Spontaneous Liver Tumors Research Articles

Hepatic transcriptome profiling reveals early signatures associated with disease transition from non-alcoholic steatosis to steatohepatitis

Background and aimNon-alcoholic fatty liver disease (NAFLD) is becoming a leading cause of chronic liver disease worldwide. The molecular events that influence disease progression from non-alcoholic fatty liver (NAFL) to aggressive non-alcoholic steatohepatitis (NASH) remain incompletely understood, leading to lack of mechanism-based targeted treatment options for NASH. This study aims to identify early signatures associated with disease progression from NAFL to NASH in mice and humans. Materials and methodsMale C57BL/6J mice were fed a high-fat, -cholesterol, and -fructose (HFCF) diet for up to 9 months. The extent of steatosis, inflammation, and fibrosis was evaluated in liver tissues. Total RNA sequencing (RNA-seq) was conducted to determine liver transcriptomic changes. ResultsAfter being fed the HFCF diet, mice sequentially developed steatosis, early steatohepatitis, steatohepatitis with fibrosis, and eventually spontaneous liver tumor. Hepatic RNA-seq revealed that the key signatures during steatosis progression to early steatohepatitis were pathways related to extracellular matrix organization and immune responses such as T cell migration, arginine biosynthesis, C-type lectin receptor signaling, and cytokine-cytokine receptor interaction. Genes regulated by transcription factors forkhead box M1 (FOXM1) and negative elongation factor complex member E (NELFE) were significantly altered during disease progression. This phenomenon was also observed in patients with NASH. ConclusionsIn summary, we identified early signatures associated with disease progression from NAFL to early NASH in a mouse model that recapitulated key metabolic, histologic, and transcriptomic changes seen in humans. The findings from our study may shed light on the development of novel preventative, diagnostic, and therapeutic strategies for NASH.

Chronic liver disease enables gut Enterococcus faecalis colonization to promote liver carcinogenesis.

Gut dysbiosis is observed in chronic hepatobiliary diseases and is frequently associated with liver carcinogenesis; however, the extent and specific mechanisms triggered by alterations in the microbiota mediating tumorigenesis in these patients remain unclear. Here we show that Enterococcus faecalis is abundant in the microbiota of patients with hepatitis C virus-related chronic liver disease. Xenotransplantation of gut microbiota from these patients increased the number of spontaneous liver tumors in mice and enhanced susceptibility to liver carcinogens. Hepatic colonization by gelE-positive E. faecalis increased liver expression of proliferative genes in a TLR4-Myd88-dependent manner, leading to liver tumorigenesis. Moreover, decreased fecal deoxycholic acid levels were associated with colonization by E. faecalis. Overall, these data identify E. faecalis as a key promoter of liver carcinogenesis.

ZHX2 Inhibits SREBP1c-Mediated <i>De Novo</i> Lipogenesis in Hepatocellular Carcinoma <i>via</i> miR-24-3p

