Hepatocellular Carcinoma Development In Mice Research Articles

HBV-related HCC development in mice is STAT3 dependent and indicates an oncogenic effect of HBx

Although most hepatocellular carcinoma (HCC) cases are driven by hepatitis and cirrhosis, a subset of patients with chronic hepatitis B develop HCC in the absence of advanced liver disease, indicating the oncogenic potential of hepatitis B virus (HBV). We investigated the role of HBV transcripts and proteins on HCC development in the absence of inflammation in HBV-transgenic mice. HBV-transgenic mice replicating HBV and expressing all HBV proteins from a single integrated 1.3-fold HBV genome in the presence or absence of wild-type HBx (HBV1.3/HBVxfs) were analyzed. Flow cytometry, molecular, histological and invitro analyses using human cell lines were performed. Hepatocyte-specific Stat3- and Socs3-knockout was analyzed in HBV1.3 mice. Approximately 38% of HBV1.3 mice developed liver tumors. Protein expression patterns, histology, and mutational landscape analyses indicated that tumors resembled human HCC. HBV1.3 mice showed no signs of active hepatitis, except STAT3 activation, up to the time point of HCC development. HBV-RNAs covering HBx sequence, 3.5-kb HBV RNA and HBx-protein were detected in HCC tissue. Interestingly, HBVxfs mice expressing all HBV proteins except a C-terminally truncated HBx (without the ability to bind DNA damage binding protein 1) showed reduced signs of DNA damage response and had a significantly reduced HCC incidence. Importantly, intercrossing HBV1.3 mice with a hepatocyte-specific STAT3-knockout abrogated HCC development. Expression of HBV-proteins is sufficient to cause HCC in the absence of detectable inflammation. This indicates the oncogenic potential of HBV and in particular HBx. In our model, HBV-driven HCC was STAT3 dependent. Our study highlights the immediate oncogenic potential of HBV, challenging the idea of a benign highly replicative phase of HBV infection and indicating the necessity for an HBV 'cure'. Although most HCC cases in patients with chronic HBV infection occur after a sequence of liver damage and fibrosis, a subset of patients develops HCC without any signs of advanced liver damage. We demonstrate that the expression of all viral transcripts in HBV-transgenic mice suffices to induce HCC development independent of inflammation and fibrosis. These data indicate the direct oncogenic effects of HBV and emphasize the idea of early antiviral treatment in the 'immune-tolerant' phase (HBeAg-positive chronic HBV infection).

Hepatocyte phosphatase DUSP22 mitigates NASH-HCC progression by targeting FAK

Nonalcoholic steatohepatitis (NASH), a common clinical disease, is becoming a leading cause of hepatocellular carcinoma (HCC). Dual specificity phosphatase 22 (DUSP22, also known as JKAP or JSP-1) expressed in numerous tissues plays essential biological functions in immune responses and tumor growth. However, the effects of DUSP22 on NASH still remain unknown. Here, we find a significant decrease of DUSP22 expression in human and murine fatty liver, which is mediated by reactive oxygen species (ROS) generation. Hepatic-specific DUSP22 deletion particularly exacerbates lipid deposition, inflammatory response and fibrosis in liver, facilitating NASH and non-alcoholic fatty liver disease (NAFLD)-associated HCC progression. In contrast, transgenic over-expression, lentivirus or adeno-associated virus (AAV)-mediated DUSP22 gene therapy substantially inhibit NASH-related phenotypes and HCC development in mice. We provide mechanistic evidence that DUSP22 directly interacts with focal adhesion kinase (FAK) and restrains its phosphorylation at Tyr397 (Y397) and Y576 + Y577 residues, subsequently prohibiting downstream activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) cascades. The binding of DUSP22 to FAK and the dephosphorylation of FAK are indispensable for DUSP22-meliorated NASH progression. Collectively, our findings identify DUSP22 as a key suppressor of NASH-HCC, and underscore the DUSP22-FAK axis as a promising therapeutic target for treatment of the disease.

Hypoxia-induced macropinocytosis represents a metabolic route for liver cancer

Hepatocellular carcinoma (HCC) invariably exhibits inadequate O2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.

Coinfection of Helicobacter pylori and Hepatitis C Virus in the Development of Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis.

