Preneoplastic Liver Research Articles

Flavonoid luteolin supplementation inhibits diethylnitrosamine‐initiated alcohol‐promoted hepatic inflammation and precancerous lesions in mice (829.7)

Chronic and excessive alcohol consumption is an established risk for hepatic inflammation and carcinogenesis. Luteolin is one of the most common flavonoids present in plants and has potential beneficial effects against chronic diseases. In this study, we examined the effect and potential mechanisms of luteolin supplementation in a carcinogen (diethylnitrosamine [DEN])‐initiated alcohol‐promoted pre‐neoplastic liver lesion mouse model. C57BL/6 mice were injected with DEN (i.p. 25 mg/kg of body weigth [BW]) at 14 days of age. At 8 weeks of age mice were group pair‐fed with Lieber‐DeCarli liquid control diet or alcoholic diet (EtOH diet, 27% total energy from ethanol) and supplemented with a dose of 30 mg luteolin/kg BW/day for a treatment period of 21 days. Results show a significant induction in the incidence and multiplicity of basophilic and eosinophilic foci, markers of pre‐neoplastic liver lesions, in DEN‐injected mice fed EtOH diet. The presence of pre‐neoplastic lesions was significantly correlated with the induction of steatosis and inflammatory foci in these mice. Supplementation with luteolin inhibited the presence of pre‐neoplastic liver lesions in DEN‐injected mice fed EtOH diet. Dietary luteolin significantly reduced the severity of hepatic inflammatory foci with an associated non‐significant reduction in expression of hepatic inflammatory cytokines TNF‐α, IL‐6 and IL‐1β. There was no difference on hepatic protein levels of sirtuin 1 (SIRT1), however, luteolin supplementation significantly reversed alcohol‐reduced SIRT1 activity assessed by the ratio of acetylated and total FoXO1. The study demonstrated that luteolin prevents EtOH diet‐promoted hepatic inflammation and pre‐neoplastic lesion, potentially mediated by SIRT1‐FoxO1 signaling pathway. Grant Funding Source: USDA/ARS grant 1950‐51000‐074S

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-β/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)+ liver cell foci and the numbers of Ki-67+ proliferating cells within GST-P+ foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P+ foci (area) to 48.9–49.4% and Ki-67+ cells to 55.5–61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN– foci, phospho-Akt substrate+ foci, phospho-Smad3– foci and Smad4– foci in GST-P+ foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN– foci in GST-P+ foci to 59.8–72.2% and Smad4– foci to 62.4–71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate+ foci in GST-P+ foci to 75.2–75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-β/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells.

Transcriptomic responses provide a new mechanistic basis for the chemopreventive effects of folic acid and tributyrin in rat liver carcinogenesis

The steady increase in the incidence and mortality of hepatocellular carcinoma (HCC) signifies a crucial need to understand better its pathogenesis to improve clinical management and prevention of the disease. The aim of this study was to investigate molecular mechanisms for the chemopreventive effects of folic acid and tributyrin alone or in combination on rat hepatocarcinogenesis. Male Wistar rats were subjected to a classic "resistant hepatocyte" model of liver carcinogenesis and treated with folic acid and tributyrin alone or in combination for 5 weeks during promotion stage. Treatment with folic acid and tributyrin alone or in combination strongly inhibited the development of glutathione-S-transferase placental form (GSTP)-positive foci. Microarray analysis showed significant changes in gene expression. A total of 498, 655 and 940 of differentially expressed genes, involved in cell cycle, p53-signaling, angiogenesis and Wnt pathways, was identified in the livers of rats treated with folic acid, tributyrin or folic acid and tributyrin. A detailed analysis of these differentially expressed genes revealed that treatments inhibited angiogenesis in the preneoplastic livers. This was evidenced by the fact that 30 out of 77 differentially expressed genes common to all three treatments are involved in the regulation of the angiogenesis pathway. The inhibition of angiogenesis was confirmed by reduced levels of CD34 protein. In conclusion, the tumor-suppressing activity of folic acid and tributyrin is associated with inhibition of angiogenesis at early stages of rat liver carcinogenesis. Importantly, the combination of folic acid and tributyrin has stronger chemopreventive effect than each of the compounds alone.