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Lipogenesis has been considered as a critical player in HCC initiation and progression. However, the underlying mechanism is still not fully understood. METHODS: ZHX2 overexpression and knockdown were introduced into HCC cell lines. BODIPY staining and D-Glucose- 13C6 labeled lipid mass spectrometry were involved to estimate lipogenesis in HCC cell lines. Colony formation and cell proliferation assays were used to determine HCC development. Hepatocytes specific Zhx2 knockout mice (Zhx2-KOhep) were included to induce spontaneous liver tumor. qRT-PCR, western blotting and dual luciferase assays were used to detect gene regulation. FINDINGS: Ectopic expression of ZHX2 significantly inhibited de novo lipogenesis in HCC cells and decreased expression of FASN, ACL, ACC and SCD1. In accordance, ZHX2 was negatively associated with SREBP1c, the master regulator of de novo lipogenesis, in HCC cell lines and human specimens. Further studies demonstrated that ZHX2 inhibited de novo lipogenesis and consequent HCC progression via repression of SREBP1c. Furthermore, treatment with SREBP1c inhibitor, fatostatin, dampened the spontaneous tumor formation in Zhx2-KOhep mice. Mechanically, ZHX2 transcriptionally increased expression of miR-24-3p, which targets on SREBP1c. INTERPRETATION: ZHX2 inhibits de novo lipogenesis in HCC cells by down-regulating the expression of SREBP1c via miR-24-3p, and further inhibits the development of HCC. These new findings may provide a new strategy for the treatment of HCC. FUNDING STATEMENT: This work was supported by grants from the National Science Foundation of China (Key project 81830017 and 81902443,81702647,81902051,81972819), Taishan Scholarship (No.tspd20181201), National Key Research and Development Program (2018YFE0126500), National Science Foundation for Distinguished Young Scholars of China (81425012), Shandong Provincial Key Innovation project (No.2018FYJH0503), Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, Key Research and Development Program of Shandong (2019GSF108238) . DECLARATION OF INTERESTS: All authors declare no potential conflicts of interest. ETHICS APPROVAL STATEMENT: The Medical Ethics Committee of Shandong University approved the use of human specimens in accordance with the Helsinki Declaration. All mice were housed under conditions free of specific pathogens and euthanized according to regulations established by the Animal Care Committee of Shandong University.

Tumor suppressor ZHX2 inhibits NAFLD\u2013HCC progression via blocking LPL-mediated lipid uptake

Non-alcoholic fatty liver disease (NAFLD) leads to hepatocellular carcinoma (HCC). However, the underlying mechanism remains largely unclear. Here, we investigated the role of the tumor suppressor Zinc fingers and homeoboxes 2 (ZHX2) in the progression of NAFLD to HCC. ZHX2 expression was significantly decreased in fatty liver tissues, especially in the liver with NAFLD–HCC. ZHX2 overexpression disturbed lipid homeostasis of cultured HCC cells, and inhibited lipid deposition in hepatocytes both in vitro and in vivo. Moreover, ZHX2 inhibited uptake of exogenous lipids through transcriptional suppression of lipid lipase (LPL), leading to retarded proliferation of HCC cells. Importantly, LPL overexpression significantly reversed ZHX2-mediated inhibition of HCC cell proliferation, xenograft tumor growth, lipid deposition, and spontaneous liver tumor formation. Consistently, IHC staining demonstrated a negative correlation of ZHX2 with LPL in an HCC cohort. Collectively, ZHX2 protects hepatocytes from abnormal lipid deposition in NAFLD through transcriptional repression of LPL, which subsequently retards cell growth and NAFLD–HCC progression. These findings illustrate a novel mechanism of NAFLD progression into HCC.

Transcriptional analysis and differentially expressed gene screening of spontaneous liver tumors in CBA/CaJ mice

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide due to its frequent metastasis, tumor recurrence, and lack of curative treatment. However, the underlying molecular mechanisms involved in HCC progression remain unclear. Here, we analyzed the global gene expression of spontaneous liver tumor tissue from CBA/CaJ mice by RNA-Seq and identified 10,706 and 10,374 genes in the normal and liver tumor groups, respectively. Only 9793 genes were expressed in both, 913 genes were identified in only the liver tumor group, and 581 genes were found in normal liver tissues. There were 2054 differentially expressed genes (DEGs), with 975 down-regulated genes and 1079 up-regulated genes. Gene ontology (GO) term enrichment analysis showed that 43 up-regulated genes were significantly associated with cell cycle regulation and hundreds of up-regulated genes were related to cell migration, adhesion, or metabolic processes. KEGG pathway enrichment also demonstrated that some DEGs were tightly associated with the cell cycle, extracellular matrix (ECM)-receptor interactions, as well as protein digestion and absorption pathways, indicating that the activation of these oncogenic cascades was closely related to tumor liver progression in CBA/CaJ mice. Ninety-three genes with elevated expression levels preferentially localized in microtubules, kinetochores, and spindles play an important role during mitosis and meiosis and are associated with the reorganization of the cytoskeleton in cancer cells during migration and invasion. Some ECM-related genes were significantly different in the tumor group, including collagen types I, III, IV, V, and VI, non-collagenous glycoproteins, laminin, and fibronectin. We further validated the functions of upregulated genes, such as cyclin-dependent kinase 1 (CDK1) and polo-like kinase 1 (PLK1), with regards to cell cycle regulation, apoptosis, and proliferation in normal human liver or liver tumor-derived cell lines. Our results indicated that the cell cycle dysregulation, ECM-receptor interaction, and cytoskeleton-associated genes in mouse livers may promote HCC progression and deciphering the function of the genes will help investigators understand the underlying molecular mechanism of HCC.

