Introduction

Hepatocellular carcinoma (HCC) is the most common liver cancer and worldwide it represents the fifth most common primary cancer1. HCC is also the third leading cause of cancer related mortality globally2. In the United States, the incidence of HCC has tripled over the last two decades3,4. Unlike many other cancers with known associated risk factors, the underlying molecular pathophysiology for HCC is still not completely known. Most commonly, the incidence of HCC is linked to known risk factors including hepatitis B and C, aflatoxin and chronic alcohol consumption5. Each of these factors alone poses a significant risk to development of liver cirrhosis6. In fact, independent of the initial insult, cirrhosis remains the single common precursor of HCC development.

Hepatocarcinogenesis is a multistep, multistage process that involves genetic and epigenetic alterations that ultimately lead to malignant transformation of hepatocytes7. Several animal models have been reported that mimic different steps leading to HCC8; however, no animal model of alcohol-related HCC exists that represents human alcoholic liver with features of steatoheptitis and liver fibrosis. Chemically induced HCC models such as the N-nitrosodiethylamine (DEN) induced HCC model are the most widely used and accepted8. DEN acts as an alkylating agent for DNA bases which initiates the formation of neoplasms9. A recent study combined DEN with alcohol administration in the drinking water10; however this form of alcohol feeding fails to result in liver steatosis or inflammation that are characteristics of human alcoholic liver disease11. Human HCC is poorly understood and molecular markers and checkpoints in HCC have recently received attention. It has been shown that the pro-inflammatory cell and cytokine environment, defects in cell proliferation and stem cell-mediated repair all contribute to the multistep process of HCC8,12,13.

In this study, we administered 6 doses of DEN to 4 week old adult C57bl/6 male mice followed by 6 weeks of Lieber-DeCarli alcohol diet. We report a synergistic effect of alcohol with DEN resulting in up-regulation of inflammatory and fibrotic markers and a remarkable induction of early hepatobiliary cancers in mice receiving alcohol-diet and DEN. At the molecular level, livers of alcohol-fed DEN-injected mice show significant up-regulation of markers of cell proliferation, stemness (CD133, nanog), as well as tissue inflammation and up-regulation of hypoxia inducible factor-1α and its target, VEGFR1. These results indicate that chronic alcohol accelerates hepatobiliary cancer via multiple key mechanisms in carcinogenesis.

Results

Combination of alcohol and DEN induces sustained liver injury

Chronic alcohol use is a major risk factor for HCC development in humans4. Here we hypothesized that the alcoholic liver environment accelerates liver tumor development after repeated administration of a chemical carcinogen, DEN. In this study, male C57bl/6 mice received 6 weekly DEN injections, starting at age of 4 weeks followed by chronic alcohol administration (Supplementary Fig. S1). Chronic alcohol exposure induces inflammation that is driven by secretion of pro-inflammatory cytokines14. We analyzed TNFα, MCP-1 and IL-6 expression at the protein and mRNA levels in the liver tissue and observed that expression of these pro-inflammatory cytokines at the protein level was significantly higher in alcohol-fed DEN-injected mice as compared to pair-fed DEN-injected or alcohol-fed saline-injected mice (Fig. 1a). Interestingly, the expression of TNFα and IL-6 at the mRNA level in alcohol-fed DEN-injected mice was lower than alcohol-fed saline-injected mice (Fig. 1b). IL-17A is an inflammatory and immunoregulatory cytokine secreted by Th17 cells, γδ T cells and has been recently reported to promote tumor growth in hepatocellular carcinoma15. While both alcohol and DEN, respectively increased IL-17A levels in our experimental model, IL-17A induction was additively increased by combination of DEN and alcohol in the liver at the tissue protein (Fig. 1a) and mRNA (Fig. 1b) levels, compared to pair-fed DEN-injected and alcohol-fed saline-injected mice. Serum ALT was significantly higher in alcohol-fed DEN-injected mice compared to any other group (Fig. 1c). Liver to body weight ratio at sacrifice was significantly higher in alcohol-fed DEN-injected mice as compared to alcohol-fed saline-injected and pair-fed DEN-injected mice (Supplementary Fig. S2). Alcohol induced oxidative stress as measured by thiobarbituric acid reactive substances (TBARS) was significantly higher in alcohol-fed DEN-injected mice compared to other experimental groups (Fig. 1d).

