Activating KRAS mutations are prevalent in human cancer and are associated with poor prognosis. Among these malignancies is pancreatic ductal adenocarcinoma (PDAC), which is universally KRAS-driven and almost universally lethal. PDAC tumors are extremely fibrotic and hypovascularized, limiting perfusion of nutrients into the tumor, and tumor cells exhibit an altered metabolic program to support survival and proliferation in this environment. One metabolic activity upregulated in these tumor cells is the uptake and catabolism of serum protein. This process yields a substantial alternative source of amino acids and can support the proliferation of cultured cells lacking free essential amino acids. However, existing methods for assaying the uptake and degradation of intact protein provide qualitative outputs, and as such, do not yield accurate estimates of flux from serum protein to amino acid monomers. Here, we present a method for quantitative measurement of the catabolic production of amino acids from serum protein in cultured cells. By culturing cells in medium containing fully 13C-labeled glucose and amino acids supplemented with unlabeled albumin, we distinguish amino acids taken up as monomers from the medium from serum protein-derived amino acids. Using a variant of classical metabolic flux analysis, we derive flux estimates for serum protein catabolism from measurements of amino acid abundance and isotopic labeling over time. Our method is highly sensitive (i.e. can estimate “baseline” serum protein catabolism in KRAS wild-type cell lines) and yields precise estimates of amino acid influx from serum protein for all proteinogenic amino acids. We have measured serum protein catabolism in a variety of cultured cell lines and find that protein catabolism yields amino acids in comparable amounts to conventional uptake in various pancreatic and non-pancreatic lines. This approach enables estimation of protein catabolic flux in any cell line and can be used to assay the effects of various genetic and pharmacological perturbations on serum protein catabolism with high sensitivity and while accounting for growth rate differences.

Citation Format: Michel Nofal, Kevin Zhang, Josh Rabinowitz. Quantitative flux measurements of serum protein catabolism in PDAC and other KRAS-mutant cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-279. doi:10.1158/1538-7445.AM2015-LB-279