FoxO3a modulation and promotion of apoptosis by interferon‐α2b in rat preneoplastic liver

FoxO3a, a member of the FOXO family of transcription factors, is expressed in adult liver and modulates the expression of genes involved in apoptosis. FoxO3a is post-translationally regulated, negatively by PI3K/Akt and MAPK/Erk and positively by oxidative stress/JNK pathways. In previous works, we have demonstrated that interferon-α2b (IFN-α2b) induces apoptosis of hepatic preneoplastic foci through the production of reactive oxygen species (ROS). To investigate the post-translational signal events triggered by the oxidative stress induced by IFN-α2b and the modulation of FoxO3a transcriptional activity during these events in rat preneoplastic liver. Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis. A group of animals received IFN-α2b and another group received IFN-α2b and ascorbic acid (ASC), by intraperitoneal injection. Lipid peroxidation, immunohistochemistry, immunoblotting, co-immunoprecipitation and sqRT-PCR assays were performed to explore the role of ROS, JNK, Akt, Erk, FoxO3a, β-catenin and PUMA in the IFN-α2b-mediated apoptotic mechanism. In vivo IFN-α2b treatment induced endogenous production of ROS which activated JNK. IFN-α2b blocked the activation of Akt and Erk, avoiding FoxO3a activity repression. Activated JNK was responsible for the nuclear translocation and transcriptional activity of FoxO3a which positively modulated the expression of PUMA, a proapoptotic player. In addition, nuclear FoxO3a competed for the nuclear β-catenin associated to TCF, inhibiting the canonical Wnt signalling pathway. The data presented here propose a model in which in vivo IFN-α2b treatment induces nuclear translocation and transcriptional activity of FoxO3a, triggering the mitochondrial apoptotic pathway in hepatic preneoplastic foci.

Coffee consumption delays the hepatitis and suppresses the inflammation related gene expression in the Long-Evans Cinnamon rat

Large-scale epidemiological studies have shown that drinking more than two cups of coffee per day reduces the risks of hepatitis and liver cancer. However, the heterogeneity of the human genome requires studies of experimental animal models with defined genetic backgrounds to evaluate the coffee effects on liver diseases. We evaluated the efficacy of coffee consumption with one of experimental animal models for human disease. We used the Long Evans Cinnamon (LEC) rat, which onsets severe hepatitis and high incidence of liver cancer, due to the accumulation of copper and iron in livers caused by the genetic mutation in Atp7B gene, and leading to the continuous oxidative stress. We determined the expression of inflammation related genes, and amounts of copper and iron in livers, and incidence of the pre-neoplastic foci in the liver tissue of LEC rats. Coffee administration for 25 weeks delayed the occurrence of hepatitis by two weeks, significantly improved survival, reduced the expression of inflammatory cytokines, and reduced the incidence of small pre-neoplastic liver foci in LEC rats. There was no significant difference in the accumulation of copper and iron in livers, indicating that coffee administration does not affect to the metabolism of these metals. These findings indicate that drinking coffee potentially prevents hepatitis and liver carcinogenesis through its anti-inflammatory effects. This study showed the efficacy of coffee in the prevention of hepatitis and liver carcinogenesis in the LEC model.

Abstract 201: (-)-Epigallocatechin-3-gallate inhibits the development of hepatic preneoplastic lesions in obese and hypertensive rats, a novel model of non-alcoholic steatohepatitis-related liver tumorigenesis.

Abstract Background: Non-alcoholic steatohepatitis (NASH), which involves hepatic inflammation and fibrosis, is associated with liver carcinogenesis. The activation of the renin-angiotensin system (RAS), which plays a key role in blood pressure regulation, promotes hepatic fibrogenesis. In this study, we investigated the effects of (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea catechins, on the development of hepatic preneoplastic lesions in SHRSP.Z-Leprfa/IzmDmcr (SHRSP-ZF) obese and hypertensive rats. Methods: Male 7-week-old SHRSP-ZF rats and control non-obese and normotensive WKY/Izm rats were fed a high fat diet and received intraperitoneal injections of carbon tetrachloride twice a week for 8 weeks. The rats were also provided tap water containing 0.1% EGCG during the experiment. Results: SHRSP-ZF rats presented with obesity, insulin resistance, dyslipidemia, an imbalance of adipokines in the serum, and hepatic steatosis. The development of hepatic preneoplastic lesions and liver fibrosis was markedly accelerated in SHRSP-ZF rats compared to that in control rats. Additionally, in SHRSP-ZF rats, RAS was activated and inflammation and oxidative stress were induced. Administration of EGCG, however, inhibited the development of hepatic premalignant lesions by improving liver fibrosis, inhibiting RAS activation, and attenuating inflammation and oxidative stress in SHRSP-ZF rats. Conclusions: Obese and hypertensive SHRSP-ZF rats treated with a high fat diet and carbon tetrachloride displayed the histopathological and pathophysiological characteristics of NASH and developed hepatic premalignant lesions, suggesting the model might be useful for the evaluation of NASH-related liver tumorigenesis. EGCG might also be able to prevent NASH-related liver fibrosis and tumorigenesis. Citation Format: Takahiro Kochi, Masahito Shimizu, Takuji Tanaka, Hisataka Moriwaki. (-)-Epigallocatechin-3-gallate inhibits the development of hepatic preneoplastic lesions in obese and hypertensive rats, a novel model of non-alcoholic steatohepatitis-related liver tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 201. doi:10.1158/1538-7445.AM2013-201

