Mouse Model Of Hepatocarcinogenesis Research Articles

Thyroid hormone inhibits hepatocellular carcinoma progression via induction of differentiation and metabolic reprogramming

Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor β (TRβ). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRβ axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. Collectively, these findings suggest that reactivation of the T3/TRβ axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRβ, could be useful tools in HCC therapy. Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.

Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin

Hepatocellular carcinoma (HCC) cells are metabolically distinct from normal hepatocytes by expressing the high-affinity hexokinase (HK2) and suppressing glucokinase (GCK). This is exploited to selectively target HCC. Hepatic HK2 deletion inhibits tumor incidence in a mouse model of hepatocarcinogenesis. Silencing HK2 in human HCC cells inhibits tumorigenesis and increases cell death, which cannot be restored by GCK or mitochondrial binding deficient HK2. Upon HK2 silencing, glucose flux to pyruvate and lactate is inhibited, but TCA fluxes are maintained. Serine uptake and glycine secretion are elevated suggesting increased requirement for one-carbon contribution. Consistently, vulnerability to serine depletion increases. The decrease in glycolysis is coupled to elevated oxidative phosphorylation, which is diminished by metformin, further increasing cell death and inhibiting tumor growth. Neither HK2 silencing nor metformin alone inhibits mTORC1, but their combination inhibits mTORC1 in an AMPK-independent and REDD1-dependent mechanism. Finally, HK2 silencing synergizes with sorafenib to inhibit tumor growth.

Chemopreventive effects of pequi oil (Caryocar brasiliense Camb.) on preneoplastic lesions in a mouse model of hepatocarcinogenesis.

Pequi (Caryocar brasiliense Camb.), a fruit from Brazil's central region, was evaluated for its chemopreventive effects on preneoplastic liver lesions induced by the carcinogen diethylnitrosamine (DEN) in mice. BALB/c mice, 14 days of age, received an intraperitoneal injection at 10 µg/g of DEN. The mice received either of two doses of pequi oil (100 or 400 mg/kg) daily from the age of 30 days and were killed at the age of 189 days. Stereological parameters, including the volume density (Vv) and the total volume (Vtot) of the lesions (preneoplastic and adenomas), were measured and the expression of cytokeratins CK8/18 was evaluated. The total volume of lesions and adenomas was reduced by 51% in the group treated with the carcinogen and 400 mg/kg of pequi oil administered daily by an oral gavage for 25 consecutive weeks. In addition, some mice in this group did not develop lesions. Among the remaining preneoplastic lesions in this group, the number of remodelled profiles increased by 2.4-fold in the 400-mg pequi oil-treated mice relative to the 100-mg-treated mice. Our results show that pequi oil exerts a hepatoprotective effect against DEN-induced development of preneoplastic lesions and adenoma in mice and the potential for its use in the prevention of liver cancer.

Abstract 104: New liver cancer genes identified by lentiviral vector-based insertional mutagenesis in mice are associated to differential survival in hepatocellular carcinoma patients

