Abstract LB-193: Ganglioside GD2 identifies cancer stem cells and inhibition of GD2 biosynthesis by targeting GD3 synthase exerts antitumor effects
Authors: Battula Venkata Lokesh , Shi Yuexi , Evans Kurt , Wang Rui-Yu , Speath Erika L. , Jacamo Rodrigo , Guerra Rudy , Sahin Aysegul , Marini Frank , Mani Sendurai , Andreeff Micheal 
Published: August 2012

Currently, only very few markers either as single or in combination are available to identify cancer stem cells (CSCs). In this report, we identified that the ganglioside GD2, a marker known to express on mesenchymal stromal cells (MSCs) is expressed on a small fraction (5.5% ± 3.4%) of transformed human mammary epithelial cells (HMLER) and breast cancer patient tumors. FACS sorted GD2+ cells appear spindle shaped and proliferate 5 fold slower compared to GD2- cells in-vitro. Analysis of breast cancer cell lines (n=12) indicated that GD2 expression varies and that the basal breast cancer cell lines have a higher percentage of GD2+ cells (median 9%, range 1.2-17%, n=6), as compared to their luminal counterparts (median 0.2%, range 0-3%, n=6, p&lt;0.001). Functional analysis revealed that GD2+ HMLER and MDA-MB-231 cells produced 2-5 fold more mammospheres in-vitro (p&lt;0.003) and tumors in-vivo than GD2- cells (p&lt;0.04). Recent reports suggest that breast tumor cells with CD44highCD24low phenotype exhibit CSCs characteristics. Interestingly, the majority (94% ± 3.5%) of GD2+ cells were characterized by the CD44highCD24low phenotype in HMLER cells. Analysis of primary breast cancer samples (n=10) revealed that GD2 is variably expressed in these samples (median 4.35%, range 0.5%-35.8%) and 95.5% ± 2.7% of GD2+ cells co-segregated with CD44highCD24lowCD45- phenotype. In contrast, only 2.4%± 0.4% of GD2- cells displayed this phenotype. Gene-chip and quantitative RT-PCR analysis revealed that GD3 synthase (GD3S), the enzyme responsible for synthesis of GD3, the precursor of GD2, was expressed 10-fold higher in GD2+ cells compared to GD2- HMLER and MDA-MB-231 cells. Analysis of GD2 expression in HMLER cells induced to undergo EMT revealed that the percentage of GD2+ cells increased 6-7 fold and the expression of GD3S &gt; 10 fold. Moreover, we observed spontaneous generation of GD2+ from GD2- cells and vice versa in both in-vitro and in-vivo, suggesting a role of EMT in this process. Stable knock-down of GD3S in MDA-MB-231 cells using shRNA impaired in-vitro matrigel invasion by more than 10-fold and completely abolished tumor growth in-vivo. Importantly, Triptolide, an anti-inflammatory and anti-cancer drug, which was recently shown to inhibit GD3S expression in melanoma cells, also inhibited GD3S expression in MDA-MB-231 and SUM159 cells by &gt;95% in a dose dependent manner and thereby inhibited growth of GD2+ cells in a time dependent manner. Intra-peritoneal administration of Triptolide (0.15mg/Kg/day) in NOD/SCID mice bearing MDA-MB-231 breast tumors completely eliminated tumors in 50% and reduced the tumor volume 7- to 8-fold in 25% of the mice. In conclusion, we identified GD2 as a new CSC specific cell surface marker and GD3 synthase as a potential therapeutic target for CSCs, with the potential of improving survival and cure rates of patients with breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-193. doi:1538-7445.AM2012-LB-193