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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: J Hepatol. 2013 Oct 2;60(2):331–338. doi: 10.1016/j.jhep.2013.09.024

Role of Myc in hepatocellular proliferation and hepatocarcinogenesis

Aijuan Qu 1, Changtao Jiang 1, Yan Cai 1, Jung-Hwan Kim 1, Naoki Tanaka 1, Jerrold M Ward 2, Yatrik M Shah 1,3, Frank J Gonzalez 1
PMCID: PMC3909877  NIHMSID: NIHMS544138  PMID: 24096051

Abstract

Background & Aims

MYC is involved in cell growth, proliferation, apoptosis, energy metabolism, and differentiation. Whether it is essential for hepatocellular proliferation and carcinogenesis is unclear due to a lack of an efficient hepatocyte-specific Myc disruption model. This study used a novel genetic model to investigate the involvement of MYC in hepatocellular proliferation and hepatocarcinogenesis in mice.

Methods

Temporal hepatocyte-specific Myc disruption was achieved by use of the tamoxifen-inducible Cre-ERT2 recombinase system under control of the serum albumin promoter. Hepatocyte proliferation was assessed by administering peroxisome proliferator-activated receptor α(PPARα) agonist Wy-14,643. A diethylnitrosamine-induced liver cancer model was used to evaluate the role of Myc in hepatocarcinogenesis.

Results

Tamoxifen administration induced recombination of Myc specifically in hepatocytes of Mycfl/fl,ERT2-Cre mice. When treated with a known hepatocellular proliferative stimulus Wy-14,643, Mycfl/fl,ERT2-Cre mice showed a lower liver/body weight ratio and suppressed hepatocyte proliferation as compared to Mycfl/fl mice. Hepatic expression of cell cycle control genes, DNA repair genes, and Myc target gene miRNAs were upregulated in Wy-14,643-treated Mycfl/fl mouse livers, but not in Wy-14,643-treated Mycfl/fl,ERT2-Cre livers. However, no differences were observed in the lipid-lowering effect of Wy-14,643 between Mycfl/fl,ERT2-Cre and Mycfl/fl mice, consistent with no differences in the expression of several PPAR α target genes involved in fatty acid β-oxidation. Moreover, when subjected to the diethylnitrosamine liver cancer bioassay, Mycfl/fl,ERT2-Cre mice exhibited a markedly lower incidence of tumor formation compared with Mycfl/fl mice.

Conclusion

Myc plays an essential role in hepatocellular proliferation and liver tumorigenesis.

Keywords: MYC, cell proliferation, tumorigenesis, BrdU, cell cycle control

INTRODUCTION

c-MYC, encoded by proto-oncogene Myc, is a pleiotropic transcription factor that belongs to the basic helix-loop-helix leucine zipper (bHLH) family of transcription factors [1, 2]. MYC binds to a 6-nucleotide DNA consensus sequence CACGTG (E-box) by forming a heterodimer with MAX, another bHLH protein to participate in the control of a wide variety of genes that are involved in the regulation of cell growth, cell proliferation, apoptosis, and differentiation by stimulating metabolism and protein synthesis [2].

MYC expression promotes the transition from G0/G1 to S phase of the cell cycle in multiple cell types by regulating cyclin/cyclin-dependent kinase (CDK) complexes [3, 4]. Rapid MYC induction is observed in hepatocytes of proliferating livers induced by peroxisome proliferator-activated receptor α (PPARα) activation [5, 6], which is tightly correlated with G0/G1 to S-phase transition. Dysregulation of MYC is commonly found in several cancers [7–9]. In the liver, forced overexpression of MYC leads to spontaneous hepatocellular carcinomas [10]. In transgenic mice with inducible MYC, withdrawal of ectopic MYC expression in liver results in regression of tumors [11, 12]. However, a direct role for MYC in hepatocellular proliferation and carcinogenesis has not been established. Although most studies demonstrate that overexpression of MYC induces hepatocellular proliferation and tumorigenesis, direct evidence for an essential role of endogenous MYC in hepatocellular proliferation and tumorigenesis is lacking.

