GALNT6/ER-α interaction could be a promising therapeutic approach to ER-α–positive breast cancer patients.

<p>Cosmc Disruption-Mediated Aberrant O-glycosylation Suppresses Breast Cancer Cell Growth via Impairment of CD44</p>

“…31,32 Aberrant O-glycosylation (characterized by the expression of Tn antigen) has been frequently observed in many human cancers, including breast cancer. 5,33,34 There are a few proposed mechanisms that may influence the O-glycosylation process and lead to Tn antigen expression. 35 Of these, dysfunction of T-synthase and/or its specific molecular chaperone-Cosmc constitute the prevailing causes.…”

Nucleocytoplasmic O-glycosylation in protists

“…Extensive monosaccharide modifications of nucleocytoplasmic proteins in regions rich in Ser, Thr and Pro are an emerging theme across all kingdoms of life. A third monosaccharide, O-αMan, was recently reported on numerous nucleocytoplasmic proteins of yeast, though a responsible nucleocytoplasmic glycosyltransferase has not been identified [13], and hints for nuclear O-α-GalNAc in mammalian cells have recently appeared [14,15]. Taken together, these findings suggest the importance of monosaccharide modifications of nucleocytoplasmic proteins throughout eukaryotic evolution.…”

Endocrine Resistance in Breast Cancer: The Role of Estrogen Receptor Stability

“…The ER is regulated by a range of PTMs including ubiquitylation, SUMOylation, phosphorylation, palmitoylation, acetylation, methylation and glycosylation [41][42][43][44]. These modifications are proposed to regulate the activity, stability, and interactions of ER with other proteins or DNA, and ESR1 mutations may influence PTMs and hence ER stability and function ( Figure 3).…”

“…Glycosylation of S573, facilitated by a polypeptide known as N-acetylgalactosaminyltransferase 6 (GALNT6), promotes nuclear localisation, transcriptional activity and stability of the ER (Table 1). In T47D and MCF-7 cells, siRNA knockdown of GLANT6, results in reduced ER nuclear localisation and expression of ER target genes (MYC, CCND1 and CTSD) and this was generally irrespective of estrogen presence [41]. Hence, S573 glycosylation by GLANT6 is an important regulator of ER localisation and transcriptional activity [41].…”