Glycosylation of estrogen receptor alpha by N-acetylgalactosaminyltransferase 6 in breast cancer

Abstract Alteration of protein O-glycosylation in various human cancers including breast cancer is well known, but molecular mechanisms of such aberrant modifications and their effects on cancer development have not been fully understood. We previously reported the critical roles of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and is responsible for initiating mucin-type O-glycosylation. Suppression of GALNT6 expression by small interfering RNA to GALNT6 significantly enhanced cell-cell adhesion, induced mesenchymal-epithelial transition, and suppressed the growth of breast cancer cells. Here we further analyzed molecular functions of GALNT6 and found that GALNT6 could glycosylate an estrogen receptor alpha (ER-α) protein, a molecule playing a central role in proliferation of hormone-dependent breast cancer cells. We have used two breast cancer cell lines, T47D and MCF7, in which both the estrogen receptor and GALNT6 were highly expressed. Knockdown of GALNT6 expression by siRNA could significantly attenuate expression of ER-α at transcriptional and protein levels in these breast cancer cell lines in a condition with or without estradiol (P<0.05). In addition, immunocytochemical analysis clearly showed the dramatic decrease of the ER-α protein in the nucleus of cancer cells. Accordingly, the downstream genes of the ER-α pathway, such as MYC, CCND1, and CTSD were significantly downregulated after the GALNT6 knockdown (P=0.002, P=0.018, and P=0.026, respectively). Moreover, to evaluate the GALNT6 function in the breast cancer cell lines, we used a small molecular compound targeting the GALNT6 activity. Interestingly, as similar to siRNA, the ER-α protein expression level was decreased in a dose-dependent manner with this GALNT6 inhibitor. To further investigate the GALNT6-ER-α pathway, we transfected Hela cell lines with mock vector, wild-type GALNT6 or enzyme-dead GALNT6 (mock, GALNT6-WT, GALNT6-H271D, respectively) as well as ER-α-expressing plasmid, and examined the O-glycosylation of ER-α by an IP pull-down assay in combination of a Vicia villosa agglutinin (VVA)-lectin assay. The ER-α glycosylation band was detectable in GALNT6-WT cells, but not in mock cells or GALNT6-H271D cells, indicating GALNT6-dependent ER-α glycosylation. Subsequent LC-MS analysis confirmed that S573 in an F domain of ER-α was glycosylated by GALNT6. Our results clearly indicate that the glycosylation of ER-alpha at S573 by GALNT6 is essential for protein stability and nuclear localization of ER-alpha and that GALNT6 may play important oncogenic roles through glycosylation ER-alphain breast cancer cells. We suggest that targeting GALNT6 may be a promising therapeutic approach to ER/GALNT6-positive breast cancer patients. Citation Format: Boya Deng, Yunus Emre Tarhan, Koji Ueda, Yo Matsuo, Jae-Hyun Park, Yusuke Nakamura. Glycosylation of estrogen receptor alpha by N-acetylgalactosaminyltransferase 6 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 977.