DNA Damage, Liver Injury, and Tumorigenesis: Consequences of DDX3X Loss.

The pleiotropic roles of DEAD-box helicase 3, X-linked (DDX3X), including its functions in transcriptional and translational regulation, chromosome segregation, DNA damage, and cell growth control, have highlighted the association between DDX3X and tumorigenesis. However, mRNA transcripts and protein levels of DDX3X in patient specimens have shown the controversial correlations of DDX3X with hepatocellular carcinoma (HCC) prevalence. In this study, generation of hepatocyte-specific Ddx3x-knockout mice revealed that loss of Ddx3x facilitates liver tumorigenesis. Loss of Ddx3x led to profound ductular reactions, cell apoptosis, and compensatory proliferation in female mutants at 6 weeks of age. The sustained phosphorylation of histone H2AX (γH2AX) and significant accumulation of DNA single-strand breaks and double-strand breaks in liver indicated that the replicative stress occurred in female mutants. Further chromatin immunoprecipitation analyses demonstrated that DDX3X bound to promoter regions and regulated the expression of DNA repair factors, DDB2 and XPA, to maintain genome stability. Loss of Ddx3x led to decreased levels of DNA repair factors, which contributed to an accumulation of unrepaired DNA damage, replication stress, and eventually, spontaneous liver tumors and DEN-induced HCCs in Alb-Cre/+;Ddx3xflox/flox mice. IMPLICATIONS: These data identify an important role of DDX3X in the regulation of DNA damage repair to protect against replication stress in liver and HCC development and progression.

Emerging Players in Autophagy Deficiency-Induced Liver Injury and Tumorigenesis.

Studies using genetic mouse models that have defective autophagy have led to the conclusion that macroautophagy/autophagy serves as a tumor suppressor. One of such models is the liver-specific Atg5 or Atg7 knockout mice, and these knockout mice develop spontaneous liver tumors. It has been generally agreed that p62-mediated Nrf2 activation plays a critical role in promoting autophagy deficiency-induced liver injury and liver tumorigenesis. The mechanisms of how persistent Nrf2 activation induces liver injury and tumorigenesis are incompletely known. We discuss the recent progress on the new roles of HMGB1 and Yap in regulating liver injury and tumorigenesis in mice with liver-specific autophagy deficiency.

PO-319 Mutational signatures of 1,2-dichloropropane and dichloromethane identified in mouse carcinogenicity assays