Abstract Next generation sequencing approaches are identifying a plethora of mutations in a variety of human cancers. However, the potential clinical implications of these new findings are still limited, since the identification of cancer driving mutations is hampered by the co-occurrence of several bystander and progression related events. Here we developed a forward genetics approach based on a new lentiviral vector-based insertional mutagen aimed at identifying liver cancer initiating genes that are relevant in human hepatocarcinogenesis. We generated a replication-defective lentiviral vector (LV) engineered with long terminal repeats carrying hepatospecific enhancers capable to induce hepatocellular carcinoma (HCC) upon a single administration in 3 clinically relevant mouse models of hepatocarcinogenesis. LV injection in newborn mice was able to induce HCC in 30% of Cdkn2a deficient mice (P=0.005 Vs untreated), in 27% of liver-specific Pten deficient mice (P=0.04) and in 75% of wild type mice coupled to CCl4 administration (P=0.002). From 30 LV-induced HCCs we could retrieve LV integrations that allowed the identification of Braf, Fign, Sos1 and Dlk1-Dio3 region as candidate cancer loci. The causative role of these genes in HCC was experimentally validated in vivo by forced expression in the mouse liver. Whole transcriptome gene expression analysis allowed unveiling the molecular pathways on which the new cancer genes have an impact. We showed that tumors with integration within Dlk1-Dio3 imprinted region displayed the overexpression of the paternally expressed gene Rtl1 encoded within the region and a peculiar upregulation of oxidative phosphorylation genes. Conversely, LV-mediated upregulation of Braf and Fign caused the overexpression of all maternal genes from Dlk1-Dio3 region in HCCs which display the downregulation of oxidative phosphorylation genes. Additionally, upregulation of Fign was associated to the deregulation of Wnt pathway in HCCs. We found that all the newly identified cancer genes were significantly upregulated and amplified or deleted in the human HCCs, highlighting a relevant role of these genes in human hepatocarcinogenesis. We also identified the specific molecular signature resulting from the activation of each cancer gene found in murine HCCs. Remarkably, gene expression analyses performed on 3 different human HCC collections showed that the signatures of all the 4 new cancer genes: 1) are present also in human HCCs; 2) can distinguish different HCC stages; 3) can identify different prognostic groups of patients. Moreover, SOS1 expression levels alone can discriminate HCC patients with good or poor overall survival. This study identified new clinically relevant liver cancer genes that may provide novel prognostic markers and therapeutic targets for the diagnosis and treatment of human HCCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 104. doi:1538-7445.AM2012-104

Abstract 4982: Identification of new human liver cancer genes by a novel lentiviral vector-based insertional mutagenesis approach in three mouse models of hepatocarcinogenesis

Abstract Retrovirus or transposon-based in vivo insertional mutagenesis triggers cancer by activating nearby oncogenes upon integration and led to the discovery of many candidate oncogenes important in human cancer. However, these insertional mutagens constantly undergo integration cycles throughout the life of the animal, thus resulting in several bystander and progression-related integrations that hamper the identification of cancer-initiating events. To overcome these limitations, we generated a replication-defective lentiviral vector (LV) engineered with long terminal repeats carrying hepatospecific enhancers capable to induce hepatocellular carcinoma (HCC) upon a single administration in 3 different mouse models of hepatocarcinogenesis. LV injection in newborn mice was able to induce HCC in 30% of Cdkn2a deficient mice (0 in controls, P=0.005), in 27% of liver-specific Pten deficient mice (6% in controls, P=0.04) and in 75% of wild type mice coupled to CCl4 administration (0 in controls, P=0.002). From 30 LV-induced HCCs we could retrieve 172 unique integrations that allowed the identification of Braf, Fign, Sos1 and Dlk1-Dio3 region as candidate cancer loci. The causative role of these genes in HCC was experimentally validated in vivo by forced expression in the mouse liver. The LV integration in Braf and Sos1 generated constitutively active proteins previously found in human tumors from tissues other than liver. Genome-wide gene expression analyses identified the specific molecular signature resulting from the activation of each cancer gene found in murine HCCs. Remarkably, gene expression analyses performed on 2 different human HCC collections showed that these signatures: 1) are present also in human HCCs; 2) discriminate among normal liver, non-tumoral diseased liver and HCC; 3) can distinguish different HCC stages. All the newly identified cancer genes were significantly upregulated and/or mutated in the human HCCs, underscoring a relevant role of these genes in human hepatocarcinogenesis. Integration of LV at Dlk1-Dio3 locus resulted in the upregulation of the paternally expressed gene Rtl1 and induced HCCs characterized by a peculiar upregulation of oxidative phosphorylation genes. Oppositely, LV-mediated upregulation of Braf and Fign caused the overexpression of all maternal genes from Dlk1-Dio3 locus in HCCs which display a canonical dowregulation of oxidative phosphorylation genes. The latter expression pattern was confirmed in a cohort of human HCCs. Our data indicate that the Dlk1-Dio3 locus plays a relevant role in hepatocarcinogenesis, energy metabolism and stem cell maintenance in mice and humans.This study uncovered a role in human HCCs of some previously known and other completely new oncogenes that altogether may provide novel candidate therapeutic targets and diagnostic markers for human HCCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2011-4982

Mouse models of hepatocarcinogenesis: what can we learn for the prevention of human hepatocellular carcinoma?