BackgroundThe relationship between Helicobacter pylori (H. pylori) and hepatocellular carcinoma (HCC) was firstly proposed in 1994 after Ward et al demonstrated the role of Helicobacter hepaticus in the development of HCC in mice. Studies also investigated the role of hepatitis B virus (HBV) and hepatitis C virus (HCV) coexisting with H. pylori in causing HCC. A causal relationship was never confirmed, and the relationship remains controversial. This meta-analysis aimed to summarize the research on this topic and investigate if a relationship exists between H. pylori infection and the development of HCC and if the presence of HCV and HBV along with H. pylori plays a role in liver carcinogenesis.MethodsFollowing PRISMA guidelines, we performed a systematic review of all relevant studies published in the literature using the keywords “Helicobacter pylori” and “hepatocellular carcinoma” on major literature databases, including PubMed, EMBASE, Web of Science, and Cochrane controlled trials register. A total of 656 research studies were identified between 1994 and 2020. Of those, 26 qualified under our selection criteria. Patients who were positive for HCC were classified as cases and those who did not have HCC were classified as controls. The H. pylori status and HCV status, if available, were identified for both groups. Statistical analysis was carried out by a biostatistician according to the Cochrane reviewer’s handbook.ResultsOut of the 26 studies included in the final analysis, 13 were retrospective case-control studies, 11 were cross-sectional studies, and two were prospective case-control and cohort studies. Overall, the prevalence of H. pylori infection was 64.78% (561 of 866) amongst HCC cases and 47.92% (1,718 of 3,585) in the non-HCC control group. The summary odds ratio (OR) for the association of H. pylori infection with the risk for HCC (using the random-effects model, which accounted for the heterogeneity across the 26 studies) was determined to be 4.75 (95% confidence interval (CI): 3.06 - 7.37, I2 = 63%). We also performed a subgroup analysis to determine the odds of developing HCC in the presence of H. pylori and HCV coinfection. The summary OR of it was 12.76 (95% CI: 4.13 - 39.41, I2 = 78%). The summary OR for the risk of developing HCC in the presence of HCV infection without H. pylori infection was 2.21 (95% CI: 0.70 - 6.94, I2 = 79%). Whereas, the odds of developing HCC in the presence of only H. pylori infection without HCV was found to be 0.54 (95% CI: 0.11 - 2.63, I2 = 80%). There was inconsistency in the data presented in some studies regarding HCV infection status. Since data were extracted from different study designs, subgroup analysis by study design was performed which showed no significant difference between the study groups (P = 0.5705).ConclusionThis meta-analysis demonstrates a positive association between H. pylori infection and the development of HCC. There is a significantly higher risk of developing HCC in the presence of HCV infection along with H. pylori. Further prospective cohort studies are needed to prove the causal relationship, especially in cases of HBV and HCV coinfection, and cirrhotic patients.

The Consumption of Cholesterol-Enriched Diets Conditions the Development of a Subtype of HCC with High Aggressiveness and Poor Prognosis.

Simple SummaryIt is well known that non-alcoholic fatty liver disease is an important risk factor in the development of hepatocellular carcinoma, but the implication of cholesterol in this subject remains unclear, especially in western countries where its consumption is particularly elevated. This work provides evidence of a cholesterol-related transcriptional fingerprint and its implications in the progression and aggressiveness of hepatocellular carcinoma with remarkable interest in clinical practice.Non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH) result as a consequence of diverse conditions, mainly unbalanced diets. Particularly, high-fat and cholesterol content, as well as carbohydrates, such as those commonly ingested in Western countries, frequently drive adverse metabolic alterations in the liver and promote NAFLD development. Lipid liver overload is also one of the main risk factors for initiation and progression of hepatocellular carcinoma (HCC), but detailed knowledge on the relevance of high nutritional cholesterol remains elusive. We were aimed to characterize HCC development in mice fed with a Western diet (high in lipids and cholesterol) and to identify molecular alterations that define a subtype of liver cancer induced by lipid overload. Mice under western or high cholesterol diets more frequently developed tumors with a more aggressive phenotype than animals fed with a chow diet. Associated changes involved macrophage infiltration, angiogenesis, and stemness features. RNA-seq revealed a specific gene expression signature (Slc41a; Fabp5; Igdcc4 and Mthfd1l) resembling the adverse phenotypic features and poor clinical outcomes seen in patients with HCC. In conclusion; consumption of lipid enriched diets; particularly cholesterol; could accelerate HCC development with an aggressive phenotype and poor prognosis

