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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2025 May 9;196(1):209–222. doi: 10.1016/j.ajpath.2025.04.014

The Patatin-Like Phospholipase Domain–Containing 3 148M Variant Exacerbates Alcohol-Induced Liver Injury and Tumorigenesis in Mice

Jung-Hyo Cho ∗,†, Hyeong-Geug Kim ∗, Menghao Huang ∗,‡,§, Shen Wang ∗, Sheng Liu §,, Alex Lu ‖, Kyle McCrocklin ∗∗, Yang Zhang ∗, Zhigang Fang ∗, Juexin Wang ∗∗,††, Wanqing Liu ‡‡, Jun Wan §,¶,∗∗,††, X Charlie Dong ∗,‡,¶,∗∗,††,∗
PMCID: PMC12413239  NIHMSID: NIHMS2105577  PMID: 40350061

Abstract

The patatin-like phospholipase domain–containing 3 (PNPLA3) protein 148M variant is strongly associated with cirrhosis and hepatocellular carcinoma (HCC). However, the underlying mechanisms remain elusive. This study aimed to elucidate the role of the PNPLA3148M variant in alcohol-related HCC development. Control and humanized PNPLA3148M transgenic mice were fed with an ethanol-containing diet for 12 weeks and examined for liver tumors. After the alcohol feeding, the PNPLA3148M mice had twofold higher liver cancer incidence rates and larger tumor sizes than the control mice. Cancer stem cell markers in the PNPLA3148M mouse livers were elevated relative to those in the control mouse livers. Alcohol detoxification was impaired in the PNPLA3148M mouse livers. Hepatic oxidative stress and DNA damage were elevated in the PNPLA3148M mice. Wnt/β-catenin and Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1/TAZ) were activated in the PNPLA3148M mouse livers. The data suggest that the PNPLA3148M variant had a strong interaction with alcohol in HCC development through attenuation of alcohol detoxification and promotion of oncogenic pathways. Targeting the PNPLA3148M variant might be useful for the prevention or treatment of alcohol-associated HCC in patients carrying this variant.

Graphical abstract

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Alcohol use is a significant risk factor for human morbidity and mortality in the world. Approximately 3 million deaths were associated with alcohol use in 2016 alone.1 Alcohol is a common cause of liver diseases, including hepatic steatosis, hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HCC is a predominant form of liver cancer that is a leading cause of cancer deaths worldwide. A recent study of Chinese patients with alcohol-associated liver disease revealed a marked increase in alcohol-associated liver disease–associated HCC from 5.8% to 30.7% between 2002 and 2018.2 Ethanol is classified as a group 1 human carcinogen by the International Agency for Research on Cancer; however, how ethanol causes cancer is not fully understood. Ethanol is primarily metabolized in the liver by two key enzymes—alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). ADH catalyzes the first reaction from ethanol to acetaldehyde, which is highly reactive and can form adducts with proteins and DNAs.3 Under normal conditions, acetaldehyde is rapidly converted to acetate by ALDH. ALDH2 is the predominant ALDH in the liver.4

Numerous genes and pathways have been implicated in HCC development. Among them, the Wnt/β-catenin and the Hippo pathways play a significant role in HCC tumorigenesis.5 In the absence of Wnt ligands, β-catenin (CTNNB1) is cytosolic and subject to ubiquitin-mediated degradation. After Wnt ligand binding to the Frizzled receptors, β-catenin is skipped from degradation and translocated to the nucleus for transcriptional function. It has been suggested that 30% to 40% of HCC tumors have aberrant activation of the Wnt/β-catenin pathway.5 For the Hippo pathway, Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1, alias TAZ) are two major downstream effectors.6 During Hippo signaling, YAP and TAZ can be phosphorylated by large tumor suppressor 1/2 (LATS1/2) and subject to proteasomal degradation. In the absence of Hippo signaling, non-phosphorylated YAP/TAZ can be translocated to the nucleus and coactivate TEA domain transcription factors (TEAD1 to TEAD4). In HCC, YAP is generally not mutated; instead, gene amplification and post-translational modifications lead to elevated YAP protein levels.7

Numerous genome-wide association studies have identified a single-nucleotide polymorphism (rs738409, C→G) in the human patatin-like phospholipase domain–containing protein 3 (PNPLA3) gene, which results in isoleucine (I) to methionine (M) substitution at amino acid 148. This is the most significant gene variant for the alcohol-related cirrhosis and HCC.8, 9, 10 The PNPLA3148M variant is associated with twofold to threefold higher risk for alcohol-related cirrhosis and twofold to fourfold higher risk for alcohol-related HCC in carriers than noncarriers, respectively.8, 9, 10 PNPLA3 is a lipid droplet-associated protein that is highly abundant in human livers. Biochemical studies have shown that PNPLA3 has triglyceride lipase and retinyl palmitate hydrolase activities; however, the pathophysiological function of the PNPLA3148M variant remains elusive.11 In this study, humanized PNPLA3 transgenic mouse models were used to investigate the role of the PNPLA3148M variant in alcohol-related HCC pathogenesis.

