Functional and genomic analyses reveal therapeutic potential of targeting ß-catenin/CBP activity in head and neck cancer.

BACKGROUND: Head 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 ß-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 ß-catenin activity in HNSCC has not been explored. METHODS: We utilized a combination of computational and experimental profiling approaches to examine the effects of blocking the interaction between ß-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. RESULTS: ICG-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 ß-catenin/CBP transcriptional activity tracked with tumor status, advanced tumor grade, and poor overall patient survival. CONCLUSIONS: Collectively, our results identify ß-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.

The Integrated Role of Wnt/ß-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics.

The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/ß-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, ß-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/ß-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/ ß-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 ß-catenin. We confirmed the role of axin in ß-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 ß-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic ß-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.