Cysteine catabolism and the serine biosynthesis pathway support pyruvate production during pyruvate kinase knockdown in pancreatic cancer cells

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with limited treatment options. Pyruvate kinase, especially the M2 isoform (PKM2), is highly expressed in PDAC cells, but its role in pancreatic cancer remains controversial. To investigate the role of pyruvate kinase in pancreatic cancer, we knocked down PKM2 individually as well as both PKM1 and PKM2 concurrently (PKM1/2) in cell lines derived from a Kras ^G12D/- ; p53 ^-/- pancreatic mouse model.

Methods: We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine metabolic profiles of wildtype and PKM1/2 knockdown PDAC cells. We further used stable isotope-labeled metabolic precursors and LC-MS/MS to determine metabolic pathways upregulated in PKM1/2 knockdown cells. We then targeted metabolic pathways upregulated in PKM1/2 knockdown cells using CRISPR/Cas9 gene editing technology.

Results: PDAC cells are able to proliferate and continue to produce pyruvate despite PKM1/2 knockdown. The serine biosynthesis pathway partially contributed to pyruvate production during PKM1/2 knockdown: knockout of phosphoglycerate dehydrogenase in this pathway decreased pyruvate production from glucose. In addition, cysteine catabolism generated ~ 20% of intracellular pyruvate in PDAC cells. Other potential sources of pyruvate include the sialic acid pathway and catabolism of glutamine, serine, tryptophan, and threonine. However, these sources did not provide significant levels of pyruvate in PKM1/2 knockdown cells.

Conclusion: PKM1/2 knockdown does not impact the proliferation of pancreatic cancer cells. The serine biosynthesis pathway supports conversion of glucose to pyruvate during pyruvate kinase knockdown. However, direct conversion of serine to pyruvate was not observed during PKM1/2 knockdown. Investigating several alternative sources of pyruvate identified cysteine catabolism for pyruvate production during PKM1/2 knockdown. Surprisingly, we find that a large percentage of intracellular pyruvate comes from cysteine. Our results highlight the ability of PDAC cells to adaptively rewire their metabolic pathways during knockdown of a key metabolic enzyme.

Keywords: Liquid chromatography mass spectrometry; Metabolism; PKM; Pancreatic cancer; Pyruvate kinase.

Fig. 1

PKM2 and PKM1/2 knockdown do not decrease proliferation of pancreatic cancer cells. a, b PDACs proliferate at the same rate with (+Dox) or without (−Dox) PKM1/2 knockdown. Proliferation was assessed by counting cell numbers in triplicates for 4 days following 7 days of vehicle (−Dox) or 1 μg/ml doxycycline (+Dox) treatment to maximize PKM2 or PKM1/2 knockdown. Western blot confirms PKM1/2 knockdown in PDACs after 7 days of vehicle (−Dox) or doxycycline (+Dox) treatment. c Relative intracellular metabolites levels are represented by peak intensities and are displayed relative to −Dox averages. Values are the average of three biological replicates. Error bars represent standard deviation. Statistically significant differences (p value < 0.05) are marked with asterisks (*). d ¹³C-glucose labeling of intracellular metabolites for 24 h in A13M13 PDAC cells with vehicle (−Dox) or PKM1/2 knockdown (+Dox). The y-axis for all graphs is the percent labeling of indicated ¹³C-isotopologue

Fig. 2

Pancreatic cancer cells upregulate the serine biosynthesis pathway following PKM1/2 knockdown. a Pool sizes of intracellular metabolites in PDAC cells treated with (+Dox) or without (−Dox) PKM1/2 knockdown were detected using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). b ¹³C-Glucose flux to serine is upregulated in A13M13 PDAC cells with PKM1/2 knockdown (+Dox). Statistically significant differences (p value < 0.01) are marked with asterisks (**). c Metabolic pathways that may generate pyruvate from upstream glycolytic intermediates during PKM knockdown. NPL, N-acetylneuraminate pyruvate lyase; SDH, serine dehydratase

Fig. 3

PHGDH knockout depletes intracellular serine, slightly decreases pyruvate generation, but does not affect cell proliferation. a Proliferation rates of wild-type A13M13 (PHGDH WT) or PHGDH knockout (PHGDH KO) populations of PDACs with vehicle (−Dox) or PKM1/2 knockdown (+Dox). Cell counts were measured daily (n = 3) after vehicle or doxycycline treatment. Western blot confirms PHGDH knockout in PDACs. b Relative intracellular metabolites levels are represented by peak intensities and are displayed relative to WT metabolite intensity averages. Cells are collected after 7 days of doxycycline treatment. Values are the average of three biological replicates. Error bars represent standard deviation. c ¹³C-Glucose labeling of intracellular metabolites for 24 h in WT and PHGDH KO PDAC clone P24 cells with PKM1/2 knockdown. The y-axis for all graphs is the percent labeling of indicated full labeled ¹³C-isotopologue. Experiments were performed in triplicates and all data are displayed as the mean values ± standard error. Statistically significant differences (p value) are marked with asterisks (*p < 0.05, **p < 0.01)

Fig. 4

PHGDH and NPL dual KO does not impact growth rate and pyruvate generation from glucose. a Average proliferation rates of 3 PHGDH/NPL dual knockouts (dual KO) clones or wild-type PDACs with vehicle (−Dox) or PKM1/2 knockdown (+Dox). Cell counts were measured daily (n = 3). b ¹³C-Glucose labeling of intracellular metabolites for 60 min in wild-type (WT) or PHGDH/NPL dual knockout clone D7 cells with PKM1/2 knockdown for 60 min. The y-axis for all graphs is the percent labeling of indicated ¹³C-isotopologue. Experiments were performed in triplicates, and all data are displayed as the mean values ± standard error

Fig. 5

Investigation of additional pathways for pyruvate generation. a Metabolic pathways that may generate pyruvate from other intermediates during PKM1/2 knockdown. PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase. b ¹³C-Glutamine labeling of intracellular metabolites for 60 min in wild-type (WT) and PHGDH/NPL dual KO D7 PDAC cells with PKM1/2 knockdown. c ¹³C-Serine labeling of intracellular metabolites for 60 min in PHGDH/NPL dual KO D7 PDAC cells with PKM1/2 knockdown. d ¹³C-Tryptophan, ¹³C-threonine, and ¹³C-cysteine labeling of intracellular metabolites for 60 min in PHGDH/NPL dual KO D7 PDAC cells with PKM1/2 knockdown. The y-axis for all graphs is the percent labeling of indicated ¹³C-isotopologue. Experiments were performed in triplicates and all data are displayed as the mean values ± standard error. Ser, serine; Trp, tryptophan; Thr, threonine; Cys, cysteine

Fig. 6

Labeling with multiple tracers shows that ~ 20% of pyruvate is generated from cysteine. Multi-tracer of 1,2-¹³C₂-glucose, 5-¹³C₁-glutamine, and ¹³C-cysteine labeling of intracellular metabolites in PHGDH/NPL dual knockout D7 PDAC cells following with vehicle (−Dox) or PKM1/2 knockdown (+Dox). The y-axis for all graphs is the percent labeling of indicated ¹³C-isotopologue. Experiments were performed in triplicates and all data are displayed as the mean values ± standard error