In this study, hepatocyte-specific temporal disruption of Myc was accomplished using the albumin promoter (SA)-expressing Cre-ERT2 system that yields hepatocyte-specific expression and activation Cre recombinase in the presence of tamoxifen [13]. Disruption of Myc suppressed hepatocellular proliferation induced by the PPARα agonist Wy-14,643. Microarrays revealed that MYC regulates a large array of genes important for cellular proliferation and cell cycle progression that is not found in Wy-14,643-treated mice with hepatic Myc disruption. Disruption of MYC expression resulted in a lower incidence of diethylnitrosamine (DEN)-induced liver tumors. These data demonstrate that MYC is required for hepatocellular proliferation and liver tumorigenesis.

MATERIALS & METHODS

Animal and diets

Mycfl/fl mice were provided by John M. Sedivy (Brown University) and described previously [14]. For temporal hepatocyte-specific disruption, Mycfl/fl mice were crossed with mice harboring the Cre-ERT2 recombinase driven by the SA, designated SA-Cre-ERT2 [15], obtained from Pierre Chambon and Daniel Metzger (University of Strasbourg), to yield the Mycfl/fl,ERT2-Cre mouse line. Mycfl/fl,ERT2-Cre and Mycfl/fl mice were maintained as littermates. For activation of the SA-Cre-ERT2 driven Cre recombinase, 6- to 8-week-old male mice were injected intraperitoneally with 2 mg tamoxifen and then fed a tamoxifen diet (1 mg/kg, Dyets Inc., Bethlehem, PA). For Wy-14,643 treatment, mice were fed for 3 days with tamoxifen diet before exposure to pelleted chow containing 0.1% Wy-14,643 (Bioserv, Frenchtown, NJ) and receiving 2 mg of tamoxifen injection every other day for the indicated time. All mice were housed in light and temperature-controlled rooms with free access to water and pelleted chow ad libitum. All animal experiments were performed in accordance with the AAALAC international guidelines and approved by the National Cancer Institute Animal Care and Use Committee.

DEN-induced hepatocarcinogenesis

Mycfl/fl and Mycfl/fl,ERT2-Cre mice were injected intraperitoneally with 50 µg/g of tamoxifen at 10 days old and with 25µg/g of DEN (Sigma-Aldrich, St. Louis, MO) at 15 days old as described [16]. After weaning at 21 days old, mice were fed the tamoxifen diet for 5 days followed by a single dose of tamoxifen injection (2 mg/mice) every other week until killing and necropsy at 9 months. The livers were then separated into individual lobes, analyzed for the presence of tumors, and subjected to histological analysis.

Triglyceride and cholesterol analysis

Serum triglycerides and cholesterol levels were measured using Serum Triglyceride Determination Kit and Serum Cholesterol Determination Kit according to manufacturer’s recommendation (Wako, Richmond, VA).

Quantitative real-time PCR

Total RNA extraction and quantitative real-time PCR (qPCR) were performed as previously described [15]. Primers were designed using qPrimerDepot and sequences were listed in Supplementary Table 1. Values were quantified using the comparative CT method, and normalized to β-actin mRNA. To determine the expression levels of microRNAs, Taqman microRNA assays (Invitrogen) directed to mmu-mir-17-5p, mmu-mir17-3p, mmu-mir-18a-5p, mmu-mir-19a, mmu-mir-290-5p, mmu-mir-291-3p, mmu-mir-293, mmu-mir-295, mmu-mir-100, mmu-let-7a-2and an endogenous reference snoRNA55, were used following the manufacture’s protocol (Applied Biosystems).

cDNA microarray analysis

Dye-coupled cDNAs were purified with MiniElute PCR purification kit (Qiagen) and hybridized to an Agilent 44K mouse 60-mer oligo microarray (Agilent Technologies, Santa Clara, CA). The data were processed and analyzed by Genespring GX software package (Agilent Technologies).

Western blot analysis

Liver whole-cell extracts were subjected to Western blot using primary monoclonal anti-MYC antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA) as previously described [15], and the signals obtained normalized to GAPDH (Santa Cruz Biotechnology Inc).