A novel splicing variant of rat Pregnone X receptor down regulates CYP gene expression.

Pregnane X receptor (PXR) is a nuclear receptor that regulates transcription of both phase I and phase II drug metabolize enzymes. PXR regulate a wide range of biological and physiological processes including drug metabolism and homeostasis of numerous endobiotics. Many alternatively spliced variants of nuclear receptors have been shown to have various effects on the transactivation of the wild‐type receptor. Multiple splicing variants were also identified for the PXR mRNA. Here we report the novel splicing variant mRNA of the rat PXR (PXR‐v). PXR‐v contains 5th intron sequence in the mRNA and it translated into a c‐terminal truncated protein, which lacks a half of the ligand binding domain. This variant mRNA is predominantly expressed in the pre‐neoplastic liver. PXR‐v cDNA transfected hepatoma cells properly expressed variant mRNA and variant protein. The CYP gene, a target gene of PXR, expression was markedly down regulated in these transformants.

Involvement of multiple cell cycle aberrations in early preneoplastic liver cell lesions by tumor promotion with thioacetamide in a two-stage rat hepatocarcinogenesis model

Thioacetamide (TAA) induces oxidative stress and hepatocarcinogenicity in rats. We previously reported that TAA promotion caused various disruptions in cell cycle protein expression in rats, including downregulation of p16(Ink4a), which is associated with intraexonic hypermethylation in hepatocellular proliferative lesions. This study further investigated the contribution of cell cycle aberrations associated with early hepatocarcinogenic processes induced by TAA using antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), in a two-stage rat hepatocarcinogenesis model. TAA-promotion after initiation with N-diethylnitrosamine increased the number and area of hepatocellular foci immunoreactive for glutathione S-transferase placental form (GST-P) and the numbers of proliferating and apoptotic cells. Co-treatment with EMIQ and ALA suppressed these increases. TAA-induced formation of p16(Ink4a-) foci in concordance with GST-P(+) foci was not suppressed by co-treatment with EMIQ or ALA. TAA-promotion increased cellular distributions of cell proliferation marker Ki-67, G2/M and spindle checkpoint proteins (phosphorylated checkpoint kinase 1 and Mad2), the DNA damage-related protein phosphorylated histone H2AX, and G2-M phase-related proteins (topoisomerase IIα, phosphorylated histone H3 and Cdc2) within GST-P(+) foci, and co-treatment with EMIQ or ALA suppressed these increases. These results suggest that downregulation of p16(Ink4a) may allow selective proliferation of preneoplastic cells by TAA promotion. However, antioxidants did not counteract this gene control. Moreover, effective suppression of TAA-induced cellular population changes within preneoplastic lesions by antioxidants may reflect facilitation of cell cycling and accumulation of DNA damage causing the activation of cell cycle checkpoints, leading to G2 and M phase arrest at the early stages of hepatocarcinogenesis promoted by TAA.

Effect of enzymatically modified isoquercitrin on preneoplastic liver cell lesions induced by thioacetamide promotion in a two-stage hepatocarcinogenesis model using rats