Figure 1
figure 1

Alcohol and DEN are additive in induction of liver inflammation and fibrosis.

(a) Tissue protein levels of pro-inflammatory cytokines (n ≥ 5mice per group). (b) Fold changes in mRNA levels of pro-inflammatory cytokines in liver tissue. (c) Serum ALT levels at sacrifice. (d) Liver tissue ROS measured by TBARS assay. (e) Representative Sirius Red staining images from all experimental groups. Bars inside the images indicate 100 μm. (f) For quantification, at least 3 different microscope fields at 10x magnification were scored for each mouse (n ≥ 5mice per group). Bar graph shows percent Sirius red positive area quantified using ImageJ. (g) Fold changes in mRNA levels of fibrosis markers in liver tissue. In all graphs, values are given as average ± SD, ANOVA and Dunnett’s multiple comparison were used to compare the means of multiple groups; (*p < 0.05).

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HCC in human alcoholics develops in fibrotic and cirrhotic livers16. The Sirius Red staining suggested a higher degree of fibrosis in alcohol-fed DEN-injected mice compared to other groups (Fig. 1e,f). To estimate the extent of fibrosis, we evaluated α-SMA, procollagen1α and TGFβ expression in the liver. As shown in Fig. 1g, all markers of fibrosis were significantly up-regulated in alcohol-fed DEN-injected mice (Fig. 1g). These data indicated significant inflammation and fibrosis in our experimental model.

Alcohol accelerates HCC development after DEN insult

Having confirmed the inflammation status, fibrotic injury in our samples, next we assessed HCC markers using 3 complementary strategies: MRI, histology and serum markers. First, the T2 weighted MRI scan of alcohol-fed DEN-injected mice showed significantly higher number of characteristic intrahepatic biliary cysts visualized as bright spots while the pair-fed DEN-injected mice had very few cysts (Fig. 2a). Pixel quantification of these MRI scans using ImageJ revealed significantly higher number of nodules in alcohol-fed DEN-injected mice compared to the pair-fed plus DEN group (Fig. 2b). Nodules were not found in pair-fed saline-injected and alcohol-fed saline-injected mice.

Figure 2
figure 2

Alcohol accelerates HCC development after DEN insult.

(a) T2-weighted MRI of liver in coronal section. Arrows denote cysts. (b) Quantification of cyst area using ImageJ. (c) Representative H & E stained liver sections from all treatment groups. Bars inside the images indicate 100 μm. Biliary cysts were observed in both DEN-injected groups while the hepatic hyperplasia (encircled in black dotted line) was exclusively observed in alcohol + DEN mice. Bar graph shows average number of biliary cysts and hepatic hyperplastic nodules counted at 10× magnification for each mouse (n ≥ 5 mice per group). (d) Fold changes in serum AFP levels. In all graphs, values are given as average ± SD, ANOVA and Dunnett’s multiple comparison were used to compare the means of multiple groups; (*p < 0.05).

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Second, histopathology examination of the liver sections independently confirmed the presence of intrahepatic biliary cysts and hepatic hyperplastic nodules in alcohol-fed DEN-injected mice (Fig. 2c). In addition to higher number of intrahepatic biliary cysts, the alcohol-fed DEN-injected mice exclusively showed hepatic hyperplastic nodules pointing towards the role of alcohol as a tumor promoter. Quantification of the cysts and nodules in liver sections revealed significantly higher number of cysts and nodules, respectively, in livers after chronic alcohol plus DEN injection compared to any other experimental groups (Fig. 2c).

