Abstract 3342: Specific inhibition of IKKβ induces caspase-independent cell death in the ovarian cancer stem cells

Abstract

Abstract Introduction: Current studies show that the cancer stem cells represent the chemo-resistant phenotype and therefore may survive chemotherapy to eventually rebuild the tumor. Our group previously identified and characterized the ovarian cancer stem cells using CD44 and MyD88 as a marker and showed that these cells have a constitutively active IKKβ/NFκB axis. We hypothesize that specific targeting of this pathway may affect survival of the ovarian cancer stem cells. Methods: A panel of CD44+/MyD88+ ovarian cancer stem cells were treated with increasing concentrations of the specific IKKβ inhibitor, BAY 11-7082 (0.1, 0.5 1, 5, and 10 μg/ml). Effect on cell growth was monitored using the Incucyte real time imaging system and cell viability was determined using the Celltiter 96 assay. Specific inhibition of IKKβ was determined by: (a) the effect on the mTOR pathway by analyzing levels of phospho-S6 kinase and (b) by determining the effect on TNFα-induced NFκB activity by measuring levels of IκB. Activity of caspases- 3/7, −8, and −9 was measured using Caspase-Glo assay and levels of XIAP monitored using western blot analysis. Effect on mitochondrial membrane potential was measured using flow cytometry after staining with JC1 dye. In vivo activity was monitored using a s.c. ovarian cancer xenograft model in nude mice. The effect of BAY 11-7082 (10 mg/kg every other day given i.p.) was compared to carboplatin (40mg/kg q7d given i.p.) and paclitaxel (10mg/kg q3d given i.p.). Results: Treatment with BAY 11-7082 inhibited cell growth and decreased cell viability in all ovarian cancer stem cell cultures tested in a dose and time dependent manner with GI50 at ∼ 5μg/ml. Specific inhibition of IKKβ was observed at this dose by a decrease in phospho-p70S6 kinase and inhibition of TNFα-induced IκB degradation. Cell death was caspase-independent with no significant increase in caspases 3/7, 8, nor 9 and no effect on the levels of XIAP. Cell death was associated with loss of mitochondrial membrane potential with more than 90% of cells showing a shift in JC1 fluorescence from red to green in 4h. In vivo studies showed that BAY 11-7082 is able to inhibit tumor growth with T/C of 64%, significantly better than carboplatin and paclitaxel (T/C 87% and 74%, p=0.009 and 0.0025, respectively). Conclusion: The capacity of the cancer stem cells to survive chemotherapy and rebuild the tumor represent a major target to inhibit recurrence in ovarian cancer patients. The demonstration that specific targeting of IKKβ can induce cell death in the ovarian cancer stem cells opens new therapeutic options for ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3342.