Defining the Nutrient Inputs that Support Pancreatic Cancer Metabolism

Abstract

Pancreatic ductal adenocarcinoma (PDA) is a particularly deadly form of cancer with a long-term survival rate of merely 12%. PDA is often detected in late stages and has few effective therapeutic options. This treatment evasion can be attributed largely in part to its surrounding tumor microenvironment (TME). PDA exists in a dense, highly fibrotic TME that results in hypoxia and a lack of serum-derived nutrients. To survive in these harsh conditions PDA rewires its metabolism and adapts its nutrient inputs. Understanding these cancer-specific pathways is vital in better characterizing PDA and identifying future therapeutic targets. The aim of this work was to profile the nutrients capable of fueling PDA metabolism. In achieving this aim, we first employed a large-scale unbiased nutrient screen called the Biolog Mammalian Phenotyping Assay®, in which we cultured 20 PDA and two control cell lines in various nutrient-depleted conditions while providing them a wide array of substrates to measure metabolic activity rescue. Through this assay we profiled carbon and nitrogen-containing metabolites, a panel of ions, metabolic efforts, and chemotherapy drugs. From these data we chose to further investigate the carbon and nitrogen sources as potential rescue options for PDA metabolism. Through these data we found that uridine consumption is markedly increased in PDA under nutrient-depleted conditions, and this strongly correlates to high expression of the enzyme uridine phosphorylase-1 (UPP1) which is responsible for catabolizing uridine into uracil and ribose 1-phosphate. Using metabolomics and several orthogonal methods, we demonstrate that uridine-derived ribose enters central carbon metabolism to fuel glycolysis and oxidative phosphorylation, restoring PDA growth. Further, we show that PDA UPP1 expression is regulated by the KRAS-MAPK pathway, and expression is further increased upon nutrient-deprivation of glucose or uridine. Importantly, we demonstrate that UPP1 is active in mouse models, and following UPP1 knockout, uridine-derived carbons no longer enter oxidative metabolism, resulting in reduced tumor growth in an in vivo mouse model. These studies highlight the ability of PDA to utilize alternative substrates within the TME, while also providing a potential target for disrupting a cancer-specific pathway. To further explore nutrients that support PDA growth, we looked at the effect of nitrogen-containing substrates in the Biolog assay. To achieve this aim, we used the statistical processing language R to collate and process the raw metabolic data from the large-scale assay. We calculated the maximum catabolic rate of each nitrogen-containing substrate and found glutamine-containing metabolites to consistently rescue PDA metabolism under nutrient-depleted conditions. Glutamine is the most abundant non-essential amino acid in circulation and provides a valuable source of both carbon and nitrogen in biochemical processes. Plotting the distribution of all glutamine-containing substrates, we observed a broad spectrum of catabolic rates, with glutamine-glutamine dipeptides being among the most rapidly consumed, and glutamine-glutamate among the slowest. These findings emphasize the role and importance of glutamine-derived carbon and glutamine-derived nitrogen and provoke future studies. Overall, this work aims to profile the nutrient inputs that PDA is capable of consuming to support metabolism. By uncovering these alternate energy sources and pathways, we provide new and much needed insight into the characteristics of PDA and present future potential targets for metabolic therapies.