Abstract 52: Glycogen synthase kinase inhibition associated with Notch-1 reduction in cholangiocarcinoma
Authors: Sokolowski Kevin M. , Kunnimalaiyaan Selvi , Gamblin T Clark , Kunnimalaiyaan Muthusamy 
Published: August 2015

Introduction: Cholangiocarcinoma (CCA) is the most common biliary cancer and the second most common hepatic malignancy after hepatocellular carcinoma with incidence in the United States steadily increasing. It is a highly malignant adenocarcinoma characterized by poor prognosis and chemotherapeutic inefficacy with 5-year survival rates remaining at less than 10%. Currently, surgical resection is the only effective strategy; however, the majority of patients (90%) are not candidates due to metastasis and concomitant liver disease. Furthermore, local and distant recurrence and high morbidity rates are common following resection. Therefore, new targeted molecular strategies against CCA are imperative. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target as it is overexpressed in various cancer types including CCA acting as an oncogene in these cancers. However, the role of the GSK-3 isoform and their interaction with Notch-1 remains unclear in CCA. Our findings and other reports demonstrate that the inhibition of Notch-1 in CCA leads to growth suppression. We hypothesize that GSK-3 stabilizes Notch-1 and thereby promoting cellular proliferation in CCA. In this study, we investigated the effect of the GSK-3 inhibitor AR-A014418 (AR) on the levels of GSK-3 isoforms and further delineated the effect of specific GSK-3 inhibition on Notch-1 signaling and growth in CCA.

Methods: The effects of AR on CCA cell lines (CCLP-1 and SG-231) were assessed by MTT assay and live cell imaging. Cell lysates were analyzed via Western blotting for active and total GSK-3, β-catenin, Notch-1, pro-apoptotic and anti-apoptotic proteins. Transient knockout against Notch-1 was used to determine the interaction of Notch-1 in CCA whereas knockout of GSK-3α or β was used to analyze the interaction with Notch-1 and delineate isoform specificity in CCA.

Results: Increasing AR treatment (0μM - 20μM) had a significant dose-dependent growth reduction (p&lt;0.001); starting at 5μM in CCA cells, compared to control. Upon Western analysis, growth suppression due to apoptosis was evidenced by increased expression of pro-apoptotic and reduced anti-apoptotic proteins. Inhibition of GSK-3 by AR reduced the levels of active GSK-3 expression with concomitant reduction in Notch-1 expression in a dose-dependent manner. Importantly, specific knock-down of GSK-3α resulted in Notch-1 reduction compared to GSK-3β knock-down. However, shRNA against Notch-1 had no effect on the expression of either GSK-3 isoform.

Conclusion: AR-A014418, a GSK-3 inhibitor, effectively inhibits CCA growth in cell culture through both GSK-3 and Notch-1 pathways. Transient knockout of GSK-3 isoforms demonstrated specificity toward GSK-3α in reducing Notch-1 suggesting GSK-3α may be required for Notch-1 stability. This is the first study to effectively correlate GSK-3α with Notch-1 in cholangiocarcinoma.

Citation Format: Kevin M. Sokolowski, Selvi Kunnimalaiyaan, T Clark Gamblin, Muthusamy Kunnimalaiyaan. Glycogen synthase kinase inhibition associated with Notch-1 reduction in cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 52. doi:10.1158/1538-7445.AM2015-52