Carcinogenesis

Carcinogenesis Advance Access originally published online on December 20, 2006
Carcinogenesis 2007 28(5):940-946; doi:10.1093/carcin/bgl249

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Published by Oxford University Press 2006.

Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice

Insook Kim, Keiichirou Morimura, Yatrik Shah, Qian Yang, Jerrold M. Ward and Frank J. Gonzalez^*

Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

^* To whom correspondence should be addressed. Tel: +301 496 9067; Fax: +301 496 8409; Email: fjgonz{at}helix.nih.gov

	Abstract

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The farnesoid X receptor (FXR) controls the synthesis and transport of bile acids (BAs). Mice lacking expression of FXR, designated Fxr-null, have elevated levels of serum and hepatic BAs and an increase in BA pool size. Surprisingly, at 12 months of age, male and female Fxr-null mice had a high incidence of degenerative hepatic lesions, altered cell foci and liver tumors including hepatocellular adenoma, carcinoma and hepatocholangiocellular carcinoma, the latter of which is rarely observed in mice. At 3 months, Fxr-null mice had increased expression of the proinflammatory cytokine IL-1ß mRNA and elevated ß-catenin and its target gene c-myc. They also had increased cell proliferation as revealed by increased PCNA mRNA and BrdU incorporation. These studies reveal a potential role for FXR and BAs in hepatocarcinogenesis.

Abbreviations: BA, bile acid; FXR, farnesoid X receptor; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; PBS, phosphate-buffered saline; qPCR, quantitative polymerase chain reaction; RT, room temperature; WT, wild-type

	Introduction

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The farnesoid X receptor (FXR, NR1H4), a member of the nuclear receptor superfamily, controls the synthesis and transport of bile acids (BAs) in liver and gut (1). In response to BA binding, it positively regulates a number of genes in these tissues that serve to decrease cellular levels of BAs. In mice, FXR induces the small heterodimer partner (SHP) in liver that downregulates Cyp7a1 and Cyp8b1 genes encoding enzymes that synthesize BAs from cholesterol and the Na⁺-taurocholate pump that transports BAs from the serum to the liver. It also controls transporters such as the bile salt export pump that transports BAs from the liver into the bile canaliculi. In the gut, FXR induces the ileal BA-binding protein and the organic solute and steroid /ß transporter that serve to transport BAs from the gut to the circulation where they are transported back to the liver. In addition, in response to BAs, FXR activates fibroblast growth factor 15/19 gene; increased fibroblast growth factor 15/19 is transported to the liver where, through the FGFR4 receptor signal transduction pathway, it induces expression of Na⁺-taurocholate pump and downregulates the Cyp7a1 and Cyp8b1 genes (1).

Studies on the Fxr-null mice revealed that FXR is central to control of BA homeostasis and enterohepatic circulation of BAs (2). Due to the absence of FXR expression, the Fxr-null mice accumulate high hepatic levels of BAs resulting in steatosis, one of the pathologies of cholestasis in humans. These mice also have elevated serum BAs, cholesterol and triglycerides, the latter of which contribute to insulin resistance (3,4). A consequence of hepatosteatosis is liver cell injury that can result in increased oxidative stress and hepatocyte proliferation that can possibly lead to liver cancer. Indeed, in the present study, the Fxr-null mice were found to spontaneously develop hepatocellular lesions, adenomas and carcinomas. This was due in part to increased cell proliferation and expression of cytokines and oncogenes.

	Materials and methods

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Mice
Wild-type (WT) and strain-matched Fxr-null mice on a C57BL/6N mixed background were maintained in the National Cancer Institute vivarium with food and water provided ad libitum. Mice were killed by carbon dioxide asphyxiation. All animal studies were carried out in accordance with the Institute of Laboratory Animal Resources guidelines and approved by the National Cancer Institute Animal Care and Use Committee.

