Genomic and structural analyses reveal that β-catenin/CBP signaling represses epidermal growth factor receptor (EGFR) N-glycan antennary fucosylation in oral cancer.
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 >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.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy in the world with oral squamous cell carcinomas (OSCC) accounting for the majority of HNSCC cases. A major driver of OSCC is the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) with 12 N-glycosylation sites. Fucosylated N-linked glycans on EGFR are associated with survival e.g., they suppress receptor dimerization and signaling. High levels of fucosylated glycan epitopes have been observed in OSCC, where invasive regions lose expression of linkage-specific fucosylated epitopes, suggesting that fucosylated glycans are involved in the suppression of cell growth and invasion. Previously, it was shown that inhibition of the interaction between nuclear β-catenin and CREB-binding protein (CBP) in human OSCC cells and in mouse tumor xenografts with a small molecule inhibitor ICG-001, interfered with OSCC proliferation and aggressive features in cellular, zebrafish, and murine models. E7386, a novel β-catenin/CBP modulator displays activity profile that closely overlaps with that of ICG-001 and exhibits ~50 - 100-fold lower EC50 values. Treatment with ICG-001 and E7386, increased expression of two glycosyltransferases, FUT2 and FUT3 coincident with decreased EGFR abundance that was accompanied by higher fucosylation of EGFR and upregulated expression of E-cadherin and junctional β-catenin. Further, genomic analyses showed a positive correlation between the ICG-001 and E7386 treatments and EGFR inhibition, suggesting that higher expression of antennary fucosyltransferase genes suppresses EGFR signaling. We now show using nLC-MS/MS analyses that EGFR from metastatic HSC-3 cells had low levels of fucosylated N-glycans, while EGFR from indolent CAL27 cells displayed higher levels of fucosylation at multiple EGFR N-linked glycosylation sites, and that these changes were statistically significant. In-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry of EGFR glycopeptides revealed new insights into the identity of fucosylated glycan epitopes. Collectively, these results suggest that the β-catenin/CBP axis promotes EGFR signaling through downregulation of fucosyltransferase expression and activity. We conclude that inhibition of β-catenin/CBP signaling with a novel small molecule E7386 may serve as a therapeutic approach to downregulate EGFR pro-tumorigenic activity in HNSCC patients. Citation Format: Kevin B. Chandler, Khalid Alamoud, Bac-Cuc Nguyen, Vanessa L. Stahl, Takashi Owa, Kenichi Nomoto, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Inhibition of β-catenin/CBP signaling alters EGFR fucosylation status in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5940.
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