Center for Biomedical Mass Spectrometry, Department of Biochemistry, Boston University School of Medicine, 670 Albany Street, rm 511, Boston, MA 02118, USA.
Mol Omics. 2020 Jun 1;16(3):195-209. doi: 10.1039/d0mo00009d. Epub 2020 Mar 23.
Epidermal growth factor receptor (EGFR) is a major driver of head and neck cancer, a devastating malignancy with a major sub-site in the oral cavity manifesting as oral squamous cell carcinoma (OSCC). EGFR is a glycoprotein receptor tyrosine kinase (RTK) whose activity is upregulated in >80% OSCC. Current anti-EGFR therapy relies on the use of cetuximab, a monoclonal antibody against EGFR, although it has had only a limited response in patients. Here, we uncover a novel mechanism regulating EGFR activity, identifying a role of the nuclear branch of the Wnt/β-catenin signaling pathway, the β-catenin/CBP axis, in control of post-translational modification of N-glycans on the EGFR. Genomic and structural analyses reveal that β-catenin/CBP signaling represses fucosylation on the antennae of N-linked glycans on EGFR. By employing nUPLC-MS/MS, we determined that malignant human OSCC cells harbor EGFR with a paucity of N-glycan antennary fucosylation, while indolent cells display higher levels of fucosylation at sites N420 and N579. Additionally, treatment with either ICG-001 or E7386, which are both small molecule inhibitors of β-catenin/CBP signaling, leads to increased transcriptional expression of fucosyltransferases FUT2 and FUT3, with a concomitant increase in EGFR N-glycan antennary fucosylation. In order to discover which fucosylated glycan epitopes are involved in the observed effect, we performed in-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry analysis of the EGFR tryptic glycopeptides. Data are available via ProteomeXchange with identifier PXD017060. We propose that β-catenin/CBP signaling promotes EGFR oncogenic activity in OSCC by inhibiting its N-glycan antennary fucosylation through transcriptional repression of FUT2 and FUT3.
表皮生长因子受体（EGFR）是头颈部癌症的主要驱动因素，这是一种恶性肿瘤，口腔是其主要的亚部位，表现为口腔鳞状细胞癌（OSCC）。EGFR 是一种糖蛋白受体酪氨酸激酶（RTK），其活性在超过 80%的 OSCC 中上调。目前的抗 EGFR 治疗依赖于使用西妥昔单抗，这是一种针对 EGFR 的单克隆抗体，但在患者中仅有有限的反应。在这里，我们发现了一种调节 EGFR 活性的新机制，确定了 Wnt/β-连环蛋白信号通路的核分支，即β-连环蛋白/CBP 轴，在控制 EGFR 上 N-糖链的翻译后修饰中的作用。基因组和结构分析表明，β-连环蛋白/CBP 信号抑制 EGFR 上 N-连接糖链触角的岩藻糖基化。通过采用 nUPLC-MS/MS，我们确定恶性人 OSCC 细胞中 EGFR 的 N-糖链触角岩藻糖基化程度较低，而惰性细胞中 N420 和 N579 位点的岩藻糖基化水平较高。此外，用 ICG-001 或 E7386 处理，这两种都是β-连环蛋白/CBP 信号的小分子抑制剂，可导致 FUT2 和 FUT3 的转录表达增加，同时 EGFR N-糖链触角岩藻糖基化增加。为了发现参与观察到的效应的哪些岩藻糖化糖表位，我们通过对 EGFR 胰蛋白酶糖肽的串联质谱分析对多岩藻糖化 N-聚糖进行了深入表征。数据可通过 ProteomeXchange 以标识符 PXD017060 获得。我们提出，β-连环蛋白/CBP 信号通过转录抑制 FUT2 和 FUT3 来抑制 EGFR 的 N-糖链触角岩藻糖基化，从而促进 OSCC 中的 EGFR 致癌活性。
