Background and Aims Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. Methods We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficientethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. Results CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. Conclusion These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.
Hepatocellular carcinoma (HCC) is the fastest growing cancer worldwide in part due to the obesity epidemic and fatty liver disease, particularly nonalcoholic steatohepatitis (NASH). Chronic inflammation with the release of cytokines and chemokines with activation of hepatic stellate cells results in changes of the liver extracellular matrix (ECM) that predisposes to the development of HCC. Blood levels of the gastrointestinal peptide cholecystokinin (CCK) are increased in humans and mice consuming a high-fat diet. We found that the CCK-B receptor (CCK-BR) expression increased in the livers of mice with NASH. Treatment of mice with a CCK-BR antagonist, proglumide, prevented NASH, lowered hepatic inflammatory cytokines and chemokines, reduced oxidative stress, decreased F4/80+ hepatic macrophages, and prevented HCC. CCK-AR and CCK-BR expression was increased in both murine and human HCC cell lines compared with that of normal liver, and CCK stimulated the growth of wild-type and CCK-A receptor knockout HCC cells in vitro, but not CCK-BR knockout cells suggesting that the CCK-BR mediates proliferation. Proglumide therapy significantly reduced growth by 70% and 73% in mice bearing Dt81Hepa1–6 or in RIL-75 HCC tumors, respectively. IHC of a human liver tissue array with a selective CCK-BR antibody revealed staining of human HCC and no staining in normal liver. Prevention Relevance: This investigation demonstrates the role of the gastrointestinal peptide cholecystokinin (CCK) in hepatocellular carcinoma (HCC) and how CCK-BR blockade reverses the premalignant state of the hepatic extracellular matrix hence, rendering it less susceptible to the development of HCC. Thereby, CCK-BR blockade is a novel approach for the prevention/treatment of HCC.
Pancreatic cancer is resistant to chemotherapy in part due to the dense desmoplastic fibrosis surrounding the tumor, the immunosuppressive cells in the tumor microenvironment (TME), and the early rate of metastases. In this study, we examined the effects of a CCK receptor antagonist, proglumide, alone and in combination with gemcitabine in murine models of pancreatic cancer. Tumor growth rate, metastases, and survival were assessed in mice bearing syngeneic murine or human pancreatic tumors treated with PBS (control), gemcitabine, proglumide, or the combination of gemcitabine and proglumide. Excised tumors were evaluated histologically for fibrosis, immune cells, molecular markers, and uptake of chemotherapy by mass spectroscopy. Peripheral blood was analyzed with a microRNAs biomarker panel associated with fibrosis and oncogenesis. Differentially expressed genes between tumors of mice treated with gemcitabine monotherapy and combination therapy were compared by RNAseq. When given in combination the two compounds exhibited inhibitory effects by decreasing tumor growth rate by 70%, metastases, and prolonging survival. Proglumide monotherapy altered the TME by decreasing fibrosis, increasing intratumoral CD8+ T-cells, and decreasing arginase-positive cells, thus rendering the tumor sensitive to chemotherapy. Proglumide altered the expression of genes involved in fibrosis, epithelial–mesenchymal transition, and invasion. CCK-receptor antagonism with proglumide renders pancreatic cancer susceptible to chemotherapy.
<p>Supplemental Table S1 and Supplemental Figures S1-S3</p>
OBJECTIVES/GOALS: NASH increases the risk of cirrhosis and liver cancer. High-fat diets increase CCK levels and CCK receptors have been identified on fibroblasts and immune cells. We hypothesized that CCK receptor blockade could prevent NASH by altering the hepatic microenvironment and macrophage activation. METHODS/STUDY POPULATION: Female mice were fed a Choline Deficient Ethionine supplemented (CDE) saturated fat diet or control high-fat diet for 18 weeks. Mice in each group were treated with a CCK receptor antagonist, proglumide (0.1 mg/ml) in the drinking water or regular water. Resected livers were stained for H&E for features of NASH and F4/80 for macrophages analysis. Liver RNA was evaluated for the expression of cytokines and chemokines using an 84-gene Profiler array (Qiagen). Oxidative stress was analyzed by qRT-PCR for heat shock proteins (HSPs) 27, 60, 70 and 90 and for glutathione by a fluorometric assay. Differences in CDE fed and CDE/proglumide-treated mouse livers were evaluated. RESULTS/ANTICIPATED RESULTS: Livers from mice on the CDE diet displayed histologic features of NASH that were prevented by proglumide. Cytokines and chemokines expression, especially CCL20 and CCL2, were increased in the CDE fed mice and these levels were reduced greater than 20-fold with proglumide. Infiltration of F4/80+ macrophages was markedly increased in the CDE livers and these were reduced by > 50% (p < 0.0001) with proglumide. RNA expression of HSP70 (p = 0.006) and HSP27 (p = 0.011) were reduced with proglumide. Hepatic glutathione concentration more than doubled in the CDE/proglumide treated mice compared to CDE mice. CCK-B receptor expression increased in the CDE-fed mouse livers compared to controls. DISCUSSION/SIGNIFICANCE OF IMPACT: CCK receptor blockade decreases NASH by reducing hepatic macrophages, oxidative stress, and blocking inflammatory cytokines and chemokines. This data supports our novel hypothesis that CCK receptors play a role in NASH and proglumide may provide an innovative treatment for this condition.
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