Molecular profiling of hepatocellular carcinomas developing spontaneously in acyl-CoA oxidase deficient mice: comparison with liver tumors induced in wild-type mice by a peroxisome proliferator and a genotoxic carcinogen
Kirstin Meyer1,4,
Ju-Seog Lee3,
Patricia A. Dyck2,
Wen-Qing Cao1,
M.Sambasiva Rao1,
Snorri S. Thorgeirsson3 and
Janardan K. Reddy1
1 Department of Pathology, Northwestern University, the Feinberg School of Medicine, Chicago, IL 60611-3008, USA
2 Center for Genetic Medicine, Northwestern University, the Feinberg School of Medicine, Chicago, IL 60611-3008, USA
3 Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4262, USA
4 To whom correspondence should be addressed Email: jkreddy{at}northwestern.edu
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Abstract
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By using cDNA microarrays, we studied the expression profiles of 26 hepatocellular carcinomas (HCC) developing spontaneously in peroxisomal fatty acyl-CoA oxidase null (AOX-/-) mice. The development of liver tumors in AOX-/- mice is due to sustained activation of peroxisome proliferator-activated receptor
(PPAR
) by the unmetabolized substrates of AOX, which serve as natural PPAR
ligands. We then compared the AOX-/- liver tumor expression profiles with those induced by ciprofibrate, a non-genotoxic peroxisome proliferator, or by the genotoxic carcinogen diethylnitrosamine (DENA) to discern differences in gene expression patterns that may predict or distinguish PPAR
-mediated liver tumors from genotoxically derived tumors. Our results show that HCCs developing in AOX-/- mice share a number of deregulated (up- or down-regulated) genes with ciprofibrate-induced liver tumors. The overall commonality of expression between AOX-/- and ciprofibrate-induced liver tumors but not with DENA-induced tumors strongly implicates the activation of PPAR
and PPAR
-regulated genes in liver, including those participating in lipid catabolism, as key factors in the development of HCC in AOX-/- and in ciprofibrate-treated mice. Northern blot analysis confirmed the differential expression of some of the genes identified in the present study, and also some genes identified previously as PPAR
regulated, such as CD36, lymphocyte antigen 6 complex locus (Ly-6D), and C3f. We found a panel of 12 genes upregulated in all three classes of liver tumors, namely AOX-/-, ciprofibrate-induced and DENA-induced. These include an uncharacterized RIKEN cDNA, lipocalin 2, insulin-like growth factor-binding protein 1, Ly-6D and CD63 among others. In conclusion, these results identify distinguishing features between non-genotoxic and genotoxic carcinogen derived liver tumors as well as genes that are upregulated in both types and suggest that RIKEN cDNA, Ly-6D and lipocalin 2 in particular appear to be desirable molecular markers for further study in liver carcinogenesis and progression.
Abbreviations: AOX, acyl-CoA oxidase; CYP, microsomal cytochrome P450; DENA, diethylnitrosamine; Ets, E26 avian leukemia oncogene; GST, glutathione S-transferase; HCC, hepatocellular carcinoma; Igfbp, insulin-like growth factor-binding protein; Ly-6D, lymphocyte antigen 6 complex locus; MUP, major (mouse) urinary protein; PPAR
, peroxisome proliferator-activated receptor
.
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Introduction
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Peroxisome proliferators form a broad spectrum of compounds of industrial, pharmaceutical and agricultural importance and include certain phthalate ester plasticizers, herbicides, leukotriene D4 receptor antagonists and lipid-lowering drugs, such as Wy-14, 643 and ciprofibrate, among others (1,2). They induce qualitatively predictable pleiotropic responses, including hepatomegaly, and peroxisome proliferation in liver parenchymal cells of rats and mice (14). Peroxisome proliferators transcriptionally activate genes encoding for the classical inducible peroxisomal straight chain fatty acid ß-oxidation system, microsomal cytochrome P450 4A (CYP4A) fatty acid
-oxidation and some of the genes involved in the mitochondrial ß-oxidation (5,6). The inducible classical peroxisomal ß-oxidation system consists of H2O2-generating fatty acyl-CoA oxidase (AOX), enoyl-CoA- hydratase/L-3-hydroxyacyl-CoA dehydrogenase (peroxisomal bifunctional enzyme; L-PBE) and 3-ketoacyl-CoA thiolase (7). The transcriptional activation of peroxisome proliferator-induced genes is mediated by peroxisome proliferator-activated receptor
(PPAR
), a member of the nuclear receptor superfamily (8). Liganded PPAR
heterodimerizes with the retinoid X receptor (RXR), and this PPAR
RXR heterodimer binds to peroxisome proliferator-activated receptor response elements (PPREs) in the promoter region of target genes to initiate transcriptional activity (9).