Third, serum AFP levels were significantly higher in alcohol-fed DEN-injected mice as compared to pair-fed DEN-injected and alcohol-fed saline-injected mice (Fig. 2d). Taken together, the MRI, histopathology and the serum AFP data provided evidence for increased number of biliary cysts and accelerated development of hepatic hyperplasia in mice receiving alcohol plus DEN.

Chronic alcohol induces stemness

Two recent independent studies suggested that cancer growth is mediated by a small population of stem-like cells, referred to as cancer stem cells (CSCs) or tumor initiating cells (TICs) that are characterized by expression of two stem cell markers, CD133 and nanog17,18. In our model, expression of CD133 and nanog was significantly higher in alcohol-fed DEN-injected mice compared to pair-fed DEN-injected or alcohol-fed saline-injected mice (Fig. 3a). CD133+ liver tumor initiating cells have been shown to promote tumor growth via IL-8/CXCL1 signaling in humans17. Expression of CXCL1 was significantly higher in alcohol-fed DEN-injected mice suggesting that CXCL1 signaling may be involved in promoting tumor growth in this experimental model (Fig. 3a).

Figure 3
figure 3

Chronic alcohol induces stemness.

(a) Fold changes in mRNA levels of stem cell markers in liver tissue (n ≥ 5 mice per group). (b) Representative immunostaining images for AFP showing more positive cells in alcohol + DEN mice, (c) CK7 and (d) CK19, respectively. Bars in (b–d) indicate 10 μm. In all graphs in panel (a) values are given as average ± SD, ANOVA and Dunnett’s multiple comparison were used to compare the means of multiple groups; (*p < 0.05).

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The expression of AFP is directly associated with hepatocyte differentiation19. Immunohistochemistry staining showed an abundance of AFP positive cells in alcohol-fed DEN-injected mice compared to all other groups (Fig. 3b). Bipotent progenitor cells or stem cells within the liver, termed as oval cells, are implicated in the pathogenesis of hepatocellular carcinoma and cholangiocarcinoma in animal models and may be important in the development of hepatocellular carcinoma in human chronic liver diseases20. In addition, oval cells can also directly de-differentiate from mature liver cells such as hepatocytes21. These oval cells have been reported to express dual lineage markers, such as alphafetoprotein (AFP) as hepatocyte marker22 and cytokeratins 7 and 19 as markers of bile duct epithelium23. Immunostaining for CK7 and CK19 revealed increased number of these markers of biliary progenitors (CK7 positive cells) and hepatic stem cells or oval cells (CK19 positive cells) in the livers of alcohol-fed DEN-injected mice, respectively (Fig. 3c,d).

Chronic alcohol up-regulates hepatocyte proliferation and Epithelial Mesenchymal Transition (EMT)

First, we evaluated the expression of cyclin D1 and p53, signature molecules implicated in HCC progression24. As shown in Fig. 4a, the expression of both cyclin D1 and p53 was significantly higher in alcohol-fed DEN-injected mice at the mRNA and protein levels compared to all other groups (Fig. 4a). To provide evidence of cell proliferation, we injected the mice with bromodeoxyuridine (BrdU), the S phase marker before sacrifice. As shown in Fig. 4b, higher number of cells was stained BrdU positive in alcohol-fed DEN-injected mice (Fig. 4b).

Figure 4
figure 4

Chronic alcohol up-regulates proliferation, EMT markers.

(a) Liver mRNA and protein levels of cyclin D1 and p53. β-tubulin and GAPDH used as loading controls for western blots. Relative density of signal is shown for each protein blot. (b) Representative immunostaining images for BrdU incorporation. Arrows point to positive staining. BrdU positive nuclei were scored in at least 3 different microscopic fields for each mouse. Bars indicate 10 μm. (n ≥ 5 mice per group). (c) Liver mRNA and protein levels of vimentin with relative density of vimentin protein. GAPDH used as loading control for western blots. Liver mRNA levels of N-cadherin and E-cadherin. In all graphs, values are given as average ± SD, ANOVA and Dunnett’s multiple comparison were used to compare the means of multiple groups; (*p < 0.05).

