Abstract P4-03-06: Selection of Rare and Novel Breast Cancer Cell Variants Based on Glutamine Metabolism

Background: Breast cancer is a heterogeneous disease composed of tumor cells that have different characteristics. Although there are several different ways to explain cellular heterogeneity, it is clear that only rare cancer cells out of the overall population 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. The purpose of this study was to test a novel function-based, rather than biomarker-based, selection strategy using glutamine (Gln) metabolism (or Gln-dependence to be more specific) since it is a prominent feature of cancer cells. 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 as a nutrient, thus suggesting a simple robust selection method for “tumorigenic” cancer cells. Methods: We chose an aggressive inflammatory breast cancer cell line SUM149 and SUM149-FP76 cell line that was recently derived by “passaging” SUM149-Luc cells (luciferase transfected for imaging in mice) in nude mice in our laboratory, first selecting for growth subcutaneously and then in inguinal fat pad. Gln-independent variants were selected by growing cells in a medium without Gln, supplemented with dialyzed fetal bovine serum. We evaluated protein level of glutaminase (GLS), an enzyme that converts glutamine to glutamate and usually correlates with Gln addiction in cancer cells, by western blotting. Expression of COX-2, an enzyme associated with inflammation, metastasis, and with cellular hierarchy, was measured by western blotting and compared between parental and Gln-independent lines. Tests were repeated in triplicate and evaluated using paired T-test. Results: We were able to select rare variants (one in 35,000 cancer cells) from the SUM149 inflammatory breast cancer cell line that grow long-term in Gln-free medium. We found that Gln-independent variants produced a significantly lower level of GLS (1/5 th ) and a significantly higher level (5-fold) of COX-2 than did the parental cell line. To provide further support for a link between Gln metabolism and tumorigenesis, we were also able to select the rare Gln-independent variants from the SUM149-FP76 cell line. Further experiments involving siRNA-mediated COX-2 knockdown indicated that the high level of COX-2 protein present in Gln-independent variants was not a direct regulator of the reduced GLS level, but rather COX-2 may offer indirect selective advantage by influencing processes such as apoptosis and “developmental hierarchy”. Conclusions: Since COX-2 is very important in breast cancermetastasis, our results suggest the possibility of a strong link between a critical metabolic adaptation (decreased GLS to maintain a sufficient Gln pool) that may be essential for cell survival and growth, and metastatic ability (via increased COX-2) in cancer cells. Our study also revealed a deep connection between glutamine metabolism and metastasis that could be exploited for developing novel models of cancer cell function and for targeted therapy development against these cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-03-06.