Abstract 2633: Deciphering the role of protein glycosylation in oral cancer: insights into tumor biology and treatment
Authors: Chandler Kevin B. , Stahl Vanessa L. , Alamoud Khalid , Nguyen Bach-Cuc , Kartha Vinay , Sadykov Khikmet , Monti Stefano , Kukuruzinska Maria A. , Costello Catherine E. 
Published: August 2019

Abstract
Head and neck cancer is a debilitating malignancy, with the majority of cases arising in the oral cavity as oral squamous cell carcinoma (OSCC). A major driver of OSCC is the epidermal growth factor receptor (EGFR), whose activity is aberrantly upregulated in &gt;80% of tumors. EGFR is highly modified with N-linked glycans; fucosylation of N-glycans interferes with EGFR dimerization and activation. Thus, post-transcriptional changes may govern EGFR activity. In OSCC, EGFR signaling converges on Wnt/β-catenin activity, known to play pivotal roles in the pathobiology of this malignancy through the interaction of nuclear β-catenin with the histone acetyltransferase CREB-binding protein (CBP). We have shown that a small molecule inhibitor of β-catenin-CBP interaction, ICG-001, interferes with OSCC proliferation and aggressive features in cellular, zebrafish and murine models. Also, OSCC-cell line derived mouse tumor xenografts exhibit reduced EGFR abundance, and genomic analyses show a positive correlation between ICG-001 and EGFR inhibition. Given that modification of EGFR with N-glycans impacts its cell-surface localization and signaling, we hypothesized that ICG-001 affects EGFR N-glycosylation. To determine the effect of inhibition of β-catenin/CBP activity on cellular N-glycosylation programs, N-glycans from CAL27 and HSC3 cells treated with ICG-001 or vehicle control were released, permethylated, and analyzed via MALDI-TOF MS. Next, EGFR glycopeptides from CAL27 and HSC3 cells treated with ICG-001 or vehicle control, were analyzed with an Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo Scientific) using EThcD. In CAL27 cells, we observed higher levels of high mannose (less processed) N-glycans and complex fucosylated N-glycans, whereas in HSC3 cells we observed complex, afucosylated N-glycans. After ICG-001 treatment, HSC3 cells displayed higher levels of fucosylated N-glycans, suggesting that ICG-001, via inhibition of β-catenin/CBP signaling, promotes a more indolent-like glycan profile. Similarly, EGFR from CAL27 cells had highly fucosylated N-glycans, while EGFR from HSC3 cells displayed N-glycans with a paucity of fucose. Treatment of HSC3 cells with ICG-001 led to higher fucosylation, potentially inhibiting EGFR signaling. Parallel analyses of gene expression signatures in response to ICG-001 treatment in HSC-3 cells showed increased transcriptional expression of fucosyltransferases, FUT2 and FUT3. Our studies suggest that the β-catenin/CBP axis promotes EGFR signaling through downregulation of FUT2 and FUT3 expression and activity. Thus, inhibition of β-catenin/CBP signaling with ICG-001 may serve as a therapeutic approach to downregulate EGFR pro-tumorigenic activity in OSCC. Supported by NIH grants P41 GM104603 (CEC), F32 CA196157 (KBC), and by the Evans Center for Interdisciplinary Biomedical Research ARC #9950000118 (MAK).
Citation Format: Kevin B. Chandler, Vanessa L. Stahl, Khalid Alamoud, Bach-Cuc Nguyen, Vinay Kartha, Khikmet Sadykov, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Deciphering the role of protein glycosylation in oral cancer: insights into tumor biology and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2633.