IntroductionThe analysis of somatic mutational signatures in a single tumour can reveal clues on the natural history of this tumour, including past exposures to carcinogens. Characterising the mutational signatures of potential carcinogens used in industry may thus help identify cancers that are due to occupational exposure. Here, we used experimental models to define the mutational signatures of dichloromethane (DCM) and 1,2-dichloropropane (DCP), two solvents suspected to cause cholangiocarcinomas in occupational settings.Material and methodsExperimentally induced tumours and matching normal tissues from mice exposed to DCP (by gavage), DCM (by gavage or inhalation) or DCP +DCM (by gavage), and spontaneous tumours from vehicle/sham-exposed mice were analysed by whole-exome sequencing. Somatic mutation calling was performed to define exome-wide mutation patterns induced by DCP and DCM in these assays. Mutation data obtained in biliary tract cancers of workers in the printing industry who have been exposed to DCP and DCM as single agents or as mixtures, as well as with public somatic mutation data from biliary tract cancers were mined for the presence of the experimentally defined DCP and DCM mutational signatures.Results and discussionsLiver tumours from DCP and DCM exposed mice had distinct somatic mutations patterns compared to spontaneous liver tumours from vehicle/sham-exposed mice. While mutations in DCP-exposed mice were dominated by C:G>T:A transitions, the most frequent types of mutations in DCM-exposed mice were T:A>A:T and T:A>C:G substitutions. An average of 10.3 somatic mutations was observed per Mb in tumours from DCM-exposed mice, approximately 3-fold higher than in DCP-exposed or sham-treated mice. The mutation patterns found in DCP-exposed mice, but not DCM-exposed mice, presented some similarities with the mutational signature observed in cholangiocarcinomas of Japanese patients with occupational DCP/DCM exposure history.ConclusionThese results show that the analysis of tumour genomes from mouse carcinogenicity assays can support the characterisation of mutational signatures of carcinogenic compounds relevant to human exposures.

The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

BackgroundC3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). Epigenetic modifications, including DNA methylation, are among pivotal control mechanisms of gene expression leading to carcinogenesis. Although information on somatic mutations in liver tumors of C3H mice is available, epigenetic aspects are yet to be clarified.MethodsWe performed next generation sequencing-based analysis of DNA methylation and microarray analysis of gene expression to explore genes regulated by DNA methylation in spontaneous liver tumors of C3H mice. Overlaying these data, we selected cancer-related genes whose expressions are inversely correlated with DNA methylation levels in the associated differentially methylated regions (DMRs) located around transcription start sites (TSSs) (promoter DMRs). We further assessed mutuality of the selected genes for expression and DNA methylation in human HCC using the Cancer Genome Atlas (TCGA) database.ResultsWe obtained data on genome-wide DNA methylation profiles in the normal and tumor livers of C3H mice. We identified promoter DMRs of genes which are reported to be related to cancer and whose expressions are inversely correlated with the DNA methylation, including Mst1r, Slpi and Extl1. The association between DNA methylation and gene expression was confirmed using a DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) in Hepa1c1c7 cells and Hepa1-6 cells. Overexpression of Mst1r in Hepa1c1c7 cells illuminated a novel downstream pathway via IL-33 upregulation. Database search indicated that gene expressions of Mst1r and Slpi are upregulated and the TSS upstream regions are hypomethylated also in human HCC. These results suggest that DMRs, including those of Mst1r and Slpi, are involved in liver tumorigenesis in C3H mice, and also possibly in human HCC.ConclusionsOur study clarified genome wide DNA methylation landscape of C3H mice. The data provide useful information for further epigenetic studies of mice models of HCC. The present study particularly proposed novel DNA methylation-regulated pathways for Mst1r and Slpi, which may be applied not only to mouse HCC but also to human HCC.

Increased liver tumor formation in neutral sphingomyelinase-2-deficient mice

Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C16-ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplets, suggesting that nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs) and higher levels of phosphorylated signal transducer and activator of transcription 3, an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs, leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis.

IGFBP7 Deletion Promotes Hepatocellular Carcinoma.