Prolonged exposure to peroxisome proliferators vis a vis sustained activation of PPAR
in liver leads to the development of hepatocellular carcinomas (HCC) in rats and mice despite the fact that these agents, either directly or after metabolic activation, fail to exhibit mutagenic or DNA-damaging potential (1012). Since livers with sustained PPAR
activation manifest substantial increases in the expression of H2O2-generating enzymes, namely AOX and CYP4A, it has been proposed that disproportionate increases in H2O2-generating enzymes and reductions in H2O2-degrading enzymes catalase and glutathione peroxidase activity lead to sustained oxidative stress in liver (2,1316). These changes result in progressive accumulation in hepatocytes of lipofuscin, a byproduct of reactive oxygen species-induced lipid peroxidation, and formation of 8-hydroxydeoxyguanosine adducts in liver DNA (1721). This metabolically induced oxidative DNA damage together with subtle increases in hepatocellular proliferation can contribute to the non-genotoxic hepatocarcinogenesis in animals with chronic PPAR
activation (2,1315). Concurrent administration of antioxidants such as ethoxyquin with peroxisome proliferators retard liver tumorigenesis (22). These observations further attest to the importance of oxidative damage in peroxisome proliferator-induced liver carcinogenesis. Also of interest is that hepatic neoplasms induced in rats by the non-genotoxic peroxisome proliferators fail to express the well characterized phenotypic markers
-glutamyltranspeptidase and glutathione-S-transferase
, whereas these two phenotypic markers are typically expressed in putative preneoplastic and neoplastic lesions in liver induced by genotoxic carcinogens (23,24). Accordingly, different mechanisms may become operative in the development of liver tumors following exposure to genotoxic and non-genotoxic agents and the tumors may reflect such hallmarks. Genotoxic chemicals or their electrophilic metabolites exert their effect by directly causing DNA damage and mutagenic action, whereas tumorigenesis with non-genotoxic agents depends upon their biological functional class and resultant metabolic perturbations that contribute to target organ specific neoplastic conversion (13,15). In this regard functional class of peroxisome proliferators, which includes structurally diverse chemicals, became the first major prototype of non-genotoxic carcinogens (25).
Recently, we have shown that disruption of the gene encoding AOX, the first and rate-limiting step of the inducible classical peroxisomal ß-oxidation system, leads to sustained activation of PPAR
in the mouse resulting in spontaneous peroxisome proliferation in liver cells as well as induction of genes that are regulated by PPAR
(26,27). These findings imply that PPAR
is activated in AOX-/- mice by the unmetabolized very-long chain fatty acyl-CoAs and other putative substrates of AOX, which then serve as endogenous ligands of this receptor (27). Therefore, AOX functions as modulator of PPAR
activity (19,27). AOX-/- mice develop HCC between 10 and 15 months of age as a result of sustained hyperactivation of PPAR
in liver by its natural ligands (27). Thus, chronic PPAR
activation either by natural ligands as in AOX-/- mice, or by synthetic ligands as in rats and mice chronically exposed to peroxisome proliferators, leads to the development of liver tumors by non-genotoxic mechanism(s) (11,13,14,27). Furthermore, mice lacking PPAR
are refractory to the induction of peroxisome proliferation, PPAR
-dependent changes in gene expression, and the development of HCC, indicating that PPAR
downstream events play a critical role in peroxisome proliferator-induced and PPAR
-mediated liver carcinogenesis (28,29). These non-genotoxic PPAR
-dependent liver tumor models represent potentially valuable systems for gene expression profiling.
