Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis
Venkata Lokesh Battula, … , Sendurai A. Mani, Michael Andreeff
Venkata Lokesh Battula, … , Sendurai A. Mani, Michael Andreeff
Published May 15, 2012
Citation Information: J Clin Invest. 2012;122(6):2066-2078. https://doi.org/10.1172/JCI59735.
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Research Article Oncology

Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis

Abstract

Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have increased resistance to conventional therapies and are capable of establishing metastasis. However, only a few biomarkers of CSCs have been identified. Here, we report that ganglioside GD2 (a glycosphingolipid) identifies a small fraction of cells in human breast cancer cell lines and patient samples that are capable of forming mammospheres and initiating tumors with as few as 10 GD2+ cells. In addition, the majority of GD2+ cells are also CD44hiCD24lo, the previously established CSC-associated cell surface phenotype. Gene expression analysis revealed that GD3 synthase (GD3S) is highly expressed in GD2+ as well as in CD44hiCD24lo cells and that interference with GD3S expression, either by shRNA or using a pharmacological inhibitor, reduced the CSC population and CSC-associated properties. GD3S knockdown completely abrogated tumor formation in vivo. Also, induction of epithelial-mesenchymal transition (EMT) in transformed human mammary epithelial cells (HMLER cells) dramatically increased GD2 as well as GD3S expression in these cells, suggesting a role of EMT in the origin of GD2+ breast CSCs. In summary, we identified GD2 as a new CSC-specific cell surface marker and GD3S as a potential therapeutic target for CSCs, with the possibility of improving survival and cure rates in patients with breast cancer.

Authors

Venkata Lokesh Battula, Yuexi Shi, Kurt W. Evans, Rui-Yu Wang, Erika L. Spaeth, Rodrigo O. Jacamo, Rudy Guerra, Aysegul A. Sahin, Frank C. Marini, Gabriel Hortobagyi, Sendurai A. Mani, Michael Andreeff

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Figure 5

Knockdown of GD3S reduces cell proliferation, mammosphere formation, and tumor initiation in MDA-MB-231 cells.

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Knockdown of GD3S reduces cell proliferation, mammosphere formation, and...
(A) To measure knockdown of GD3S, vector control, or GD3S-KD-MDA231 cells were analyzed for mRNA expression of GD3S by real-time TaqMan RT-PCR. Relative expression of GD3S is shown. *P < 0.0001. (B) To measure levels of GD2 on the cell surface, vector control or GD3S-KD-MDA231 cells were stained with anti-GD2 antibody and analyzed on an LSR II flow cytometer (BD). GD2 expression is shown on the y axis and FSC on the x axis. (C) Percentage of GD2+ cells in vector control and GD3S-KD-MDA231 cells. *P < 0.01. (D) To measure cell migration, vector control and GD3S-KD-MDA231 cells were cultured in the presence or absence of 30% serum in a Transwell migration chamber. The average number of cells per microscopic field is shown. *P < 0.001. (E) Mammosphere formation assay using either vector control or GD3S-KD-MDA231 cells was performed by seeding 1,000 cells per well in 24-well cell culture dishes containing mammosphere growth medium. After 10 days, the mammospheres were counted under a light microscope. Scale bar: 200 μm. Numbers of mammospheres formed from either vector control or GD3S-KD-MDA231 cells are shown. *P < 0.0001. (F) To examine tumor initiation potential, 1 × 106 vector control or GD3S-KD-MDA231 cells were transplanted subcutaneously into flanks of NOD/SCID mice. At the end of the ninth week, mice were shaved to remove excess hair on the tumors, and photographs were taken. (G) The tumors size was measured between 4 and 9 weeks. P < 0.000001.