Abstract PR11: Tissue-of-origin dictates the metabolic fate of branched chain amino acids in mutant Kras-driven cancers
Authors: Mayers Jared R. , Torrence Margaret E. , Davidson Shawn M. , Papagiannakopoulos Thales , Lau Allison N. , Jacks Tyler , Heiden Matthew G. Vander 
Published: May 2016

Abstract
Introduction: Growth signaling is associated with changes in metabolism in cell culture, but the relatively homogenous nature of culture systems leaves open the question of how specific mutations might behave differently in different tissue contexts. Indeed, previous work has demonstrated that tumor metabolic gene expression most closely resembles the tissue of origin for that tumor1, and that the same oncogenic driver can cause different metabolic phenotypes in different tissues2. We recently found that pancreatic cancers (PDAC) are associated with increased plasma branched-chain amino acid (BCAA) levels resulting from increased whole body protein turnover, while a model of non-small cell lung cancer (NSCLC) driven by the same genetic lesions displays the opposite changes in plasma BCAA levels3. Based on these data, we hypothesized that changes in plasma BCAAs might reflect a difference in the utilization of BCAA by tumors arising in each tissue.
Methods: C57B6/J male mice heterozygous for the lox-stop-lox (LSL)-KrasG12D allele and homozygous for the Trp53flox/flox allele were exposed to an inhaled viral Cre-recombinase or crossed to a pancreas-specific Pdx-1-cre to generate models of NSCLC and PDAC respectively. Cell lines previously derived from these mouse models were used for in vitro and allograft studies. To identify the metabolic fates of BCAAs in vitro, we performed tracing studies utilizing stable-isotope 13C-Leu or 15N-Leu in place of the abundant 12C or 14N isotopomer. Similarly, we used amino acid defined diets in which stable isotope BCAAs were included to trace amino acid fate in vivo.
Data: We first confirmed that plasma BCAA levels change in opposite directions early in disease in the isogenic mouse models of PDAC and NSCLC. Consistent with different metabolic fates of BCAAs, 13C stable isotope tracing in vivo revealed increased incorporation of BCAAs into tissue protein in NSCLC tumors, with decreased incorporation observed in PDAC tumors. NSCLC tumors also displayed increased generation ofα-ketoisocaproate, the transamination product of leucine, suggesting an additional role for BCAAs a nitrogen source in NSCLC. Further supporting this possibility, we found increased expression of branched-chain amino acid transferase 2 (Bcat2), but not other BCAA catabolic genes, in NSCLC tumors. To directly examine the fate of BCAA-derived nitrogen, we used 15N-BCAA tracing studies and CRISPR/Cas9-mediated knockout of BCAA catabolic genes. Tracing in vitro revealed increased transamination of 15N-Leu in NSCLC cells compared with PDAC cells. We also generated NSCLC and PDAC cells deficient in Bcat isoforms to assess the effects of Bcat knockout in both allograft and autochthonous models.
Conclusions: Despite sharing an identical combination of mutations, mouse models of NSCLC and PDAC exhibit distinct phenotypes with regards to BCAA metabolism in tumors. The different fates of BCAAs in each of these tumor types might explain previously observed alterations in plasma BCAA levels and provides further insight into how tumors can influence whole body metabolic phenotypes in early cancer.
1. Hu, J., et al. Heterogeneity of tumor-induced gene expression changes in the human metabolic network. Nature biotechnology 31, 522-529 (2013).
2. Yuneva, M.O., et al. The Metabolic Profile of Tumors Depends on Both the Responsible Genetic Lesion and Tissue Type. Cell Metabolism 15, 157-170 (2012).
3. Mayers, J.R., et al. Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development. Nature Medicine (2014).
Citation Format: Jared R. Mayers, Margaret E. Torrence, Shawn M. Davidson, Thales Papagiannakopoulos, Allison N. Lau, Tyler Jacks, Matthew G. Vander Heiden. Tissue-of-origin dictates the metabolic fate of branched chain amino acids in mutant Kras-driven cancers. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstractnr PR11.