There is growing evidence that chronic inflammatory processes are involved in triggering the sequence from chronic liver injury to liver fibrosis, ultimately leading to liver cancer. In the last years this process has been recapitulated in a growing number of different mouse models. However, it has remained unclear whether and how these mouse models reflect the clinical reality of human hepatocellular carcinoma (HCC). Research with animal models but also human liver specimens has indicated that the NF-κB signaling pathway might withhold a crucial function in the mediation of chronic hepatic inflammation and the transition to HCC in humans. However, previous studies led to divergent and partly conflicting results with regards to the functional role of NF-κB in hepatocarcinogenesis. Here, we discuss a new genetic mouse model for HCC, the liver-specific TAK1 knockout mouse, which lacks the NF-κB activating kinase TAK1 specifically in parenchymal liver cells. Molecular findings in this mouse model and their possible significance for chemopreventive strategies against HCC are compared to other murine HCC models.

Mouse models of hepatocarcinogenesis: what can we learn for the prevention of human hepatocellular carcinoma?

There is growing evidence that chronic inflammatory processes are involved in triggering the sequence from chronic liver injury to liver fibrosis, ultimately leading to liver cancer. In the last years this process has been recapitulated in a growing number of different mouse models. However, it has remained unclear whether and how these mouse models reflect the clinical reality of human hepatocellular carcinoma (HCC). Research with animal models but also human liver specimens has indicated that the NF-κB signaling pathway might withhold a crucial function in the mediation of chronic hepatic inflammation and the transition to HCC in humans. However, previous studies led to divergent and partly conflicting results with regards to the functional role of NF-κB in hepatocarcinogenesis. Here, we discuss a new genetic mouse model for HCC, the liver-specific TAK1 knockout mouse, which lacks the NF-κB activating kinase TAK1 specifically in parenchymal liver cells. Molecular findings in this mouse model and their possible significance for chemopreventive strategies against HCC are compared to other murine HCC models.

Hepatic oval cell response to the choline-deficient, ethionine supplemented model of murine liver injury is attenuated by the administration of a cyclo-oxygenase 2 inhibitor

Oval cell proliferation precedes neoplasia in many rodent models of hepatocellular carcinoma and prevention of this proliferative response can reduce the risk of subsequent carcinoma. This study aimed to determine whether a selective cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, affects (i) the oval cell response to liver injury in a mouse model of hepatocarcinogenesis and (ii) an oval cell line. Four-week-old mice were fed either normal chow or a choline deficient, ethionine supplemented (CDE) diet in the presence or absence of SC-236. Liver histology and oval cell numbers were determined after 2, 4, 12 and 52 weeks of treatment. Oval cells were scored using morphological criteria and positive immuno-staining for the M(2)-isozyme of pyruvate kinase (M2PK) or A6. An immortalized oval cell line (PIL-2) was used to study the in vitro effects of SC-236 on oval cell proliferation, apoptosis and Akt phosphorylation. The percentage of M2PK-positive oval cells and COX-2-positive cells was reduced by 80% and 45%, respectively, in CDE-fed mice receiving SC-236 compared with CDE-fed animals not receiving SC-236. Some M2PK-positive oval cells were also COX-2 positive. The percentage of A6-positive cells was not affected by SC-236 administration to CDE-fed mice. Administration of SC-236 increased apoptosis as evidenced by a 73% increase in the number of TUNEL-positive cells at 2 weeks in CDE-fed mice. Primary oval cells and PIL-2 cells expressed COX-2. In vitro treatment of PIL-2 cells with SC-236 resulted in a dose-dependent preferential death of A6-negative cells. Administration of 25 and 50 microM Prostaglandin E(2) partially attenuated SC-236 induced cell death by 25%. In vitro oval cell death was associated with apoptosis and a 70% reduction in Akt phosphorylation. These results suggest that the SC-236 induced reduction of M2PK-positive oval cell numbers may be due to COX-2 dependent inhibition of Akt phosphorylation and induction of apoptosis.

