Pancreatic ductal adenocarcinoma (PDAC) is one of the most potent and perilous diseases known, with a median survival rate of 3-5 months due to the combination of only advanced stage diagnosis and ineffective therapeutic options. Metformin (1,1-Dimethylbiguanide hydrochloride), the leading drug used for type 2 diabetes mellitus, emerges as a potential therapy for PDAC and other human cancers. Metformin exerts its anticancer action via a variety of adenosine monophosphate (AMP)-activated protein kinase (AMPK)- dependent and/or AMPK-independent mechanisms. We present data here showing that metformin down- regulated pancreatic transcription factor pancreatic duodenal homeobox-1 (PDX-1), suggesting a potential novel mechanism by which metformin exerts its anticancer action. Metformin inhibited PDX-1 expression at both protein and mRNA levels and PDX-1 transactivity as well in PDAC cells. Extracellular signal-regulated kinase (ERK) was identified as a PDX-1-interacting protein by antibody array screening in GFP-PDX-1 stable HEK293 cells. Co-transfection of ERK1 with PDX-1 resulted in an enhanced PDX-1 expression in HEK293 cells in a dose-dependent manner. Immunoprecipitation/Western blotting analysis confirmed the ERK-PDX-1 interaction in PANC-1 cells stimulated by epidermal growth factor (EGF). EGF induced an enhanced PDX-1 expression in PANC-1 cells and this stimulation was inhibited by MEK inhibitor PD0325901. Metformin inhibited EGF-stimulated PDX-1 expression with an accompanied inhibition of ERK kinase activation in PANC- 1 cells. Taken together, our studies show that PDX-1 is a potential novel target for metformin in PDAC cells and that metformin may exert its anticancer action in PDAC by down-regulating PDX-1 via a mechanism involving inhibition of ERK signaling.

was defined as a dim colored invagination, resembling a cup or pocket shaped, measuring ≥25% of the nuclear diameter. Two independent examiners, after counting 200 blasts on peripheral blood or bone marrow slides, determined the % of CL blasts (discrepant counts were settled with the aid of a third examiner). The results were then compared with clinical, laboratory, immunophenotypic, cytogenetic [G Band Karyotype(KT)] and molecular (FLT3-ITD and NPM1) findings obtained at diagnosis. CL % were classified into 3 groups: A(<5%), B(5-9%), C(≥10%). Statistical analysis with T-Student, X 2 and Fisher exact tests were performed. Results: CLs varied from 0-30%; 63 patients (85%) had < 5% (group A); 4 (5,4%) were B and 7 (9%) had ≥10% (C). Group C patients were mostly female (5 of 7). Median white blood counts were higher in group C (69x109/L, range 1.9-202x109/L) vs B (22.7 x109/L, range 12-28.4 x109/L) vs A (12.8 x109/L, range 1.2 -212 x109/L) (CxA p = 0.002). Immunophenotyping studies showed that cases with higher CL percentages were less likely CD34 positive: [C (1 of 7) vs B (100%, 4/4) vs A (77%, 48/62) (CxA, p < 0.002)] and that they also tended to be more likely HLA-DR negative [C (3/7) vs B (4/4) vs A (47/54), (CxA, p = 0.06)]. KTs were normal in 16/45 of group A, 2/4 of group B and in 1/3 of group C. FLT3-ITD was present in 15% (11/74) of this cohort. These mutations were most frequent in group C [(3/7, 43%) vs. B (1/4, 25%) vs A (7/63, 11%) (CxA p = 0.055)]. NPM1 mutations were identified in 12/70 (17%) of the cases. These mutations were also more frequent in group C [6/7; 85.7%) vs B (0/4) vs A (6/59, 10%), (CxA/B p < 0.001)]. Summary/Conclusion: It is important to rapidly identify features related to cytogenetic and molecular abnormalities in patients with AML. Thus, a morphological evaluation is an important step of acute leukemia diagnosis. Previous studies had reported the association of CL morphology with higher blood counts, a more mature immunophenotype (negativity for CD34 and HLA-DR), normal KT and FLT3-ITD/NPM1 mutations. Notably in our study, these associations were only statistically significant when CLs were >10% of the blasts, with the exception of normal KT (probably due to the small number of evaluable cases). Curiously, at least in 3 other studies, CL morphology was also more frequent in females. Recent studies using electron-microscopy have demonstrated mitochondrial accumulation at the CL region which could be related to the pathogenesis of theses AMLs. In summary, identifying CL morphology is not difficult, and our study can validate a method to predict phenotypic features and NPM1 mutations with a high degree of confidence.