Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development

Abstract

Most patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with advanced disease and survive less than 12 months. PDAC has been linked with obesity and glucose intolerance, but whether changes in circulating metabolites are associated with early cancer progression is unknown. To better understand metabolic derangements associated with early disease, we profiled metabolites in prediagnostic plasma from individuals with pancreatic cancer (cases) and matched controls from four prospective cohort studies. We find that elevated plasma levels of branched-chain amino acids (BCAAs) are associated with a greater than twofold increased risk of future pancreatic cancer diagnosis. This elevated risk was independent of known predisposing factors, with the strongest association observed among subjects with samples collected 2 to 5 years before diagnosis, when occult disease is probably present. We show that plasma BCAAs are also elevated in mice with early-stage pancreatic cancers driven by mutant Kras expression but not in mice with Kras-driven tumors in other tissues, and that breakdown of tissue protein accounts for the increase in plasma BCAAs that accompanies early-stage disease. Together, these findings suggest that increased whole-body protein breakdown is an early event in development of PDAC.

Fig. 1. Plasma metabolites and risk of future pancreatic cancer diagnosis

P–values of the log–transformed, continuous metabolite levels from human plasma comparing pancreatic cancer cases and controls in conditional logistic regression models conditioned on matching factors and adjusted for age at blood draw (years, continuous), fasting time (<4, 4–8, 8–12, ≥12 hours, missing) and race/ethnicity (White, Black, other, missing). The dashed green line indicates the statistically significant P–value threshold after Bonferroni correction for multiple–hypothesis testing, P–trend ≤0.0006 (0.05/83). The dashed blue line indicates P– trend of 0.05.

Fig. 2. Plasma BCAAs are elevated during subclinical disease

a, Graph of odds ratio (95% confidence interval) for future pancreatic cancer diagnosis among cohort cases and matched controls comparing highest versus lowest quintiles of circulating BCAAs stratified by time from blood collection to the case's cancer diagnosis. Odds ratio (95% confidence interval) from conditional logistic regression models conditioned on matching factors and adjusted for age at blood draw (years, continuous), fasting time (<4, 4–8, 8–12, ≥12 hours, missing) and race/ethnicity (White, Black, other, missing). Red horizontal line marks an odds ratio of 1.0. b. Graph of total plasma BCAAs in KPC mice over time and in littermate controls. Each control data point is an average for one mouse over the course of the study (Supplementary Fig. 3b) and values for KPC mice living longer than 19 weeks are averaged for the >19 weeks time point. For weeks 15-17, n = 6 KPC and n = 9 control, t-test, P=0.001. c, H&E staining of pancreatic tissue obtained from KP^–/–C mice and littermate controls at 3-4 weeks of age. Tissue are shown from a control mouse with histologically normal pancreas (left); a KP^–/–C mouse with areas of PDAC adjacent to areas of normal pancreas (middle); and a KP^–/–C mouse with areas of PDAC and pancreatic intraepithelial neoplasia (closed arrow heads) (right); scale bar = 50μM. d, Mean (±SEM) body weights at 3-4 weeks of age for KP^–/–C mice and littermate controls (n = 7 KP^–/–C, n = 11 control mice). e, Mean (±SEM) total plasma branched chain amino acid levels from KP^–/–C mice and littermate controls at 3-4 weeks of age (n = 10 KP^–/–C, n = 14 control mice, t-test, P=0.002). f, P–values for comparison of circulating amino acid levels in KP^–/–C mice and littermate controls at 3-4 weeks of age, cys = cystine (n = 10 KP^–/–C, n = 14 control mice), The dashed red line indicates P–value of 0.05. g, top, glucose tolerance test in KP^–/–C mice and littermate controls at the time of weaning (n = 7 KP^–/–C, n = 11 control mice) and bottom, insulin tolerance test in KP^–/–C mice and littermate controls at four weeks of age (n = 7 KP^–/–C, n = 15 control mice). h, Mean (±SEM) fasting plasma insulin levels from KP^–/–C mice and littermate controls at four weeks of age (n = 7 KP^–/–C, n = 11 control mice).

Fig. 3. BCAA elevations are derived from a long–term pool of amino acids

a, Plasma levels (mean ± SEM) of ¹³C–labeled leucine and valine normalized to food intake over time following a two–hour exposure to diets containing ¹³C–labeled leucine and valine. The time points correspond to the red arrowheads in the diagram. b, Diagram of experiment using labeled diets to investigate contributions to plasma BCAA levels from long–term pools. Two cohorts of mice were used for these experiments, one sacrificed in the fed state and a second sacrificed in the fasted state at the time points indicated by the red arrowheads. c, Fractional labeling of total amino acids in protein hydrolysate of gastrocnemius muscle from fasted KP^–/–C mice and control littermates (n = 8 KP^–/–C, n = 6 control). d, Fractional labeling of plasma amino acids in KP^–/–C and control mice in the fed state (n = 3 KP^–/–C, n = 4 control). e, The calculated contribution of the short– and long–term BCAA pools to the BCAAs present in plasma . f, Mean (±SEM) gastrocnemius weight (left panel, t-test, P=0.01), a predominantly fast–twitch muscle, and heart weight (right panel) normalized to body weight (n = 6 KP^–/–C, n = 10 control).