Sarani Ghoshal

Metformin prevents hepatocellular carcinoma development by suppressing hepatic progenitor cell activation in a rat model of cirrhosis.

Hepatocellular carcinoma (HCC)‐associated mortality is increasing at an alarming rate, and there is a readily identifiable cohort of at‐risk patients with cirrhosis, viral hepatitis, nonalcoholic fatty liver disease, and diabetes. These patients are candidates for chemoprevention. Metformin is an attractive agent for chemoprevention because it is inexpensive, has a favorable safety profile, and is well tolerated over long time periods.

STAT3 is a key transcriptional regulator of cancer stem cell marker CD133 in HCC

Cancer stem cell (CSC) marker CD133 was found to be upregulated in many cancers including hepatocellular carcinoma (HCC). However, the molecular mechanism of CD133 regulation in the liver tumor microenvironment has remained elusive. In this study Won and colleagues report that interleukin-6 (IL-6) mediated signal transducer and activator of transcription factor 3 (STAT3) signaling and hypoxia enhance the expression of CD133 and promote the progression of HCC.

Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells

Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.

Molecular magnetic resonance imaging accurately measures the antifibrotic effect of EDP‐305, a novel farnesoid X receptor agonist

We examined a novel farnesoid X receptor agonist, EDP‐305, for its antifibrotic effect in bile duct ligation (BDL) and choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) models of hepatic injury. We used molecular magnetic resonance imaging with the type 1 collagen‐binding probe EP‐3533 and the oxidized collagen‐specific probe gadolinium hydrazide to noninvasively measure treatment response. BDL rats (n = 8 for each group) were treated with either low or high doses of EDP‐305 starting on day 4 after BDL and were imaged on day 18. CDAHFD mice (n = 8 for each group) were treated starting at 6 weeks after the diet and were imaged at 12 weeks. Liver tissue was subjected to pathologic and morphometric scoring of fibrosis, hydroxyproline quantitation, and determination of fibrogenic messenger RNA expression. High‐dose EDP‐305 (30 mg/kg) reduced liver fibrosis in both the BDL and CDAHFD models as measured by collagen proportional area, hydroxyproline analysis, and fibrogenic gene expression (all P < 0.05). Magnetic resonance signal intensity with both EP‐3533 in the BDL model and gadolinium hydrazide in the CDAHFD model was reduced with EDP‐305 30 mg/kg treatment (P < 0.01). Histologically, EDP‐305 30 mg/kg halted fibrosis progression in the CDAHFD model. Conclusion: EDP‐305 reduced fibrosis progression in rat BDL and mouse CDAHFD models. Molecular imaging of collagen and oxidized collagen is sensitive to changes in fibrosis and could be used to noninvasively measure treatment response in clinical trials. (Hepatology Communications 2018;2:821‐835)

Orthotopic and heterotopic murine models of pancreatic cancer and their different responses to FOLFIRINOX chemotherapy

ABSTRACT Syngeneic, immunocompetent allograft tumor models recapitulate important aspects of the tumor microenvironment and have short tumor latency with predictable growth kinetics, making them useful for trialing novel therapeutics. Here, we describe surgical techniques for orthotopic and heterotopic pancreatic ductal adenocarcinoma (PDAC) tumor implantation and characterize phenotypes based on implantation site. Mice (n=8 per group) were implanted with 104 cells in the pancreas or flank. Hy15549 and Han4.13 cell lines were derived from primary murine PDAC (Ptf1-Cre; LSL-KRAS-G12D; Trp53 Lox/+) on C57BL/6 and FVB strains, respectively. Single-cell suspension and solid tumor implants were compared. Tumors were treated with two intravenous doses of FOLFIRINOX and responses evaluated. All mice developed pancreatic tumors within 7 days. Orthotopic tumors grew faster and larger than heterotopic tumors. By 3 weeks, orthotopic mice began losing weight, and showed declines in body condition requiring euthanasia starting at 4 weeks. Single-cell injection into the pancreas had near 100% engraftment, but solid tumor implant engraftment was ∼50% and was associated with growth restriction. Orthotopic tumors were significantly more responsive to intravenous FOLFIRINOX compared with heterotopic tumors, with greater reductions in size and increased apoptosis. Heterotopic tumors were more desmoplastic and hypovascular. However, drug uptake into tumor tissue was equivalent regardless of tumor location or degree of fibrosis, indicating that microenvironment differences between heterotopic and orthotopic tumors influenced response to therapy. Our results show that orthotopic and heterotopic allograft locations confer unique microenvironments that influence growth kinetics, desmoplastic response and angiogenesis. Tumor location influences chemosensitivity to FOLFIRINOX and should inform future preclinical trials. This article has an associated First Person interview with the first author of the paper. Summary: Orthotopic and heterotopic allograft locations confer unique microenvironments that influence growth kinetics, desmoplasia and angiogenesis. These differences translate to differential sensitivities to FOLFIRINOX and should inform future preclinical study design.