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HCC development is characterized by epithelial mesenchymal transition (EMT)25. Consistent with this, the epithelial mesenchymal transition markers, N-cadherin and vimentin, were significantly up-regulated at the mRNA level in alcohol-fed DEN-injected mice (Fig. 4c). Up regulation of vimentin in alcohol-fed DEN-injected mice was also seen at the protein level (Fig. 4c). Loss of E-cadherin has been shown to promote liver carcinogenesis26. The expression of E-cadherin was significantly reduced in alcohol-fed DEN-injected mice as compared to alcohol-fed saline-injected and pair-fed DEN-injected mice (Fig. 4c).

Chronic alcohol induces hedgehog signaling

Progression of hepatobilliary cancer involves activation of multiple intracellular regulatory pathways7. We analyzed the hedgehog (Hh) signaling, a major signaling pathway reported to be dysregulated in HCC27. Upon activation of the surface receptor PTCH1/2 by Hh ligands, downstream transcription factors Gli1/2/3 are activated which drive the expression of several Hh target genes including Cyclin D2, OPN and CD44. The expression of Gli1 was significantly higher in alcohol-fed DEN-injected mice as compared to alcohol-fed saline-injected or pair-fed DEN-injected mice (Fig. 5a). Hh targets, CCND2, OPN and CD44 were significantly up-regulated affirming the activation of Hh pathway in this model (Fig. 5b). Sonic hedgehog (Shh), the ligand for Hh pathway showed significant protein induction in liver from alcohol-fed DEN-injected mice (Fig. 5c). These data suggest that alcohol alters the expression of Hh target genes and dysregulates Hh signaling contributing to progression of HCC.

Figure 5
figure 5

Up-regulation of hedgehog signaling in experimental HCC model.

(a) Liver mRNA levels of Gli-1. (b) Liver mRNA levels of hedgehog target genes, Ccnd2 (cyclinD2), Opn (osteopontin) and CD44. (c) Liver protein levels of Shh, the ligand for hedgehog pathway with relative band intensity. In all graphs, values are given as average ± SD, ANOVA and Dunnett’s multiple comparison were used to compare the means of multiple groups; (*p < 0.05).

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Altered liver microRNA-122 and HIF-1α correlates with HCC in mice

MicroRNAs are small non-coding RNA molecules that regulate post transcriptional gene expression via RNA silencing. miRs have been reported to control the liver tumor development and aggressiveness28. The most abundant miRNA in the liver is miR-122. Decreases in liver tissue miR-122 have been correlated with gain of metastatic properties of liver cancer and increased mortality29. We found that the expression of miR-122 in the liver tissue was significantly lower in alcohol-fed DEN-injected mice compared to any other groups in this study (Fig. 6a). miR-122 regulates the expression of cyclin G1, whose high levels have been reported in several human cancers30. In addition, by modulating cyclin G1, miR-122 influences p53 protein stability and transcriptional activity and reduces invasion capability of HCC-derived cell lines31. We observed an up-regulation of cyclin G1 (Fig. 6b) and p53 (Fig. 4a) in the liver. Bcl-w, an anti-apoptotic gene and a target of miR-122, was significantly up-regulated in alcohol-fed DEN-injected mice (Fig. 6b).

Figure 6
figure 6

Altered microRNA-122 and HIF-1α expression in liver confirms HCC in mice.

(a) miR-122 levels in liver. (b) Targets of miR-122, Ccng1 (cyclinG1) and Bcl-w mRNA in liver. (c) Liver HIF-1α mRNA levels, HIF-1α DNA binding activity assayed by EMSA and target gene Vegfr1 mRNA levels (n ≥ 5 mice per group). In all graphs, values are given as average ± SD, ANOVA and Dunnett’s multiple comparison were used to compare the means of multiple groups; (*p < 0.05).