To investigate the protective effect of enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and thioacetamide (TAA)-promoted rats. We examined the modifying effect of co-administration with EMIQ on the liver tissue environment including hepatic macrophages and lymphocytes and on the induction mechanism of preneoplastic cell apoptosis during early stages of hepatocellular tumor promotion. TAA increased the number and area of glutathione S-transferase placental form (GST-P)+ liver cell foci and the numbers of proliferating and apoptotic cells in randomly selected areas in liver sections. Co-administration with EMIQ suppressed these effects. TAA also increased the numbers of ED2+, cyclooxygenase-2+, and heme oxygenase-1+ liver cells, as well as the number of CD3+ lymphocytes. These effects were also suppressed by EMIQ. EMIQ increased liver levels of thiobarbituric acid-reactive substance and 8-hydroxydeoxyguanosine, and TUNEL+ apoptotic cells, death receptor 5 (DR5)+ cells and 4-hydroxy-2-nonenal+ cells within GST-P+ foci. Outside the GST-P+ foci, EMIQ decreased the numbers of apoptotic cells and DR5+ cells. These results suggest that TAA-induced tumor promotion involves activation of hepatic macrophages producing proinflammatory factors. EMIQ may suppress the TAA-induced tumor-promoting activity by an anti-inflammatory mechanism mediated by suppressing the activation of these macrophages. Furthermore, EMIQ may suppress tumor-promoting activity differentially between the inside and outside of GST-P+ foci. Within GST-P+ foci, EMIQ facilitates the apoptosis of preneoplastic cells through the upregulation of DR5. Outside the GST-P+ foci, EMIQ suppresses apoptosis and the subsequent regeneration of non-transformed liver cells.

Abstract PR1: Oncogenic driver mutations shape the nature of immune responses in tumors.

Abstract The composition of tumor-associated leukocytes and their pro- or anti-tumor polarization are heterogeneous among different tumor types. In addition, the abundance of leukocyte subtypes can vary even among tumors of the same type. Understanding what determines this heterogeneity is important since the immune cell contexture of tumors can serve as a strong predictor of clinical outcomes and response to anti-cancer therapy. Given that tumorigenesis is driven by mutations in various types of proto-oncogenes and tumor-suppressor genes, we postulated that the identity of driver mutation could shape the leukocytic composition of tumors. To test this, we used the established mouse models for liver tumorigenesis induced by hydrodynamic transfection of c-MYC (MYC) or myr-AKT1+N-RasV12 (AKT+RAS) oncogenes. The resulting somatic integration and long-term overexpression of MYC or AKT+RAS in the liver leads to development of hepatoblastoma and hepatocellular carcinoma, respectively. We profiled liver-infiltrated CD45+ leukocytes in these two models by staining liver single-cell suspensions with lineage-specific leukocyte markers, followed by polychromatic FACS analysis. A massive increase in the CD11b+/F4/80-/Ly6Ghi/Ly6Cmed fraction of CD45+ leukocytes was detected in the AKT+RAS, but not in the MYC model. Compared to the vehicle-injected controls, AKT+RAS livers exhibited a 3-fold increase in this leukocyte population both in pre-neoplastic stages and in tumors. Overexpression of either oncogenes alone did not trigger such increase, suggesting that CD11b+/F4/80-/Ly6Ghi/Ly6Cmed response resulted from cooperation between AKT and RAS. Consistent with the FACS results, IHC staining of liver tissue sections with the neutrophil marker 7/4 antigen revealed an abundant positive staining both in pre-neoplastic livers and in tumors in the AKT+RAS model, while only a small infiltration of 7/4 antigen-positive cells was observed the MYC model only at tumor stage. To address if CD11b+/F4/80-/Ly6Ghi/Ly6Cmed play a role in tumorigenesis, we treated mice with the rat monoclonal Gr-1 antibody to systemically deplete Ly6G/Ly6C-expressing cells. Treatment with Gr-1 starting on day -1 before oncogene transfection ablated CD11b+/F4/80-/Ly6Ghi/Ly6Cmed cells from the livers and showed a tendency to increase the tumor burden in AKT+RAS mice for nearly 50%, suggesting that these cells act in early stages to suppress AKT+RAS-induced tumorigenesis. In contrast, Gr-1 treatment starting on day 4 after oncogene transfection no longer had an effect on AKT+RAS-induced tumorigenesis. Nevertheless, the same protocol significantly extended survival of mice and reduced tumor burden in the MYC model, suggesting a tumor-promoting role of CD11b+/F4/80-/Ly6Ghi/Ly6Cmed in MYC-induced tumorigenesis. In conclusion, our results demonstrate that the leukocytic composition and the nature of immune responses during tumorigenesis are shaped, at least in part, by tumor-driving mutations. We are currently elucidating the mechanism by which CD11b+/F4/80-/Ly6Ghi/Ly6Cmed cells promote or suppress tumorigenesis driven by MYC and AKT+RAS. This abstract is also presented as Poster A46. Citation Format: Vladislava Juric, Brian Ruffell, J. Michael Bishop. Oncogenic driver mutations shape the nature of immune responses in tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR1.

