BackgroundHead and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy characterized by tumor heterogeneity, locoregional metastases, and resistance to existing treatments. Although a number of genomic and molecular alterations associated with HNSCC have been identified, they have had limited impact on the clinical management of this disease. To date, few targeted therapies are available for HNSCC, and only a small fraction of patients have benefited from these treatments. A frequent feature of HNSCC is the inappropriate activation of β-catenin that has been implicated in cell survival and in the maintenance and expansion of stem cell-like populations, thought to be the underlying cause of tumor recurrence and resistance to treatment. However, the therapeutic value of targeting β-catenin activity in HNSCC has not been explored.MethodsWe utilized a combination of computational and experimental profiling approaches to examine the effects of blocking the interaction between β-catenin and cAMP-responsive element binding (CREB)-binding protein (CBP) using the small molecule inhibitor ICG-001. We generated and annotated in vitro treatment gene expression signatures of HNSCC cells, derived from human oral squamous cell carcinomas (OSCCs), using microarrays. We validated the anti-tumorigenic activity of ICG-001 in vivo using SCC-derived tumor xenografts in murine models, as well as embryonic zebrafish-based screens of sorted stem cell-like subpopulations. Additionally, ICG-001-inhibition signatures were overlaid with RNA-sequencing data from The Cancer Genome Atlas (TCGA) for human OSCCs to evaluate its association with tumor progression and prognosis.ResultsICG-001 inhibited HNSCC cell proliferation and tumor growth in cellular and murine models, respectively, while promoting intercellular adhesion and loss of invasive phenotypes. Furthermore, ICG-001 preferentially targeted the ability of subpopulations of stem-like cells to establish metastatic tumors in zebrafish. Significantly, interrogation of the ICG-001 inhibition-associated gene expression signature in the TCGA OSCC human cohort indicated that the targeted β-catenin/CBP transcriptional activity tracked with tumor status, advanced tumor grade, and poor overall patient survival.ConclusionsCollectively, our results identify β-catenin/CBP interaction as a novel target for anti-HNSCC therapy and provide evidence that derivatives of ICG-001 with enhanced inhibitory activity may serve as an effective strategy to interfere with aggressive features of HNSCC.Electronic supplementary materialThe online version of this article (10.1186/s13073-018-0569-7) contains supplementary material, which is available to authorized users.

The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability of E-cadherin junctions in response to DPAGT1 inhibition. We show the impact of pathway dysregulation through measurements of cell migration in scratch-wound assays. Collectively, our results highlight the importance of numerical analyses of cellular networks dynamics to gain insights into physiological processes and potential design of therapeutic strategies to prevent epithelial cell invasion in cancer.

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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 >90% of tumors. EGFR is highly modified with N-linked glycans; fucosylation of N-linked 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 affected EGFR N-glycosylation. We immunoprecipitated EGFR from indolent CAL27 and metastatic HSC-3 cells after treatment with ICG-001 or vehicle control and determined the effect of inhibition of Β-catenin/CBP activity on its N-glycosylation status. We subjected immunoprecipitated EGFR to proteolysis, performed glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC), analyzed glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation, and compared site-specific glycoform patterns for the two cell types +/- ICG-001. At specific N-glycosylation sites, EGFR from indolent CAL27 cells had highly fucosylated N-glycans, while EGFR from metastatic HSC-3 cells displayed N-linked glycans with a paucity of fucose. Treatment of HSC-3 cells with ICG-001 revealed higher fucosylation at sites N151, N420, suggesting that ICG-001 promoted modification with terminal fucose, 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 that fucosylate residues on the outer arms of N-linked glycans. Our studies suggest that the Β-catenin/CBP axis promotes EGFR signaling by inhibiting its fucosylation 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 protumorigenic 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, Khalid Alamoud, Vinay K. Kartha, Khikmet Sadykov, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Inhibition of Β-catenin/CBP signaling in oral cancer alters EGFR N-glycosylation and abundance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2516.

The purpose of the study was to determine if genetic and pharmacological inhibition of terminal uridyltransferases (TUTases) ZCCHC11 and ZCCHC6 could modulate Glioblastoma (GBM) cell growth in vitro. ZCCHC11 (also known as TUT4 and TENT3A) and ZCCHC6 (also known as TUT7 and TENT3B) catalyze uridylation of diverse RNA species. Recent evidence indicates that genetic perturbation of ZCCHC11/6 expression can disrupt cell proliferation of both immortalized and patient-derived primary GBM cell lines (Kim et al., Mol Cell 2020). We confirmed that genetic inhibition of ZCCHC11/6 with siRNA or CRISPR can decrease viability of U-87 MG and A-172 GBM cell lines, and we identified DK-MG as another GBM cell line with sensitivity to ZCCHC11/6 knockdown. Herein, we report the first novel, potent, and selective inhibitor of ZCCHC11/6, TS-1. Biochemically, TS-1 can block ZCCHC11/6-mediated uridylation of an RNA substrate in vitro (IC50 = 0.65 nM for recombinant ZCCHC11 protein; IC50 = 9.6 nM for recombinant ZCCHC6 protein). ZCCHC11/6 inhibitor TS-1 could decrease cell viability in U-87 MG, A-172 and DK-MG cells (cell viability IC50s ranging between 0.7-1.5 μM) and induce apoptosis and cell cycle arrest. A cellular assay detecting uridylation of miR-191 as a marker for ZCCHC11/6 activity was developed and validated. TS-1 strongly decreased uridylation of miR-191 in U-87 MG, A-172 and DK-MG cells (IC50 ranging between 10-40 nM). In contrast, TS-2, a weakly active enantiomer of TS-1 (enzyme IC50 = 0.15 μM and 1.8 μM against ZCCHC11 and ZCCHC6, respectively), had less impact on cell viability, apoptosis, cell cycle and uridylation in cells. In conclusion, we have verified ZCCHC11/6-dependency in a set of GBM cell lines and we have also developed a first-in class potent and selective small molecule that reduces in vitro Glioblastoma cell proliferation through selective inhibition of TUTases activity. Citation Format: Robinson Triboulet, Khikmet Sadykov, Jessica L. Johnson, Andrew R. Snyder, Sarah K. Knutson, Pavan Kumar, Christopher B. Mayo, Dillon Hawley, Andrew Madanjian, Ross L. Stein, David M. Wilson, Darren M. Harvey, Shomir Ghosh, Robert M. Campbell. Selective inhibition of ZCCHC11/ZCCHC6 TUTases with genetic and pharmacological tools supports a role in glioblastoma cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1822.