NONO promotes hepatocellular carcinoma progression by enhancing fatty acids biosynthesis through interacting with ACLY mRNA

BackgroundDysregulation of fatty acid (FA) metabolism is involved in hepatocellular carcinoma (HCC) development. Non-POU domain-containing octamer binding protein (NONO), known as the component of nuclear paraspeckles, has recently been found to promote HCC progression. In this study, we investigated the functions of NONO in regulating de novo FA synthesis and its underling mechanism during HCC development.MethodsThe roles of NONO in HCC development by applying gene function loss analysis in HCC cells were detected by quantitative real-time polymerase chain reaction, cell proliferation, and cell invasion assays. The underlying mechanism of NONO in HCC development was examined by western blotting, subcellular fractionation, RNA-binding protein immunoprecipitation-sequencing, chromatin immunoprecipitation, co-immunoprecipitation and mass spectrometry. The effect of NONO on tumorigenesis in vivo was performed with a subcutaneous xenograft mouse model of HCC.ResultsNONO promotes HCC progression by interacting with and increasing ATP-citrate lyase (ACLY) mRNA to enhance FA biosynthesis. Furthermore, NONO promotes ACLY expression through enhancing nuclear ACLY mRNA stability in Diethylnitrosamine-stimulated HCC cells, not related to nuclear paraspeckles. Moreover, we find that NONO/SFPQ (Splicing factor proline and glutamine rich) heterodimer is essential for NONO interacting with ACLY mRNA in DEN stimulated HCC cells. In addition, NONO, Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) and ACLY expressions contribute HCC development in mice and are related to poor survival.ConclusionNONO promotes HCC progression by enhancing FA biosynthesis through interacting with ACLY mRNA and provide a novel potential target for HCC therapy.

FBP1 Is a Tumor Suppressor Linking Hepatic Metabolism to Tumorigenesis

Loss of the gluconeogenic enzyme FBP1 promoted hepatocellular carcinoma development in mice.

Oncogenic potential of N-terminal deletion and S45Y mutant \u03b2-catenin in promoting hepatocellular carcinoma development in mice

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with limited treatment options. Mutation of β-catenin is one of the most frequent genetic events along hepatocarcinogenesis. β-catenin mutations can be in the form of point mutation or large N-terminal deletion. Studies suggested that different β-catenin mutations might have distinct oncogenic potential.MethodsWe tested the oncogenic activity of β-cateninS45Y, one of the most frequent point mutations of β-catenin, and ∆N90-β-catenin, a form of β-catenin with a large N-terminal deletion, in promoting HCC development in mice. Thus, we co-expressed β-cateninS45Y or ∆N90-β-catenin together with c-Met into the mouse liver using hydrodynamic injection.ResultsWe found that both β-catenin mutations were able to induce HCC formation in combination with c-Met at the same latency and efficiency. Tumors showed similar histological features and proliferation rates. However, immunohistochemistry showed predominantly nuclear staining of β-catenin in c-Met/∆N90-β-catenin HCC, but membrane immunoreactivity in c-Met/β-cateninS45Y HCC. qRT-PCR analysis demonstrated that both ∆N90-β-catenin and β-cateninS45Y induced the same effectors, although at somewhat different levels. In cultured cells, both ∆N90-β-catenin and β-cateninS45Y were capable of inducing TCF/LEF reporter expression, promoting proliferation, and inhibiting apoptosis.ConclusionsOur study suggests that β-cateninS45Y and ∆N90-β-catenin, in combination with the c-Met proto-oncogene, have similar oncogenic potential. Furthermore, nuclear staining of β-catenin does not always characterize β-catenin activity.

AIM2 deficiency reduces the development of hepatocellular carcinoma in mice.

Chronic liver inflammation is crucial in the pathogenesis of hepatocellular carcinoma (HCC). Activation of the inflammasome complex is a key inflammatory process that has been associated with different liver diseases, but its role in HCC development remains largely unexplored. Here we analyzed the impact of different inflammasome components, including absent in melanoma 2 (AIM2) and NOD-like receptor family pyrin domain containing 3 (NLRP3), in the development of diethylnitrosamine (DEN)-induced HCC in mice. Genetic inactivation of AIM2, but not NLRP3, reduces liver damage and HCC development in this model. AIM2 deficiency ameliorates inflammasome activation, liver inflammation and proliferative responses during HCC initiation. We also identified that AIM2 is highly expressed in Kupffer cells, and that AIM2-mediated production of IL-1β by these cells is enhanced after DEN-induced liver damage. Our data indicate that AIM2 promotes inflammation during carcinogenic liver injury and that it contributes to genotoxic HCC development in mice, thereby recognizing AIM2 as a potential therapeutic target in this disease.