In an effort to identify genes, which might play a role in hepatocarcinogenesis resulting from sustained activation of PPAR
, we investigated the gene expression profiles of HCC developing in AOX-/- mice using cDNA microarrays. Our objective in this study was to begin to compare gene expression profiles in AOX-/- mouse tumors with those induced either by ciprofibrate, a synthetic peroxisome proliferator, which is a non-genotoxic hepatocarcinogen (22), or by the genotoxic carcinogen diethylnitrosamine (DENA) (30), in an effort to identify specific genes or gene clusters that reflect peroxisome proliferator-induced liver cancer. These studies provide some clues regarding the general markers for mouse HCC and also identify few genes that appear to be unique for tumors caused by endogenous and exogenous PPAR
ligands.
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Materials and methods
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Animals and tissue samples
AOX-/- null mice (C57Bl/6 J), 1519 months of age, derived from a colony maintained in this laboratory, were used to harvest HCC (26). A total of 26 HCC were obtained from 12 male and three female AOX-/- mice. These tumors ranged in size from 1 to 2 cm in diameter. To ensure the same genetic background, we also obtained three HCC from wild-type (C57BL/6 J) male mice that were fed ciprofibrate, a synthetic peroxisome proliferator in powdered chow at a concentration of 0.025% (w/w) for 19 months. To obtain HCC induced by a genotoxic hepatocarcinogen, wild-type C57BL/6 J mice (23 months old) were injected once intraperitoneally with DENA (20 mg/kg body wt). Three HCC obtained from these mice were used for analysis. At necropsy, livers were grossly evaluated for tumors and portions were fixed in 10% phosphate-buffered formalin, or 4% paraformaldehyde, for morphological studies or frozen in liquid nitrogen and stored at -80°C until further use. Mice were housed in a controlled environment with a 12-h light/dark cycle. All animal experimentation was carried out according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals.
RNA isolation
Total RNA was isolated from frozen liver tissue using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For cDNA microarrays, RNA was purified a second time using RNeasy-kit (Qiagen, Valencia, CA). Integrity of RNA was assessed by denaturing agarose gel electrophoresis (visual presence of sharp 28S and 18S bands), and spectrophotometry. Tumor mRNA was analyzed using individual tumor samples. RNA from the livers of six wild-type mice (5, 7 and 15 months old) were pooled and used as control in all experiments. We decided to pool the RNA from wild-type mice to reflect the composite of ages of tumor-bearing mice and to obtain a single representative liver mRNA expression profile in mice 315 months old.
Preparation of fluorescence-labeled cDNA and microarray hybridization
To prepare fluorescence-labeled cDNA targets 20 mg of total RNA were reverse transcribed (Superscript II reverse transcription kit; Invitrogen) using aminoallyl-derivatized dUTP incorporation followed by amino-coupling of mono-reactive dyes Cy3 and Cy5 (Amersham Pharmacia Biotech, Piscataway, NJ) as described (31). Labeled targets were purified and concentrated using YM-30 Microcon columns (Millipore, Bedford, MA). The appropriate Cy3 and Cy5 targets were combined, along with 1 µl (10 µg) of mouse COT-1 DNA, 1 µl (810 mg) of poly(A), 1 µl (4 µg) of yeast t-RNA, 15 µl of formamide, 3.2 µl of 20x SSC, and 0.9 µl of 10% SDS in a final volume of 30 µl. After denaturation, labeled targets were added to mouse cDNA arrays containing (Advanced Technology Center, National Institutes of Health, Bethesda, MD). Of the 9180 unique sequences analyzed, 7102 were named genes. After hybridization for 1416 h at 42°C, the slides were washed for 1 min in 1x SSC and 0.1% SDS, for 1 min in 1x SSC and 0.2x SSC, respectively, for 10 s in 0.05x SSC, and then spin dried. Fluorescence images were captured using a GenePixA 4000 scanner (Axon Instruments, Foster City, CA). To eliminate any dye bias, all samples were analyzed at least twice by switching fluorescent dyes Cy5 and Cy3.