Hepatitis C virus acts as a tumor accelerator by blocking apoptosis in a mouse model of hepatocarcinogenesis†

We developed hepatitis C virus (HCV) core-E1-E2 and HCV core transgenic mice on a common genetic background to assess the contribution of HCV structural proteins to hepatocarcinogenesis. Eight-week-old core-E1-E2, core, and nontransgenic mice inbred on the FVBxC57Bl/6 background were treated with diethylnitrosamine (DEN) and sacrificed at 32 weeks old. Proliferation and apoptosis were assessed by immunohistochemistry. The effect of viral proteins on apoptosis was evaluated in HepG2 cells in which apoptosis was induced by anti-Fas antibody. HCCs were identified at 32 weeks in the majority of DEN-treated mice from all three groups. The mean size of HCCs was significantly larger in core-E1-E2 transgenic (4.63 +/- 1.48 mm), compared with core transgenic (0.78 +/- 0.26 mm, P = .01), and nontransgenic (1.0 +/- 0.19 mm, P = .002) mice. While there were no differences in proliferation, the apoptotic index in core-E1-E2 transgenic HCCs was significantly lower than those found in core and non-transgenic HCCs. Core-E1-E2 transfected HepG2 cells demonstrated a significantly lower apoptotic index (0.35 +/- 0.11) compared with that of core transfected cells (0.74 +/- 0.07, P = .0103). Analysis of a Fas-induced apoptosis model in HCV transgenic mice confirmed that core-E1-E2 transgenic liver underwent significantly less apoptosis than transgenic tissue expressing core only. In conclusion, HCV core-E1-E2 transgenic mice develop significantly larger tumors than transgenic mice expressing core alone or nontransgenic mice. The accelerated tumor phenotype is attributable to suppression of apoptosis rather than enhanced proliferation. These data implicate HCV E1 and/or E2 in conjunction with core as antiapoptotic, tumor accelerator proteins.

Altered differentiation of hepatocytes in a transgenic mouse model of hepatocarcinogenesis.

Transgenic mice carrying the SV40 T antigen (TAg) gene, which develop hepatocellular and biliary cell tumors by 4 mo of age, show ductular structures in the neonatal liver. Coexpression of c-myc with TAg increases the extent and persistence of ductular lesions and also accelerates tumor development. To analyze possible links between altered gene expression and cell differentiation and to determine the relationship between the ductular structures and tumor development in these mice, ductular cells in single (TAg) and bitransgenic (TAg x c-myc) mice were characterized for biliary and hepatocellular differentiation, transgene expression, and proliferation activity. The results show that the ductular cells in these transgenic mice have characteristics of biliary cells, including basement membrane formation, positive laminin staining, and bile duct-specific lectin (Dolichos biflorus agglutinin and peanut agglutinin) binding, and characteristics of hepatocytes, including albumin expression and ultrastructural features such as round nuclei with 1 or 2 nucleoli and well-developed cytoplasmic organelles. However, differences in transgene expression and cell proliferation between the ductular cells and nonductular hepatocytes were not apparent. Thus, the ductular cells could not be defined as tumor progenitor cells in these mouse livers. However, this model suggests that manipulation of gene expression can alter differentiation of hepatic parenchymal cells.

Altered Heme Pathway Regulation and Drug Metabolizing Enzyme System in a Mouse Model of Hepatocarcinogenesis: Effect of Veronal

1. Male CF 1 mice were fed p-dimethylaminoazobenzene (DAB) for 35 days and received 5,5-diethylbarbituric acid, before or after DAB treatment, with the purpose of investigating whether the onset of the preinitiation stage of carcinogenesis alters the natural regulatory mechanism of the heme pathway. 2. Changes detected in drug metabolizing enzymes are likely to be the consequence of a primary deregulation mechanism of heme metabolism, shown by an increase in δ-aminolevulinic acid synthetase activity and a decrease in microsomal heme oxygenase, which would finally lead to a great enhancement of cytochrome P450 levels. 3. The alterations found here would give rise to a pattern distinctive to that usually observed in the so-called resistant hepatocyte.