Histological analysis

Hematoxylin and Eosin (H&E) staining were performed on formalin fixed paraffin embedded sections. Immunohistochemistry staining for BrdU was performed as previously described using antibodies against BrdU (AbDSerotec, Raleigh, NC) [17].

Data analysis

Data are expressed as means ± standard deviations (SD). P values were calculated by independent t-test between two groups or one-way ANOVA analysis with Tukey correction between multiple groups using Prism 5. A P < 0.05 was considered significant.

RESULTS

Generation of a mouse model with temporal hepatocyte-specific disruption of Myc

Mycfl/fl mice were crossed with SA-Cre-ERT2 transgenic mice to generate a temporal and conditional disruption of Myc (Mycfl/fl,ERT2-Cre). To confirm hepatocyte-specific disruption of Myc, Mycfl/fl and Mycfl/fl,ERT2-Cre mice were treated with one dose of vehicle or tamoxifen, and livers and extrahepatic tissues analyzed at 24 hours post-treatment. Myc recombination was only observed in livers from tamoxifen-treated Mycfl/fl,ERT2-Cre mice but not from Mycfl/fl or vehicle-treated Mycfl/fl and Mycfl/fl,ERT2-Cre mice (Figure 1A). A decrease in Myc mRNA level was also achieved in livers only from tamoxifen-treated Mycfl/fl,ERT2-Cre mice and not from Mycfl/fl mice; Myc mRNA was not changed in extrahepatic tissues of tamoxifen-treated Mycfl/fl,ERT2-Cre mice (Figure 1B). Western blot analysis of liver extracts revealed diminished MYC protein levels in tamoxifen-treated Mycfl/fl,ERT2-Cre mice (Figure 1C). These data indicate that an efficient inducible liver-specific disruption of Myc was achieved using the SA-Cre-ERT2 system.

Fig. 1. Conditional and temporal disruption of Myc in hepatocytes.

Fig. 1

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(A) PCR diagnostic for activated ERT2-Cre-mediated recombination of Myc allele in genomic DNA isolated from kidney (lanes1) and liver (lanes 2) of Mycfl/fl or Mycfl/fl,ERT2-Cre mice treated with vehicle (VEH) or tamoxifen (TM) for 24 hours. (B) qPCR analysis measuring Myc mRNA level in liver, lung, heart, kidney and skeletal muscle from Mycfl/fl or Mycfl/fl,ERT2-Cre mice treated with tamoxifen and killed 24 hours post-treatment. n=6–8. ***p<0.001 versus Mycfl/fl group. (C) Western blot of MYC for whole liver extracts from Mycfl/fl and Mycfl/fl,ERT2-Cre mice treated with TM and killed 24 hours post treatment.

Myc disruption represses hepatocellular proliferation and hepatomegaly but not β-oxidation induced by PPARα agonist Wy-14,643

Administration of PPARα agonists results in hepatocyte proliferation and hepatomegaly in mice and rats [18]. Previous studies demonstrated that PPARα agonists induce Myc mRNA in rats and mice and that increased MYC protein levels correlated with hepatocellular proliferation [6]. When treated with Wy-14,643 for two weeks, Mycfl/fl,ERT2-Cre mice showed a lower number of BrdU-positive hepatocytes compared to Mycfl/fl mice (Figure 2A). The labeling index was 21±4.7% for hepatocytes in Wy-14,643-treated Mycfl/fl,ERT2-Cre mice for hepatocytes compared to 67±7.1% in Wy-14,643-treated Mycfl/fl mice (Figure 2B), demonstrating that Myc disruption markedly suppresses Wy-14,643-induced hepatocyte proliferation. It was also noteworthy that the BrdU labeling index of non-parenchymal cells was comparable between Wy-14,643-treated Mycfl/fl and Mycfl/fl,ERT2-Cre mice (Figure 2B), indicating that proliferation of non-parenchymal cells is not affected by disruption of Myc in hepatocytes. The decrease in hepatocellular proliferation was further revealed by a lower liver/body weight ratio in the Mycfl/fl,ERT2-Cre mice as compared to the Mycfl/fl mice (Figure 2C). However, the serum triglyceride-lowering extent was similar in the mouse lines (Figure 2D), and the induction of well-characterized PPAR α target genes, acyl-CoA oxidase (Acox1), cytochromes P450 4a10 and 4a14 (Cyp4a10 and Cyp4a14) did not differ between Mycfl/fl and Mycfl/fl,ERT2-Cre mice (Figure 2E), indicating that MYC is not required for PPARα-dependent fatty acid β-oxidation.

