Combined Simvastatin and Metformin Treatment Targets Growth and Fibroinflammatory Responses in Pancreatic Stellate Cells (PaSC) 1 Cedars - Sinai Medical Center, Los Angeles, CA; 2 University of California, Los Angeles, CA Richard T. Waldron, 1, 2 Lihong Huo1, 1 Saumya Mehta, 1 Enrique Rozengurt, 2 Guido Eibl, 2 Stephen J. Pandol 1,2 , Aurelia Lugea 1,2 Conclusions Our data highlight chemopreventive potential of Met/Sim targeting PaSC protumor and inflammatory responses to restrain the development of pancreatic cancer and the progression of chronic pancreatitis Figure 8 Transcriptome analysis by RNA – Seq reveals marked effects of Metformin and Simvastatin in PaSC RNAseq was performed in cells treated for 24 h with Met ( 1 mM) and Sim ( 1 μM) alone or in combination (A) Violin Plots showing data distribution in the control and treatments groups (B) Venn diagrams show differentially expressed genes found upregulated or downregulated in the different treatment groups compared to the control group (C) Heatmap shows top 20 genes found significantly up - (purple) or downregulated (orange) in the indicated treatment groups compared to the control group Data was analyzed in R ; n= 3 per group Background Pancreatic stellate cells (PaSC) mediate pro - and anti - tumorigenic responses in pancreatic cancer (PC) and participate in chronic pancreatitis progression We reported that obesity and diabetes activate PaSC and accelerate PC progression in pancreas - specific Kras - G 12 D mutant ( Ptf 1 Cre/+ ; Kras G 12 D/+ ; KC) mice In obese KC mice, we found chemopreventive effects of the anti - diabetic drug Metformin (Met) and the statin Simvastatin (Sim), which decreased stromal expansion, progression of precancerous lesions and PC incidence both independently and in combination : Chang HH et al, Sci Rep 2018 ; Hao F et al ; PLoS One 2019 ; Teper Y et al, Sci Rep 2023 Met and Sim are widely prescribed FDA - approved drugs Metformin is used in the treatment of diabetes ; it reduces glycemia and improves insulin sensitivity by regulating gluconeogenesis in liver At the cellular level, metformin can activate AMPK resulting in inhibition of the cell growth regulator mTOR Simvastatin is a widely used statin with pleotropic effects via inhibition of HMG - coA reductase resulting in reduced levels of cholesterol and prenylated substrates Aims Here, we investigated in cultured PaSC the effects of these treatments on PaSC pathobiology Mechanistically, Met and Sim can alter PaSC phenotype via modulation of mTOR and YAP signaling (Fig 1 ) YAP is a transcriptional regulator we reported is upregulated in PaSC within PC tumors and is required to maintain the activation state and fibro - inflammatory responses (Fig 2 ; Hu et al ; Front Physiol 2019 ) We also found that insulin via mTOR/Akt has growth effects in PaSC and potentiates the synthesis and secretion of collagens and protumor cytokines ( Yang et al, Am J Physiol 2016 ) Figure 1. Potential effects of Met and Sim in PaSC Modified from: KRAS, YAP, and obesity in pancreatic cancer: A signaling network with multiple loops. G. Eibl and E. Rozengurt, Semin Cancer Biol. 2017 Simvastatin Metformin (1,1 - dimethylbiguanide hydrochloride) A B C D 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 fold over basal Ctgf mRNA # * * # * YAP siRNA NT TGF β 1 _ _ TGF β 1 0 1 2 3 4 fold over basal IL6 mRNA # * # * * YAP siRNA NT TGF β 1 _ _ TGF β 1 E Figure 2. Yes - Associated Protein 1 Plays Major Roles in PaSC activation and fibroinflammatory responses . In cultured PaSC, YAP knockdown by siRNA transfection (A) reduces mPaSC numbers & proliferation (48 h post transfection; B - C). YAP siRNA also alters expression levels of genes involved in matrix remodeling (D ), and markedly prevents TGF β - induced Connective tissue growth factor ( Ctgf ) and IL6 expression, two key regulators of fibrogenesis and inflammation. NT, control. Hu et al; Front Physiol 2019 Figure 3 Metformin activates AMPK and inhibits mTORC 1 pathway and YAP in PaSC (A & C) Representative western blots showing levels of phosphorylated AMPK and p 70 S 6 K, YAP and α - SMA in activated PaSC treated for 1 or 24 h with Metformin at the indicated concentrations (B & C) Graphs show densitometry values (OD) for immunoblots depicted in A and C, respectively Data in graphs are mean ± SEM ; n= 3 independent experiments Western bl. B Western bl. