Figure 5 from PPARδ Orchestrates a Prometastatic Metabolic Response to Microenvironmental Cues in Pancreatic Cancer

PPARδ controls the balance between OXPHOS and glycolysis, linked to EMT and metastasis. A,In vitro invasion in PDAC-215, 253, and 354 cells stably transduced with inducible lentiviral vectors expressing either a nontargeting short hairpin RNA (NT shRNA) or three different shRNAs against PPARD (sh#1, sh#2, and sh#3). Transduced cells were pretreated with doxycycline for 24 hours, then incubated with MCM, etomoxir (Eto), or L-165 for 48 hours, and finally plated in modified Boyden chambers for 16 hours (n = 7). B, Top, ZsGreen expression by RT-qPCR in liver homogenates from an in vivo metastasis assay of PDAC-354 cells stably expressing either the NT or the sh#1 against PPARD. Cells were pretreated with doxycycline and/or 20 μmol/L etomoxir for 48 hours. After intrasplenic implantation, mice were treated with oral doxycycline (2 mg/mL; drinking water) and etomoxir (15 mg/kg, i.p. daily) for 7 days, when splenectomies were performed. Bottom, numbers indicate the percentage and total number of micrometastases in each experimental group. C, PDAC-215, 253, and 354 transduced cells as in A were pretreated with doxycycline for 24 hours, then incubated with MCM, etomoxir, or L-165, and then tested for ATP-linked respiration (top) and glycolytic capacity (bottom) after additional 24 hours (n = 8). D, Mitochondrial stress test (top row) and glycolysis test (bottom row) following treatment with control (Ctrl) or the PPARδ agonists L-165 or GW0742. Left column, representative OCR and extracellular acidification rate (ECAR) profiles for PDAC-253. Right column, pooled data for PDAC-215, 253, and 354 cells (n = 6–9). Glyco, glycolytic; Max res, maximum respiration; O, ATP synthase inhibitor oligomycin; F, mitochondrial OXPHOS uncoupler FCCP carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; A+R, complex III inhibitor antimycin A + electron transport change inhibitor rotenone. G, glucose; 2DG, glycolysis inhibitor 2-deoxy-glucose. E, ATP-linked respiration (left) and maximal respiration (right) for control vs. GW0742-treated cells following treatment with or without palmitate-BSA (FAO assay). PDAC-354 cells were treated with 10 μmol/L GW0742 for 48 hours prior to the assay (n = 5). In A, C, and D, the bars represent pooled data from PDAC-215, 253, and 354, showing individual data points corresponding to each PDX. All data are represented as the mean ± SEM. *, P P P P