Activation of IGF signaling is a major oncogenic event in diverse cancers, including hepatocellular carcinoma (HCC). In this setting, the insulin-like growth factor binding protein IGFBP7 inhibits IGF signaling by binding the IGF1 receptor (IGF1R), functioning as a candidate tumor suppressor. IGFBP7 abrogates tumors by inhibiting angiogenesis and inducing cancer-specific senescence and apoptosis. Here, we report that Igfbp7-deficient mice exhibit constitutively active IGF signaling, presenting with proinflammatory and immunosuppressive microenvironments and spontaneous liver and lung tumors occurring with increased incidence in carcinogen-treated subjects. Igfbp7 deletion increased proliferation and decreased senescence of hepatocytes and mouse embryonic fibroblasts, effects that were blocked by treatment with IGF1 receptor inhibitor. Significant inhibition of genes regulating immune surveillance was observed in Igfbp7-/- murine livers, which was associated with a marked inhibition in antigen cross-presentation by Igfbp7-/- dendritic cells. Conversely, IGFBP7 overexpression inhibited growth of HCC cells in syngeneic immunocompetent mice. Depletion of CD4+ or CD8+ T lymphocytes abolished this growth inhibition, identifying it as an immune-mediated response. Our findings define an immune component of the pleiotropic mechanisms through which IGFBP7 suppresses HCC. Furthermore, they offer a genetically based preclinical proof of concept for IGFBP7 as a therapeutic target for immune management of HCC. Cancer Res; 77(15); 4014-25. ©2017 AACR.

Abstract B20: EnCore-LNP mediated tumor delivery of MYC and CTNNB1 Dicer Substrate RNAs (DsiRNAs)

Abstract MYC and CTNNB1 are well-characterized drivers of numerous tumor types. Human and preclinical genetic evidence suggest that pharmacological intervention to reduce transactivation of MYC and CTNNB1-regulated genes would yield therapeutic benefit to many cancer patients. Since the proteins encoded by these genes are challenging to target via conventional modalities, progress in new therapeutic agents has been slow despite decades of research. RNA interference technology has enabled the inhibition of previously-undruggable genetic targets at the mRNA level, and has advanced to clinical development for several indications. DCR-MYC is a Phase I-stage lipid nanoparticle (LNP)-formulated Dicer substrate siRNA (DsiRNA), representing a potent class of RNAi triggers being developed by Dicerna Pharmaceuticals. Here we describe new preclinical data that increase our understanding of the parameters that impact tumor delivery and activity of DsiRNA. We demonstrate that the cationic lipid and PEG-lipid components of Dicerna's unique EnCore LNP platform can be modulated to improve delivery of DsiRNA to both orthotopic and spontaneous liver tumors, as well as xenograft tumors of diverse non-hepatic tissue origin. Characterization of LNP formulations with respect to plasma PK, tissue exposure and target mRNA knockdown was employed towards understanding the pharmacology of LNP-mediated tumor delivery. Citation Format: Marc Abrams, Shanthi Ganesh, Bo Ying, Girish Chopda, Utsav Saxena, Anee Shah, Martin Koser, Rokhand Arvan, Dongyu Chen, Serena Shui, Rohan Diwanji, Wei Zhou, Benjamin Holmes, Boyoung Kim, Hailin Yang, Mihir Patel, Wendy Cyr, Wendy Cyr, Natalie Pursell, Nicole Avitahl-Curtis, Hank Dudek, Cheng Lai, Weimin Wang, Bob D. Brown. EnCore-LNP mediated tumor delivery of MYC and CTNNB1 Dicer Substrate RNAs (DsiRNAs). [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B20.

Mouse Hepatic Tumor Vascular Imaging by Experimental Selective Angiography.