Imaging and microarray analysis
Resulting images were analyzed using Gene Pix Pro v3.0 (Axon Instruments). Cy3:Cy5 intensity ratios from each gene were calculated and subsequently normalized to ratios of overall signal intensity from the corresponding channel in each hybridization. The ratio of distribution extracted from microarray images exhibited a normal pattern, constant coefficient-of-variation, and sufficient positivity.
After inverting Cy3:Cy5 ratio values of the reciprocal experiment, statistics were calculated to quantify consistency of up- or down-regulation between the HCC and wild-type mouse liver. Under the null hypothesis there would be no change in gene expression and the log ratio would be zero. Therefore, in our calculations we considered each individual log ratio as a distance from zero and thus used T-distribution applied to the average and standard error to calculate probability values. Genes with low probability values and a fold change >2 in either direction (log ratio >1) were considered to be specific to HCC.
The next step was to associate the genes specific to the HCC with biological knowledge. Gene Ontology (http://www.geneontology.org) classifications were computationally mined from public databases such as the Mouse Genome Informatics at Jackson Laboratories to provide an unbiased characterization of genetic function.
For comparison of gene expression patterns among different tumors average fluorescence ratios of each gene were calculated from duplicated experiments of each sample after statistical analysis according to the above-mentioned criteria. Clustering of the samples from the different tumors was performed using a hierarchical clustering algorithm and Euclidean distance as a measure of difference between samples. Dendogram and heat map were generated using Spotfire software (Spotfire, Somerville, MA). The expression level of each gene in HCC was represented by pseudo-color in matrix format, with red representing expression greater than in normal liver, green representing expression lower than in normal liver, and color intensity representing the magnitude of expression ratio. Comparison of different sized tumor samples did not influence our cluster analysis. For the drug treated groups the n used in the distribution is 2, whereas for the AOX-/- mice the n was 25. Although the probability values for the mutant were lower but the same ratio and P-value were employed for all three groups.
Northern blotting
Total RNA (20 µg) was glyoxylated, electrophoresed on a 0.8% agarose gel and transferred to nylon membranes. Hybridization was carried out at 42°C in 50% formamide hybridization solution using 32P-labeled cDNA probes as described previously (26). Equal loading and transfer were assessed by hybridization with 18S rRNA probe.
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Results
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Analysis of differential gene expression
To ascertain if gene expression patterns provide clues to distinguish HCC induced by non-genotoxic mechanisms from those induced by a genotoxic agent, we used cDNA microarrays to first obtain gene expression profiles of liver tumors induced by sustained activation of PPAR
by its natural ligands in AOX-/- mice. For this purpose, we compared the data derived from 25 liver tumors obtained from 12 AOX-/- mice with expression profiles of liver tumors induced in wild-type mice after prolonged exposure to ciprofibrate, a synthetic non-genotoxic peroxisome proliferator (an exogenous PPAR
ligand), or by the classical liver carcinogen DENA to gain insights into hepatocarcinogenic mechanisms. Conventional methods such as differential display RTPCR and representational difference do provide important clues, but such analyses are usually time consuming and provide a limited amount of information. cDNA microarray technology provides a better tool with a global view in monitoring gene expression although number of genes on a given microarray remains a limitation. In this study, we were able to monitor mRNA expression of 9180 unique murine genes. Our means of identifying differentially expressed genes was based on consistent fold-change and statistical significance assessed by T-distribution applied to the average and standard error to calculate probability values in each tumor/control liver pair.