Fig. 2. Myc disruption suppresses hepatocyte proliferation but not the lipid-lowering effect of Wy-14,643.

Fig. 2

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(A) Representative BrdU staining of liver sections from vehicle or Wy-14,643-treated Mycfl/fl and Mycfl/fl,ERT2-Cre mice. (B) BrdU labeling index for hepatocytes and non-parenchymal cells. n=6–8. **p<0.01 versus Mycfl/fl+Wy. (C) Liver/body weight ratios at two weeks post-treatment. n=6–8. **p<0.01 versus Mycfl/fl+Wy. (D) Serum triglyceride levels in vehicle or Wy-14,643-treated Mycfl/fl and Mycfl/fl,ERT2-Cre mice. (E) mRNA levels of PPARα target genes Acox1, Cyp4a10 and Cyp4a14 in Mycfl/fl and Mycfl/fl,ERT2-Cre mice determined by qPCR.

Myc controls cellular growth and proliferation networks in hepatocytes

To further explore the expression profile of genes controlled by MYC, microarray analysis was conducted on liver RNA from Mycfl/fl and Mycfl/fl,ERT2-Cre mice on chow diet or Wy-14,643- containing diet. The data revealed that 1247 genes were significantly decreased and 825 genes increased in Mycfl/fl,ERT2-Cre mice compared with Mycfl/fl mice (Supplementary Fig. 1, microarray data in http://www.ncbi.nlm.nih.gov/geo/, GEO # GSE43842). Thirty-three of the decreased genes are known to be involved in cell cycle control and 13 are involved in DNA replication, recombination and repair (Table 1). Pathway analysis revealed that the top down-regulated network was associated with cellular growth and proliferation (Table 2). These data reinforce a role for MYC in driving hepatocellular growth and proliferation by controlling the cell cycle, and DNA replication, recombination and repair.

Table 1.

Top Biological Functions Regulated in Liver by Myc in Hepatocytes

Name p-value # Molecules
Downregulated
Cell cycle 5.57E-05 – 3.83E-02 27
DNA replication, recombination, and repair 5.57E-05 -4.53E-02 13
Protein trafficking 5.26E-04 -3.83E-02 6
Cell death 6.84E-04 -3.83E-02 18
Cell-to-cell signaling and interaction 1.40E-03 -4.99E-02 33
Upregulated
Carbohydrate metabolism 7.81E-04 -4.25E-02 11
Cell morphology 7.81E-04 -4.97E-02 21
Cell-to-cell signaling and interaction 7.81E-04 -4.95E-02 19
Lipid metabolism 7.81E-04 -4.28E-02 16
Molecular transport 7.81E-04 -4.97E-02 19
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Table 2.

Associated Networks Regulated in Liver by Myc in Hepatocytes

Down-regulated
ID Associated network functions Score
1 Cellular development, cell death, cellular growth and proliferation 31
2 Cellular growth and proliferation, hematological system development and function, cell-to cell signaling and interaction 29
3 Tissue development, embryonic development, organ development 18
4 Cellular growth and proliferation, cell death, post-translational modification 15
5 Lipid metabolism, small molecule biochemistry, vitamin and mineral metabolism 15
Up-regulated
1 Cellular movement, cell-to-cell signaling and interaction, nervous system development and function 36
2 Cell death, cell-mediated immune response, cellular development 30
3 Inflammatory response, amino acid metabolism, molecular transport 15
4 Cell cycle, DNA replication, recombination, and repair, cell death 15
5 Tissue development, cellular development, reproductive system disease 13
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The score was calculated by Ingenuity Pathway Analysis software. Details are given in the Supplementary Information file.

