The FOXO3/PGC-1β signaling axis is essential for cancer stem cell properties of pancreatic ductal adenocarcinoma

Abstract

In 95% of patients with pancreatic ductal adenocarcinoma, recurrence is observed following chemotherapy. Findings from several studies have indicated that cancer stem cells (CSCs) are resistant to anticancer agents and may be involved in cancer recurrence and metastasis. The CD44 protein is a major CSC marker, and CD44 also plays an indispensable role in the CSC properties in several cancers, including pancreatic cancer; however, no clinical approach exists to inhibit CD44 activity. Here, we have performed knock-in/knockdown experiments, and we demonstrate that the forkhead box O3 (FOXO3)/liver kinase B1 (LKB1)/AMP-activated protein kinase/peroxisome proliferator-activated receptor-γ co-activator-1β (PGC-1β)/pyruvate dehydrogenase-A1 pathway is essential for CD44 expression and CSC properties. We observed that patients exhibiting high pyruvate dehydrogenase-A1 expression have a poor prognosis. Systemic PGC-1β knock-out mice are fertile and viable and do not exhibit an overt phenotype under normal conditions. This suggests that cGMP induction and PGC-1β inhibition represent potential strategies for treating patients with pancreatic ductal adenocarcinoma.

Keywords: CD44; FOXO; PGC-1β; liver kinase B1 (LKB1); pancreatic cancer; stem cells.

Figure 1.

LKB1 is indispensable for pancreatic cancer stem cells. A, pancreatic cancer cells were transfected with FOXO3 siRNA for 48 h, and LKB1 levels were determined by Western blotting (n = 3). S, scrambled siRNA; F, FOXO3 siRNA. B, Panc-1 cells were transfected with FOXO3 siRNA for 24 h, and STK11 (LKB1) levels were determined by quantitative RT-PCR analysis (n = 3). C, LKB1 expression in Panc-1 cells was compared with that in CD44⁺ and CD44⁻ cells by flow cytometry. D, quantitative RT-PCR showing that mRNA of STK11 (LKB1) was overexpressed in spheroid cells compared with that in adherent cells (n = 3). E and F, pancreatic cancer cells were transfected with LKB1 siRNA, and spheroid formation was assessed (n = 3). G and H, Panc-1 cells were transfected with LKB1 siRNA, and colony formation was assessed (n = 3). Error bar represents S.E. Scr, scrambled.

Figure 2.

LKB1 is essential for CD44 expression. A, Panc-1 cells were treated with Bay 41-2272 (5 μm) for 36 h. Quantitative RT-PCR shows the mRNA expression of STK11 (LKB1) (n = 3). B, Panc-1 cells were treated with Bay 41-2272 for 48 h, and LKB1 expression was examined by Western blotting (n = 3). C, Panc-1 cells were transfected with LKB1 siRNA for 48 h and examined by Western blotting. D, pancreatic cancer cells were transfected with LKB1 siRNA for 48 h and examined by flow cytometry. E, Panc-1 cells were transfected with LKB1 siRNA for 48 h and examined by Western blotting. F, spheroid assay of FOXO3 (F) siRNA, LKB1 (L) siRNA, and LKB1 expression vector (Vec.) (n = 3) is shown. G, Panc-1 cells were transfected with LKB1 siRNA for 48 h and examined for the phospho-AMPK (P-AMPK) (Thr-172) level by Western blotting (n = 3). Lanes were run on the same gel but were noncontiguous (white lines). H, the effect of AMPK inhibitor dorsomorphin (Dor.) (5 μm; 48 h) on CD44 expression in Panc-1 cells was determined by Western blotting (n = 3). Lanes were run on the same gel but were noncontiguous (white lines). I, the effect of AMPK inhibitor dorsomorphin (5 μm; 21 days) on spheroid formation was assessed (n = 3). Error bar represents S.E. Scr, scrambled; Cont., control; Rel., relative; β-act, β-actin.

Figure 3.

LKB1 plays a crucial role in PGC-1β expression. A, the effect of FOXO3 knockdown on mitochondrial membrane potential of each PDAC cell line was determined by flow cytometry using JC-1 (n = 3). Δψm, mitochondrial membrane potential. B, the impact of LKB1 knockdown on mitochondrial membrane potential was determined by flow cytometry using JC-1 (n = 3). C, pancreatic cancer cells were transfected with LKB1 siRNA for 48 h. PGC-1β expression was examined by Western blotting (n = 3). S, scrambled siRNA; L, LKB1 siRNA. Error bar represents S.E. Scr, scrambled.

