A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells

Abstract

Epithelial ovarian cancer (EOC) is a disease responsible for more deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade serous EOC tumors, compared to low malignant potential EOC tumors and normal ovarian tissues. This data also suggested for a role of GALNT3 in aberrant EOC glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology. The GALNT3-dependent glycoproteins identified by this metabolic labeling approach support the oncogenic role of GALNT3 in EOC dissemination and may be pursued as novel EOC biomarkers and/or therapeutic targets. Biological significance: Knowledge of the O-glycoproteome has been relatively elusive, and the functions of the individual polypeptide GalNAc-Ts have been poorly characterized. Alterations in GalNAc-Ts expression were shown to provide huge variability in the O-glycoproteome in various pathologies, including cancer. The application of a chemical biology approach for the metabolic labeling and subsequent characterization of O-glycoproteins in EOC using the Ac4GalNAz metabolite has provided a strategy allowing for proteomic discovery of GalNAc-Ts specific functions. Our study supports an essential role of one of the GalNAc-Ts — GALNT3, in EOC dissemination, including its implication in modulating PTMs and EOC metabolism. Our approach validates the use of the applied metabolic strategy to identify important functions of GalNAc-Ts in normal and pathological conditions.

Altered expression of different GalNAc-transferases is associated with disease progression and poor prognosis in women with high-grade serous ovarian cancer

“…We have previously identified the GalNAc-T3 (GALNT3) gene as a potential EOC oncogene, highly expressed in advanced disease, as GALNT3 expression was significantly associated with poor outcome (15). We also demonstrated that in EOC cells, GALNT3 might directly alter biosynthesis and/or aberrant glycosylation of different O-glycoproteins that could play an essential role in EOC dissemination (15)(16)(17). However, to date, no other studies have comprehensively assessed the implications of other members of the GalNAc-Ts gene family in EOC dissemination.…”

LY75 Ablation Mediates Mesenchymal-Epithelial Transition (MET) in Epithelial Ovarian Cancer (EOC) Cells Associated with DNA Methylation Alterations and Suppression of the Wnt/β-Catenin Pathway

“…Despite treatment improvements, long-term survival rates for patients with advanced disease remain disappointing [3]. EOC lethality primarily stems from the inability to detect the disease at an early, organ-confined stage, and the lack of effective therapies for advanced-stage disease (e.g., metastasis) [4]. Indeed, despite advances in cytotoxic therapies [5,6], only 30% of patients with advanced-stage EOC survive 5 years after initial diagnosis [4].…”

“…One way to resolve this problem is to target metastasis-specific pathways with novel therapies. Hence, focused identification of novel prometastatic EOC pathways and molecules could improve the chances of discovering new and more effective EOC therapies [4].…”

Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells

“… This article contains raw and processed data related to research published in “ Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation ” [1] . The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s.…”