To confirm these pathways, qPCR analysis of mRNAs encoded by cell cycle control genes was performed in Mycfl/fl and Mycfl/fl,ERT2-Cre mice treated with Wy-14,643 for 48 hours. Ablation of MYC expression significantly down-regulated a series of cyclins, including cyclin mRNAs, Ccnb1, Ccne1 and Ccne2, and cyclin-dependent kinase 1 (Cdk1) that are critical for control of the G1/S phase in hepatocytes (Figure 3A). Loss of MYC in hepatocytes also suppressed genes that are crucial for the G2/M DNA damage checkpoint, such as checkpoint kinase 1 homologue (Chek1) and RAD51 homolog (S. cerevisiae) (Rad51). Moreover, a family of minichromosome maintenance (Mcm) genes required for the initiation and elongation of DNA replication, were dramatically reduced by liver-specific Myc disruption (Figure 3B). These data demonstrate that disruption of Myc suppresses Wy-14,643-induced hepatocellular proliferation via inhibition of genes responsible for G1/S cell cycle, checkpoint and DNA damage repair.

Fig. 3. Altered expression of cell cycle control, DNA repair genes and miRNAs in mice lacking hepatic expression of MYC.

Fig. 3

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(A) qPCR analysis of cell cycle control genes-encoded mRNAs. (B) qPCR analysis for mRNAs encoding proteins involved in DNA damage repair and checkpoints. n=6–8. *p<0.05 and **p<0.01. (C) qPCR analysis of the mir-17–92 cluster, mir-290–295 cluster and mir-100/let-7a-2 cluster in livers from vehicle or Wy-14,643-treated Mycfl/fl and Mycfl/fl,ERT2-Cre mice. Expression was normalized to endogenous reference snoRNA55. n=6–8. **p< 0.01 and ***p<0.001 compared to all the other three groups.

MYC regulates miR17-92 cluster in hepatocytes

PPARα activation in mice leads to an increase in mir-17-92 polycistronic cluster [6]. A critical mechanism by which MYC induces proliferation and tumorigenesis is direct regulation of the mir17-92 miRNA cluster [19]. To test whether PPARα-dependent induction of mir-17-92 cluster is MYC dependent, qPCR analysis was performed. Mir-17-5p was rapidly induced by over 5-fold after two days of Wy-14,643 exposure in Mycfl/fl mice, but not in Mycfl/fl,ERT2-Cre mice (Figure 3C). Disruption of hepatic Myc could also inhibit the induction of other mir-17-92 cluster family members such as mir-17-3p, mir-18a-5p, and mir-19a. However, no significant changes were observed in expression of the mir-290-295 cluster (homolog of human miR-371-3 cluster in mouse) or mir-100/let-7a2 (Figure 3C). This is consistent with previous studies that MYC activates this miRNA cistron [6].

