Liver-Specific Expressions of <i>HBx</i> and <i>src</i> in the <i>p53</i> Mutant Trigger Hepatocarcinogenesis in Zebrafish

<div><p>Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of <i>src</i> in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and <i>src</i> transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential <i>in vivo</i> platforms for drug screening for therapies for human liver cancer.</p> </div

Comparison of the histopathology of hepatocytes among wild-type and p53 mutant fish overexpressing HBx or src from 1.5 to 11 months of age.

<p>(A) Liver fibrosis was determined by Sirius Red staining. (B) Glycogen accumulation was identified by periodic acid-Schiff (PAS) staining. (C) Apoptosis was examined using the TUNEL assay. (D) Activated caspase 3a was detected by IHC staining. (E) Nuclear PCNA expression was assessed using IHC staining. For each figure, panel 1 represents GFP-mC and p53 mutant control fish, and panel 2 represents HBx and HBx(p53^-) transgenic fish. The different colors denote different scores. For A and C, there are four scores in total, as follows: gray-0, yellow-1, orange-2, and red-3. For B, D and E, there are five scores in total, as follows: gray-0, green-1, yellow-2, orange-3, and red-4.</p

Quantitative RT-PCR analyses of selected marker genes in HBx transgenic fish of the wild-type and p53 mutant backgrounds.

<p>(A) Lipid metabolism-associated genes, including lipogenic factors, lipogenic enzymes, PPAR-gamma targets, and lipid beta-oxidation; (B) fibrosis markers and cell cycle/division-associated genes; and (C) tumor markers and metastasis markers were analyzed by Q-PCR. RNAs from the liver samples at five different ages (3, 5, 7, 9, and 11 months) in the Tg(<i>l-fabp</i>:HBx-mCherry)(TG11) and Tg(<i>l-fabp</i>:HBx-mCherry;<i>cmcl2</i>:GFP) transgenic fish (TG2) were analyzed. Each Ct value was normalized using β-actin and was then compared with the Ct value for the GFP-mCherry fish. The delta-delta Ct values were converted into fold differences. Multiple replicates were performed, and the mean values are shown with standard deviations. The differences among the variables were assessed using a two-tailed Student’s <i>t</i>-test. The symbol “_*” represents significance between HBx(WT) and GFP-mCherry control or between HBx(p53^-) and GFP-mCherry control, and the “#” represents significance between HBx(p53^-) and HBx(WT). P<0.05 was considered to be statistically significant; _*, #: 0.01**, ##: 0.001***, ###: P≤0.001.</p

Cumulative frequency showing the fraction of fish that had developed the various liver diseases.

<p>The cumulative frequency of transgenic fish from multiple lines with chronic inflammation (A), steatosis (B), bile duct dilation (C), hyperplasia (D), dysplasia (E) and HCC (F) were analyzed by Kaplan-Meier analysis. The following different colors denote the different fish lines: black-GFP-mC control, orange-p53 mutant, blue-HBx transgenic fish in wild-type background, red-HBx(p53^-) transgenic fish, green-src transgenic fish in wild-type background, and purple-src(p53^-) transgenic fish. P<0.05 was considered to be statistically significant.</p

Assessment of p-erk, p-akt, myc, vegf and jnk1 signaling in <i>Src</i>-overexpressing wild-type and p53 mutant zebrafish.

<p>Immunohistochemical analyses of phosphorylated erk1/2 (p-erk), phosphorylated akt (p-akt), myc, vegf and jnk1 were performed in liver sections prepared from src-overexpressing wild-type zebrafish (A) or p53 mutant zebrafish (B) that displayed hyperplasia, dysplasia, HCC and sarcomatoid HCC (x 400). GFP-mCherry transgenic fish that were 11 months of age were used as controls and are shown in the first row of each panel. Scale bars: 50 μm. (C) Western blot analysis for the activation of erk and akt in the livers of transgenic fish at different stages of HCC development. After measuring the band intensity using the UVP VisionWorks LS software, the relative density was normalized to β–actin. Ratios of p-erk/erk and p-akt/akt were analyzed, and the data are expressed as relative fold changes for src or src(p53^-) transgenic fish relative to GFP-mC controls.</p

Histopathology of the hepatocytes in <i>Src</i>-overexpressing transgenic zebrafish in the wild-type and p53 mutant backgrounds.

