Metabolic plasticity imparts erlotinib-resistance in pancreatic cancer by upregulating glucose-6-phosphate dehydrogenase

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant forms of cancer. Lack of effective treatment options and drug resistance contributes to the low survival among PDAC patients. In this study, we investigated the metabolic alterations in pancreatic cancer cells that do not respond to the EGFR inhibitor erlotinib. We selected erlotinib-resistant pancreatic cancer cells from MiaPaCa2 and AsPC1 cell lines. Metabolic profiling of erlotinib-resistant cells revealed a significant downregulation of glycolytic activity and reduced level of glycolytic metabolites compared to the sensitive cells. The resistant cells displayed elevated expression of the pentose phosphate pathway (PPP) enzymes involved in ROS regulation and nucleotide biosynthesis. The enhanced PPP elevated cellular NADPH/NADP+ ratio and protected the cells from reactive oxygen species (ROS)-induced damage. Inhibition of PPP using 6-aminonicotinamide (6AN) elevated ROS levels, induced G1 cell cycle arrest, and sensitized resistant cells to erlotinib. Genetic studies identified elevated PPP enzyme glucose-6-phosphate dehydrogenase (G6PD) as an important contributor to erlotinib resistance. Mechanistically, our data showed that upregulation of inhibitor of differentiation (ID1) regulates G6PD expression in resistant cells thus contributing to altered metabolic phenotype and reduced response to erlotinib. Together, our results highlight an underlying role of tumor metabolism in PDAC drug response and identify G6PD as a target to overcome drug resistance.

Keywords: Erlotinib resistance; Metabolic reprogramming; Pancreatic cancer.

Fig. 1

Erlotinib-resistant cells display downregulated glycolysis. a Effect of erlotinib treatment (48 h) on the survival of erlotinib-sensitive (MiaPaCa2 and AsPC1) and erlotinib-resistant (MiaPaCa/Erlo and AsPC/Erlo) cells was analyzed using clonogenic assay (n = 3). b Representation of central carbon metabolism. c, d Immunoblot and real-time PCR analyses depicting glycolytic enzyme levels in drug-sensitive and drug-resistant cells. HK2, hexokinase 2; GPI, glucose phosphate-isomerase; PFK, phosphofructokinase; PKM, pyruvate kinase M; PGM, phosphoglycerate mutase; PGK, phosphoglycerate kinase; LDHA, lactate dehydrogenase A (n = 2) (n = 3). e The metabolic phenotype was assessed in cells using Seahorse metabolic analyzer. Graph showing ECAR (glycolysis) levels in sensitive and resistant cells (n = 3). f Pyruvate levels were assessed in cells using LC-MS analysis. Graph showing pyruvate levels relative to MiaPaCa2 cells (n = 2). Data presented as average ± SEM (*p < 0.05, ^#p < 0.01)

Fig. 2

Upregulated PPP protects resistant cells from oxidative stress. a Real-time PCR analysis was performed to determine the levels of pentose phosphate pathway (PPP) enzymes. Graph represents enzyme levels in MiaPaCa/Erlo cells relative to MiaPaCa2 cells. G6PD, glucose 6-phoshate dehydrogenase; PGLS, 6-Phosphogluconolactonase; 6PGD, 6-phosphogluconate dehydrogenase; RPE, ribulose-phosphate 3-epimerase; RPI, ribulose-phosphate 4-isomerase; TKT, transketolase; TALDO, transaldolase (n = 2). b Drug-sensitive and drug-resistant cells were analyzed for G6PD levels (immunoblot) and glutathione content (glutathione detection kit) (n = 3). c DCFDA assay was performed to determine overall ROS levels in cells (top) (n = 3). Cells treated with hydrogen peroxide (H₂O₂) for 10 min at indicated concentration were analyzed for clonogenic survival (bottom) (n = 2). d Untreated and 6AN-treated (48 h) cells were analyzed for NADPH/NADP+ content using commercial kit (n = 2). e, f Effect of PPP inhibition (48 h 6AN treatment at indicated concentrations) was determined on cellular ROS and glutathione content in MiaPaCa/Erlo cells (n = 3). g Erlotinib-treated (48 h) cells were analyzed for ROS levels using DCFDA dye (n = 3). Data presented as average ± SEM (*p < 0.05, ^#p < 0.01)

Fig. 3

Resistant cells are sensitive to PPP inhibition. a Graph depicting clonogenic survival of resistant and sensitive cells treated with 6AN for 48 h at indicated concentrations (n = 4). b Clonogenic survival assay was performed on drug-sensitive and drug-resistant cells treated with erlotinib in combination with 6AN (48-h combination treatment) (n = 3). c Cell cycle analysis was performed on resistant cells treated with 6AN (48 h) by flow cytometry using propidium iodide stained cells (n = 4). Effect of 6AN on cyclins was ascertained using immunoblot analysis (n = 3). d Effect of G6PD knockdown (72-h post siRNA transfection) on MiaPaCa/Erlo cell cycle distribution and cyclin levels was determined (n = 2). e Effect of G6PD knockdown on MiaPaCa/Erlo cell sensitivity to erlotinib was determined using clonogenic assay. Comparative effect of G6PD siRNA on survival of MiaPaCa2 and MiaPaCa/Erlo cell was also determined in same experiment (n = 2). f MiaPaCa2 cells transfected with G6PD overexpression plasmid (G6PD/pRK5) were analyzed for their sensitivity to erlotinib using clonogenic survival assay (n = 3). The results were compared with empty vector transfected MiaPaCa/Erlo cells that were treated with erlotinib. Data presented as average ± SEM (*p < 0.05, ^#p < 0.01)

Fig. 4

Myc regulates G6PD levels in resistant cells. a Immunoblot depicting c-myc levels in the resistant cells. Effect of siRNA-mediated downregulation of c-myc on G6PD levels was assessed using immunoblot analysis (n = 3). b Effect of c-myc inhibition, c-myci (10058F4, c-myc inhibitor), was analyzed on G6PD levels (n = 3). c Effect of acute c-myci treatment on extracellular acidification rate of MiaPaCa/Erlo cells was analyzed using Seahorse metabolic analyzer (n = 2). d Levels of ID1 were compared in drug-sensitive and drug-resistant cells using immunoblot analysis (n = 3). e Effect of ID1 knockdown was assessed on levels of c-myc and G6PD in MiaPaCa/Erlo cells using immunoblot analysis (n = 3)