Hepatic Lesions Observed in Hepatitis C Virus Transgenic Mice Infected by Helicobacter hepaticus

The heterogeneity of hepatitis C virus (HCV) infection cannot always be explained by HCV genotypes or host genetic factors, raising the issue of possible cofactors. A new form of hepatitis leading to liver cancer was discovered in 1992 in mice, owing to an infection by Helicobacter hepaticus. Moreover, several studies showed an association between the presence of HCV and Helicobacter in the liver of patients with severe liver diseases suggesting a possible synergism between the two pathogens. In an HCV transgenic mouse model with a B6C3F1 background, the combination of H. hepaticus infection and the HCV transgene resulted in a significantly greater incidence and multiplicity of preneoplastic and neoplastic liver foci in males. Because the mouse genetic background is a major determinant in the development of liver disease, our aim was to test the synergism between HCV and H. hepaticus infection using transgenic mice with a more sensitive genetic background to H. hepaticus infection. For this purpose, four groups of mice were followed up to 14 months, the presence of H. hepaticus was monitored by PCR and hepatic lesions were looked for. We found that H. hepaticus, but not the HCV transgene, increased the number of hepatic lesions. The presence of carcinoma was more likely to occur on a background of hepatitis, and the overall lesions were more frequent in the presence of steatosis. The effect of the mouse genetic background was greater than the effect of the HCV transgene and was sufficient to promote lesions particularly via its sensitivity to H. hepaticus infection. Genetic susceptibility may be a more important factor than expected. Indeed, the synergism between HCV and H. hepaticus infection involved in liver disease may be highly host dependent.

Disruptive cell cycle regulation involving epigenetic downregulation of Cdkn2a (p16Ink4a) in early-stage liver tumor-promotion facilitating liver cell regeneration in rats

Cell cycle aberration was immunohistochemically examined in relation to preneoplastic liver cell foci expressing glutathione S-transferase placental form (GST-P) at early stages of tumor-promotion in rats with thioacetamide (TAA), a hepatocarcinogen facilitating liver cell regeneration. Immunoexpression of p16Ink4a following exposure to other hepatocarcinogens/promoters and its DNA methylation status were also analyzed during early and late tumor-promotion stages. GST-P+ liver cell foci increased cell proliferation and decreased apoptosis when compared with surrounding liver cells. In concordance with GST-P+ foci, checkpoint proteins at G1/S (p21Cip1, p27Kip1 and p16Ink4a) and G2/M (phospho-checkpoint kinase 1, Cdc25c and phospho-Wee1) were either up- or downregulated. Cellular distribution within GST-P+ foci was either increased or decreased with proteins related to G2-M phase or DNA damage (topoisomerase IIα, phospho-histone H2AX, phospho-histone H3 and Cdc2). In particular, p16Ink4a typically downregulated in GST-P+ foci and regenerative nodules at early tumor-promotion stage with hepatocarcinogens facilitating liver cell regeneration and in neoplastic lesions at late tumor-promotion stage with hepatocarcinogens/promoters irrespective of regenerating potential. Hypermethylation at exon 2 of Cdkn2a was detected at both early- and late-stages. Thus, diverse disruptive expression of G1/S and G2/M proteins, which allows for clonal selection of GST-P+ foci, results in the acquisition of multiple aberrant phenotypes to disrupt checkpoint function. Moreover, increased DNA-damage responses within GST-P+ foci may be the signature of genetic alterations. Intraexonic hypermethylation may be responsible for p16Ink4a-downregulation, which facilitates cell cycle progression in early preneoplastic lesions produced by repeated cell regeneration and late-stage neoplastic lesions irrespective of the carcinogenic mechanism.

