Transcriptional regulation of alcohol induced liver fibrosis in a translational porcine hepatocellular carcinoma model

Abstract

Hepatocellular carcinoma (HCC) is the 5th most common and 2nd deadliest cancer worldwide. HCC risk factors include alcohol induced liver cirrhosis, which prompts hepatic inflammation, cell necrosis, and fibrosis deposition. As 25% of HCC cases are associated with alcohol induced liver disease, understanding the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic responses are critical. This study utilized the Oncopig Cancer Model-a transgenic pig model that recapitulates human HCC through induced expression of KRAS^G12D and TP53^R167H driver mutations-to investigate the molecular mechanisms underlying alcohol induced liver disease. Oncopigs (n = 5) underwent fibrosis induction via infusion of ethanol and ethiodized oil (1:3 v/v dosed at 0.75 mL/kg) into the hepatic arterial circulation. Eight-weeks post induction, liver tissue samples from fibrotic and age-matched control (n = 5) Oncopigs were collected for histological evaluation and transcriptional profiling. Increased hepatic inflammation and fibrosis was observed in fibrotic Oncopigs via pathological assessment. Transcriptional profiling (RNA-seq) resulted in the identification of 4387 differentially expressed genes between Oncopig fibrotic and control livers. GO term enrichment analysis identified pathway alterations associated with cirrhosis progression in humans, including cell proliferation, angiogenesis, extracellular matrix deposition, and oxidation-reduction. Key alterations include activation of hepatic stellate cells, increased matrix metalloproteinase production, and altered expression of ABC and SLC transporter genes involved in transport of anticancer drugs.These results demonstrate Oncopig liver fibrosis recapitulates transcriptional hallmarks of human cirrhosis, making the Oncopig an ideal model for studying the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic response.

Keywords: Alcoholic liver disease; Fibrosis; Matrix metalloproteinases; Pig cancer model; Translational research.

Fig. 1.. Alcohol exposure results in fibrosis induction in Oncopig livers.

Representative images of Masson’s trichrome-stained Oncopig liver sections (2.5X) histologically graded for fibrosis using a porcine adapted METAVIR scheme. (A) Control, histologically normal Oncopig liver with normal pre-existing fibrous septa and associated classic hepatic-lobule architecture. (B) F3 fibrosis grade demonstrating numerous inter- and intra-lobular dissecting fibrous septa present at 8 weeks post-induction. (C) METAVIR inflammation, fibrosis scores, and percent fibrosis for each samples 8 weeks post-induction.

Fig. 2.. Alcohol exposure results in reproducible alteration of hepatic gene expression profiles.

(A) PCA plot demonstrating clustering of samples by group based on the relative expression of 21,748 genes for which expression information was available for each sample. (B) Total number of DEGs, including those up- and down-regulated in the fibrotic compared to control group. (C) PCA plot demonstrating samples cluster by group based on the 4387 DEGs. (D) Heatmap of the normalized expression level of the 4387 DEGs for each sample, represented as z-scores. Dendrograms represent relationships between samples (columns) based on complete linkage clustering.

Fig. 3.. GO terms enriched for DEGs associated with liver cirrhosis progression.

GO terms enriched for the 4387 DEGs are associated with biological processes involved in human liver cirrhosis progression.

Fig. 4.. Downregulation of MYC associated with inactivation of HSCs.

(A) Reduced expression of MYC was observed in the Oncopig fibrotic liver samples. (B) Diagram depicting upregulation of MYC due to chronic alcohol abuse resulting in hepatic apoptosis and activation and proliferation, which contributes to accelerated liver fibrosis. Adopted from Nevzorova et al. [37]. * denotes q-value = 9.5 × 10⁻⁴.

Fig. 5.. Differential MMP and TIMP expression in fibrotic Oncopig livers.

Altered expression of MMPs and TIMPs in Oncopig fibrotic compared to control livers. * denotes q-value < 0.05.

Fig. 6.. Increased expression of proangiogenic factors.

Increased expression of proangiogenic factors in Oncopig fibrotic compared to control livers.

Fig. 7.. DEGs associated with oxidation-reduction processes.

Up-regulated and down-regulated DEGs associated with the oxidation-reduction GO term in Oncopig fibrotic compared to control livers.

Fig. 8.. Altered expression of ABC and SLC transporters.

Altered expression of (A) ABC and (B) SLC transporter genes in Oncopig fibrotic compared to control liver samples. Dendrograms represent relationships between samples (columns) based on complete linkage clustering.