Abstract 1250: Selection of rare and novel breast cancer cell variants based on their plasticity of glutamine metabolism
Authors: Singh Balraj , Tai Karen , Braunlin Megan , Madan Simran , Irving LaTashia R. , Bajaj Ankur , Lucci Anthony 
Published: June 2012

Abstract
Breast cancer is a heterogeneous disease and only rare cancer cells succeed in metastasizing. The current approach of classifying subpopulations of cancer cells based on certain biomarkers derived from the bulk of primary tumor is unsatisfactory for identifying the rare cells that cause metastasis. One attribute of metastatic cells is their ability to stop proliferating and survive long-term under unfavorable conditions. While the majority of cells present in metastatic breast cancer cell lines are predicted to be dependent on Gln and thus will die upon Gln withdrawal, we hypothesized that some rare cells would survive and grow without Gln, thus suggesting a simple robust function-based selection method for metabolically plastic cancer cells. We analyzed several aggressive breast cancer cell lines including SUM149, SUM149-FP76 (variant cells cultured from a fat pad tumor in nude mice), SUM190, MDA-MB-231, MDA-MB-231-BSC60 (a metastatic clone), 168FARN, 4T07, and 4T1. Gln-independent variants were selected by growing cells in a medium without Gln. We evaluated protein level of glutaminase (GLS) that correlates with Gln addiction in cancer cells, Myc that drives Gln catabolism, and COX-2 that is involved in metastasis, by western blotting. We evaluated the sensitivity of cell lines to chemotherapeutic drugs, doxorubicin and paclitaxel, by a cell proliferation assay. We utilized the two-sample t-test for statistical analysis. We were able to select rare variants (one in 35,000 cancer cells) from the SUM149-Luc cell line that grow long-term in Gln-free medium. We found that Gln-independent variants produced a significantly lower level of GLS (1/5th) and a significantly higher level (5-fold) of COX-2 than did the parental cell line. The Gln-ind variants produced a high level of Myc protein similar to the Gln-addicted bulk population of cells. The Gln-ind phenotype in the rare variants is stable since exposure to Gln for multiple (&gt; 10) passages did not lead to Gln addiction, although they were able to utilize Gln when available. Of interest, the Gln-ind variants were significantly more resistant to doxorubicin and paclitaxel than the parental cell lines, requiring approximately 10X drug concentrations for inhibiting the Gln-ind variants. The Gln-ind variants are unique in another significant manner- the variants, but not the parental SUM149 cell line, yielded colonies in a chemically defined medium that is devoid of serum. We were successful in isolating rare metabolically plastic Gln-ind variants from all the cell lines mentioned above. Our results revealed a link between metabolic plasticity (i.e., decreased GLS to maintain a sufficient Gln pool for cell growth) and metastatic ability (via increased COX-2 level) that could be exploited for selecting novel subpopulations of cancer cells and for targeted therapy development against these cells.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1250. doi:10.1158/1538-7445.AM2011-1250