Figure 2. PDAC expresses proline metabolic enzymes.

(a) Free proline is metabolized in the mitochondrial matrix, first by proline oxidase (PRODH1), to generate 1-pyrroline-5-carboxylic acid (P5C) before conversion into glutamic-γ-semialdehyde (GSA). P5C dehydrogenase (P5CDH) metabolizes GSA into glutamate and NADH. Free proline is synthesized from P5C via pyrroline carboxylases 1 and 2 (PYCR1, 2) in the mitochondrion and PYCR3 in the cytosol. Proline incorporated into collagen (proline-Col) can be modified into hydroxyproline (OH-proline) by prolyl-4-Hydroxylases (P4H). (b,c) Localization of PRODH1 and P4HA3 in human and PKI mice PDAC by IHC (b) and in epithelial compartment by IF co-staining with the wide-spectrum cytokeratin (Ws KRT) epithelial marker in PKI mice (c). Representative images from n=4 patients (b) and n=3 PKI mice (b,c). Tumour glands and disseminated cells are indicated by asterisks and arrows, respectively. Scale bar, 100 μm. (d) Quantification of PRODH1 and P4HA3 protein levels in PKI mouse PDAC and in KI mouse control pancreas revealed by western blot analysis (upper panel, with marker size indicated in kDa). Protein levels in each tissue samples (histograms, lower panel, from n=3 control KI mice and n=3 PDAC PKI mice) are normalized to total loaded-proteins (Amido black staining). Data expressed as mean±s.e.m. (***P<0.001; two-tailed unpaired Student’s t-test). Uncropped images of blots are shown in Supplementary Fig. 10. (e) Quantification of PRODH1 protein levels in normal human pancreas (n=11) and human PDAC samples (n=10). PRODH1 levels are normalized to one normal pancreas used as a reference. Data are illustrated as mean±95% CI, *** P<0.001; Mann–Whitney test. Images of human PDAC and normal pancreas macroarray are displayed in Supplementary Fig. 1. CI, confidence interval.