The genes found differentially expressed in tumors versus normal liver belong to a variety of different categories. The up-regulated genes (Table I), and the down-regulated genes (data not shown) include genes encoding for signal molecules, tumor associated genes, molecules involved in cell adhesion, immune reaction and different metabolic pathways, among others. Analysis of the mRNAs that are deregulated (up- or down-regulated) by 2-fold in tumors compared with normal liver tissue, revealed the following: 60 genes up-regulated and 105 genes down-regulated in AOX-/- mouse tumors; 136 genes up-regulated and 156 down-regulated in ciprofibrate-induced tumors, and 61 genes up-regulated and 105 genes down-regulated in DENA-induced liver tumors. Cluster analysis of all tumor samples revealed 143 genes, which match the arbitrary criterion of 2-fold change in either direction with a statistical significance of P < 0.01 (Figure 1). Of considerable interest is that each tumor class revealed a somewhat unique expression pattern. It should be noted that 25 of 26 liver tumors derived from AOX-/- mice formed a distinct subtype. Ciprofibrate-induced tumor expression profile is closer to that of AOX-/- liver tumors indicating a definable gene expression profile of liver tumors developing as a result of sustained PPAR
-activation either by endogenous or by exogenous ligands. DENA-induced liver tumor expression profile appeared distinct from AOX-/- and ciprofibrate-induced liver tumors. Although the number of ciprofibrate and DENA-induced liver tumors used in this study for comparison is rather small, they clearly reveal distinctive tumor types and different sample sizes were not major contributing factors in Figure 1.

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Fig. 1. Comparison of gene expression patterns in the different HCC. Expression data for 143 genes, from 26 liver tumors derived from AOX-/- mice, and three tumors from ciprofibrate-treated mice, and three tumors induced by DENA in wild-type mice, were analyzed by hierarchical clustering. Data are presented in pseudocolored matrix format; each column represents a tumor and each row a cDNA. Red represents expression greater than reference; green is less reference; and gray is missing or excluded data. Hierarchial clustering dendrogram (green for AOX-/-), red for ciprofibrate-induced, and blue for DENA-induced liver tumors are used to distinguish three major classes of liver tumors. Data seem to distinguish genotoxic (DENA-induced) from nongenotoxic (AOX-/- and ciprofibrate-induced) liver carcinogenesis.
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Comparison of alterations in gene expression in three different tumor classes allowed the identification of genes, which can be considered as general liver tumor markers, independent of the mechanistic origin of the tumors (Table II). Of the 38 commonly deregulated genes in all three classes of tumors, 12 genes were up-regulated and 26 were down-regulated. Lipocalin 2, serum amyloid P-component, insulin-like growth factor-binding protein 1 (Igfbp1), CD63 (3235), lipoprotein lipase and an EST were up-regulated
4-fold or higher in all three classes of liver tumors. The down-regulated genes shared by all three tumors are more diverse and include several members of the cytochrome P450 family (Cyp2c29, Cyp7b1, Cyp4f14 and Cyp26a1) as well as enzymes involved in protein metabolism. The list also includes several ESTs (Table II).
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Table II. Genes with altered expression in all three classes of liver tumors (AOX-/-, ciprofibrate-induced, and DENA-induced)
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Genes upregulated in liver tumors developing in AOX-/- mice and in ciprofibrate-induced tumors but not in DENA tumors
Comparison of gene expression profiles in AOX-/- tumors with ciprofibrate-induced tumors revealed 65 genes, which are deregulated in both these tumor types, but unchanged in DENA tumors (Table III). We found 26 genes upregulated in AOX-/- and ciprofibrate tumors. Besides the known PPAR
-regulated genes such as Cyp4a14, Cyp4A10, carnitine acetyltransferase, fatty acid-binding protein and Pex11
(7), this group includes several tumor-associated genes namely cathepsin L, histone deacetylase 1 and E26 avian leukemia oncogene (Ets2). These two tumor types share 40 genes, whose expression is down-regulated (Table III). We found down-regulation of the placental form of glutathione S-transferase (GST
) by 3.5- and 7.0-fold in AOX-/- mouse tumors and ciprofibrate tumors, respectively. GST
is one of the known phenotypic markers for tumors induced by prolonged peroxisome proliferator treatment (23,24). Several of the genes underexpressed in HCC in AOX-/- and ciprofibrate-treated mice are involved in steroid metabolism and sex-related responses. Reduced expression of 17-ß-hydroxysteroid dehydrogenase (Hsd17b5), 11-ß-hydroxysteroid dehydrogenase (Hsd11b1), and isoenzymes of the Cyp2d family leads to lower levels of male sex hormones. The strongest down-regulation in both PPAR
-related liver tumors was that of major urinary protein (Mup1). Furthermore, reduced expression of some acute-phase response proteins, such as complement component 9 (C9) and serum amyloid A 3 (Saa3) was also noted. A large number of genes shared by tumors developing in AOX-/- mice and induced by ciprofibrate attests to the possible similarities in hepatocarcinogenic mechanisms emanating from chronic PPAR
activation by endogenous and exogenous ligands.
