Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is an extremely lethal disease due to late diagnosis, aggressiveness and lack of effective therapies. Considering its intrinsic heterogeneity, patient stratification models based on transcriptomic and genomic signatures, with partially overlapping subgroups, have been established. Besides molecular alterations, PDAC tumours show a strong desmoplastic response, resulting in profound metabolic reprogramming involving increased glucose and amino acid consumption, as well as lipid scavenging and biosynthesis. Interestingly, recent works have also revealed the existence of metabolic subtypes with differential prognosis within PDAC, which correlated to defined molecular subclasses in patients: lipogenic subtype correlated with a classical/progenitor signature, while glycolytic tumours associated with the highly aggressive basal/squamous profile. Bioinformatic analyses have demonstrated that the representative genes of each metabolic subtype are up-regulated in PDAC samples and predict patient survival. This suggests a relationship between the genetic signature, metabolic profile, and aggressiveness of the tumour. Considering all this, defining metabolic subtypes represents a clear opportunity for patient stratification considering tumour functional behaviour independently of their mutational background.

Lipid droplets as metabolic determinants for stemness and chemoresistance in cancer

The pronounced desmoplastic response in pancreatic ductal adenocarcinoma (PDAC) contributes to the development of a microenvironment depleted of oxygen and nutrients. To survive in this hostile environment, PDAC cells use various adaptive mechanisms that may represent therapeutic targets. In this study, we showed that nutrient starvation and microenvironmental signals commonly present in PDAC tumors activate PPARδ to rewire cellular metabolism and promote invasive and metastatic properties both in vitro and in vivo. Mild mitochondrial inhibition induced by low-dose etomoxir or signals from tumor-associated macrophages altered the lipidome and triggered the downstream transcriptional program of PPARδ. Specifically, PPARδ reduced mitochondrial oxygen consumption and boosted the glycolytic capacity by altering the ratio of MYC and PGC1A expression, two key regulators of pancreatic cancer metabolism. Notably, genetic or pharmacologic inhibition of PPARδ prevented this metabolic rewiring and suppressed both invasiveness in vitro and metastasis in vivo. These findings establish PPARδ as a central driver of metabolic reprogramming in response to starvation and tumor microenvironmental cues that promotes a prometastatic phenotype in PDAC, suggesting that PPARδ inhibition could serve as a therapeutic strategy to combat PDAC progression. Significance: Nutrient starvation and microenvironmental signals activate PPARδ in pancreatic cancer to support survival and metastasis by promoting metabolic plasticity and invasiveness, providing a strong rationale for developing PPARδ-targeted therapies for pancreatic cancer.

Induction of EMT-like phenotype and metabolic switch in PDAC cells upon starvation. A, Representative images illustrating morphologic changes for PDAC-354 cells in response to treatment for 72 hours with the complex I inhibitor metformin (3 mmol/L), the β-oxidation inhibitor etomoxir (20 μmol/L), the complex II inhibitor malonate (5 mmol/L), the pyruvate carrier inhibitor UK5099 (100 μmol/L), or tumor-like conditions [low pH (HCl 50 μmol/L) + low glucose (1 mmol/L) + 3%O2]. Scale bar, 50 μm. B, Expression of EMT-associated genes (ZEB1, SNAI1, SNAI2, LOXL2, and VIM) was determined by RT-qPCR after cells were treated for 48 hours as indicated in A or with MCM. Pooled data for PDAC-185, A6L, 215, 253, and 354 (n ≥ 4 for each cell type). Data are normalized to HPRT1. Eto, etomoxier; Mal, malonate; Met, metformin. C, PDAC-215, 253, and 354 cells were treated with MCM or 20 μmol/L etomoxir for 48 hours and seeded in modified Boyden invasion chambers containing 20% FBS in the lower compartment. The number of invasive cells was analyzed after 16 hours (n = 7–11). D, GFP+-Luciferase+-PDAC-354 cells were treated with control, MCM, or 20 μmol/L etomoxir for 48 hours and then injected intrasplenically to assess their metastatic capacity (n = 9 mice/group). Representative photographs of liver metastasis (top) and subsequent hematoxylin and eosin staining (bottom). Scale bar, 200 μm. E, Representative OCR profile for PDAC-253 cells treated for 48 hours as indicated (mitochondrial stress test). 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. F, Maximal and ATP-linked respiration (resp) in non-CSC vs. CSC cells. Bars represent pooled data from PDAC-215, 253, and 354, showing individual data points corresponding to each PDX (n = 5–7). G, Representative extracellular acidification rate (ECAR) profile for PDAC-253 cells treated for 48 hours as indicated (glycolysis test). G, glucose; O, ATP synthase inhibitor oligomycin; 2DG, glycolysis inhibitor 2-deoxy-glucose. H, Glycolysis, glycolytic capacity, and glycolytic reserve in adherent (non-CSC) vs. sphere-derived cells (CSC). Bars represent pooled data from PDAC-215, 253, and 354, showing individual data points corresponding to each PDX (n = 4–5). All data are represented as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. See also Supplementary Figs. S1–S3.

Expression of the PPAR family members and downstream targets after incubation with etomoxir and MCM

PPARδ rewires cellular metabolism regulating MYC/PGC1A balance. A, Expression of MYC, PGC1A, and MYC/PGC1A ratio in PDX-354 after mitochondrial energy deprivation during 48 to 72 hours (n = 4–7). B,MYC and PGC1A reporter assay. Promoter activity was estimated as luciferase bioluminescence at the indicated times following treatment with PPARδ agonist GW0742 or PPARD overexpression (PPARD OE; n = 3–5). C, PDAC-354 cells were transduced with inducible lentiviral vectors expressing either a nontargeting short hairpin RNA (NT shRNA) or two different shRNAs against MYC (sh#1 and sh#2) or the complete cDNA of PGC1A. Effect of MYC knockdown (shMYC, pooled data for sh#1 and sh#2) or PGC1A overexpression (PGC1A OE) on invasiveness in response to treatment with 5 μmol/L PPARδ agonist L-165 for 48 hours (n = 6–8). D, CUT&Tag analysis of MYC protein binding at the CCNE, PGC1A, SNAI2, and VIM loci. WashU Epigenome Browser tracks showing CUT&TAG signals at the mentioned loci with the indicated transcription start site (TSS). Blue signals represent MYC binding in control (Ctrl) conditions, and red signals represent MYC binding upon 24 hours of etomoxir (Eto) treatment in PDAC-002 cells. E–H, PDAC-215 and 354 cells were transduced as in A, pretreated with doxycycline for 48 hours, and then incubated with MCM or etomoxir. E, OCR changes for maximal respiration (Max resp; left) and ATP-linked respiration (n = 4; right). F, Glycolytic (Glyco) capacity (left) and reserve (n = 4; right). G,ZEB1 gene expression. H, Invasive capacity (n = 10). I, PDAC-354 cells were treated with MCM or 20 μmol/L etomoxir for 48 hours in the presence or absence of the MYC/Max interaction inhibitor Mycro3 (25 μmol/L). Cells were then seeded in modified Boyden invasion chambers containing 20% FBS in the lower compartment. The number of invasive cells was assessed after 16 hours (n = 5). In E, F, and H, the bars represent pooled data from PDAC-215 and 354, showing individual data points corresponding to each PDX. All data are represented as the mean ± SEM. #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. unstimulated control. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. NT. See also Supplemenatry Figs. S10–S13.