Figure 4.

PGC1-β plays a crucial role in CD44 expression and pancreatic cancer stem cells. A, the effect of AMPK inhibitor dorsomorphin (5 μm; 48 h) on PGC-1β expression in Panc-1 cells was determined by Western blotting (n = 3). C, control (Cont.); D, dorsomorphin. Lanes were run on the same gel but were noncontiguous (white lines). B, immunofluorescence staining for PGC-1β and CD44 on pancreatic cancer patient tissues (magnification, ×60). Scale bar, 50 μm. Representative data from eight patients are shown. C, PGC-1β expression in Panc-1 cells was compared with that in CD44⁺ and CD44⁻ cells by flow cytometry. D, Panc-1 cells were transfected with PGC-1β siRNA for 48 h, and a colony assay was performed (n = 3). E, Panc-1 cells were transfected with PGC-1β siRNA for 48 h, and a spheroid assay was performed (n = 3). F, pancreatic cancer cells were transfected with FOXO3 siRNA for 48 h. PGC-1β expression in Panc-1 cells was determined by Western blotting (n = 3). G, Panc-1 cells were transfected with PGC-1β siRNA for 48 h, and expression levels were determined by Western blotting (n = 3). H, Panc-1 cells were treated with Bay 41–2272 (5 μm) for the indicated times, and PPARGC1B (PGC-1β) and PPARGC1A (PGC-1α) mRNA levels were measured (n = 3). I, Panc-1 cells were treated with Bay 41-2272 for 48 h, and PGC-1β expression was examined by Western blotting (n = 3). The same samples are used in Fig. 2B. C, control (Cont.); B, Bay 41-2272. J, spheroid assay of FOXO3 siRNA (F), scrambled siRNA (S), LKB1 siRNA (L), and PGC-1β expression vector (Vec.) (n = 3). Lanes were run on the same gel but were noncontiguous (white lines). Error bar represents S.E. Scr, scrambled; Rel., relative.

Figure 5.

PGC-1β plays a crucial role in PDHA1 expression. A, pancreatic cancer cells (Panc-1) were transfected with FOXO3 siRNA, and lactate levels were determined (n = 3). B, immunofluorescence staining for PDHA1 and CD44 in pancreatic cancer patient tissues and pancreatic cell lines Panc-1, BxPC-3, and MIAPaCa-2 cells (magnification, ×60). Representative data from eight patients are shown. Scale bar, 50 μm. C, quantitative RT-PCR showing the mRNA expression of PDHA1 (adherent versus spheres; n = 3). D, Panc-1 cells were transfected with FOXO3 siRNA, and PDH activity was measured (n = 3). E, pancreatic cancer cells were transfected with PGC-1β siRNA for 48 h, and PDHA1 expression was examined by Western blotting (n = 3). S, scrambled siRNA; P, PGC-1β siRNA. F, Western blot of FOXO3 siRNA-transfected Panc-1 cells. Error bar represents S.E. Scr, scrambled; Rel., relative.

Figure 6.

PDHA1 plays a crucial role in pancreatic stem cell properties. A and B, spheroid assay following transfection of pancreatic cancer cells with PDHA1 siRNA (n = 3) (magnification, ×20). Scale bar, 50 μm. C and D, colony assay following transfection of pancreatic cancer cells with PDHA1 siRNA. Error bar represents S.E. Scr, scrambled.

Figure 7.

Commonality of FOXO3 and PDHA1 knockdown by evaluating the effect of the PDHA1 knockdown on FOXO3-regulated genes. A and B, the effect of a PDHA1 knockdown on FOXO3-regulated genes was evaluated by gene set enrichment analysis. C, pancreatic cancer cells (Panc-1) were transfected with PDHA1 siRNA for 48 h and examined by Western blotting (n = 3). S, scrambled siRNA; P, PDHA1 siRNA. D, the impact of PDHA1 expression on the prognosis of patients with PDAC is shown. The difference in the overall survival was analyzed using the Kaplan-Meier survival model with a log-rank test. E, a novel pathway that impairs pancreatic carcinoma stem cancer cells is shown. Error bar represents S.E. Scr, scrambled.