Statins Inhibit Proliferation of Pancreatic Ductal Adenocarcinoma Cancer (PDAC) Cells Acting Synergistically with Metformin and Inhibitors of MEK and MTOR

of metformin on PDAC cell proliferation are mediated through AMPK. Recently, AMPK has been shown to phosphorylate YAP at a site (Ser94) that inhibits its activity. We therefore hypothesized that statins and metformin synergistically inhibit PDAC cell proliferation, thereby increasing the potency of these inhibitory drugs. Results: To determine whether statin act synergistically with metformin on PDAC cell proliferation, we examined the effect of statins without and with metformin on the ability of PANC-1 and MiaPaCa-2 cells to form colonies (>50 cells/colony), using culture medium containing a physiological concentration of glucose (5 mM). Under these conditions, metformin, at 50-100 mM, dose-dependently decreased colony formation by these PDAC cells. The key point is that a combination of metformin (100 mM) with a low concentration of cerivastatin (0.01 mM) dramatically inhibited colony formation. Similarly, a low concentration of metformin (50 mM) acted synergistically with simvastatin (0.3 mM) to inhibit colony formation by either PANC-1 or MiaPaCa-2 cells. These results underpin a new combinatory approach for PDAC chemoprevention using FDA-approved drugs. In previous studies, we found that active-site mTOR inhibitors induce ERK over-activation in PDAC cells and that co-targeting these pathways enhanced inhibition of PDAC cell proliferation (Mol Cancer Ther. 14:1014-23, 2015). Accordingly, treatment of PANC-1 cells with the dual PI3K/mTOR inhibitor BEZ235 or the MEK inhibitor PD0325901 (each at a concentration as low as 5 nM) modestly inhibited colony formation while their combination resulted in a more pronounced inhibition. Strikingly, a combination of nanomolar concentrations of BEZ235, PD0325901 and cerivastatin dramatically inhibited colony formation by PANC-1 or MiaPaCa-2 cells. Collectively, our results revealed synergistic inhibition of PDAC colony formation by statins in combination with metformin or dual PI3K/mTOR inhibitors + MEK inhibitors. Conclusions: Many studies using statins in model systems in vitro have been questioned for using high concentrations of these agents (10-100 mM). The salient feature of our results is that the potentiating effects of metformin or mTOR inhibitors and MEK inhibitors on the ability of statins to inhibit long-term PDAC cell proliferation (colony forming assay), rendered statins biologically active at clinically relevant concentrations.

Mo2059 Figure 2. Network of eligible comparisons The size of circle is proportional to the number of participants received the treatment (sample size). A) Overall recurrence of colorectal adenomas; B) Recurrence of advanced adenomas; C) Serious adverse events.Every line links direct comparable treatments; The width of the lines reflect the number of trials.

STATINS POTENTLY INHIBIT YAP ACTIVITY AND PROLIFERATION OF MOUSE PANCREATIC CELLS ISOLATED FROM KC AND KPC MICE Fang Hao, Jen-Kuan Chang, James Sinnett-Smith, Guido Eibl, Hui-Hua Chang, Enrique Rozengurt Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human diseases. The highly conserved Hippo pathway is a key regulator of development, organsize, tissue regeneration, tumorigenesis, PDAC development and a novel target for the statins. The conditional KrasG12D mouse model (LSL-KrasG12D; p-48-Cre; referred as KC) of PDAC development is characterized by the presence of an activating Kras mutation, which is conditionally expressed in pancreatic progenitor cells during embryologic development. Coexpression of a p53 mutant (p53R172H) greatly accelerates the development of invasive PDAC (model referred as KPC mice). To extend our studies on the effect of statins on PDAC (see our other Abstracts), we examined the effect of statins on pancreatic cells derived from KC and KPC mice. Results: We examined the effects of cerivastatin and simvastatin on YAP localization, YAP/TEAD-regulated gene expression and colony formation in a pancreatic cell line established from KC mice. We found that treatment of KC cells with either cerivastatin or simvastatin induced striking translocation of YAP from the nucleus to the cytoplasm, thereby blunting the ability of YAP to act as a co-activator of TEAD transcription factors. In line with this possibility, exposure to cerivastatin or simvastatin inhibited the expression of YAP/TEAD-regulated genes in a dose-dependent manner, as shown by decreased mRNA levels of Ctgf, Cyr61 and Birc5. Half-maximal effect (IC50) was produced by cerivastatin at a concentration as low as 0.03 mM. At the highest concentration tested (0.3 mM), these statins inhibited Ctgf, or Cyr61 mRNA levels by 90%. Importantly, treatment with cerivastatin or simvastatin markedly inhibited colony formation by KC cells in a dose-dependent manner. In accord with the results obtained with human PDAC cells, treatment of KC cells with metformin markedly increased the inhibitory effect of cerivastatin or simvastatin on colony formation. Additional studies showed that treatment with cerivastatin strikingly inhibited the expression of YAP/TEAD-regulated genes, including Ctgf, Cyr61 and Birc5 and the colonyforming ability of KPC cells. Cerivastatin inhibited the proliferation of KPC cells at lower concentrations than KC cells (IC50=0.01 mM), suggesting that the expression of the p53 mutant enhances the sensitivity of PDAC cells to statins. Conclusions: Our results show that statins, specific inhibitors of the 3-hydroxy-methylglutaryl CoA reductase (HMGCR), potently inhibited YAP function and proliferation in KC and KPC cells. The results, together with our accompanying Abstracts using human cells and recent epidemiological findings linking statin administration with reduced incidence of PDAC, raise the possibility of repurposing FDA-approved statins for the prevention and therapy of PDAC, a devastating disease with a dismal prognosis.

