Pyruvate kinase M1 regulates butyrate metabolism in cancerous colonocytes

Abstract

Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non-cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re-addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 suppresses glycolysis and inhibits hypoxia-inducible factor-1 alpha. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.

Figure 1

Expression of PKM isoform in human non-cancerous and cancerous colorectal tissues. (A) Immunofluorescence staining for PKM2 and PKM1 expression on non-cancerous colorectal tissue and colorectal cancer tissue. (B) Quantification of immunofluorescence data with relative expression of PKM2 and PKM1 in each group. Error bars are mean ± SEM for ten independent sections.

Figure 2

Butyrate oxidation in PKM2 knockdown HCT116 colorectal cancer cells. (A) Western blot analysis of PKM1 and PKM2 expression in the positive control (PC; Mouse White Adipose Tissue), HCT116, scramble (SCR), and different PKM1/2 targeted knockdown cells with β-actin as a loading control. Quantification of the western blot is shown in the right panel. For statistical analysis, western blot was conducted 5 times. Error bars are mean ± SEM. (B) Percent change in oxygen consumption rate (OCR) relative to baseline in which SCR and PKM2 KD C5 cells treated with and without butyrate (5 mM). Total contribution of butyrate toward OCR (%) is observed after injection of 2-deoxyglucose (2DG). The right panel shows the area under the curve (AUC) analysis from OCR measurements taken after 2DG injection but before azide injection. These measurements represent butyrate oxidation (arbitrary units). (C) Percent change in OCR relative to baseline in which SCR and PKM2 KD C4 cells treated with and without butyrate (5 mM). The right panel shows AUC analysis from OCR measurements taken after 2DG injection but before azide injection. These measurements represent butyrate oxidation (arbitrary units). Data points represent the average OCR (%) over 3–5 replicates per condition for butyrate oxidation measurements. Error bars are mean ± SEM.

Figure 3

Butyrate oxidation is PKM1 dependent rather than PKM2 in HCT116 colorectal cancer cells. (A) Western blot analysis of PKM1 and PKM2 expressions in HCT116, SCR, and re-expressed PKM1 (M1R) and PKM2 (M2R) cells with β-actin as a loading control. Quantification of the western blot is shown in the right panel. For statistical analysis, western blot was conducted 5 times. (B) Percent change in oxygen consumption rate (OCR) relative to baseline in which C4 and M1R cells treated with and without butyrate (5 mM). Total contribution of butyrate toward OCR (%) is observed after injection of 2-deoxyglucose (2DG). The right panel shows the area under the curve (AUC) analysis from OCR measurements taken after 2DG injection but before azide injection. These measurements represent butyrate oxidation (arbitrary units). (C) Percent change in OCR relative to baseline in which C4 and M2R cells treated with and without butyrate (5 mM). The right panel shows the area under the curve analysis from OCR measurements taken after 2DG injection but before azide injection. These measurements represent butyrate oxidation (arbitrary units). Data points represent the average OCR (%) over 3–5 replicates per condition for butyrate oxidation measurements. Error bars are mean ± SEM.

Figure 4

PKM1 is responsible for mitochondrial function in HCT116 colorectal cancer cells. (A) Percent change in oxygen consumption rate (OCR) relative to baseline in which SCR and C4 cells respond to oligomycin, FCCP, and Antimycin A/Rotenone. The right panel shows the calculated electron transport chain (ETC) accelerator response and basal respiration. (B) Percent change in OCR relative to baseline in which C4 and M1R cells respond to oligomycin, FCCP, and Antimycin A/Rotenone. The right panel shows the calculated ETC accelerator response and basal respiration. For these experiments, each cell line was treated with or without butyrate (5 mM). These measurements represent the mitochondrial function. Each data point represents the average OCR (%) over 3–5 replicates per condition. Error bars are mean ± SEM.

Figure 5

Molecular consequences in PKM1- and PKM2-diminished HCT116 colorectal cancer cells. (A) Western blot analysis of phospho-AMPK (Thr172) and total AMPK levels in HCT116, SCR, C4, C5, and M1R cells with β-actin as a loading control. The right panel shows the quantification of p-AMPK levels relative to total AMPK levels. (B) Western blot analysis of phospho-PDH (Ser293) and total PDH levels in HCT116, SCR, C4, C5, and M1R cells with β-actin as loading controls. The right panel shows the quantification of p-PDH levels relative to total PDH levels. For statistical analysis, western blot was conducted 5 times. Error bars are mean ± SEM.

Figure 6

The expression of HIF1α and SCAD is affected by PKM1. (A) Western blot analysis of HIF1α levels in HCT116, SCR, C4, and M1R cells with β-actin as a loading control. The right panel shows the quantification of HIF1a expression. (B) Western blot analysis of HIF1α levels in HCT116, SCR, and C4 cells treated with and without pyruvate (5 mM). β-actin served as a loading control. (C) Western blot analysis of SCAD levels in HCT116, SCR, C4, and M1R cells with PDH as loading controls. (D) Western blot analysis of HIF1α and SCAD levels in HCT116 cells with and without hypoxic conditions for 24 h. β-actin served as loading control for HIF1α, and PDH served as loading control for SCAD. For statistical analysis, western blot was conducted 5 times. Error bars are mean ± SEM.

Figure 7

The role of HIF1α in regulating butyrate oxidation. (A) Percent change in oxygen consumption rate (OCR) relative to baseline in which HCT116 and HIF1α KO cells treated with and without butyrate (5 mM). Total contribution of butyrate toward OCR (%) is observed after injection of 2-deoxyglucose (2DG). The right panel shows the area under the curve (AUC) analysis from OCR measurements taken after 2DG injection but before azide injection. These measurements represent butyrate oxidation (arbitrary units). Data points represent the average OCR (%) over 3–5 replicates per condition for butyrate oxidation measurements. (B) Percent change in extracellular acidification rate (ECAR) relative to baseline in which HCT116 and HIF1α KO cells respond to glucose, 2DG, and azide. The right panel shows AUC from ECAR measurements taken after glucose injection but before 2DG injection. These measurements represent glycolysis (arbitrary units). Data points represent the average ECAR (%) over 3–5 replicates per condition for glycolysis measurements. Error bars are mean ± SEM.

Figure 8

Model of PKM1 regulation of cellular metabolism in cancerous colonocyte. Working model of pathways regulating butyrate oxidation. In this model, the downregulated metabolic enzyme PKM1 is a key factor in the shift of cancerous colonocytes from oxidative metabolism toward aerobic glycolysis. The loss of PKM1 in cancerous colonocytes leads cells to increase glycolysis through reduction of SCAD along with an increase in HIF1α due to a decrease in pyruvate levels. The increase in HIF1α and the decrease in SCAD together explain the lower butyrate oxidation in CRC cells.