PurposeHuman hepatocellular carcinoma (HCC) has unique vascular features, which require selective imaging of hepatic arterial perfusion and portal venous perfusion with vascular catheterization for sufficient evaluation. Unlike in humans, vessels in mice are too small to catheterize, and the importance of separately imaging the feeding vessels of tumors is frequently overlooked in hepatic tumor models. The purpose of this study was to perform selective latex angiography in several mouse liver tumor models and assess their suitability.Materials and MethodsIn several ectopic (Lewis lung carcinoma, B16/F10 melanoma cell lines) and spontaneous liver tumor (Albumin-Cre/MST1fl/fl/MST2fl/fl, Albumin-Cre/WW45fl/fl, and H-ras12V genetically modified mouse) models, the heart left ventricle and/or main portal vein of mice was punctured, and latex dye was infused to achieve selective latex arteriography and/or portography.Results H-ras12V transgenic mice (a HCC and hepatic adenoma model) developed multiple liver nodules that displayed three different perfusion patterns (portal venous or hepatic artery perfusion predominant, mixed perfusion), indicating intra-tumoral vascular heterogeneity. Selective latex angiography revealed that the Lewis lung carcinoma implant model and the Albumin-Cre/WW45fl/fl model reproduced conventional angiography findings of human HCC. Specifically, these mice developed tumors with abundant feeding arteries but no portal venous perfusion.ConclusionDifferent hepatic tumor models showed different tumor vessel characteristics that influence the suitability of the model and that should be considered when designing translational experiments. Selective latex angiography applied to certain mouse tumor models (both ectopic and spontaneous) closely simulated typical characteristics of human HCC vascular imaging.

TH-C-17A-12: Integrated CBCT and Optical Tomography System On-Board a Small Animal Radiation Research Platform (SARRP)

Purpose: We present a newly developed on-board optical tomography system for SARRP. Innovative features include the compact design and fast acquisition optical method to perform 3D soft tissue radiation guidance. Because of the on-board feature and the combination of the CBCT, diffusive optical tomography (DOT), bioluminescence and fluorescence tomography (BLT and FT), this integrated system is expected to provide more accurate soft tissue guidance than an off-line system as well as highly sensitive functional imaging in preclinical research. Methods: Images are acquired in the order of CBCT, DOT and then BLT/FT, where the SARRP CBCT and DOT are used to provide the anatomical and optical properties information to enhance the subsequent BLT/FT optical reconstruction. The SARRP stage is redesigned to include 9 imbedded optical fibers in contact with the animal's skin. These fibers, connected to a white light lamp or laser, serve as the light sources for the DOT or FT, respectively. A CCD camera with f/1.4 lens and multi-spectral filter set is used as the optical detector and is mounted on a portable cart ready to dock into the SARRP. No radiation is delivered during optical image acquisition. A 3-way mirror system capable of 180 degree rotation around the animal reflects the optical signal to the camera at multiple projection angles. A special black-painted dome covers the stage and provides the light shielding. Results: Spontaneous metastatic bioluminescent liver and lung tumor models will be used to validate the 3D BLT reconstruction. To demonstrate the capability of our FT system, GastroSense750 fluorescence agent will be used to imaging the mouse stomach and intestinal region in 3D. Conclusion: We expect that this integrated CBCT and optical tomography on-board a SARRP will present new research opportunities for pre-clinical radiation research. Supported by NCI RO1-CA 158100

Mechanisms of STAT3 activation in the liver of FXR knockout mice

Farnesoid X receptor (FXR, Nr1h4) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. FXR is essential in maintaining bile acid (BA) homeostasis, and FXR(-/-) mice develop cholestasis, inflammation, and spontaneous liver tumors. The signal transducer and activator of transcription 3 (STAT3) is well known to regulate liver growth, and STAT3 is feedback inhibited by its target gene, the suppressor of cytokine signaling 3 (SOCS3). Strong activation of STAT3 was detected in FXR(-/-) mouse livers. However, the mechanism of STAT3 activation with FXR deficiency remains elusive. Wild-type (WT) and FXR(-/-) mice were used to detect STAT3 pathway activation in the liver. In vivo BA feeding or deprivation was used to determine the role of BAs in STAT3 activation, and in vitro molecular approaches were used to determine the direct transcriptional regulation of SOCS3 by FXR. STAT3 was activated in FXR(-/-) but not WT mice. BA feeding increased, but deprivation by cholestyramine reduced, serum inflammatory markers and STAT3 activation. Furthermore, the Socs3 gene was determined as a direct FXR target gene. The elevated BAs and inflammation, along with reduced SOCS3, collectively contribute to the activation of the STAT3 signaling pathway in the liver of FXR(-/-) mice. This study suggests that the constitutive activation of STAT3 may be a mechanism of liver carcinogenesis in FXR(-/-) mice.