244 - Collagen type I Protects from Hepatocellular Carcinoma Development in Mice

244 - Collagen type I Protects from Hepatocellular Carcinoma Development in Mice

Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA accumulation and SASP in senescent cells

Accumulating evidence indicates that the senescence-associated secretory phenotype (SASP) contributes to many aspects of physiology and disease. Thus, controlling the SASP will have tremendous impacts on our health. However, our understanding of SASP regulation is far from complete. Here, we show that cytoplasmic accumulation of nuclear DNA plays key roles in the onset of SASP. Although both DNase2 and TREX1 rapidly remove the cytoplasmic DNA fragments emanating from the nucleus in pre-senescent cells, the expression of these DNases is downregulated in senescent cells, resulting in the cytoplasmic accumulation of nuclear DNA. This causes the aberrant activation of cGAS-STING cytoplasmic DNA sensors, provoking SASP through induction of interferon-β. Notably, the blockage of this pathway prevents SASP in senescent hepatic stellate cells, accompanied by a decline of obesity-associated hepatocellular carcinoma development in mice. These findings provide valuable new insights into the roles and mechanisms of SASP and possibilities for their control.

Loss of Pten synergizes with c-Met to promote hepatocellular carcinoma development via mTORC2 pathway

Hepatocellular carcinoma (HCC) is a deadly malignancy with limited treatment options. Activation of the AKT/mTOR cascade is one of the most frequent events along hepatocarcinogenesis. mTOR is a serine/threonine kinase and presents in two distinct complexes: mTORC1 and mTORC2. While mTORC1 has been extensively studied in HCC, the functional contribution of mTORC2 during hepatocarcinogenesis has not been well characterized, especially in vivo. Pten expression is one of the major mechanisms leading to the aberrant activation of the AKT/mTOR signaling. Here, we show that concomitant downregulation of Pten and upregulation of c-Met occurs in a subset of human HCC, mainly characterized by poor prognosis. Using CRISPR-based gene editing in combination with hydrodynamic injection, Pten was deleted in a subset of mouse hepatocytes (sgPten). We found that loss of Pten synergizes with overexpression of c-Met to promote HCC development in mice (sgPten/c-Met). At the molecular level, sgPten/c-Met liver tumor tissues display increased AKT and mTOR signaling. Using Rictor conditional knockout mice, we demonstrate that sgPten/c-Met-driven HCC development strictly depends on an intact mTORC2 complex. Our findings therefore support the critical role of mTORC2 in hepatocarcinogenesis. sgPten/c-Met mouse model represents a novel valuable system that can be used for the development of targeted therapy against this deadly malignancy.

Acquisition of Cholangiocarcinoma Traits during Advanced Hepatocellular Carcinoma Development in Mice

Past studies have identified hepatic tumors with mixed hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) characteristics that have a more aggressive behavior and a poorer prognosis than classic HCC. Whether this pathologic heterogeneity is due to a cell of origin of bipotent liver progenitors or the plasticity of cellular constituents comprising these tumors remains debated. In this study, we investigated the potential acquisition of CC-like traits during advanced development of HCC in mice. Primary and rare high-grade HCC developed in a genetic mouse model. A mouse model of highly efficient HCC invasion and metastasis by orthotopic transplantation of liver cancer organoids propagated from primary tumors in the genetic model was further developed. Invasive/metastatic tumors developed in both models closely recapitulated advanced human HCC and displayed a striking acquisition of CC-related pathologic and molecular features, which was absent in the primary HCC tumors. Our study directly demonstrates the pathologic evolution of HCC during advanced tumor development, providing the first evidence that tumors with mixed HCC and CC features, or at least a subset of these tumors, represent a more advanced developmental stage of HCC. Finally, liver cancer organoid-generated high-grade tumors exhibited significantly increased extracellular vesicle secretion, suggesting that identifying tumor-specific extracellular vesicle proteins in plasma may be a promising tool for liver cancer detection.

