Hepatocellular carcinoma chemoprevention by targeting the angiotensin-converting enzyme and EGFR transactivation.

Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis-induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.

Fibrotic Response to Neoadjuvant Therapy Predicts Survival in Pancreatic Cancer and Is Measurable with Collagen-Targeted Molecular MRI.

PURPOSE: To evaluate the prognostic value of posttreatment fibrosis in human PDAC patients, and to compare a type I collagen targeted MRI probe, CM-101, to the standard contrast agent, Gd-DOTA, for their abilities to identify FOLFIRINOX-induced fibrosis in a murine model of PDAC. EXPERIMENTAL DESIGN: Ninety-three chemoradiation-treated human PDAC samples were stained for fibrosis and outcomes evaluated. For imaging, C57BL/6 and FVB mice were orthotopically implanted with PDAC cells and FOLFIRINOX was administered. Mice were imaged with Gd-DOTA and CM-101. RESULTS: In humans, post-chemoradiation PDAC tumor fibrosis was associated with longer overall survival (OS) and disease-free survival (DFS) on multivariable analysis (OS P = 0.028, DFS P = 0.047). CPA increased the prognostic accuracy of a multivariable logistic regression model comprised of previously established PDAC risk factors [AUC CPA (-) = 0.76, AUC CPA (+) = 0.82]. In multiple murine orthotopic PDAC models, FOLFIRINOX therapy reduced tumor weight (P < 0.05) and increased tumor fibrosis by collagen staining (P < 0.05). CM-101 MR signal was significantly increased in fibrotic tumor regions. CM-101 signal retention was also increased in the more fibrotic FOLFIRINOX-treated tumors compared with untreated controls (P = 0.027), consistent with selective probe binding to collagen. No treatment-related differences were observed with Gd-DOTA imaging. CONCLUSIONS: In humans, post-chemoradiation tumor fibrosis is associated with OS and DFS. In mice, our MR findings indicate that translation of collagen molecular MRI with CM-101 to humans might provide a novel imaging technique to monitor fibrotic response to therapy to assist with prognostication and disease management.

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.

Pioglitazone Reduces Hepatocellular Carcinoma Development in Two Rodent Models of Cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the deadliest malignancies worldwide due to the lack of effective treatments. Chemoprevention in high-risk patients is a promising, alternative strategy. In this study, pioglitazone was investigated for its ability to prevent hepatocarcinogenesis in two rodent models of cirrhosis. METHODS: In the first model, male Wistar rats were given repeated, low-dose injections of diethylnitrosamine (DEN) to accurately recapitulate the progression of fibrosis to cirrhosis and HCC. In the second model, a single dose of DEN was administered to male C57Bl/6 pups at day fifteen followed by administration of a choline-deficient, L-amino acid defined, high-fat diet (CDAHFD) at week six for 24 weeks. Pioglitazone treatment started at the first signs of fibrosis in both models. RESULTS: Pioglitazone effectively reduced fibrosis progression and HCC development in both models. Gross tumor nodules were significantly reduced after pioglitazone treatment (7.4 ± 1.6 vs. 16.6 ± 2.6 in the rat DEN model and 5.86 ± 1.82 vs. 13.2 ± 1.25 in the mouse DEN+CDAHFD model). In both models, pioglitazone reduced the activation of mitogen-activated protein kinase (MAPK) and upregulated the hepato-protective AMP-activated protein kinase (AMPK) pathway via increasing circulating adiponectin production. CONCLUSION: Pioglitazone is an effective agent for chemoprevention in rodents and could be repurposed as a multi-targeted drug for delaying liver fibrosis and hepatocarcinogenesis.

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

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.

Molecular Liver Cancer Prevention in Cirrhosis by Organ Transcriptome Analysis and Lysophosphatidic Acid Pathway Inhibition.

Cirrhosis is a milieu that develops hepatocellular carcinoma (HCC), the second most lethal cancer worldwide. HCC prediction and prevention in cirrhosis are key unmet medical needs. Here we have established an HCC risk gene signature applicable to all major HCC etiologies: hepatitis B/C, alcohol, and non-alcoholic steatohepatitis. A transcriptome meta-analysis of >500 human cirrhotics revealed global regulatory gene modules driving HCC risk and the lysophosphatidic acid pathway as a central chemoprevention target. Pharmacological inhibition of the pathway in vivo reduced tumors and reversed the gene signature, which was verified in organotypic ex vivo culture of patient-derived fibrotic liver tissues. These results demonstrate the utility of clinical organ transcriptome to enable a strategy, namely, reverse-engineering precision cancer prevention.

RPTOR, a novel target of miR-155, elicits a fibrotic phenotype of cystic fibrosis lung epithelium by upregulating CTGF.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the most frequent of which is F508del-CFTR. CF is characterized by excessive secretion of pro-inflammatory mediators into the airway lumen, inducing a highly inflammatory cellular phenotype. This process triggers fibrosis, causing airway destruction and leading to high morbidity and mortality. We previously reported that miR-155 is upregulated in CF lung epithelial cells, but the molecular mechanisms by which miR-155 affects the disease phenotype is not understood. Here we report that RPTOR (regulatory associated protein of mTOR, complex 1) is a novel target of miR-155 in CF lung epithelial cells. The suppression of RPTOR expression and subsequent activation of TGF-ß signaling resulted in the induction of fibrosis by elevating connective tissue growth factor (CTGF) abundance in CF lung epithelial cells. Thus, we propose that miR-155 might regulate fibrosis of CF lungs through the increased CTGF expression, highlighting its potential value in CF therapy.

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.

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

BACKGROUND: 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. METHODS: The authors studied the efficacy of metformin as a prevention agent in a clinically relevant rat model of HCC, in which tumors develop in the setting of chronic inflammation and cirrhosis. Repeated injections of diethylnitrosamine were used to induce sequential cirrhosis and HCC, and metformin was administered at the first signs of either fibrosis or cirrhosis. RESULTS: Prolonged metformin exposure was safe and was associated with decreases in fibrotic and inflammatory markers, especially when administered early at the first signs of fibrosis. In addition, early metformin treatment led to a 44% decrease in HCC incidence, whereas tumor burden was unchanged when metformin was administered at the first signs of cirrhosis. It is noteworthy that activation of the hepatic progenitor/stem cell compartment was first observed at the onset of cirrhosis; therefore, only early metformin treatment suppressed receptor for advanced glycation end products and inhibited the activation of hepatic progenitor cells. CONCLUSIONS: The current results are the first to demonstrate an effect on progenitor/stem cells in the setting of chemoprevention and provide further rationale to explore metformin as an early intervention in clinical trials of patients with chronic liver disease at high risk for HCC.

Progressive decrease of phosphocreatine, creatine and creatine kinase in skeletal muscle upon transformation to sarcoma.

In vertebrates, phosphocreatine and ATP are continuously interconverted by the reversible reaction of creatine kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, creatine-rich skeletal muscle, are good source materials to study the status of creatine and creatine kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphocreatine and creatine kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and creatine kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type creatine kinase was found to be downregulated, whereas ubiquitous mitochondrial creatine kinase was upregulated. These significantly decreased levels of creatine and creatine kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.