Mycfl/fl,ERT2-Cre mice are resistant to DEN-induced hepatocarcinogenesis

To investigate the direct role of MYC in liver tumorigenesis, the susceptibility of Mycfl/fl and Mycfl/fl,ERT2-Cre mice to DEN-induced liver cancer was determined. Injection of 25 µg/g DEN resulted in 100% incidence of visible tumors in Mycfl/fl mice whereas only 33% incidence in Mycfl/fl,ERT2-Cre mice (Figure 4A); the number and size of tumors were also decreased in Mycfl/fl,ERT2-Cre mice (Figure 4B and 4C). Histological analysis demonstrated that Mycfl/fl,ERT2-Cre mice had a 55% incidence of preneoplastic foci and 33% adenomas but no carcinomas, whereas 100% of Mycfl/fl mice had both preneoplastic foci and adenomas, with 67% of them developing carcinomas.(Figure 4D and 4E). Ki67 immunostaining showed a significant increase in of hepatocyte proliferation in DEN-treated Mycfl/fl that was markedly reduced in Mycfl/fl,ERT2-Cre livers (Figure 4F). qPCR analysis further demonstrated a significant increase of mir-290-295 RNA in DEN-induced liver tumors from the DEN-treated Mycfl/fl mice as compared to but not in DEN-treated Mycfl/fl,ERT2-Cre mice; normal liver tissues from saline-treated Mycfl/fl and Mycfl/fl,ERT2-Cre mice were used as controls (Figure 4G), which is consistent with a previous study showing that miR-371-3 (homolog of mir-290-295 in humans) could be a direct target of MYC in HCC [20]. To investigate whether the 55% incidence of preneoplastic foci and 33% adenomas developed in Mycfl/fl,ERT2-Cre mice was due to the residual Myc-floxed allele, Myc recombination was assessed in a few tumors. PCR analysis showed that a slight band for Myc-floxed allele was detected in adenomas from Mycfl/fl,ERT2-Cre mice suggesting that a small amount of MYC could be produced in these tumors, but likely less that that in tumors from Mycfl/fl mice (Supplementary Fig. 2). This indicates that DEN-induced tumor hepatocytes from Mycfl/fl,ERT2-Cre mice could escape Myc disruption through the transformation of residual hepatocytes or stem cells that did not undergo recombination of the Myc-floxed allele.

Fig. 4. Mycfl/fl,ERT2-Cre mice are resistant to DEN-induced liver tumorigenesis.

Fig. 4

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(A) Incidence of visible tumors from in Mycfl/fl and Mycfl/fl,ERT2-Cre mice treated with 25 µg/g of DEN at age of 15 days and killed at 9 months later. (B) The number of tumors counted from the surface of liver lobes. n=6–9 per group. (C) Tumor size measured on the surface of liver lobes. n=9 per group. (D) Incidence of preneoplastic foci, hepatocellular adenoma, and hepatocellular carcinoma in DEN-treated Mycfl/fl and Mycfl/fl,ERT2-Cre mice. Data are expressed as percentage of total number of mice (n=9). (E) Representative images of H.E. staining from DEN-treated Mycfl/fl and Mycfl/fl,ERT2-Cre mice. “T” denotes tumor area. (F) Representative images of Ki67 staining from DEN-induced tumors in Mycfl/fl and Mycfl/fl,ERT2-Cre mice. (G) qPCR analysis of mir-290–295 cluster and mir-100/let-7a-2 cluster in saline-treated livers and DEN-induced tumors in Mycfl/fl and Mycfl/fl,ERT2-Cre mice. Expression was normalized to endogenous reference snoRNA55. n=6 for saline treatment, n=9 for Mycfl/fl+DEN and n=3 for Mycfl/fl,ERT2-Cre+DEN. *p<0.05 and **p< 0.01, ***P<0.001.

DISCCUSION

MYC, a transcription factor encoded by proto-oncogene c-myc, is thought to contribute to hepatocyte proliferation, liver regeneration and tumorigenesis in response to hepatic injury and carcinogens exposure. Given the potential key role of Myc, it would be of value to determine whether MYC is essential for these processes using a genetic model in vivo. In the current study, mice with a temporal hepatocyte-specific Myc disruption were developed using SA-Cre-ERT2 system [13]. Continuous treatment with tamoxifen led to maintenance of Myc disruption in hepatocytes. Disruption of Myc in hepatocytes represses hepatocellular proliferation induced by PPARα agonist Wy-14,643. Mice with hepatocyte-specific Myc disruption were also largely resistant to DEN-induced hepatocarcinogenesis, thus demonstrating that MYC is essential in hepatocellular proliferation and tumorigenesis.