Abstract 310: Oncogene-specific regulation of immune responses during tumorigenesis

Abstract Oncogenes can induce inflammatory signaling, leading to the recruitment of immune cells to tumor site. Immune cells can further influence tumor development by either restraining or facilitating tumorigenesis. Here, we investigated how distinct oncogenes shape the immune responses during tumorigenesis in vivo. We utilized several established mouse models of liver cancer in which tumorigenesis is driven by the overexpression of MYC, MET, or AKT+RAS oncogenes. We evaluated the immune cells at early vs. late stages of tumor development by immunohistochemical staining of liver tissues using lineage-specific immune cell markers. In addition, we assessed activation of the pro-inflammatory NFκB pathway by immunoblot detection of IkBα and phospho-Ser(536)-NFκB levels in liver lysates, as well as by immunohistochemical analysis of NFκB (p65) nuclear localization. Histological analysis revealed leukocytic clustering around hepatocytes in pre-neoplastic livers in MYC and AKT+RAS, but not in the MET liver tumor model. Moreover, the leukocyte profiles identified in pre-neoplastic livers and liver tumors were different in MYC vs. AKT+RAS model. Finally, we found that the NFκB pathway was inactive in MYC and AKT+RAS tumors, but hyperactivated in MET tumors. In summary, our results suggest that the type of tumor-initiating oncogenic lesion profoundly influences the identity of recruited immune cells and activation of NFκB pathway in the course of tumorigenesis. We are currently in the process of elucidating the mechanisms behind oncogene-mediated regulation of immune responses, which may reveal novel targets for anti-cancer therapy. 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 310. doi:1538-7445.AM2012-310

V-AKT murine thymoma viral oncogene homolog/mammalian target of rapamycin activation induces a module of metabolic changes contributing to growth in insulin-induced hepatocarcinogenesis

Mounting epidemiological evidence supports a role for insulin-signaling deregulation and diabetes mellitus in human hepatocarcinogenesis. However, the underlying molecular mechanisms remain unknown. To study the oncogenic effect of chronically elevated insulin on hepatocytes in the presence of mild hyperglycemia, we developed a model of pancreatic islet transplantation into the liver. In this model, islets of a donor rat are transplanted into the liver of a recipient diabetic rat, with resulting local hyperinsulinism that leads to the development of preneoplastic lesions and hepatocellular carcinoma (HCC). Here, we investigated the metabolic and growth properties of the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (AKT/mTOR) pathway, a major downstream effector of insulin signaling, in this model of insulin-induced hepatocarcinogenesis. We found that activation of insulin signaling triggers a strong induction of the AKT/mTOR cascade that is paralleled by increased synthesis of fatty acids, cholesterol, and triglycerides, induction of glycolysis, and decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic and neoplastic liver lesions, when compared with the healthy liver. AKT/mTOR metabolic effects on hepatocytes, after insulin stimulation, were found to be mTORC1 dependent and independent in human HCC cell lines. In these cells, suppression of lipogenesis, glycolysis, and the pentose phosphate pathway triggered a strong growth restraint, despite insulin administration. Noticeably, metabolic abnormalities and proliferation driven by insulin were effectively reverted using the dual PI3K/mTOR inhibitor, NVP-BEZ235, both in vitro and in vivo. The present results indicate that activation of the AKT/mTOR cascade by unconstrained insulin signaling induces a defined module of metabolic alterations in hepatocytes contributing to aberrant cell growth. Thus, inhibition of AKT/mTOR and related metabolic changes might represent a novel preventive and therapeutic approach to effectively inhibit insulin-induced hepatocarcinogenesis.

A multi-mineral natural product inhibits liver tumor formation in C57BL/6 mice.

C57BL/6 mice were maintained for up to 18 months on high-fat and low-fat diets with or without a multi-mineral supplement derived from the skeletal remains of the red marine algae Lithothamnion calcareum. Numerous grossly observable liver masses were visible in animals on the "western-style" high-fat diet sacrificed at 12 and 18 months. The majority of the masses were in male mice (20 out of 100 males versus 3 out of 100 females; p = 0.0002). There were more liver masses in animals on the high-fat diet than on the low-fat diet (15 out of 50 on high-fat versus 5 out of 50 on low-fat; p = 0.0254). The multi-mineral supplement reduced the number of liver masses in mice on both diets (3 out of 25 male mice in the low-fat diet group without the supplement versus 1 out of 25 mice with supplement; 12 of 25 male mice in the high-fat diet group without the supplement versus 3 of 25 mice with supplement [p = 0.0129]). Histological evaluation revealed a total of 17 neoplastic lesions (9 adenomas and 8 hepatocellular carcinomas), and 18 pre-neoplastic lesions. Out of eight hepatocellular carcinomas, seven were found in unsupplemented diet groups. Steatosis was widely observed in livers with and without grossly observable masses, but the multi-mineral supplement had no effect on the incidence of steatosis or its severity. Taken together, these findings suggest that a multi-mineral-rich natural product can protect mice against neoplastic and pre-neoplastic proliferative liver lesions that may develop in the face of steatosis.