Pathology
Histological analysis was carried out on livers of 3-month-old and 12-month-old Fxr-null mice. Tissues were carefully investigated for the existence of tumors after being weighed, and small portions of liver tissues without any macroscopic nodules were snap frozen in liquid nitrogen and stored at –80°C until further analysis. The remaining liver tissues were fixed in 10% phosphate-buffered formalin, embedded in paraffin and prepared for routine hematoxylin and eosin staining for histological examination. Serial sections of the liver tumor were also stained by Masson's trichrome staining to detect collagen.

RNA analysis
RNA was extracted from liver using TRIzol reagent (Invitrogen, Carlsbad, CA). Quantitative real-time polymerase chain reaction (qPCR) was performed using cDNA generated from 1 µg total RNA with SuperScript III reverse transcriptase kit (Invitrogen). Primers were designed for qPCR using the Primer Express software (Applied Biosystems, Foster City, CA), and sequences are available upon request (Supplementary Table). qPCR reactions were carried out using SYBR Green PCR master mix (Applied Biosystems) in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Values were quantified using the comparative CT method, and samples normalized to ß-actin.

Western blot analysis
Total cell extracts were prepared from frozen livers by homogenizing in lysis buffer [20 mM MOPS pH 7.0, 0.5% Triton X-100, 1 mM ethylenediaminetetraacetic acid, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 30 mM Na₃VO₄, 1 mM NaF, 20 mM Na₄O₇P₂, protease inhibitors (Complete protease inhibitor cocktail, Roche)]. The protein concentration of the total extracts was determined by Bradford method (Bio-rad protein assay reagent) using bovine serum albumin as standard. For immunoblot analysis, 25 µg of proteins were electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes blocking in tris-buffered saline containing 0.5% Tween 20 (Sigma, St Louis, MO) and 3% non-fat dry milk for 1 h at room temperature (RT). Membrane was overnight incubated with primary antibodies diluted in the blocking buffer: c-myc (9E10) (1:500, sc-40, Santa Cruz), ß-catenin (1:1000, C7270, Sigma), GAPDH (1:1000, Chemicon) at 4°C and anti-mouse horseradish peroxidase-conjugated IgG (Jackson ImmunoResearch) for 1 h at RT. Immunoreactive bands were identified with chemiluminescence detection system, as described by the manufacturer's protocol (PerkinElmer Lab.).

Hepatocyte proliferation
For the BrdU incorporation study, mice were anesthetized with 2.5% avertin and implanted subcutaneously with an osmotic pump (Alzet model 2001, DURECT Corporation, Cupertino, CA) containing 200 µl of 16 mg/ml BrdU (Sigma). After 1 week of treatment, mice were killed by overexposure to carbon dioxide and livers were removed and fixed in 10% phosphate-buffered formalin (Fisher Scientific, Fair Lawn, NJ). Serial sections were prepared for BrdU staining and TUNEL assay. Sections from the duodenum were also obtained and fixed in 10% phosphate-buffered formalin in order to verify uniform flow of BrdU up to the time the animals were killed. Immunostaining for BrdU (DakoCytomation, Carpinteria, CA) was performed using monoclonal antibody labeled with biotin by the ARK kit (DakoCytomation) prior to application to tissues. In brief, sections were rinsed and incubated sequentially in primary antibody [diluted 1:100 in phosphate-buffered saline (PBS) containing 1% bovine serum albumin] for 2 h at RT and in avidin-biotinylated peroxidase complex (Vector Laboratory, Burlingame, CA) in PBS for 30 min. The bound antibody was visualized by 3, 3'-diaminobenzidine (DAB) as a peroxidase substrate. Sections were rinsed in water, counterstained with hematoxylin (Sigma), dehydrated and mounted in permanent mounting medium. The proliferating cells were quantitated as the number of brown stained nuclei, an indication of BrdU incorporation out of the total hematoxylin-stained cells (blue nuclei).

