Antidiabetic Drug Metformin Prevents Progression of Pancreatic Cancer by Targeting in Part Cancer Stem Cells and mTOR Signaling

Abstract

Epidemiologic studies have shown that diabetes mellitus is associated positively with increased risk of pancreatic ductal adenocarcinoma (PDAC), and recent meta-analysis studies showed that metformin, reduces the risk of pancreatic cancer (PC). We tested the effects of metformin on pancreatic intraepithelial neoplasia (PanIN) and their progression to PDAC in p48Cre/+.LSL-KrasG12D/+ transgenic mice. Mice fed control diet showed 80% and 62% incidence of PDAC in males and females, respectively. Male mice showed 20% and 26%, and female mice showed 7% and 0% PDAC incidence with 1000- and 2000-ppm metformin treatments, respectively. Both doses of metformin decreased pancreatic tumor weights by 34% to 49% (P < 0.03-0.001). The drug treatment caused suppression of PanIN 3 (carcinoma in situ) lesions by 28% to 39% (P < .002) and significant inhibition of carcinoma spread in the pancreas. The pancreatic tissue and/or serum of mice fed metformin showed a significant inhibition of mammalian target of rapamycin (mTOR), extracellular signal-regulated kinases (ERK), phosphorylated extracellular signal-regulated kinases (pErk), and insulin-like growth factor 1 (IGF-1) with an increase in phosphorylated 5' adenosine monophosphate kinase (pAMPK), tuberous sclerosis complex 1 (TSC1, TSC2), C-protein and an autophagy related protein 2 (ATG2). The cancer stem cell (CSC) markers were significantly decreased (P < 0.04-0.0002) in the pancreatic tissue. These results suggest that biologic effects of metformin are mediated through decreased CSC markers cluster of differentiation 44 (CD44 and CD133), aldehyde dehydrogenase isoform 1 (ALDH1), and epithelial cell adhesion molecule (EPCAM) and modulation of the mTOR signaling pathway. Our preclinical data indicate that metformin has significant potential for use in clinical trials for PC chemoprevention.

Figures

Figure 1

(A and B) Effect of metformin on pancreas weights at the termination of the experiment in male (A) and female (B) mice. Both doses ofmetformin significantly reduced the pancreatic tumor weights. (C and D) Effect of metformin on the incidence (percentage of mice with carcinomas) of PDAC in male (C) and female (D) mice. Effect of metformin on survival and mortality of male (E) and female (F) mice.

Figure 2

(A and B) Effect of metformin on the PanIN multiplicity (means ± SE; A—male, B—female). (C and D) Percentage of carcinoma spread per pancreas (C—male, D—female; 0—0 ppm, LD—1000 ppm, HD—2000 ppm). (E and F) Effect of metformin on normal pancreas (E—male, F—female). The data in the panels were analyzed by unpaired t test with Welch's correction; values are considered statistically significant at P < .05.

Figure 3

Effect of metformin on mTOR and pAMPK in pancreatic tumors. IHC and immunofluorescence analyses were performed with paraffin-embedded and microsectioned pancreatic tissues as described in the Materials and Methods section. A significantly decreased expression of mTOR and an increased pAMPK expression were seen with metformin treatment.

Figure 4

(A–F) Effect of metformin on the expression of mRNA for mTOR, Rheb, TSC1, TSC2, cyclin D1, and ATG2, as determined with quantitative real-time PCR. (G) Effect of metformin on the expression of ERK, pERK, cMYC, CD133, and p70-S6 proteins in PDAC. (H) Effect of metformin on IGF-1 and C-protein in the serum. Protein expression was analyzed by Western blot analysis as described in the text.

Figure 5

Effect of metformin on CSC markers in pancreatic tumors. (A) Immunofluorescence analysis for CD24 and CD44 was performed on paraffin-embedded and microsectioned pancreatic tissues as described in the Materials and Methods section. A significantly decreased expression of CD24, CD44, and CD44/CD244 ratio was seen with metformin treatment. (B-F) Effect of metformin on the expression of mRNA for the CSC markers CD44, EPCAM, CD133, ALDH1, and DclK1, as determined with quantitative real-time PCR.

Figure 6

Effect of metformin on apoptosis and CSC markers. Dual immunofluorescence analysis for annexin V/CD44 (green/red) and annexin V/EPCAM (green/red) was performed with paraffin-embedded and microsectioned pancreatic tissues as described in the Materials and Methods section. A significant decrease in CSC markers (CD44 and EPCAM) was associated with an increased apoptosis (annexin V—green) on metformin treatment. Effect of metformin on the expression of mRNA for the apoptotic markers caspase-3 and Bax and anti-apoptotic marker Bcl-2 as determined with quantitative real-time PCR.