A major driver of oral squamous cell carcinoma (OSCC), the most prominent head and neck cancer, is the epidermal growth factor receptor (EGFR), whose activity is upregulated in &gt;90% of tumors. Post-translational changes, including N-linked glycosylation may strongly impact EGFR activity. In OSCC, EGFR signaling focuses on Wnt/ Β-catenin activity that plays pivotal roles in the pathobiology of this malignancy through the interaction of nuclear Β-catenin with the histone acetyltransferase CREB-binding protein (CBP). Modification of EGFR with N-glycans impacts its cell-surface localization, dimerization, and signaling. Fucosylation interferes with EGFR dimerization and activation. Upon treatment with the drug candidate ICG-001, OSCC-cell line derived mouse tumor xenografts exhibited reduced levels of EGFR that are consistent with genomic analyses. We hypothesized that ICG-001 affected EGFR N-glycosylation. We determined the effect of ICG-001 on the EGFR N-glycosylation status by immunoprecipitating the receptor from indolent CAL27 and metastatic FaDu and HSC-3 cells (following treatment +/- ICG-001). After subjecting the immunoprecipitate to proteolysis, we enriched the glycopeptide fraction via hydrophilic interaction liquid chromatography (HILIC), and analyzed glycopeptides by tandem MS, using both collision-induced and electron transfer dissociation. We also released and permethylated the glycans, and analyzed these by MALDI-MS and tandem MS. We then compared the results for the overall glycan pools and the site-specific glycosylation patterns +/- ICG-001. At specific N-glycosylation sites, EGFR from indolent CAL27 cells had highly fucosylated N-glycans, whereas the corresponding N-linked glycans on EGFR from metastatic HSC-3 cells displayed much lower levels of fucosylation. Our MS/MS data suggested that ICG-001 promoted modification with terminal fucose, potentially inhibiting EGFR signaling. Sialylation patterns were also changed by treatment with ICG-001. These results suggest that that inhibition of β-catenin/CBP signaling by treatment with ICG-001 may serve as a therapeutic approach to downregulate EGFR pro-tumorigenic activity in OSCC.

Drug candidate may slow metastasis in oral cancer by affecting N-linked glycosylation on the epidermal growth factor receptor (EGFR)

2019 Spring National Meeting

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