TUNEL assay
TUNEL assay was performed on serial sections obtained from BrdU study using DeadEnd Colorimetric TUNEL System (Promega, WI) according to manufacturer's protocol. Briefly, after deparaffinization, sections were treated with proteinase K (Sigma) in PBS (20 µg/ml) for 15 min at RT, fixed in 10% phosphate-buffered formalin for 5 min and incubated with biotinylated nucleotide in rTdT reaction buffer for 1 h at 37°C. After terminating the reaction with 2x SSC for 15 min at RT, endogenous peroxidase activity was blocked using 3% H₂O₂ in distilled water for 10 min. Sections were incubated with streptavidin–horseradish peroxidase (1:500 in PBS) for 30 min at RT and the bound conjugates were visualized by 3, 3'-diaminobenzidine as a peroxidase substrate. Sections were counterstained with hematoxylin (Sigma), dehydrated and mounted in permanent mounting medium. Positively stained hepatocyte nuclei were counted in at least 10 randomly chosen fields (x200) from three mice. The TUNEL-positive nuclei were quantitated as a percentage of hematoxylin-stained nuclei (blue nuclei).

BA analysis
For serum BA analysis, WT and Fxr-null mice were anesthetized with 2.5% avertin and trunk blood collected in a serum separator tube (Becton Dickinson, Franklin Lakes, NJ) and the serum was separated by centrifugation at 7000g for 5 min. For hepatic BA measurements, 0.1 g of liver, without macroscopic gross lesions, was homogenized in 70% ethanol, shaking incubated at 50°C for 2 h and centrifuged at 10 000 r.p.m. for 10 min. The supernatant was used for the BA assay. Total BA pool size was determined using liver, gallbladder and intestine with its contents. In brief, collected liver, gallbladder and intestine were weighed, freeze-dried and powered together and BA extract was prepared by shaking incubating in 70% ethanol at 50°C for 4 h followed by the same procedure for hepatic BA extract. The BA content of all samples was measured by colorimetric analysis using a BA analysis kit (Sigma). The BA pool size was calculated based on the weight of BA pool tissues and normalized by body weight.

Statistical analysis
Variations in liver/body weight ratio, BA levels, gene expression and BrdU analysis between the different treatments or animal strains were evaluated with Student's t-test. Variations in the incidences of tumors were evaluated with one-way factorial analysis of variance and multiple comparison tests. All the calculations for statistical analysis were performed using the Statview SE+ Graphics, version 5.0 (Abacus Concepts, Berkeley, CA).

	Results

Top Abstract Introduction Materials and methods Results Discussion Supplementary data References

 
To verify the loss of FXR expression in the Fxr-null mice, FXR mRNA levels were analyzed in 3-month-old and 12-month-old WT and Fxr-null mice (Figure 1A). FXR mRNA expression in 12-month-old WT mice was less than one-third of that of 3-month-old mice. No significant mRNA was detected in livers of Fxr-null mice or either age. As expected, expression of the FXR target gene Shp was high in young and old WT mice and low in the Fxr-null mice (Figure 1B). Conversely, levels of the SHP target gene Cyp7a1 were low in WT mice and high in Fxr-null mice with the 12-month-old mice showing twice the levels of Cyp7a1 mRNA than the 3-month-old null mice (Figure 1C).

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1. Gene expression analysis from liver tissue in 3-month-old (3mo) and 12-month-old (12mo) WT and Fxr-null mice. FXR, CYP7A1 and SHP mRNA expression was assessed by qPCR. Expression was normalized to ß-actin and each bar represents the mean value ± SD. *P < 0.05 compared with livers from age-matched WT controls.

 
Body weights of Fxr-null mice were 20% less than the corresponding WT mice regardless of the age (Figure 2A), whereas the liver size, as a percentage of body weight of the Fxr-null mice, was higher than WT mice only in 12-month-old Fxr-null mice (Figure 2B). As expected, the serum BAs were higher in young Fxr-null mice, as noted in an earlier study (2). The serum BA levels in old Fxr-null mice were 5.6-fold higher than that of control WT mice (15.6 ± 3.8 versus 91.5 ± 26.5 µM; P = 0.03) and were about three times higher than that in the serum of young Fxr-null mice (Figure 2C). Livers of the Fxr-null mice are constantly exposed to higher BA levels as indicated by the increased BA pool size in the 3-month-old Fxr-null mice (Figure 2D). Most importantly, hepatic BA levels were twice as high as that in 12-month-old Fxr-null mice reaching levels of up to 300 µM/g liver tissue (Figure 2E).