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Protein levels (fold over control) Metformin (mM) p-AMPK p-70S6K YAP ** * 1 2.5 5 10 ** ** ** * * * * p - AMPK Protein levels (OD) Met (mM) 0 0.1 0.5 1 2.5 5 p - P70S6K 0 0.1 0.5 1 2.5 5 A C D 1 h incubation 24 h incubation 1 h incubation 24 h incubation * P<0.05 vs. basal ** P<0.01 vs. basal Methods Mouse and human PaSC were treated with Sim ( 0 1 - 10 μM), Met ( 0 1 - 5 mM) or both, and their transcriptome, proteome and activation of selected signaling pathways analyzed by RNAseq, qPCR and Western blotting Cytokine/chemokine expression and release was assessed by qPCR and Proteome profiler cytokine array assay Figure 4 Metformin decreases expression of the profibrotic factors Col 1 a 1 and Fap, the inflammatory mediators IL 6 and Ccl 2 (MCP 1 ) and the growth factor Hgf that promotes cancer cell growth PaSC were treated for 24 and 48 h with Met at the indicated concentrations mRNA levels were measured by qPCR Graphs shows relative expression levels relative to basal (Met 0 ) ; mean ± SD ; n= 3 independent experiments * P < 0 05 vs Met 0 diet qPCR Acta2 Col1a1 IL - 6 Fap Ccl2 Hgf Met 0 Met 2.5 mM Met 5 mM mRNA Relative expression * * * * * * * * * * Figure 6 Simvastatin decreases YAP protein levels in activated PaSCs leading to reduced growth signaling and blunted TGF β 1 responses ( A) Dose - response effects of Sim on growth signaling (Akt ; mTOR) and YAP levels (B - C) Graphs show quantification of p - Akt, p - P 70 S 6 K and YAP protein levels depicted in panel A (D) Sim treatment significantly reduces TGFB 1 - induced IL - 6 upregulation Data in graphs are mean ± SEM ; n= 3 independent experiments * P < 0 05 vs basal ; # P < 0 05 vs TGF β 1 B A D C Western bl. qPCR 0 0.2 0.4 0.6 0.8 1 Fold over basal TNF α 0.30 0.04 1 G - CSF 0.50 0.015 CXCL11 0.50 0.002 IL - 6 0.53 0.033 CCL5 0.63 0.010 CXCL10 0.64 0.004 CXCL12 0.68 0.002 CXCL9 0.70 0.014 IL - 4 0.92 ns IL - 13 1.00 ns P value Effects of Simvastatin on the PaSC secretome t - test 24 h Figure 5 Simvastatin reduces TGFB 1 - induced secretion of several cytokines & chemokines PaSC were pre - treated with 5 ng/ml TGFβ 1 for 1 h and then treated for 24 h with 1 μM Simvastatin Graph shows mean ± SD, n= 2 Proteome profiler cytokine array Table lists cytokines/chemokines significantly reduced by simvastatin treatment ; IL - 4 and IL - 3 secretion was not affected change fold Met Sim Met+Sim A B Control Met Met+ Sim Sim Log(2) values Met Sim Met+Sim Met vs. Cont. Met+Sim vs. Cont. Sim vs. Cont. Overexpressed Underexpressed C Difference 5 - 5 0 Figure 9 Effects of Sim and Met on expression levels of PaSC signature genes RNAseq analysis shows that Sim and more dramatically Met+Sim treatment ( see Fig 8 ) results in reduced expression of PaSC activation markers and increase expression of the quiescent markers Gfap (Glial fibrillary acidic protein) and Ppargc 1 a (PPARG Coactivator 1 alpha) Control Met Met+Sim Sim Col1a1 Col1a2 Col3a1 Col6a1 Col5a2 Col4a1 Col5a1 Col6a2 Col16a1 Col4a2 Col4a5 Sparc Aspn Bgn Hspg2 Cdh11 Vcan Acta2 Fap Pdgfra Pdgfrb Mmp3 Mmp9 Mmp11 Mmp12 Mmp14 Mmp23 Serpine1 Ngf Fn1 Spp1 Dcn Has1 Ppargc1a Gfap Collagens and matrisomal proteins PaSC markers ECM degradation Quiescence & others - 2 2 0 z - Score -1.5 -1.0 -0.5 0.0 0.25 0.5 0.75 1 1.25 1.5 2 Log2FC vs. vehicle Figure 7 Dose - response effect of Sim and Met alone or in combination on PaSC viability Cells were treated for 72 h with Met ( 0 25 - 2 mM), Sim ( 0 25 - 2 μM) or Sim+Met Cell viability was determined by MTT assay Graph shows Log 2 FC, n= 4 MTT assay Effects of Sim and Met on cell viability Met+Sim Met Sim 0 1 2 3 4 5 6 - log 10 p value Upregulated Pathways Met+Sim vs. control Oxidative Stress Induced Gene Expression Via Nrf2 Positive regulation of fibroblast proliferation Response to glucose starvation Nervous system development Positive regulation of cell migration Lipid metabolic process Protein dephosphorylation Negative regulation of MAPK cascade Cell differentiation Endoplasmic reticulum unfolded protein response 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 - log 10 p value Downregulated Pathways Met+Sim vs. control Cholesterol homeostasis Regulation of canonical Wnt signaling pathway Regulation of JAK - STAT cascade Macrophage differentiation Regulation of I - kappaB kinase/NF - kappaB signaling Wound healing Receptor protein tyrosine kinase signaling pathway Regulation of interleukin - 6 production Activation of innate immune response Collagen fibril organization Angiogenesis Extracellular matrix organization Immune system process B A Figure 10 Selected pathways significantly impacted by Met+Sim treatment in PaSC RNAseq was performed in cells treated for 24 h with Met ( 1 mM) and Sim ( 1 μM) alone or in combination Davis analysis (A - B) Graphs show enriched GO biological processes (BP) by DAVID database Differentially over - and undexpressed genes were selected based on FDR> 0 05 ; Log 2 FoldChange > 2