Corroboration of microarray results
We confirmed by northern blotting up- or down-regulation of some of the known PPAR
responsive genes (Figure 2A), some of the genes recently identified as possibly PPAR
-regulated (Figure 2B), as well as some of the genes identified in the present study using cDNA microarray (Figure 2C). For northern analysis total RNA isolated from AOX-/-, ciprofibrate and DENA tumors as well as from age matched pooled wild-type mouse livers was used. It should be noted that comparison of RNA from wild-type mice of different ages did not show any difference (data not shown). As expected, L-PBE, peroxisomal 3-ketoacyl-CoA thiolase, Cyp4a10 and Cyp4a14, ACTE and Pex11
were up-regulated in ciprofibrate-induced and in AOX-/- tumors. AOX mRNA levels increased in ciprofibrate-induced tumors and not in AOX-/- tumors, because of the null mutation of this gene in AOX-/- mouse (Figure 2A). In comparison, these mRNAs were not increased in DENA-induced tumors. Up-regulation of recently identified putative PPAR
regulated genes such as CD36, PDK4, C3f and mLipase was also observed in liver tumors causally related to PPAR
activation but not in DENA-induced tumors (Figure 2B). However, mRNA levels of RIKEN cDNA 1200016E24 gene, and lymphocyte antigen 6 complex locus (Ly-6D), which were identified as putative PPAR
responsive genes in Wy-14, 643-treated mouse livers (37,52), were upregulated in all three types of tumors suggesting a general involvement of these genes in hepatocarcinogenesis. Northern blot data also confirm the increases noted in cDNA microarray of CD63, insulin-like growth factor-binding protein (Igfbp2), lipocalin 2 (Lcn2) and Ets2 in all three classes of tumors (Figure 2C). A marked reduction of MUP1 and C9 was found in all three tumors but more so in AOX-/- and ciprofibrate-induced tumors. Orosomucoid (Orm2) expression was reduced in AOX-/- and ciprofibrate tumors but not in DENA-induced liver tumors (Figure 2C).

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Fig. 2. Confirmation of microarray results by northern blot analysis with total RNA isolated from livers of 15-month-old wild-type mice (lanes 13), and tumors developed in AOX-/- mice (lanes 46), in wild-type mice fed ciprofibrate (0.125%, w/w; lanes 79), and wild-type mice 17 months after a single administration of DENA (20 mg/kg body wt; lanes 1012). Northern blots were hybridized with 32P-labeled cDNA probes as indicated. 18S ribosomal RNA was used as a loading indicator. RIKEN cDNA 1200016E24 gene belongs to a UniGene cluster.
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Discussion
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This study applied cDNA microarray technology to obtain gene expression profiles of individual liver tumors developing in AOX-/- mice from spontaneous hyperactivation of PPAR
resulting from unmetabolized endogenous PPAR
ligands (27). We then compared the expression profiles of AOX-/- liver tumors with profiles of liver tumors induced by synthetic PPAR
ligand ciprofibrate, a non-genotoxic hepatocarcinogen, and by DENA, a genotoxic liver carcinogen. These observations define gene expression modules that might reflect possible tumorigenic mechanisms. The results suggest the involvement of multiple regulatory pathways in liver carcinogenesis in mice with sustained activation of PPAR
either by endogenous or by exogenous ligands. Our results show that HCC developing in AOX-/- mice share a number of deregulated genes with ciprofibrate-induced liver tumors (Table III). The overall commonality of expression profiles between AOX-/- and ciprofibrate-induced liver tumors but not with DENA-induced tumors, strongly implicates the activation of PPAR
and possible involvement of PPAR
-regulated genes in liver as a key factor in the development of HCC in AOX-/- and ciprofibrate-induced treated mice. Microarray and northern blot analyses revealed that the expression of several genes participating in different steps of lipid metabolism is up-regulated in livers developing in AOX-/- mouse and those induced by ciprofibrate. These include the members of the inducible peroxisomal ß-oxidation (with the exception of AOX gene in AOX-/- tumors), microsomal
-oxidation enzymes Cyp4a14, and Cyp4a10, lipoprotein lipase, fatty acid-binding protein and carnitine acetyltransferase. Increased expression of these genes has been described to be associated with PPAR
-dependent peroxisome proliferation (37). This suggests that well differentiated HCC in AOX-/- and peroxisome proliferator-treated mice retain PPAR
-receptor-mediated responses somewhat similar to that seen in normal liver. It also indicates that transcriptional activation of PPAR
regulated genes is not necessarily linked to morphological parameter of peroxisome proliferation, since the size and number of peroxisomes in AOX-/- and ciprofibrate-induced liver tumors were reduced in comparison to non-tumorous liver cells (38).