Abstract A172: Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in FGF19-expressing hepatocellular carcinoma

Abstract Background: Fibroblast growth factor (FGF) / FGF receptor (FGFR) gene aberrations such as amplification, mutation and fusion are associated with many types of human cancers. Recently, FGF19 overexpression was observed in approximately 50% of hepatocellular carcinoma (HCC) patients. The FGF19-FGFR4 signaling has been implicated in the development of HCCs in mice. FGFR4 kinase inhibitors are expected to be a targeted therapy for FGF19-expressing HCC. A phase I clinical trial of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, is ongoing (NCT02038673). Method: We tested selectivity of ASP5878 among 128 kinases and sensitivity of ASP5878 on cell proliferation of HCC cell lines. Activation of FRS2 and ERK, downstream molecules of FGFR signaling, and PARP cleavage in FGF19 expressing HCC cell lines were evaluated with Western blotting. In vivo antitumor effects of ASP5878 were examined in HCC subcutaneous xenograft and orthotopic inoculation mouse models. Finally, plasma levels of FGF19 were measured after dosing ASP5878. Results: Among 128 kinases, only 9 kinases including FGFR1-4 and FGFR3/4 mutations were inhibited more than 50% by ASP5878 (200 nmol/L). The IC50 values of ASP5878 against FGFR1, 2, 3 and 4 kinases were 0.47, 0.60, 0.74 and 3.5 nmol/L, respectively. ASP5878 inhibited cell proliferation of HCC cell lines with FGF19 overexpression. IC50 values were 8.5, 27, and 21 nmol/L in Hep3B2.1-7, HuH-7 and JHH-7, respectively. ASP5878 inhibited activation of downstream signaling molecules, FRS2 and ERK, and induced apoptosis in Hep3B2.1-7 cells. Oral dosing of ASP5878 at 3 mg/kg induced sustained tumor regression in the Hep3B2.1-7 subcutaneous xenograft model, which was poorly responsive to sorafenib. In an HuH-7 orthotopic inoculation mouse model, ASP5878 induced complete tumor regression and dramatically extended the survival. In addition, oral dosing of ASP5878 reduced plasma levels of FGF19 in the HuH-7 subcutaneous xenograft model.Conclusion: These results suggest that ASP5878 is a potentially effective therapeutic agent for FGF19-expressing HCC. Citation Format: Takashi Futami, Hidetsugu Okada, Rumi Kihara, Tatsuya Kawase, Ayako Nakayama, Tomoyuki Suzuki, Minoru Kameda, Nobuaki Shindoh, Tadashi Terasaka, Masaaki Hirano, Sadao Kuromitsu. Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in FGF19-expressing hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A172.

P0075 Overexpression of niemann-pick type C2 protein inhibits cell proliferation and liver cancer development via modulating the ERK1/2 pathway

Background Primary hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related deaths. Therefore, it is important to identify new targets for the treatment of HCC. Niemann-pick type C2 (NPC2) plays an important part in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in HCC tumourigenesis. Methods To identify the NPC2-dependent mechanism, we examined the status of MAPK signalling. Findings In this study, we showed that NPC2 is abundantly expressed in normal liver, but is downregulated in human HCC tissues. Patients with NPC2 downregulation expressed much higher α-fetoprotein, multiple tumour types, vascular invasion, later pathological stage, and shorter survival. NPC2 overexpression inhibited HuH7 cells proliferation, migration, and xenograft tumourigenesis. Furthermore, administration of hepatotropic adeno-associated virus 8 delivered NPC2 significantly suppressed the spontaneous HCC development in mice. NPC2 overexpression decreases phosphorylation of ERK1/2. Interpretation Our study demonstrates that NPC2 may play an important part in negatively regulating ERK1/2 activation in liver cancer. Thus, NPC2 may represent a new treatment strategy for liver cancer.

C‐Met And β‐catenin Co‐delivery By Hydrodynamic Tail Vein Injection Promotes HCC Development In Mice