Since embryonic Myc knockout mice are lethal [21], liver-specific Myc-deficient mice were used investigate the role of MYC in liver physiology and pathology. Previously, other groups targeted Myc disruption in liver [22, 23]. Myc alleles were disrupted by injecting newborn Mycfl/fl homozygous mice with pIpC to activate the α/β interferon-inducible Mx-promoter driving Cre recombinase (Mycfl/fl Mx-Cre) revealing that lower MYC in newborn mice had decreased hepatocyte size and polyploidization as the liver develops [23]. A model of hepatocyte-specific Myc disruption was also generated by crossing Mycfl/fl mice to SA-Cre-harboring mice [22, 24]. However, the efficiency of Myc disruption was low, with Myc mRNA expression remaining higher than 30% in 10-week-old mice [22]. A similar result was obtained when the Mycfl/fl mouse was crossed with the SA-Cre line [25], where efficient disruption of Myc could not be achieved (unpublished data). This lack of efficient MYC loss in the Alb-Cre mice is likely due to the rapid proliferation of MYC-expressing hepatocytes during liver growth and the regenerative capacity of the liver with non-recombined Myc alleles. In the present model, recombination of the Mycfloxed alleles was achieved by the hepatocyte-specific Cre-ERT2 system, which temporally activates the Cre recombinase expression under the control of serum albumin promoter in the presence of tamoxifen [13, 15]. This mouse line achieved stable and efficient disruption of MYC protein specifically in hepatocytes, yielding a more suitable model for assessing its physiological and pathological roles in hepatocytes compared with the previous mouse models.

Fibrate drugs used to lower plasma triglycerides [26], are agonist for PPARα, a ligandactivated nuclear receptor that controls genes involved in fatty acid transport and catabolism, primarily in liver, but also in kidney, heart and skeletal muscle [27, 28]. In rodents, short-term administration of PPARα agonists, such as fibrate drugs and the experimental compound Wy-14,643, initiates a pleiotropic response of hepatomegaly, peroxisome proliferation, and increased fatty acid transport and oxidation in liver, whereas long-term exposure leads to tumorigenesis in a PPARα-dependent manner [5, 29]. Clues to the molecular link between PPARα and hepatocellular proliferation was recently uncovered. Given the importance of MYC in regulating cellular growth, proliferation and metabolism, MYC might serve as a critical link between PPARα and hepatocellular proliferation. Indeed, Myc mRNA and MYC protein levels were robustly induced as early as 24 hours upon Wy-14,643 treatment in mice as a result of suppression of expression of the let-7C miRNA-encoding gene [6]. Let-7C destabilizes Myc mRNA and the lower let-7C expression leads to increased Myc mRNA and MYC protein and the resultant hepatocellular proliferation. Wy-14,643 treatment for two weeks caused a significant increase of liver weight in Mycfl/fl mice which was mainly due to hepatocellular proliferation, as revealed by the BrdU labeling index, consistent with earlier studies [30]. However, this response was markedly blunted by Myc disruption in hepatocytes of Mycfl/fl,ERT2-Cre mice, as the BrdU labeling index dropped from about 68% to 21% in hepatocytes. The difference of liver/body weight ratio between Mycfl/fl and Mycfl/fl,ERT2-Cre mice occurred as early as two days post Wy-14,643 treatment. Mechanistically, several critical MYC target genes involved in cell cycle control, including Ccna2, Ccnb1, Ccne1, Ccne2 and Cdk1, were suppressed in Mycfl/fl,ERT2-Cre mice upon Wy-14,643 treatment. In addition to cell cycle control genes, minichromosome maintenance (Mcm) 2–7 genes, identified as direct MYC target genes in neuroblastoma [31], were down-regulated by Myc disruption. MCM2-7, a family of six related proteins required for the initiation and elongation of DNA replication, form heterohexameric MCM complexes and act as a replicative helicase at the DNA replication fork, a key component of the pre-replication complex [32–34]. The decreased MCM2-7 indicates that Myc disruption reduces DNA replication in hepatocytes upon Wy-14,643 challenge. Moreover, two checkpoint genes, Chek1 and Rad51, were repressed by Myc inactivation. CHEK1 kinase acts downstream of ATM/ATR kinase and plays an important role in DNA damage checkpoint control, embryonic development and tumor suppression [35, 36]. RAD51 was also reported to be involved in DNA damage signaling, recombination repair, and tumorigenesis [37]. Many of the above genes were previously found regulated and critical for PPARα-induced hepatocellular proliferation in PPARα ligand-independent constitutively-active mice [17]. This further confirms that MYC drives the proliferative response to PPARα activation in mice.