Role of epigenetic and miR‐22 and miR‐29b alterations in the downregulation of Mat1a and Mthfr genes in early preneoplastic livers in rats induced by 2‐acetylaminofluorene

Carcinogenesis is a multistep sequential process of clonal expansion of initiated cells associated with the accumulation of multiple cancer-specific heritable phenotypes. The acquisition of these heritable cancer-specific alterations may be triggered by mutational and/or non-mutational changes in the genome that affect the regulation of gene expression. Currently, cancer-specific epigenetically mediated changes in gene expression are regarded as driving events in tumorigenesis. In the present study, we investigated the role of gene-specific expression changes in the mechanism of rat hepatocarcinogenesis induced by the complete hepatocarcinogen 2-acetylaminofluorene (2-AAF). The results of the present study demonstrate significant alterations in gene expression, especially of Mat1a and Mthfr genes, during early stages of rat 2-AAF-induced liver carcinogenesis. Both of these genes were downregulated in the livers of 2-AAF-treated male rats. Inhibition of Mat1a expression was associated with an increase in histone H3 lysine 27 trimethylation and a decrease in histone H3 lysine 18 acetylation at the gene promoter/first exon region. Additionally, we demonstrate for the first time a critical contribution of miR-22 and miR-29b microRNAs in the inhibition of Mat1a and Mthfr gene expression during 2-AAF-induced rat hepatocarcinogenesis. The downregulation of Mat1a and Mthfr genes was accompanied by marked functional alterations in one-carbon metabolism. The results of the present study suggest that downregulation of the Mat1a and Mthfr genes may be one of the main driver events that promote liver carcinogenesis by causing a profound accumulation of subsequent epigenetic abnormalities during progression of the carcinogenic process.

KIF20A mRNA and Its Product MKlp2 Are Increased During Hepatocyte Proliferation and Hepatocarcinogenesis

Mitotic kinesin-like protein 2 (MKlp2), a microtubule-associated motor, is required during mitosis exit for the final step of cytokinesis. It also contributes to retrograde vesicular trafficking from the Golgi apparatus to the endoplasmic reticulum in interphase. The KIF20A gene encoding MKlp2 is controlled by the E2F-retinoblastoma protein-p16 pathway, and its widely expressed mRNA is found in fetal and proliferating adult tissues. The expression pattern and function of MKlp2 in the adult liver, however, have not been investigated. We report herein that MKlp2 transiently accumulates in vivo during mouse liver regeneration after partial hepatectomy and is strongly overexpressed in preneoplastic and neoplastic mouse liver. In vitro in mitogen-stimulated primary hepatocytes, MKlp2 accumulated in the nucleus during the G2 phase of the cell cycle coincident with the mitotic kinase Aurora B. Human hepatoma cell lines exhibited high levels of MKlp2; however, it was undetectable in normal human hepatocytes. RNAi-mediated MKlp2 knockdown in hepatoma cells induced polyploidization consistent with its essential function in promoting cytokinesis and inhibited cell proliferation without inducing apoptosis. KIF20A mRNA was strongly accumulated in a large series of human hepatocellular carcinomas, with the highest expression observed in tumors with genomic instability. Accumulation of MKlp2 in normal proliferating, preneoplastic, and transformed hepatocytes suggests that MKlp2 contributes to both normal and pathologic hepatocyte proliferation and is linked to tumor aggressiveness in human hepatocellular carcinomas.

Chronic Administration of 2-Acetylaminofluorene Alters the Cellular Iron Metabolism in Rat Liver

Dysregulated intracellular iron homeostasis has been found not only in rodent and human hepatocellular carcinomas but also in several preneoplastic pathological states associated with hepatocarcinogenesis; however, the precise underlying mechanisms of metabolic iron disturbances in preneoplastic liver and the role of these disturbances remain unexplored. In the present study, using an in vivo model of rat hepatocarcinogenesis induced by 2-acetylaminofluorene, we found extensive alterations in cellular iron metabolism at preneoplastic stages of liver carcinogenesis. These were characterized by a substantial decrease in the levels of cytoplasmic non-heme iron in foci of initiated hepatocytes and altered expression of the major genes responsible for the proper maintenance of intracellular iron homeostasis. Gene expression analysis revealed that the decreased intracellular levels of iron in preneoplastic foci might be attributed to increased iron export from the cells, driven by upregulation of ferroportin (Fpn1), the only known non-heme iron exporter. Likewise, increased Fpn1 gene expression was found in vitro in TRL1215 rat liver cells with an acquired malignant phenotype, suggesting that upregulation of Fpn1 might be a specific feature of neoplastically transformed cells. Other changes observed in vivo included the downregulation of hepcidin (Hamp) gene, a key regulator of Fpn1, and this was accompanied by decreased levels of CCAAT/enhancer binding proteins alpha and beta, especially at the Hamp promoter. In conclusion, our results demonstrate the significance of altered intracellular iron metabolism in the progression of liver carcinogenesis and suggest that correction of these alterations could possibly affect liver cancer development.