Figure 4. Median rank and SUCRA values of competing agents SUCRA=surface under the cumulative ranking curve. CrI=credibility interval. For both efficacy(recurrence colorectal adenomas) and safety(incidence of serious adverse events), SUCRA would be 1 when a treatment is certain to be the best and 0 when a treatment is certain to be the worst. The higher SUCRA, the higher probablity of the agent to be the most efficacious or safest.

Mo2060 STATINS POTENTLY INHIBIT YAP FUNCTION AND PROLIFERATION OF PANCREATIC DUCTAL ADENOCARCINOMA CANCER (PDAC) CELLS Fang Hao, Qinhong Xu, Yinglan Zhao, James Sinnett-Smith, Steven H. Young, Jan V. Stevens, Enrique Rozengurt Background: The highly conserved Hippo pathway is a key regulator of development, organsize, tissue regeneration, tumorigenesis and a novel target for the statins. Hippo signals are transduced through a kinase cascade that phosphorylates the transcriptional co-activators YAP and TAZ. Recently, YAP has been identified as a major downstream target of RAS, AMPK, growth factor signaling and cell-cell adhesion, all of which are implicated in pancreatic ductal adenocarcinoma cancer (PDAC). Here, we determined whether statins inhibit YAP function and proliferation of PDAC cells. Results: Exposure of PANC-1 and MiaPaCa-2 cells to cerivastatin, simvastatin or fluvastatin dramatically inhibited the expression of YAP/ TEAD-regulated genes, including Connective Tissue Growth Factor (CTGF) and Cysteinerich angiogenic inducer 61 (Cyr61) in a dose-dependent manner, as determined by RTqPCR. At the highest concentration tested, these statins inhibited CTGF or Cyr61 mRNA levels by 90%. Treatment with cerivastatin also blocked the increase in CTGF mRNA levels

Mo2058 STATINS INHIBIT PROLIFERATION OF PANCREATIC DUCTAL ADENOCARCINOMA CANCER (PDAC) CELLS ACTING SYNERGISTICALLY WITH METFORMIN AND INHIBITORS OF MEK AND MTOR Qinhong Xu, Fang Hao, Yinglan Zhao, James Sinnett-Smith, Enrique Rozengurt Background: The transcriptional co-activators YAP and TAZ are key regulators of development, organ-size, tissue regeneration, tumorigenesis and a novel target for the statins. Recently, YAP has been identified as a major downstream target of RAS, growth factor signaling and cell-cell adhesion, all of which are implicated in pancreatic ductal adenocarcinoma cancer (PDAC). Previously, we demonstrated that the inhibitory effects of low concentrations

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AGA Abstracts

AGA Abstracts

with presumed increased risk of CRC, moderate-to-low dose aspirin and sulindac-associated combination therapy are both effective for recurrent colorectal adenomas. Future studies are required to provide more precise estimates of the optimal NSAIDs with an effective dose and low adverse events.We also suggest the further evaluation of NSAIDs-associated combination regimens and other novel agents (e.g.metformin)in the chemoprevention of CRC.