We observed up-regulation of adipose differentiation related protein (Adfp) in AOX-/- and ciprofibrate-induced tumors. Adfp is a fatty acid-binding protein that specifically facilitates uptake of long chain fatty acids and its mRNA and protein expression levels are increased by long chain fatty acids (39). Up-regulation of Adfp in AOX-/- and ciprofibrate-treated mouse liver tumors seems necessary to provide increased amounts of long chain fatty acids for up-regulated peroxisomal ß-oxidation resulting from PPAR
-activation. In AOX-/- tumors we also noted upregulation of cathepsin L, cathepsin E, histone deacetylase 1, cystatin B, Ets2, galectin 3, phospholipid scramblase 1 and cancer-related gene-liver 1. Alterations in the expression of some of these genes in ciprofibrate and DENA-induced liver tumors were not evident in the present study, and this may reflect small sample size of these tumors. Galectin 3 is the most extensively studied member of the galectin group, a newly defined and growing family of animal lectins (40). Galectin 3 is absent in normal hepatocytes but is expressed at significant levels in HCC (40). In addition, it is also expressed in cirrhotic liver, suggesting its expression in proliferating cells and possibly a marker for pre-neoplastic lesions in liver. Overexpression of histone deacetylase 1 results in a severe delay during the G2/M phases of the cell cycle, indicating that histone acetylation/deacetylation are crucial for normal cell cycle progression (41). Ets2 is a member of the Ets family of transcription factors, downstream mediators of cellular transformation and development. It has been detected as up-regulated in prostate cancer (36) and is linked to tumor progression (42). Ets transcriptional activation can be stimulated by activated oncogenes or receptor-mediated tyrosine kinases via the Ras-Raf-MAP-kinase pathway. Ets2 may modulate the progression of tumors arising due to the expression of oncogenes, such as activated ErbB2 (43). Expression of Ets2 has direct downstream effects on sensitive target genes, like matrix metalloproteinases, which contribute to vascularization, invasive behavior, and metastasis of tumors. In AOX-/- liver tumors, we also found a significant increase in the mRNA for cancer-related gene-liver 1, a novel tumor-related gene, first identified in DENA-induced liver tumors (34). Its expression is up-regulated in murine and human HCCs and correlates with malignancy in the liver (34).
We found a number of genes downregulated in AOX-/- and ciprofibrate-induced liver tumors. Enzymes catalyzing major steps during steroid hormone synthesis were down-regulated in these two classes of tumors. These include hydroxysteroid (17-ß) dehydrogenase 5, hydroxysteroid 11-ß dehydrogenase 1, hydroxysteroid dehydrogenase-3-ß and isoenzymes of the cytochrome P450 2d family (Cyp2d9 and Cyp2d10). Reduction in hydroxysteroid 11-ß dehydrogenase 1 reflects decreased cortisol levels in liver and reduced hepatic gluconeogenesis in these tumors (37,44). The strongest down-regulated gene was major urinary protein (62.2-fold), an extracellular protein, which belongs to the lipocalin family and is thought to act as a pheromone transporter (45). Liver is the major site of MUP synthesis, and it is secreted into the serum and excreted by the kidney. Expression of MUP mRNA is under developmental and hormonal control. Expression is highly stimulated by androgens, especially testosterone. Down-regulation of MUP expression has been noted in the liver of AOX-/- mice (46). It should be noted that
- 2u-globulin, a homolog of MUP, was markedly reduced in the liver of rats treated with ciprofibrate (47). Lower levels of sex hormones and reduced pheromone excretion may, at least in part, contribute to the reduced fertility observed with AOX homozygous male mice (26).