Suppression of Amphiregulin/Epidermal Growth Factor Receptor Signals Contributes to the Protective Effects of Quercetin in Cirrhotic Rats ,

The hepatic wound-healing response to chronic noxious stimuli may lead to liver fibrosis, a key feature of the preneoplastic cirrhotic liver. Fibrogenic cells activate in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin is an epidermal growth factor receptor (EGFR) ligand specifically induced upon liver injury. We investigated the effects of quercetin on the amphiregulin/EGFR signal and on the activation of downstream pathways leading to cell growth. Rats were divided into 4 groups (8 rats/group): rats subjected to common bile duct ligation (CBDL), Sham (rats subjected to simulated CBDL), quercetin-treated sham, and quercetin-treated CBDL (CBDL-Q). Quercetin (50 mg/kg i.p. injection) was administered daily for 2 wk starting on d 14 after surgery. Overexpression of amphiregulin, EGFR, TNFa, IL-6, TGFβ, platelet-derived growth factor (PDGF), extracellular regulated kinase, protein kinase B (Akt), cycloxygenase (COX)-2, and glioma-associated oncogenes (GLI)-1 and-2 were observed in liver of CBDL rats after 4 wk of bile duct ligation. CBDL-Q rats had a significantly diminished expression of amphiregulin and EGFR compared with untreated CBDL rats. Furthermore, mRNA levels of TNFa, IL-6, TGFβ, and PDGF and the protein content of COX-2, GLI-1, and GLI-2 were significantly lower in CBDL-Q rats than in untreated CBDL rats. The findings indicate that quercetin ameliorated activation of survival pathways and downregulated the expression of genes related to inflammation and precancerous conditions. Suppression of amphiregulin/EGFR signals may contribute to this effect.

Abstract 5556: Role of the aberrant intracellular iron metabolism in rat liver carcinogenesis

Abstract Intracellular iron homeostasis in normal cells is tightly regulated by the coordination of several proteins that are responsible for the uptake, intracellular storage, and removal of iron from cells. Cancer cells, including liver cancer cells, are characterized by profound abberations in intracellular iron metabolism. Dysregulated iron homeostasis has been found not only in rodent and human hepatocellular carcinomas, but also in several preneoplastic pathological states associated with liver cancer development; however, the precise mechanistic role of iron metabolic disturbances in the initiation and early progression of liver carcinogenesis has remained unexplored. In the present study, using an in vivo model of rat hepatocarcinogenesis induced by 2-acetylaminofluorene, we found extensive alterations in cellular iron metabolism at preneoplastic stages of liver carcinogenesis characterized by a substantial decrease in the levels of cytoplasmic non-heme iron in initiated hepatocytes and altered expression of the major genes responsible for the proper maintenance of accurate intracellular homeostasis. Specifically, analysis of the gene expression patterns revealed prominent up-regulation of the ferroportin (Fpn) gene and down-regulation of the hepcidin (Hamp) gene in the preneoplastic livers. This indicates that the decreased intracellular levels of non-heme iron in preneoplastic foci may be attributed to increased iron export from the cells driven by the loss of an inhibitory effect of hepcidin on Fpn. This suggestion is supported by a substantial up-regulation of Fpn (3-fold) compared to transferrin receptor (Tfrc) gene, (1.6 fold), the latter of which is responsible for the iron intake into cell. These data indicate that in preneoplastic livers there is a disruption in the balance of regulatory networks controlling cellular iron uptake and export. Interestingly, we detected similar gene expression changes in initiated preneoplastic liver cells that were transformed by culturing of non-tumorigenic TRL-1215 rat liver cells in the presence of iron. In conclusion, our results demonstrate the significance of altered intracellular iron metabolism in the progression of liver carcinogenesis and suggest that correction of these alterations may affect liver cancer development. 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 5556. doi:10.1158/1538-7445.AM2011-5556