Gsi-1 Synergizes With Lde225 In Ovarian Cancer Cells By Inhibiting The Proteasome.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Cancer stem cells (CSCs) have been implicated in the development and recurrence of chemoresistant tumors in multiple malignancies. Previous research has suggested that targeting stem cell pathways, such as Notch and Hedgehog, may aid in overcoming the chemoresistant properties of these cells. However, attempts to target the Notch and Hedgehog pathways synergistically have had mixed results using different inhibitors, and the mechanism through which these inhibitors act is not fully known. Objective: To explore the potential synergy of concurrent targeting of the Notch and Hedgehog pathways and the mechanism through which this may occur. Methods: The Notch pathway was targeted with GSI-1 and GSI-XXI, and the Hedgehog pathway was targeted with the Smoothened small molecule inhibitor LDE225. The proteasome inhibitor Bortezomib was used alone and in combination with LDE225. Effects of these drugs on three pairs of chemosensitive and chemoresistant ovarian cancer cell lines were examined: SKOV3ip1 and SKOV3TRip2 (taxane-resistant), HeyA8 and HeyA8MDR (multi-drug resistant), and A2780ip2/A2780cp20 (platinum resistant). Cell viability was determined by the MTT assay. PI staining and PARP cleavage were used to assess cell cycle arrest and apoptosis. Proteasome activity was determined by the Proteasome-glo chymotrypsin-like cell-based assay. Results: GSI-1 significantly decreased cell viability alone and synergized with LDE225 in SKOV3TRip2 cells. However, GSI-XXI did not reduce cell viability or exhibit synergy with LDE225. In addition, specific inhibition of Notch via siRNA-mediated downregulation did not result in synergy with LDE225, suggesting a Notch-independent mechanism of synergy with GSI-1. To elucidate differences between GSI-1 and GSI-XXI, we examined effects of these drugs on the proteasome. GSI-1 inhibited the proteasome to the same extent as the conventional proteasome inhibitor bortezomib, while GSI-XXI did not affect proteasome activity. Combination treatment with bortezomib and LDE225 also resulted in synergy in SKOV3TRip2 cells. Monotherapy with both GSI-1 and bortezomib decreased transcription of the hedgehog target genes Patched, Gli1, and Gli2. This effect was enhanced when these drugs were combined with LDE225. Combination therapy also resulted in arrest of cells in the G2/M and S phases (60% of cells in S/G2/M with combination therapy vs. 38% of control, p<0.001), as well as an increase in apoptosis and PARP cleavage. Conclusion: The Notch inhibitor GSI-1 acts synergistically with Hedgehog inhibition through a Notch-independent decrease in proteasome activity. Similar synergy was noted with the proteasome inhibitor bortezomib. This synergistic combination appears to be working through S/G2/M phase arrest. Combined Hedgehog and proteasome inhibition offers a promising approach to targeting chemoresistant cells in ovarian malignancies. Citation Format: Mata R. Burke, Adam Steg, Dae Hoon Jeong, Zachary C. Dobbin, Charles N. Landen. GSI-1 synergizes with LDE225 in ovarian cancer cells by inhibiting the proteasome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3423. doi:10.1158/1538-7445.AM2013-3423