A mechanism by which PPARα regulates gene expression and hepatocellular proliferation was revealed in which MYC is the central player [6]. As noted above, activated PPARα indirectly induced Myc mRNA and protein expression via suppression of let-7C. In addition, a series of miRNAs were up-regulated upon PPARα activation, including the mir-17–92 polycistron cluster, and ChIP assays demonstrated that MYC could bind to the promoter of the gene encoding mir-17-5p. The mir-17-92 cluster is directly regulated by MYC leading to enhanced cell cycle progression and a blockade of tumor cell apoptosis [38, 39]. Consistent with this data, hepatic disruption of MYC in the current study inhibited Wy-14,643-induced expression of mir-17-5p, mir-17-3p, mir-18a-5p, and mir-19a, indicating that the mir-17-92 cluster is a direct MYC target and might be partially responsible for PPARα activation-induced hepatocellular proliferation and tumorigenesis.

Numerous studies suggest that MYC plays a role in glucose metabolism and ribosomal biogenesis, [40] and that overexpression of MYC improved glucose homeostasis in diabetic mice [41]. However, the present data suggest that Myc disruption in Mycfl/fl,ERT2-Cre mice did not alter the expression of genes involved in glucose metabolism, nor the genes responsible for glutamine metabolism under normal conditions (data not shown). In addition, Myc inactivation does not alter the induction of fatty acid-catabolizing genes induced by PPARα activation, such as Cyp4a10, Cyp4a14 and Acox1. These data indicate that MYC may not be essential for glucose, fatty acid and glutamine metabolism under these conditions. Myc disruption may increase the β-oxidation-independent carbohydrate and lipid metabolism via an indirect regulatory pathway. Whether MYC is crucial for glucose and glutamine metabolism during tumorigenesis and other hepatic pathological processes needs to be elucidated by additional experimentation.

To investigate the role of MYC in liver carcinogenesis, the DEN-induced hepatocarcinogenesis model was used. Mycfl/fl,ERT2-Cre mice were found to be resistant to development of liver adenomas and carcinomas as compared to the Mycfl/fl mice. The mechanism leading to a decrease in hepatocellular cancer is likely due to a lower proliferative capacity of hepatocytes during development resulting in less DEN-induced DNA damage being fixed as gene mutations. Dysregulation of Myc is a hallmark of many human malignancies, including liver cancers. In liver, constitutive overexpression of MYC induces tumorigenesis, whereas inhibition of MYC expression resulted in regression of established tumors in vivo [11, 12], indicating that targeting MYC is a potential approach for therapeutics of liver tumors. Further studies are required to determine how MYC contributes to multiple carcinogenic steps, such as initiation and promotion.

Supplementary Material

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Acknowledgments

We thank John M. Sedivy for providing the Mycfl/fl mice, and Pierre Chambon and Daniel Metzger for providing the SA-Cre-ERT2 mice.

Source of Funding: This work was supported by the National Cancer Institute Intramural Research Program, and National Institutes of Health Grant CA148828 (to Y.M.S.).

Abbreviations

Mycfl/fl

floxed Myc allele

Cre-ERT2

cre recombinase:tamoxifen-inducible estrogen receptor ligand-binding domain fusion protein

Mycfl/fl,ERT2-Cre

inducible liver-specific Myc knockout mouse

bHLH

basic helix-loop-helix leucine zipper

CDK

cyclin-dependent kinase

PPARα

peroxisome proliferator-activated receptor α

DEN

diethynitrosamine

SA

serum albumin promoter

BrdU

bromodeoxyuridine

Acox1

acyl-CoA oxidase

Cyp4a10 and Cyp4a14

cytochromes P450 4a10 and 4a14

Chek1

checkpoint kinase 1 homologue

Rad51

RAD51 homolog (S. cerevisiae)

Mcm

minichromosome maintenance

Mycfl/flMx-Cre

α/β interferon-inducible Mx-promoter driving Cre recombinase

Footnotes

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Conflict of interests: The authors declare no conflict of interest

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NIHMS544138-supplement-01.pdf (76.6KB, pdf)
02
NIHMS544138-supplement-02.pdf (1.3MB, pdf)

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