Abstract P1-06-03: Epithelial to mesenchymal transition (EMT) regulates the spontaneous generation of GD2+ breast cancer stem-like cells through NFκB activation
Authors: Battula VL , Sun J , Nguyen K , Hortobagyi G , Andreeff M 
Published: May 2016

Breast cancer recurrence may be a consequence of persistent breast cancer stem-like cells (BCSCs) that survive chemo- or hormonal therapy. Therefore, targeting BCSCs could complement standard chemotherapy. We discovered that the ganglioside GD2 is expressed on and defines BCSCs (Battula et al., JCI, 2012), as consequence of activation of the enzyme GD3 synthase (GD3S). Inhibition of GD3S expression inhibited breast cancer metastasis to lung. We also observed that GD2- breast cancer cells spontaneously generate GD2+ cells in vitro. As induction of EMT generates a stem cell–like phenotype, we hypothesized that EMT regulates the generation of GD2+ breast cancer cells. To test this hypothesis, MDA-MB-231and SUM159 cells were cultured in vitro and the percentage of GD2+ cells was measured over time. Interestingly, the percentage and absolute number of GD2+ cells increased in a time-dependent manner, suggesting the spontaneous generation of GD2+ cells. Concomitantly, mesenchymal-related markers including vimentin, N-cadherin, and twist increased 3 to 6 - fold. To further investigate whether this process is operational in vivo, GFP+ MDA-MB-231 cells were transplanted into mammary fat pads of NOD/SCID mice. Each week, a group of mice was sacrificed, tumors were extracted and the number of GFP+GD2+ cells was determined by flow cytometry. In line with our in vitro results, we observed significant increases in GD2+ BCSCs with increasing tumor volume from 15.1%±4.6% to 37%±8.7% over a 6 week period, suggesting that breast cancer cells spontaneously undergo EMT during tumor progression and generate GD2+ BCSCs.

To identify possible targets to inhibit EMT in breast cancer cells, proteomic analysis using Kinexus® antibody arrays revealed activation of NFκB and focal adhesion kinase (FAK) signaling in GD2+ breast cancer cells. The activation of NFκB (phospho p65) in GD2+ cells was validated by CyTOF mass cytometry using metal tagged antibodies. These data suggest that inhibition of NFκB signaling may inhibit GD2+ BCSC growth. Indeed, the IKK inhibitor BMS345541 reduced GD2+ cells by &gt;95% and inhibited GD3S expression (determined by qRT-PCR) in a dose- and time-dependent fashion. 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. In addition, treatment with BMS345541 inhibited the ability of breast cancer cells to form mammospheres by &gt;90% in vitro. In-vivo tumorigenesis assay demonstrated that BMS345541 induced a significant decrease (p &lt;0.01) in tumor volume, and increased survival of tumor bearing mice: median survival was 78 days for BMS345541-treated mice vs. 58 days for controls (p&lt;0.002).

Conclusion: GD2+ BCSCs are spontaneously produced during tumor progression by EMT and NFκB and FAK mediated signaling might regulate this process. Inhibition of NFκB and FAK signaling pathways may inhibit the spread of BCSCs and reduce breast cancer metastases.

Citation Format: Battula VL, Sun J, Nguyen K, Hortobagyi G, Andreeff M. Epithelial to mesenchymal transition (EMT) regulates the spontaneous generation of GD2+ breast cancer stem-like cells through NFκB activation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-06-03.