Hepatocellular carcinoma (HCC) isthe fifth mostfrequently diagnosed type of cancer worldwide and second most frequent cause of cancer related deaths. Thus generation of a mouse model that mimics human HCC will improve understanding of biology and provide opportunity to test novel therapies. Hepatocyte growth factor receptor c‐Met is overexpressed in HCC. Also β‐catenin mutations affecting serine 33 (S33) and S45 but not N‐terminal deletions affecting exon‐3 are frequently observed in HCC patients. Intriguingly, expression of mutant forms of β‐catenin or c‐Met alone fail to induce HCC development in mice. Previous study has shown that co‐delivery of β‐catenin N‐terminal deletion mutant with c‐Met induced HCC, but there is no study on crosstalk of β‐catenin point mutants and c‐Met. We co‐delivered c‐Met and β‐catenin point mutants (S33Y and S45Y) by hydrodynamic tail vein injection to initiate liver tumorigenesis. Microscopic nodules were apparent at 4 weeks and large tumors were evident at 9 weeks post injection of c‐Met/S45Y‐β‐catenin. Microscopic nodules were apparent at 6 weeks while large tumors were evident at 12 weeks after c‐Met/S33Y‐β‐catenin injection. Tumors in both conditions were HCCs that displayed overexpression of several downstream targets of Wnt and HGF pathways. In summary, our results demonstrate that c‐Met cooperates with point‐mutant‐β‐catenin to promote growth and development of HCC.

Tumor Progression Locus 2 Depletion Inhibits Hepatic Inflammation and Steatosis and Incidence of Hepatocellular Carcinoma

Tumor progression locus 2 (TPL2) is a serine-threonine kinase that functions as a critical regulator of inflammatory pathways and participates in oncogenic events. Non-alcoholic fatty liver disease (NAFLD) is a risk factor for hepatocellular carcinoma (HCC) development. We have recently showed that a high carbohydrates diet (HCD, 66% and 12% energy from carbohydrates and fat, respectively) induces hepatic inflammation and promotes HCC development in mice. In the present study, we investigated the role of TPL2 in the pathogenesis of HCD-associated NAFLD and HCC in Tpl2 knock out (Tpl2 KO) mice. Both Tpl2 KO mice and wild type (WT) mice were initiated by the i.p. injection of a carcinogen (diethylnitrosamine, DEN, 25 mg/kg BW) at 2 weeks of age and followed by a HCD feeding for 22 weeks. Results showed that the deletion of Tpl2 significantly reduced hepatic inflammatory foci as compared to the WT mice. This reduction was associated with decreased phosphorylation of JNK and ERK and lower expression of hepatic inflammatory genes (IL-1β, IL-18, MCP-1 and NALP3). The deletion of TPL2 also reduced steatosis which was associated with decreased hepatic expressions of ACC, SCD1, SREBP1C and AKT phosphorylation. Furthermore, the lacking of TPL2 resulted in both reduction of ER stress biomarkers and activation of mTOR signaling. Although there were no differences on tumor multiplicity and tumor volume, Tpl2 KO mice exhibited significantly lower incidence of HCC than WT mice (P<0.05). In conclusion, TPL2 plays a significant role in promoting NAFLD and the DEN-initiated HCC development. Supported by the USDA/ARS 1950-51000-074S.

Niemann-Pick type C2 protein regulates liver cancer progression via modulating ERK1/2 pathway: Clinicopathological correlations and therapeutical implications.

Primary hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related death. It is important to identify new targets for early diagnosis and treatment of HCC. Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in HCC tumorigenesis. In this study, we showed that NPC2 is abundantly expressed in normal liver, but is downregulated in human HCC tissues. The patients with NPC2 downregulation expressed much higher α-fetoprotein, multiple tumor type, vascular invasion, later pathological stage and shorter survival rate. Knockdown NPC2 in liver cancer cell lines promote cell proliferation, migration and xenograft tumorigenesis. In contrast, NPC2 overexpression inhibits HuH7 promoted tumor growth. Furthermore, administration of hepatotropic adeno-associated virus 8 (AAV8) delivered NPC2 decreased the inflammatory infiltration, the expression of two early HCC markers-glypican 3 and survivin and suppressed the spontaneous HCC development in mice. To identify the NPC2-dependent mechanism, we emphasized on the status of MAPK/ERK signaling. MEK1/2 inhibitor treatment demonstrated that the expression of NPC2 affected the activation of ERK1/2 but not MEK1/2. In addition, cholesterol trafficking inhibitor treatment did not alter the cell proliferation and the activation of MEK/ERK. In conclusion, our study demonstrates that NPC2 may play an important role in negatively regulate cell proliferation and ERK1/2 activation that were independent of cholesterol accumulation. AAV-NPC2 may thus represent a new treatment strategy for liver cancer.