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Tumor tissues are often characterized by low levels of tissue oxygen known as hypoxia, which makes tumor tissues resistant to radiotherapy32. Hypoxia induces the transcription factor HIF-1α which drives the expression of various cell proliferation and angiogenesis genes

Table 1 Primers used in this study.
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miRNA Analysis

Tissue samples were lysed in QIAzol Lysis reagent (Qiagen, Maryland, USA), homogenized with stainless steel beads in TissueLyser II (Qiagen, Maryland, USA) and incubated on ice for five minutes followed by miRNA isolation using Direct-zol RNA MiniPrep kit with on column DNase digestion (Zymo Research, Irvin, CA). Reverse transcription (30 min −16 °C; 30 min −42 °C; 5 min −85 °C) was performed in Eppendorf Mastercycler (Eppendorf, New York, USA) using 10 ng RNA, TaqMan primers and MiRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), followed by quantitative RT-PCR (10 min −95 °C; 40 cycles of 15 sec −95 °C; 1 min −60 °C) in CFX96 (Bio-Rad Laboratories, Hercules, CA) using TaqMan Universal PCR Master Mix (Biorad, Hercules, CA). All tissue results were normalized to snoRNA202 expression. Serum sample controls were spiked with Caenorhabditis elegans (cel)-miR-39, as per instructions (Qiagen, Gaithersburg, MD) and subsequently analyzed utilizing a primer pool. Briefly, serum cDNA synthesis was performed with a final 0.1x primer concentration and Applied Biosystems’s rtPCR kit followed by individual target-specific RT-qPCR analysis using Bio-Rad iTaq Universal Probes Master Mix according to manufacturer instructions.

Western blot analysis

Whole cell lysates, nuclear and cytoplasmic extracts were prepared from mouse livers as described previously60. Proteins of interest were detected by immunoblotting with specific primary antibodies against: cyclin D1 (SC-753; santacurz), p53 (ab28; abcam), vimentin (ab92547; Abcam), Shh (SC-9024, Santa Cruz), β-tubulin-HRP (ab185057; Abcam), GAPDH-HRP (ab9482; Abcam). Respective horseradish peroxidase–labeled secondary antibodies were from Santacruz Biotechnology (Dallas, TX). The specific immunoreactive bands of interest were detected by chemiluminescence (Bio-Rad, Hercules, CA). The immunoreactive bands were quantified by densitometric analysis using the UVP System (Bio-Rad Laboratories, Hercules, CA).

Histopathological analysis

Sections of formalin-fixed, paraffin-embedded livers were stained with hematoxylin and eosin (H&E), or Sirius Red and assessed for histological features of carcinoma and fibrosis. The H&E stained sections were independently examined by a veterinary pathologist, Dr. Garlick in a blinded manner (see acknowledgments). The quantitation of Sirius Red staining was performed using ImageJ software. Immunohistochemistry staining for AFP (ab46799; Abcam), CK7 (ab9021; Abcam), CK19 (ab52625; Abcam), was performed on formalin-fixed, paraffin-embedded livers according to the manufacturer’s instructions. To examine cell proliferation, mice were injected i.p. with 100 mg/kg BrdU (Sigma, St. Louise, MO) 2 hr prior to sacrifice and paraffin sections were stained using the anti BrdU antibody (ab6326, Abcam). ImageJ (NIH) was used for image analysis.

Statistical Analysis

Statistical significance was determined using two – tailed t-test; two-way ANOVA and Dunnett’s multiple comparison post-test were used to compare the means of multiple groups. Data are shown as mean ± SD and were considered statistically significant at p < 0.05. GraphPad Prism 6.02 (GraphPad Software Inc., La Jolla, CA) was used for analysis.