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2. Body weights, liver weights and BA levels in WT and Fxr-null mice. Body weights and liver weights were determined in 3-month-old and 12-month-old WT and Fxr-null mice. Serum and BA pool size determined in 3-month-old (3mo) and serum and hepatic BAs determined in 12-month-old (12mo) WT and Fxr-null mice. *P < 0.05.

 
Both male and female Fxr-null mice killed at 12 months of age were found to have liver lesions (Table I). Fxr-null livers exhibited macroscopic gross lesions (Figure 3a) and histological analysis revealed that in most 12-month-old mice, degenerative lesions were found that consisted of hypertrophic and eosinophilic hepatocytes accompanied by proliferating oval cells and lipid disposition (Figure 3b and g). Hepatocellular adenomas were observed in a number of mice characterized as well-circumscribed lesions composed of well-differentiated hepatocytes (Figure 3c). Hepatocellular carcinoma (HCC) (Figure 3d) and mixed tumors that consisted of hepatocellular and cholangiocellular components were also found in a few mice (Figure 3e). Mixed tumor involved a fibrous stroma as noted by Masson's trichrome staining (Figure 3f) and immune cell infiltration, ductule formation and fibrosis (Figure 3h). Of 53 mice, 64% of mice had preneoplastic foci, 36% had adenomas, 6% had HCCs and 9% had mixed HCCs, hepatocholangiocellular carcinoma (Table I). A total tumor incidence of 38% was found in the 12-month-old Fxr-null mice. In the control WT mice, no foci or tumors were noted. Similar percentages of foci and tumors were found in male and female mice. These data indicated that absence of FXR resulted in hepatic carcinogenesis.

View this table: [in this window] [in a new window]   Table I. Incidence of liver lesions in male and female 12 month old WT of Fxr-null mice

 

View larger version (133K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3. Representative photomicrographs of liver lesions from 12-month-old Fxr-null mice. (a) Representative Fxr-null liver with multiple macroscopic lesions. (b) Degenerative lesions including hypertrophic and eosinophilic hepatocytes accompanied by fat disposition were detected in most Fxr-null mouse livers to various extents. Altered cell foci (arrows) were detected at a high incidence in this group regardless of the gender (original magnification x100). (c) Representative photomicrograph of a hepatocellular adenoma (arrow) which was characterized as well-circumscribed lesions composed of well-differentiated hepatocytes, compressing adjacent parenchyma without normal lobular architecture (original magnification x100). (d) Photomicrograph of HCC (original magnification x400) (e) Photomicrograph of mixed tumor (original mag x100). The tumor consisted of hepatocellular (left side) and cholangiocellular (right side) components. (f) Tumor in (e) has increased fibrous stroma detected by Masson's trichrome staining (original magnification x40). The hepatocellular component showed a trabecular growth pattern and consisted of moderately differentiated neoplastic hepatocytes, and cholangiocellular components showed poorly formed tubular structures lined by low cuboidal cells. Based on these findings, this mixed tumor was diagnosed as a hepatocholangiocellular carcinoma. (g) Oval cell proliferation (arrows) and ductules (original magnification x200). (h) Mixed tumor with inflammatory cell infiltration, ductules formation and fibrosis (original magnification x200).

 
To investigate the mechanism contributing to the formation of liver tumors in Fxr-null mice, the expression of genes involved in inflammation and cell proliferation was analyzed in non-neoplastic hepatic parenchyma. In addition, the cell proliferative gene ß-catenin and its target gene c-myc were also examined in young and aging Fxr-null mice. Among the cytokines examined (interferon , IL-1ß, IL-2, IL-6, IL-10, IL-12p35, IL-12p40, IL-23p19, iNOS, MCP-1 and tumor necrosis factor ) (data not shown), only IL-1ß mRNA levels were increased in 3-month-old and 12-month-old Fxr-null mice as compared with WT mice (Figure 4A). The oncogene c-myc mRNA was also elevated in Fxr-null mice with higher levels noted in younger mice. The protein level of c-myc was accordingly increased in Fxr-null mice at both ages (Figure 4B). ß-Catenin was increased 30% over controls in 3-month-old mice, levels more than double in 12-month-old Fxr-null mice as compared with age-matched WT mice (Figure 4A). Nonetheless, the protein level of ß-catenin did not show detectable differences analyzed using total liver protein extract.