Inhibition of Acetyl-CoA Carboxylase by Phosphorylation or the Inhibitor ND-654 Suppresses Lipogenesis and Hepatocellular Carcinoma

The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.

Abstract 5248: Pioglitazone prevents hepatocellular carcinoma development in a rat model of cirrhosis

Introduction: Advanced hepatocellular carcinoma (HCC) is a leading cause of mortality worldwide with limited treatment options. There is a readily identifiable cohort of cirrhosis patients at risk and they are ideal candidates for chemoprevention. Anti-hyperglycemic agents have garnered interest for their potential anti-fibrotic as well as chemo-preventive effects. Pioglitazone, a selective PPAR-γ agonist, has been shown to reduce non-alcoholic steatohepatitis (NASH), but its role as an anti-fibrotic and chemopreventive agent has yet to be elucidated. The hypothesis of this study is that Pioglitazone reduces cirrhosis and subsequent HCC development in rats with diethylnitrosamine (DEN)-induced cirrhosis. Methods: Male Wistar received DEN 50mg/kg by intraperitoneal injection. DEN injury reliably recapitulates histological and molecular features of human HCC development with induction of hepatic fibrosis at 8 weeks, cirrhosis at 12 weeks, and HCC by 18 weeks. DEN-injured rats were randomized to receive oral gavage of pioglitazone at 3mg/kg/day (n=9) or vehicle control (n=9). Initiation of pioglitazone coincided with the development of liver fibrosis at 8 weeks. All animals were sacrificed at 18 weeks. Results: As expected, repeated injections of DEN in rats resulted in progressive fibrosis, cirrhosis, followed by HCC formation. Treatment with pioglitazone resulted in a 56% reduction of surface nodules relative to treatment with vehicle (7.4±4.9 vs. 17±7; p Conclusion: Overall our data supports the hypothesis that the anti-diabetic agent pioglitazone may be repurposed as a drug to reduce fibrosis and prevent HCC. This could be beneficiary in patient management given the low cost as well as minimal side effects. Citation Format: Shen Li, Sarani Ghoshal, Gunisha Arora, Derek J. Erstad, Michael Lanuti, Kenneth K. Tanabe, Bryan C. Fuchs. Pioglitazone prevents hepatocellular carcinoma development in a rat model of cirrhosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5248. doi:10.1158/1538-7445.AM2017-5248

Abstract 3781: Combination therapy with a liver selective acetyl-CoA carboxylase inhibitor ND-654 and sorafenib improves efficacy in the treatment of cirrhotic rats with hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is increasing in incidence worldwide. Current treatment options for HCC are limited, and as such, prognosis is extremely poor with a 5-year survival less than 12%. Sorafenib is the only FDA-approved drug for the treatment of HCC but its effects are marginal as it only extends survival by a few months. Therefore, new treatment options are urgently needed. Metabolic attenuation is a promising approach to cancer therapy, and rate-limiting steps in key biosynthetic pathways are particularly attractive targets. We recently identified ND-654, a hepatoselective (∼3000:1 liver to muscle exposure), allosteric ACC inhibitor that binds to the ACC subunit dimerization site and inhibits the enzymatic activity of both ACC1 (IC50 = 3 nM) and ACC2 (IC50 = 8 nM). Daily oral administration of 10 mg/kg ND-654 reduced tumor incidence by 55% and significantly improved median survival time in a rat model of cirrhosis and HCC. Here, we examine the effects of ND-654 alone and in combination with sorafenib on HCC development in cirrhotic rats. Methods: Male Wistar rats were treated once weekly with 50 mg/kg diethylnitrosamine (DEN) for 18 weeks to induce sequential development of fibrosis, cirrhosis and HCC. After establishment of cirrhosis and when HCCs are first developing (13 weeks), rats were treated daily by oral gavage with either 1) vehicle control, 2) 10 mg/kg ND-654, 3) 10 mg/kg sorafenib, or 4) 10 mg/kg ND-654 and 10 mg/kg sorafenib. After 18 weeks, tumor nodules were counted and liver and tumor tissue was harvested for analysis. Results: Similar to our previous study, simultaneous inhibition of ACC1 and ACC2 significantly reduced HCC incidence by 41% (p Citation Format: Lan Wei, Geraldine Harriman, Sarani Ghoshal, Omeed Moaven, Jeremy Greenwood, Sathesh Bhat, William F. Westlin, H. James Harwood, Rosana Kapeller, Kenneth K. Tanabe, Bryan C. Fuchs. Combination therapy with a liver selective acetyl-CoA carboxylase inhibitor ND-654 and sorafenib improves efficacy in the treatment of cirrhotic rats with hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3781.