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.

Abstract Pancreatic cancer (PC) is one of the most lethal malignancies and a devastating disease almost uniformly lethal with a <6% five year survival. Developing novel strategies to prevent or delay progression of PC is currently of intense interest. Epidemiologic studies show that diabetes mellitus is positively associated with increased risk for PC. Recent meta-analysis showed that metformin, an insulin-lowering agent, reduces risk of PC. Studies have clearly demonstrated that metformin elicits a protective effect by targeting mTOR signaling and tumor-initiating cancer stem cells (TICSC). However, metformin efficacy and systematic analysis of mechanisms have not been evaluated in a preclinical model that recapitulates human PC. We tested the effects of metformin on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC) in Kras-activated p48Cre/+-LSL-KrasG12D/+ transgenic mice. Six-week old male and female KrasG12D/+ mice (∼35/group) were fed AIN-76A diets containing 0, 1000 and 2000 ppm metformin for 38 weeks. Pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To evaluate molecular mechanisms, we analyzed modulation of mTOR signaling molecules: mTOR, pAMPK, IRS1, TSC1, TSC2, S6, p70-S6, Rheb, ERK, pERK, ATG2, leptin, IGF1, C-reactive protein and markers of TICSC : ALDH1, CD133, CD24, CD44, EPCAM and DCLK-1 in pancreatic tumors and/or serum by IHC, IHF, Western blotting, and/or real time-PCR. Male and female mice fed control diet had 80 and 62% incidence of PDAC, respectively. Dietary administration of 1000 or 2000 ppm metformin yielded a PDAC incidence of 20 or 26%, respectively, in males (p<0.002-0.0006) and 7 or 0%, respectively, in females (p<0.0004-0.0001).The pancreatic tumor weights were decreased by 34-49% (p<0.03-0.001) with both doses of metformin. Most importantly, metformin caused >95% (p<0.0001) inhibition of carcinoma spread in the pancreas and a significant suppression of PanIN 3 lesions (carcinoma in situ) (28-39%, p<0.002). The pancreatic tissue and/or serum of mice fed metformin showed a significant decrease in mTOR signaling molecules mTOR, S6, p70-S6, Rheb, IGF-1, C-protein, ERK and pERK with an increase in pAMPK, TSC1, TSC2, IRS1 and ATG2. The TICSC markers CD44, CD133, ALDH1 and EPCAM were reduced significantly with an increase in CD24 expression (p<0.04-0.0002). These results suggest that the anti-tumor effects of metformin are mediated through decreased TICSC markers and modulation of the mTOR signaling pathway. These studies with a transgenic mouse model of pancreatic cancer suggest that metformin has significant potential for human chemoprevention trials in individuals at high-risk for pancreatic cancer. {Supported by NCI-CN-N01-53300}. Citation Format: Altaf Mohammed, Misty Brewer, Rebekah L. Ritchie, Anuj Marya, Stan Lightfoot, Naveena B. Janakiram, Vernon E. Steele, Chinthalapally V. Rao. Metformin prevents progression of pancreatic intraepithelial neoplasia to ductal adenocarcinoma by targeting cancer stem cells and mTOR signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2268. doi:10.1158/1538-7445.AM2013-2268

Pancreatic cancer still remains a devastating disease almost uniformly lethal with a <6% five year survival, despite tremendous scientific efforts for over three decades. Developing novel strategies to prevent or delay progression of pancreatic cancer is currently of intense interest. Clinical and preclinical studies have clearly demonstrated eflornithine (DFMO) as a potential chemopreventive agent for several cancers. However, efficacy of DFMO in a preclinical model that recapitulates human pancreatic cancer has not yet been evaluated. The rationale for the use of DFMO as a cancer chemopreventive agent has been strengthened in recent years because ODC has been shown to be transactivated by the c-myc oncogene in cell/tissue types and to cooperate with the ras oncogene in malignant transformation of epithelial tissues. In the present study, we tested the effects of eflornithine on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC) in Kras activated p48Cre/+-LSL-KrasG12D/+ transgenic mice. Six-week old male and female KrasG12D/+ (∼35/group) mice were fed (AIN-76A) diets containing 0, 1000 and 2000 ppm DFMO for 38 weeks. No significant body weight differences were observed within the treatment groups. Pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To understand molecular mechanisms, we analyzed levels of proliferation, apoptosis and cell cycle makers; PCNA, p21, cyclin E, Bcl-xl, c-myc, caveolin-1, cyclin D1 and β-catenin by IHC, Western blotting, and/or RT-PCR methods. Results suggests that control diet fed mice showed 85 and 65% incidence of PDAC in male and female mice, respectively. Whereas 1000 ppm DFMO diet fed mice showed 10% (male) and 16% (female) incidence of PDAC respectively; and, 2000 ppm DFMO diet fed mice showed 6% (male) and 9% (female) incidence of PDAC respectively. The pancreatic tumor weights were decreased by 31-43% (p<0.03-0.001) with both doses of DFMO in male and female mice. Most importantly, the drug treatment showed >95% (p<0.07-0.0006) inhibition of carcinoma spread in the pancreas. Also, a significant dose dependent suppression of PanIN 3 lesions (carcinoma in-situ) (27 and 31% P<0.04 in male; 21 and 33% in female) was observed in mice fed with DFMO at 1000 and 2000 ppm, respectively. The pancreas of mice fed DFMO diets showed a significant inhibition of PCNA, cyclin D1, β-catenin, cyclin E, Bcl-xl, c-myc, cavolin-1 expression levels (p<0.05-0.0002); and increased p21 and apoptosis, when compared to the pancreatic cancer tissues derived from control diet fed mice. In summary, our preclinical data clearly indicate that DFMO has significant potential for undertaking clinical trials of pancreatic cancer chemoprevention. {Supported by NCI-CN-N01-53300}. Citation Format: Altaf Mohammed, Naveena B. Janakiram, Misty Brewer, Rebekah L. Ritchie, Anuj Marya, Stan Lightfoot, Vernon E. Steele, Chinthalapally V. Rao. Eflornithine (DFMO) prevents progression of pancreatic intraepithelial neoplasia to ductal adenocarcinoma in LSL-KrasG12D/+ mice. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B115.