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4. (A) Gene expression analysis from liver tissue in 3-month-old (3mo) and 12-month-old (12mo) WT and Fxr-null mice. IL-1ß, c-myc, ß-catenin and PCNA mRNA expression was assessed by qPCR. Expression was normalized to ß-actin and each bar represents the mean value ± SD. *P < 0.05 compared with livers from age-matched WT controls. (B) Western blot analysis of c-myc and ß-catenin was performed using total protein extract from livers without macroscopic gross lesions. GAPDH was used as an equal loading control. Bars at the right side indicate positions of size markers (Bio-rad prestained sodium dodecyl sulfate–polyacrylamide gel electrophoresis standard low range).

 
Due to the critical role of c-myc in cell proliferation, PCNA expression was examined. Indeed, PCNA mRNA was markedly elevated in 3-month-old Fxr-null mice in non-neoplastic hepatic parenchyma (Figure 4A). In 12-month-old Fxr-null mice, PCNA mRNA expression was significantly decreased. A decrease was also noted in 12-month-old WT mice as compared with 3-month-old WT mice. To confirm the extent of cell proliferation, BrdU incorporation was assessed in 3-month-old and 12-month-old WT and Fxr-null mice (Figure 5A and 5B). A marked increase in labeled nuclei was found in 3-month-old Fxr-null mice compared with age-matched WT mice. There was also an increase in BrdU incorporation in 12-month-old Fxr-null mice compared with controls, albeit the extent of labeling was very low (Figure 5B). Incorporation of BrdU into intestinal epithelial cells did not differ between the 3-month-old WT and Fxr-null mice indicating that BrdU content was similar between the two lines (data not shown).

View larger version (108K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5. Induction of hepatocyte proliferation and increase in hepatocyte TUNEL index in Fxr-null mice. (A) Immunohistochemistry of BrdU-labeled hepatocyte nuclei from livers of 3-month-old (3mo) and 12-month-old (12mo) WT and Fxr-null mice. (B) Quantitation of BrdU labeling index from livers of 3-month-old (3mo) and 12-month-old (12mo) WT and Fxr-null mice. (C) Immunohistochemistry of TUNEL-positive hepatocyte nuclei from livers of 3-month-old (3mo) and 12-month-old (12mo) WT and Fxr-null mice. (D) Quantitation of hepatocyte TUNEL index from livers of 3-month-old (3mo) and 12-month-old (12mo) WT and Fxr-null mice. Quantitation was done by counting at least 10 randomly chosen fields (x200) from three mice livers. Each bar represents the mean value ± SD. *P < 0.05 compared with livers from age-matched WT controls.

 
Hepatocyte apoptosis was evaluated by TUNEL assay using a serial section from BrdU study (Figure 5C and 5D). There was 10-fold increase in TUNEL-positive hepatocytes in 3-month-old Fxr-null mouse liver compared with that in age-matched WT mice (Figure 5D). In 12-month-old Fxr-null mice, the incidence of TUNEL-positive hepatocytes was decreased compared with 3-month-old Fxr-null mice and was 3-fold higher than that in age-matched WT controls.

