(P12-013-25) Differential Adaptability to Glutamine in Liver Versus Lung Metastasizing Breast Cancer Cells

Objectives: Our work aims to compare the glutamine metabolic adaptations between breast cancer cells that preferentially metastasize to lung versus liver.

Methods: Wnt-driven breast cancer cell models that preferentially metastasize to lung (metM-Wnt^Lung cells; MLg) or liver (metM-Wnt^Liver cells; MLr) were utilized. Cells were exposed to low (0.5 mM), normal (2 mM), or high (4 mM) glutamine levels, and cell viability (MTT assay), ¹⁴C-glutamine uptake, ¹³C₅-glutamine (100%) cellular flux, and mRNA abundance of metabolic enzymes were measured.

Results: With normal glutamine, ¹⁴C-glutamine uptake was 26% higher in MLg compared to MLr. This result was consistent with higher mRNA level of glutamine-catabolizing enzymes, glutamate synthase and glutamate dehydrogenase (GLUD), and higher enrichment of M+5-glutamate and M+5-α-ketoglutarate in MLg than the MLr. These results suggest higher glutamine flux into the TCA cycle in MLg than the MLr. On the other hand, high glutamine increased viability by 18% in MLr but had no effect in MLg. To further examine mechanisms supporting higher viability of MLr under high glutamine conditions, cells were treated with NH₄Cl (5 mM) as ammonia is a by-product of glutamine catabolism. Results showed that viability and mRNA level of the ammonia-utilizing enzyme, asparagine synthetase, were higher in MLr than MLg. These results suggest that MLr’s ability to better adapt to high glutamine is in part due to utilization of excess ammonia. Similarly, MLr were more viable by 12% than the MLg with low glutamine. Despite reduction in overall ¹³C₅-glutamine flux under low glutamine in both cell lines, MLr had higher enrichment of unlabeled TCA metabolites. MLr also had higher mRNA level of pyruvate carboxylase and GLUD, enzymes that can utilize non-glutamine carbons to reduce reliance on glutamine, suggesting that MLr’s ability to adapt to low glutamine is in part due to higher utilization of non-glutamine sources.

Conclusions: Overall, MLr adaptation, but not the MLg, to high glutamine is due to utilization of excess ammonia and to low glutamine due to utilization of non-glutamine carbons, respectively.

Funding Sources: This work is supported by Purdue University Institute for Cancer Research, Women’s Global Health Institute and NIH R01CA232589 and R01A271597.