<p>(A1) H&E staining of liver sections from wild-type fish revealed normal histology at 11 months. (A2~A6) H&E staining of liver sections from <i>src</i>-overexpressing, wild-type fish displayed steatosis, hyperplasia, dysplasia and HCC at 11 months. (B1) H&E staining of liver sections from the p53 mutant fish showed normal features at 11 months. (B2~B6) H&E staining of liver sections from src(p53^-) transgenic fish showed severe steatosis and chronic inflammation, dysplasia, HCC and sarcomatoid HCC at 11 months. (C) The hepatocytes from the double transgenic line overexpressing HBx and src in a wild-type background exhibit steatosis, chronic inflammation, hyperplasia, and HCC. All sections were stained with H&E and photographed at 400X magnification. Scale bars: 50 μm. A7, B7, and C4 show the statistical analysis of the H&E staining results. The following different colors denote the different pathological features: gray-normal, purple-chronic inflammation, blue-steatosis, light blue-bile duct dilation, green-hyperplasia, yellow-dysplasia, and red-HCC.</p

Assessment of pcna, p-erk, p-akt, myc, vegf, jnk1, p-src(tyr527), p-src(tyr416), and p-src(tyr418) signaling in HCC, sarcomatoid HCC, and adjacent normal tissue of src-overexpressing transgenic zebrafish.

<p>Liver sections from the src-overexpressing transgenic zebrafish that had developed HCC and sarcomatoid HCC, as well as adjacent normal tissue, were analyzed with H&E staining (A) and immunostaining for pcna, p-erk, p-akt, myc, vegf, jnk1, p-src(tyr527), p-src(tyr416), and p-src(tyr418) (B~J). All slides, comprising normal tissue (1), HCC (2) and sarcomatoid HCC (3), were analyzed using Panoramic Viewer under lower resolution (0) and subsequently at higher resolution. (A0-A3) H&E stain, (B0~B3) pcna protein expression, (C0~C3) p-erk expression level, (D0~D3) p-akt expression level, (E0~E3) myc protein expression, (F0~F3) vegf protein expression, (G0~G3) jnk1 protein expression, (HG0~H3) p-src(tyr527) level, (I0~I3) p-src(tyr416) level, and (J0~J3) p-src(tyr418) expression level were assessed. Scale bars: 50 μm. </p

The assessment of <i>src</i>, p-ERK and p-AKT signaling in wild-type, p53 mutant and Tg(<i>l-fabp</i>:HBx-mCherry;<i>cmcl2</i>:GFP) fish with different liver diseases.

<p>(A) Immunohistochemical analysis of <i>src</i> protein expression in hepatocytes from the GFP-mC and p53 mutant control fish and HBx(p53^-) transgenic fish at 11 months. (B) src IHC results from HBx transgenic fish at 1.5, 7, 9 and 11 months. (C) src IHC results from HBx(p53^-) transgenic fish at 1.5, 7, 9 and 11 months. (D) Immunohistochemical detection of phosphorylated ERK and phosphorylated AKT in 11-month-old Tg(<i>l-fabp</i>:HBx-mCherry;<i>cmcl2</i>:GFP) transgenic fish with hyperplasia and HCC (x 400). Scale bars: 50 μm. (E) Western blot analysis for the activation of erk and akt in the livers of transgenic fish at different stages of HCC development. After measuring the band intensity using the UVP VisionWorks LS software, the relative density was normalized to β–actin. The ratios of p-erk/erk and p-akt/akt were analyzed, and the data are expressed as fold changes of HBx(p53^-) transgenic fish relative to GFP-mC controls.</p