	Discussion

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Mice lacking expression of FXR were found to develop degenerative liver lesions and liver tumors at 12 months of age with a high incidence of 38% of mice having liver tumors. Since all mice were killed at 12 months of age, the possibility remains that the incidence would have approached 100% in older mice. In this experiment, two major histological types of tumor were detected, hepatocellular adenoma/carcinoma and hepatocholangiocellular carcinoma. Hepatocellular adenoma/carcinoma is a very common liver tumor in mice and can arise spontaneously in B6C3H mice at an incidence of up to 40% in a 2 year bioassay (5). On the other hand, hepatocholangiocellular carcinoma is a very rare tumor and the spontaneous incidence of this tumor is reported to be <0.5% in over 1300 animals, whereas in Fxr-null mice, the incidence was 9% as mixed HCC (6). Carcinogens were also reported to induce this type of tumor, but the incidence is very low compared with that found in the present study (7). Although it was speculated that malignant hepatocytes have the potential for biliary differentiation (8), the pathogenesis of hepatocholangiocellular carcinoma is not well understood. However, it is of interest to note that old Fxr-null liver had proliferating oval cells. Oval cell proliferation normally occurs following severe, prolonged liver injury and is often seen in the preneoplastic stage in liver (9). The elevated hepatic BAs and the sustained chronic inflammation in the Fxr-null mice may have stimulated proliferation of hepatic progenitor cells/oval cells which later differentiate to hepatocytes or cholangiocytes, or they may induce undifferentiated stem cells to proliferate. Interestingly, hepatic fibrosis was observed in Fxr-null liver as shown by the heavy deposit of collagen thus indicating the involvement of activated hepatic stellate cells (HSCs), which produce extracellular matrix upon activation in liver fibrogenesis (10,11). In addition to fibrogenesis, activated HSCs were suggested to induce tumor progression of neoplastic hepatocytes, and BAs at cholestatic concentrations can induce HSCs proliferation via epidermal growth factor receptor (12). Fibrogenesis in the Fxr-null liver can at least in part be attributed to the elevated hepatic BAs, while a direct role for FXR in signaling in HSCs cannot be ruled out since FXR expression was detected in HSCs (13). In this regard, investigations into whether FXR may be a potential therapeutic target for the treatment of cholestasis and fibrosis have gained interest (13–15).

Hepatocellular proliferation is a requirement for tumorigenesis. Indeed, livers of 3-month-old Fxr-null mice were found to be highly proliferative as revealed by increased PCNA expression and BrdU incorporation. The increased proliferation index was accompanied by an increase in mRNA and protein levels of the pro-oncogene c-myc in Fxr-null livers of both 3-month-old and 12-month-old mice. The parallel increase in the intact c-myc protein (63 kDa, Figure 4B) and its degradation product (41 kDa) (16) indicated that increased c-myc protein levels were due to an overall increase in c-myc protein, not due to a different rate of degradation. To see if induction of c-myc is via ß-catenin, ß-catenin expression was analyzed. Whereas expression levels of ß-catenin mRNA were induced, its protein level did not show any detectable differences. This discrepancy may be attributed to post-transcriptional regulation of ß-catenin.

A decrease in BrdU labeling index in the 12-month-old Fxr-null mice was observed despite the higher serum BA levels. This may be due to the overall decrease in liver regenerative capacity by aging especially in the absence of tumors. Notably, a previous study revealed that treatment with the tumor promoter phenobarbital resulted in an initial increase in DNA labeling index but the labeling index did not further increase or returned to control levels after prolonged treatment of 4–8 weeks, whereas liver weights continued to increase during 5 weeks of treatment (17,18). This implies that the BrdU labeling index may not be directly proportional to the duration of stimulation. Since the tumor regions of old Fxr-null livers were not examined, it remains a possibility that over time, the BrdU labeling index may have decreased despite continuous accumulation of hepatic BAs via adaptive compensatory mechanisms. This possibility remains to be proven by further experimentation.

Unpublished data revealed that in urine of 12-month-old Fxr-null mice, BA levels were also mildly elevated consistent with a previous study done in young mice (2). Interestingly, an Fxr-null mouse later diagnosed to carry HCC showed remarkably higher urinary BA excretion among old Fxr-null mice. This suggests that elevated serum BAs, especially in old mice, which correspond to increased urinary excretion, could be further enhanced by progression of hepatic disease.