Biochemical/metabolic changes associated with hepatocellular carcinoma development in mice

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality after lung and stomach cancers. This work was undertaken to investigate some of the biochemical mediators/pathways associated with or implicated in the pathogenesis of HCC. Male albino mice were classified into two groups: normal control group and HCC group. Early stage HCC was induced by injection of diethylnitrosamine (DEN) i.p. 200 mg/kg as a single dose, and after 2 weeks, the mice were given i.p. injection of thioacetamide (TAA) 100 mg/kg twice per week for 4 weeks. Mice were left for further 2 weeks without any treatment, after which, mice were sacrificed; blood and liver samples were collected. Serum was used for determination of activities of glucose-6-phosphate dehydrogenase (G6PDH) and aldolase as well as levels of insulin-like growth factor-1 (IGF-1) and epithelial cadherin (E-cadherin). One portion of the liver was used for histopathological examination and immunohistochemical staining of the tumor suppressor p53 protein. Another portion of the liver was used for determination of citrate synthase activity. Induction of HCC in mice resulted in significant increase in G6PDH and aldolase activities, and E-cadherin level, but significant decrease in IGF-1. HCC mice group showed moderate expression of p53 protein. These results suggest that the molecular pathogenesis of HCC in mice involves reduction of serum level of IGF-1 and increased serum level of E-cadherin accompanied by dysregulation of p53 protein expression. HCC was also associated with reprogrammed metabolic profile shifted toward increased glycolysis and lipogenesis.

Microbiome–Obesity–Liver Cancer Interaction: Senescence of Hepatic Stellate Cells and Bile Acids Play New Roles

Yoshimoto S, Loo TM, Atarashi K, et al. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 2013;499:97–101. The increased prevalence of obesity is a significant health concern in developed countries. Epidemiologic human studies and experiments using rodents show that obesity is associated with not only the manifestations of metabolic disease, such as diabetes and cardiovascular disease, but also with increased incidence of a number of cancers, including hepatocellular carcinoma (HCC). The incidence of HCC is increased in patients with hepatosteatosis accompanied with liver inflammation (referred to as nonalcoholic steatohepatitis) rather than patients with simple steatosis. Emerging evidences demonstrate that intestinal microbiome is among the key components in the progression of obesity and HCC (Nature 2006; 444:1027–1031; Cancer Cell 2012;21:504–516). Yoshimoto et al demonstrated that senescence-associated secretory phenotype (SASP) of hepatic stellate cells (HSC) induced by a bacterial byproduct, deoxycholic acid (DCA), promotes obesity-associated HCC progression. The study initiated to confirm that chemical toxin-induced Ras mutation causes HCC only in obese mice fed a high-fat diet. This phenotype was reproduced in genetically obese ob/ob mice. This study found that cellular senescence markers such as p21Waf1/Cip1 and p16INK4a, as well as DNA damage markers (53BP1 and γ-H2AX), were up-regulated in HCC lesions, particularly in HSC. These HSC produced SASP cytokines, such as interleukin (IL)-6, GROα, and CXCL9, in which proliferation was inhibited, demonstrating that HSC in HCC lesions has become senescent. Although HSC do not produce IL-1α, a SASP contributor, HSC induced the expression of inflammasome components and IL-1β. In IL-1β−/− mice, a high-fat diet induced hepatosteatosis and HSC senescence, as demonstrated by the expression of 53BP1 and p21; however, the production of SASP cytokines and HCC formation were suppressed, suggesting that SASP is essential for obesity-associated HCC development. The study then highlighted the contribution of intestinal microbiome to HCC development in mice. Gut sterilization by oral administration of antibiotics cocktail or vancomycin that targets Gram-positive bacteria significantly suppressed SASP of HSC and growth of HCC. They also found that serum levels of DCA and the composition of Clostridium cluster XI and XIVa that convert primary bile acids to DCA were increased in obese mice, but the DCA content was decreased by antibiotic treatment. Because DCA produces reactive oxygen species that cause DNA damage and SASP, the researchers then focused on the crucial role of DCA in obesity-associated HCC growth. High-fat diet–driven HCC growth was dramatically suppressed by lowering DCA levels by administrations of difructose anhydride III, which inhibits 7α-dehydroxylation activity or by ursodeoxycholic acid, which promotes bile acid secretion. Conversely, oral DCA feeding enhanced HCC development along with HSC senescence and SASP in obese mice. Finally, the study confirmed the presence of senescent HSC and SASP in HCC developed in nonalcoholic steatohepatitis patients.