ACB‐PCR measurement of spontaneous and furan‐induced H‐ras Codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver

Furan is a rodent liver carcinogen, but the mode of action for furan hepatocarcinogenicity is unclear. H-ras codon 61 mutations have been detected in spontaneous liver tumors of B6C3F1 mice, and the fraction of liver tumors carrying H-ras codon 61 CAA to AAA mutation increased in furan-treated mice. Allele-specific competitive blocker PCR (ACB-PCR) has been used previously to quantify early, carcinogen-induced increases in tumor-associated mutations. The present pilot study investigated whether furan drives clonal expansion of pre-existing H-ras mutant cells in B6C3F1 mouse liver. H-ras codon 61 CAA to CTA and CAA to AAA mutations were measured in DNA isolated from liver tissue of female mice treated with 0, 1, 2, 4, or 8 mg furan/kg body weight, five days per week for three weeks, using five mice per treatment group. Spontaneous levels of mutation were low, with two of five control mice having an H-ras codon 61 CTA or AAA mutant fraction (MF) greater than 10(-5) . Several furan-treated mice had H-ras codon 61 AAA or CTA MFs greater than those measured in control mice and lower bound estimates of induced MF were calculated. However, no statistically-significant differences were observed between treatment groups. Therefore, while sustained exposure to furan is carcinogenic, at the early stage of carcinogenesis examined in this study (three weeks), there was not a significant expansion of H-ras mutant cells.

1095 ROLE OF FXR IN THE ACTIVATION OF HEPATOCYTE CHEMOPROTECTION AND IN THE REFRACTORINESS OF LIVER TUMORS TO CHEMOTHERAPY

Results: We report that hURI expression in mouse hepatocytes leads to multistep tumorigenesis resulting in spontaneous and heterogeneous liver tumours, including hepatoma and HCC. We show that even in the absence of rapid proliferation, hepatocytespecific URI expression induces replicative stress and consequently genomic instability-associated DNA damage. Importantly, during early stage of tumorigenesis and prior to premalignant lesions, metabolic profiling identified alterations in pathways converging to insufficient nucleotide synthesis that fail to support normal replication and genomic stability. Finally, restoring metabolic intermediates in the liver rescued the premalignant phenotype. Conclusions: Our results identify URI as an oncogene involved in multi stage liver carcinogenesis and point to an unanticipated role of metabolic pathways alteration-dependent DNA damage leading to heterogeneous liver tumours. We finally propose that reinstating corrupted metabolic pathways could be beneficial as a preventive treatment in liver cancers. These authors contributed equally to the work: Isabel Maria Ruppen, Latifa Bakiri, Ramon Campos-Olivas, Osvaldo Grana.

Secret ingredients: who knows what's in your food?

British chef and food activist Jamie Oliver ignited a firestorm in January 2011 when he mentioned on the Late Show with David Letterman that castoreum, a substance used to augment some strawberry and vanilla flavorings, comes from what he described as “rendered beaver anal gland.”1 The next year, vegans were outraged to learn that Starbucks used cochineal extract, a color additive derived from insect shells, to dye their strawberry Frappuccino® drinks2 (eventually, the company decided to transition to lycopene, a pigment found in tomatoes3). Although substances like castoreum and cochineal extract may be long on the “yuck factor,”4 research has shown them to be perfectly safe for most people; strident opposition arose not from safety issues but from the ingredients’ origins. But these examples demonstrate that the public often lacks significant knowledge about the ingredients in foods and where they come from. This is not a new development; the public relationship to food additives has a long history of trust lost, regained, and in some cases lost again. The Federal Food, Drug, and Cosmetic (FD&C) Act of 19385 was passed shortly after the deaths of 100 people who took an untested new form of a popular drug, which contained what turned out to be a deadly additive.6 The new law was consumer oriented and intended to ensure that people knew what was in the products they bought, and that those products were safe. The law has been amended over the years in attempts to streamline and bring order to the sprawling task of assessing and categorizing the thousands of substances used in foods, drugs, and cosmetics. One result of this streamlining is that under current U.S. law, companies can add certain types of ingredients to foods without premarket approval from the thin-stretched Food and Drug Administration (FDA). In other words, there are substances in the food supply that are unknown to the FDA. In 2010 the Government Accountability Office (GAO) concluded that a “growing number of substances … may effectively be excluded from federal oversight.”7 Is this a problem? The answer depends on whom you ask.