The hepatocyte TUNEL-positive index was increased 10-fold in the 3-month-old Fxr-null liver compared with age-matched WT control, whereas it was increased 3-fold in 12-month-old Fxr-null mouse liver compared with the same age control group. The TUNEL index was significantly lowered in old mice compared with young Fxr-null mice similar to the BrdU labeling index. Because the apoptotic bodies were not clearly seen in TUNEL-positive hepatocyte, the increased TUNEL index in Fxr-null mice, especially in young mice, probably represent not only apoptotic cell death but also other types of cell death (19).

The increased TUNEL index in young Fxr-null mouse liver may in part explain the absence of obvious hepatomegaly in 3-month-old mice despite the higher proliferative activity. On the other hand, the contributions of apoptosis to tumorigenesis in the current study need further examination because we cannot conclude that the increased TUNEL index is indeed due to increased apoptosis although increase in both cell proliferation and cell death in early and later stages of hepatocarcinogenesis was reported in rat models and human livers (20).

Cytotoxic BA accumulation in liver of Fxr-null mice apparently contributed to the occurrence of degenerative lesions and liver tumor. BAs function as intracellular signaling molecules in a variety of cells and change cellular functions such as proliferation, differentiation, secretion, apoptosis (12,21–25), liver regeneration (26), and modulation of metabolic homeostasis (27).

BAs trigger proliferation of cholangiocyte, biliary epithelial cells in chronic cholestasis (28), and injure cholangiocytes in mice lacking canalicular phospholipid transporter due to the increased monomeric concentration of BAs in the absence of phospholipids in bile (29,30). The canalicular phospholipids transporter, MDR3, is positively regulated by FXR (31). Combined with the elevated hepatic BAs, the loss of adaptive regulation of phospholipids excretion in Fxr-null liver may have contributed to the mixed tumor, hepatocholangiocellular carcinoma formation in Fxr-null mice.

Interestingly, previous studies have shown that BAs can increase IL-1ß levels and elevated BA levels can induce hepatic inflammation (32). Similarly with previous reports, IL-1ß mRNA levels were elevated in young and old Fxr-null mice compared with age-matched WT controls. While the expression of a large number of genes important for inflammation was measured, only IL-1ß expression was increased significantly in Fxr-null mice. The specific increase in IL-1ß mRNA observed in Fxr-null mice may suggest a direct role for BA signaling in IL-1ß regulation. On the other hand, c-myc was suggested to mediate intestinal epithelial cell, IEC-6 cell, proliferation induced by BA treatment (25) and the activation of ß-catenin was associated with promotion of tumor formation in intestine by BAs (33,34).

It is also noteworthy that in a recent study mRNA and protein levels of IL-1ß were induced by Myc activation in ß cells and mediated angiogenesis induced by Myc activation (35). Further studies are warranted to elucidate the underlying mechanism of c-myc induction and concomitant induction of IL-1ß in Fxr-null mouse liver and their association with spontaneous hepatic tumor development in Fxr-null mice.

In conclusion, the present study proposes a mechanism where increased BA levels induce IL-1ß. IL-1ß is an important inflammatory signal that has been demonstrated to mediate cell proliferation, differentiation and apoptosis (36). Perhaps, the alteration in these events mediated by IL-1ß indirectly results in expression of ß-catenin and c-myc, which eventually leads to tumorigenesis. While the mechanism for tumorigenesis in the Fxr-null mouse model remains to be defined, the causal relationship between inflammation and cancer has been well documented (37). The present study identifies a novel role for FXR and hepatic BAs as key mediators of inflammation-induced heptocarcinogenesis.

	Supplementary data

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Supplementary table is available at Carcinogenesis Online.

	Acknowledgments

 
This study was supported by the National Cancer Institute Intramural Research Program. We thank Reiko Kurotani for helpful suggestions.

Conflict of Interest Statement: None declared.

	References

Top Abstract Introduction Materials and methods Results Discussion Supplementary data References

 

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Received August 25, 2006; revised November 27, 2006; accepted December 7, 2006.

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