Epithelial-mesenchymal transition induction is associated with augmented glucose uptake and lactate production in pancreatic ductal adenocarcinoma

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a common malignancy with dismal prognosis. Metastatic spread and therapeutic resistance, the main causes of PDAC-related mortalities, are both partially underlined by the epithelial-mesenchymal transition (EMT) of PDAC cells. While the role of Warburg metabolism has been recognized in supporting rapid cellular growth and proliferation in many cancer types, less is known about the metabolic changes occurring during EMT, particularly in the context of PDAC.

Results: In the current study, experimental models of EMT were established in the Panc-1 cell line of human PDAC via exposure to two physiologically relevant EMT inducers (tumor necrosis factor-α and transforming growth factor-β) and the metabolic consequences examined. The two EMT models displayed similar alterations in the general metabolic profile including augmented glucose uptake and lactate secretion as well as the lack of change in oxidative metabolism. Examination of molecular markers revealed differences in the pathways underlying the metabolic rewiring. ¹³C-Glucose tracer data confirmed that a major portion of accumulated lactate was derived from glucose, but subsequent flux analysis suggested involvement of non-canonical pathways towards lactate production.

Conclusions: Our results characterize the metabolic reprogramming occurring during PDAC cell EMT and highlight the common changes of increased glucose uptake and lactate secretion under different EMT conditions. Such insight is urgently required for designing metabolic strategies to selectively target cells undergoing EMT in PDAC.

Keywords: 13C Metabolomics; Epithelial-mesenchymal transition; Glucose metabolism; Pancreatic adenocarcinoma; Tumor metabolism.

Fig. 1

EMT is induced upon TNFα and TGFβ treatments. Panc-1 cells were cultured in the presence of 40 ng/ml TNFα (α) or 10 ng/ml TGFβ (β) or both (+) for 72 h. a Western blot detection of IkB, phospho-Smad2/3, total-Smad2/3, and Snail at 0, 0.25, 0.5, 1, 3, 24, 48, and 72 h time points. β-actin was used as a loading control. b Cell morphology under bright-field microscopy at the end of incubation. c Western blot detection of E-cadherin, N-cadherin, vimentin, and Snail. β-actin was used as a loading control. d, e Measurements of SNAI1 and SNAI2 mRNA levels by qPCR. β-actin was used as a housekeeper. f Cells were seeded onto six-well plates and allowed to grow to 50 % confluence in the presence of added factors for 2 days. The monolayer was wounded with a plastic tip and monitored under bright-field microscope for 48 h with continued treatments with images taken at 0, 24, and 48 h. Cell migration was quantified by assessing percentage wound closure using the ImageJ software. Results are shown as mean ± SEM with n = 3. *p<0.05, **p < 0.01, ***p < 0.001

Fig. 2

Glycolysis was promoted upon TNFα- and TGFβ-induced EMT. Panc-1 cells were first cultured in the presence of 40 ng/ml TNFα or 10 ng/ml TGFβ or both for 72 h to induce EMT. a Rate of glucose uptake was measured using the non-metabolisable glucose analogue ³H-2-deoxy-glucose tracer over an 8-min period. The amount of ³H radiation trapped in the cells were counted using a beta-counter. b Lactate assay was performed on cell culture media collected after 72 h from the respective groups. c Western blot detection of hexokinase II (HK2), fructose-1,6-bisphosphotase1 (FBP1), fructose-1,6-bisphosphotase 2 (FBP2), pyruvate kinase isoform 2 (PKM2) and lactate dehydrogenase-A (LDH-A). β-actin was used as a loading control. d-l qPCR measurements of SLC2A1 (encoding Glut1), SLC2A3 (encoding Glut3), lactate dehydrogenase B (LDH-B), monocarboxylate transporter 1, (MCT1), monocarboxylate transporter 4 (MCT4) and pyruvate dehydrogenase kinase 1-4 (PDK 1-4) transcripts using β-actin as a housekeeper. Results are shown as mean ± SEM with n = 5 (a) or n = 3 (b-l). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Oxidative metabolism is with TNFα- and TGFβ-induced EMT. Panc-1 cells were cultured in the presence of 40 ng/ml TNFα or 10 ng/ml TGFβ or both for 72 h to induce EMT. a Oxygen consumption rate (OCR) was measured using the Seahorse XF24 Bioanalyzer at basal conditions. b Western blot detection of mitochondrial electron transport chain complex III, II, and I. β-actin was used as a loading control. c Glucose oxidation was measured using the U-¹⁴C-glucose tracer over a 1-h period. The amount of ¹⁴C in CO₂ originated from ¹⁴C-glucose was counted using a beta counter to give an indication of glucose oxidation rate. d Total lipid in the cells was extracted using the chloroform/methanol method following 1 h incubation in U-¹⁴C-glucose-containing media. The amount of ¹⁴C incorporated in the total lipid was quantified using a beta counter. e Western blot detection of fatty acid synthase (FAS), phospho- and total-acetyl-CoA carboxylase (P-ACC and T-ACC), and stearoyl-CoA desaturase-1 (SCD1). β-actin was used as a loading control. Results are shown as mean ± SEM with n = 5 (a) or n = 3 (b, e) or n = 4 (c, d). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

[U-¹³C]-Glucose tracer model data and flux results. Panc-1 cells were cultured in the presence of 40 ng/ml TNFα or 10 ng/ml TGFβ or both for 72 h prior to the start of the labelling experiment. a Metabolic model used for fitting of the extracellular enrichment data, allowing for the reversible exchange of pyruvate and lactate. G6P glucose 6-phosphate, G3P glyceraldehyde 3-phosphate. b Estimated rates of lactate production and uptake of non-glucose substrates from extracellular enrichment data. c Estimated yields of pyruvate dehydrogenase (PDH) and NADP-dependent malic enzyme (ME1) from intracellular enrichment data. Box-and-whisker plots show the 2.5th, 25th, 50th, 75th, and 97.5th percentile values; optimum values shown as asterisks. d Enrichment fractions of intracellular metabolites pyruvate (PYR), lactate (LAC), alanine (ALA), malate (MAL), aspartate (ASP), α-ketoglutarate (AKG), and citrate (CIT) showing the fractions of carbon atoms of these metabolites derived from glucose