Abstract 1528: GD2+ breast cancer stem cell growth is dependent on NFκB signaling and suppressed by the IKK inhibitor BMS345541 in vitro and in vivo
Authors: Battula Venkata Lokesh , Sun Jeffrey , Hortobagyi Gabriel N. , Andreeff Michael 
Published: August 2015

Recurrence of breast cancer after treatment could be caused by breast cancer stem-like cells (BCSCs) or breast cancer-initiating cells. Therefore, targeting BCSCs is an important strategy that could complement standard chemotherapy. We recently reported that the ganglioside GD2 is exclusively expressed on BCSCs and that targeting its metabolic enzyme (GD3 synthase, GD3S), reduced breast tumor growth and metastasis in immuno-deficient mice (Battula et al., JCI, 2012). To identify signaling pathways that are activated in GD2+ BCSCs, we performed proteomic analysis in GD2+/- cells from breast cancer cell lines MDA-MB-231 and SUM159 cells using Kinexus® antibody arrays. Data analysis employing Ingenuity® pathway analysis revealed activation of NFκB signaling in GD2+ cells in both cell lines. To validate the array data, we tested activation of NFκB (phospho p65) by CyTOF mass cytometry using metal tagged antibodies and found that NFκB was indeed activated in GD2+ but not in GD2- cells, suggesting activation of canonical NFκB signaling in GD2+ BCSCs. Systemic phospho- and total-protein analyses by immuno blotting revealed that proteins involved in non-canonical signaling, including RelB, NFκB2, and Traf2, were also up-regulated (2-3 fold) in GD2+ compared to GD2- cells. These data suggest that both canonical and non-canonical NFκB signaling are active in GD2+ cells and that it may require inhibition of both pathways to block GD2+ BCSC growth. We found that BMS345541 (IC50 for IKKβ = 0.4μM (canonical) and IC50 for IKKα = 4μM (canonical and non-canonical) reduced GD2+ cells and inhibited GD3S expression (determined by qRT-PCR) in a dose- and time-dependent fashion. Treatment with BMS345541 at 5μM for 72hrs reduced GD2+ cell frequency from 18.5±2.5% to 1.7±0.4% (P &lt;0.001) which is &gt;90% reduction of GD2+ cells in a dose-dependent manner. In contrast, treatment with doxorubicin increased the percentage of GD2+ cells, from 13.5±2.5% to 21±2.6% in MDA-MB-231 cells, suggesting that GD2+ cells are resistant to doxorubicin. BCSCs generate mammospheres under low adhesion conditions. Treatment with the BMS compound inhibited the ability of breast cancer cells to form mammospheres app.12 fold in vitro. To test if BMS345541 inhibits in vivo tumor growth, NOD/SCID mice were transplanted with MDA-MB-231 cells (0.5×106) injected into the mammary fat pad. Following engraftment (2 weeks after transplantation), mice were treated with 50mg/kg of BMS345541 or with DMSO daily for only 5 days (IP, n = 5). The BMS345541-treated group showed a significant decrease (P &lt;0.01) in tumor volume, and prolonged survival compared to the control group: median survival was 78 days for the BMS345541-treated group vs. 58 days for controls (P&lt;0.002). Our data suggest that NFκB inhibition by BMS345541 reduces tumor growth in vivo and prolongs survival of tumor-bearing mice by inhibiting NFκB-mediated GD2+ BCSC growth.

Citation Format: Venkata Lokesh Battula, Jeffrey Sun, Gabriel N. Hortobagyi, Michael Andreeff. GD2+ breast cancer stem cell growth is dependent on NFκB signaling and suppressed by the IKK inhibitor BMS345541 in vitro and in vivo. [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 1528. doi:10.1158/1538-7445.AM2015-1528