Association of cyclin D and estrogen receptor α36 with hepatocellular adenomas of female mice under chronic endoplasmic reticulum stress

Hepatocellular adenomas (HCAs) are benign tumors that can lead to medical complications. Chronic inflammation and mutations in β-catenin, hepatocyte nuclear factor 1α, or glycoprotein 130 are potential causes for human HCA. However, additional causes may exist due to heterogeneity of HCA. We investigated whether HCA are caused by endoplasmic reticulum (ER) stress. Mice with a liver-specific deletion of the major chaperone BiP (LGKO) were used. Liver tumor occurrence was examined in LGKO with or without feeding of a high-fat diet (HFD) and characterized with immunohistochemistry with molecular markers of proliferation/malignancy. Molecular changes were analyzed with immunoblotting. Spontaneous monoclonal liver tumors were observed in 34% of LGKO females with constitutive hepatic ER stress. Lack of portal tracks or central veins, dilated sinusoidal spaces, hemorrhagic areas, active proliferation, and lipid deposits were observed in the liver tumors. HFD feeding induced multiclonal liver tumors in 83% of the LGKO females versus 0 in wild-type females. Hepatocytes reactive to antiglypican 3 antibodies were detected in the HFD-induced, but not spontaneous, tumors. In the liver tumors, inhibition of cyclin D and increase of the 36 kD estrogen receptor variant (estrogen receptor α36), active transcription activator 4/6, glycogen synthase kinase 3β, and extracellular signal-regulated protein kinases 1 and 2 were detected, whereas no change of hepatocyte nuclear factor 1α, β-catenin, p-53, androgen receptor α, or estrogen receptor α was detected. HFD activated Janus kinase and signal transducers and activators of transcription 3. Our evidence supports a novel link of HCA with ER stress and altered expression of cyclin D and estrogen receptor α36. Additional stress such as HFD may promote malignant transformation of HCA through the Janus kinase-signal transducers and activators of transcription pathway.

Hepatocellular carcinomas in B6C3F1 mice treated with Ginkgo biloba extract for two years differ from spontaneous liver tumors in cancer gene mutations and genomic pathways.

Ginkgo biloba leaf extract (GBE) has been used for centuries in traditional Chinese medicine and today is used as an herbal supplement touted for improving neural function and for its antioxidant and anticancer effects. Herbal supplements have the potential for consumption over extended periods of time, with a general lack of sufficient data on long-term carcinogenicity risk. Exposure of B6C3F1 mice to GBE in the 2-year National Toxicology Program carcinogenicity bioassay resulted in a dose-dependent increase in hepatocellular tumors, including hepatocellular carcinoma (HCC). We show that the mechanism of hepatocarcinogenesis in GBE exposed animals is complex, involving alterations in H-ras and Ctnnb1 mutation spectra, WNT pathway dysregulation, and significantly altered gene expression associated with oncogenesis, HCC development, and chronic xenobiotic and oxidative stress compared to spontaneous HCC. This study provides a molecular context for the genetic changes associated with hepatocarcinogenesis in GBE exposed mice and illustrates the marked differences between these tumors and those arising spontaneously in the B6C3F1 mouse. The molecular changes observed in HCC from GBE-treated animals may be of relevance to those seen in human HCC and other types of cancer, and provide important data on potential mechanisms of GBE hepatocarcinogenesis.