The present cDNA microarray and northern blot data also provides information on the genes whose expression is altered in all three classes of tumors. RIKEN cDNA 1200016E24, Ly-6D, lipocalin 2, serum amyloid associated protein 2, Igfbp1, CD63, IMAGE clone 737676 belonging to transmembrane 4 superfamily and lipoprotein lipase were among the up-regulated genes in all three classes of tumors. Lipocalin 2 (Lcn2 or oncogene 24p3) is the most up-regulated gene in all three tumors by cDNA microarray whereas RIKEN cDNA seemed to be highly prominent on northern blots. Lipocalins are small extracellular transport proteins and increased expression of lipocalin 2 has been described in pancreatic carcinoma (46,48). Recently, it has been identified as a target of estrogen in normal breast tissue (49). The exact role lipocalins play in the development of liver cancer is not entirely clear. Additional studies are needed to ascertain if lipocalin 2 can serve as a marker for liver cancer and if it exerts any functional role in liver cancer development and progression. CD63 (ME491 in humans) is a member of transmembrane 4 superfamily (TM4SF). Several members of TM4SF family appear to influence tumor progression and metastasis (32,33). CD63 is highly expressed in a variety of other tumors including mouse and human HCC (35). Expression levels of members of the TM4SF family can therefore serve as possible prognostic predictors. Igfbp1 is known to be expressed during fetal development (50). Up-regulation of this gene indicates that tumors are less differentiated than the adult liver. Ly-6D antigen is a cell surface glycoprotein that is highly expressed in tumor cells of high malignancy (51) and was identified as up-regulated in the AOX-/- mouse liver as well as in mouse liver after peroxisome proliferator exposure (52). We found upregulation of Ly-6D in all three classes of liver tumors. As pointed out above, the overexpression of RIKEN 1200016E24 gene was noted in all three tumors, whereas C3f, belonging to gene-rich cluster (53) was increased in PPAR
-related tumors.
A variety of genes were down-regulated in all three types of tumors. Of these Cyp2c29, Cyp7b1, ornithine aminotransferase, and nuclear receptor LXRa are of special interest. LXR plays an important role in controlling lipid homeostasis by activating several genes such as members of the ATP-binding cassette (ABC) superfamily of transporter proteins ABCA1 and ABCG1, apolipoprotein E, and cholesterol ester transport protein that are involved in reverse cholesterol transport. They also participate in fatty acid metabolism by activating the sterol regulatory element-binding protein 1c gene (54,55). Thus, down-regulation of LXR in AOX-/- and ciprofibrate-induced mouse liver tumors can be caused by decreased levels of cholesterol because of the activated lipid catabolism via PPAR
activation in these mice. It is possible that mice with DENA induced liver tumors may also have low serum lipids. This suggests a cross-talk between LXRa and PPAR
in regulation of lipid homeostasis.
In summary, the cDNA microarray data obtained in this study show that liver tumors developing in AOX-/- mice and those induced by ciprofibrate differ in their repertoire of altered genes from that of DENA-induced liver tumors and that all three classes of tumors also show a subset of genes whose expression is up-regulated. Thus, PPAR
-mediated liver tumors, a prototype of non-genotoxic hepatocarcinogenesis, differ from liver tumors induced by DENA, a genotoxic carcinogen in certain respects. These differences may prove to be molecular fingerprints depicting carcinogenic mechanism(s). Judicious use of cDNA microarrays and other emerging technologies, such as ProteinChip approach to delineate changes in protein profiles (56), should be useful in gaining mechanistic information about genotoxic and nongenotoxic liver carcinogenesis.
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Acknowledgments
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This work was supported by National Institutes of Health Grants GM 23750 (to J.K.R.) and CA 84472 (to M.S.R.).
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Received December 3, 2002;
revised January 27, 2003;
accepted February 28, 2003.