Materials and Methods

Animals

Human PNPLA3148I and PNPLA3148M transgenic mice were generated using a human bacterial artificial clone harboring the human PNPLA3 gene and mutagenesis for generation of the PNPLA3148M variant, as previously reported.12 The transgenic mice were on the C57BL/6 genetic background. An HCC mouse model was generated by cotreatment with a 5% (v/v) of ethanol diet (Lieber-DeCarli diet; Bio-Serv, Flemington, NJ) and CCl4. Briefly, wild-type (WT), PNPLA3148I, and PNPLA3148M mice were first acclimated to the ethanol-containing liquid diet by a gradual increase (1% per day) in ethanol concentration from 0% to 5% (v/v) during the first 6 days and then treated with the 5% ethanol (v/v) diet for 12 weeks. At the same time, the animals were intraperitoneally injected with CCl4 in corn oil weekly at a dose of 0.32 μg/g of body weight for 10 weeks. At the end of the experiment, the animals were euthanized for blood and tissue sample collections.

Histologic Analysis

Tissue samples were fixed in 10% formalin, embedded, sectioned, and stained with hematoxylin and eosin at the Histology Core of Indiana University School of Medicine (Indianapolis, IN). For immunofluorescence analysis, tissue sections were processed, as previously reported.13 The antibodies used in this work included the following: α fetoprotein (AFP; Santa Cruz Biotechnology, Dallas, TX; sc-8399), arginase 1 (ARG1; Cell Signaling Technology, Danvers, MA; 93668S), CD44 molecule (CD44; Santa Cruz Biotechnology; sc-7297), CD133 (Novus Biologicals, Centennial, CO; NBP2-44250), 4-hydroxynonenal (4-HNE; R&D Systems, Minneapolis, MN; MAB-3249), malondialdehyde (MDA; Thermo Fisher Scientific, Waltham, MA; MA5-27560), phospho-histone H2A.X (pH2A.X; Cell Signaling Technology; 9718S), tumor protein P53 binding protein 1 (TP53BP1; Bethyl Labs, Montgomery, TX; A300-272A-M), collagen 1 (COL1; Abcam, Waltham, MA; ab260043), collagen 3 (COL3; Abcam; ab7778), smooth muscle actin α 2 (ACTA2; Abcam; ab5694), TIMP metallopeptidase inhibitor 1 (TIMP1; Proteintech, Rosemont, IL; 10753-1-AP), WNT4 (Santa Cruz Biotechnology; sc-376279), CTNNB1 (Cell Signaling Technology; 8480S), YAP (Cell Signaling Technology; 14074S), and TAZ/WW domain-containing transcription regulator 1 (WWTR1) (Cell Signaling Technology; 82630S). Immunofluorescence images were taken using a Zeiss (White Plains, NY) fluorescence microscope with AxionVison Rel 4.8 software (Zeiss). Images were analyzed using ImageJ software version 1.54 (NIH, Bethesda, MD; https://imagej.net/ij).

Biochemical Analysis

Measurements of acetaldehyde, hydrogen peroxide, nitric oxide, MDA, and glutathione in liver tissue samples were performed using commercial kits from BioAssay Systems (Hayward, CA), Thermo Fisher Scientific, Abcam (Waltham, MA), and Sigma-Aldrich (St. Louis, MO), respectively, as previously described.13

Protein Analysis

Mouse tissue samples were homogenized using a T25 digital homogenizer (IKA Works Inc., Wilmington, NC) in lysis buffer (50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 10 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, 100 μmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and Complete Protease Inhibitor; Sigma-Aldrich). Protein samples were resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA). Membranes were first incubated with a blocking buffer and then with specific primary antibodies overnight at 4°C. Some of the antibodies used for immunostaining were also used for immunoblotting. Other antibodies used were described in the following: PNPLA3 (Thermo Fisher Scientific; PA5-18901), perilipin 2 (PLIN2; Proteintech; A6276), ALDH2 (Thermo Fisher Scientific; MA5-17029), actinin α (Santa Cruz Biotechnology; sc-17829), and actin β (ACTB; Abcam; ab8226). After three washes with Tris-buffered saline solution containing 0.1% Tween-20, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies at a dilution of 1:1000 (Cell Signaling Technology) for 1 hour at room temperature. Signals were detected using enhanced chemiluminescence substrates (Thermo Fisher Scientific) and imaged on a ChemiDoc MP Imaging System (Bio-Rad Laboratories). Images were analyzed using the ImageJ software version 1.54.

RNA Analysis

Total RNAs were extracted and purified from mouse tissue samples using TRI Reagent (Sigma-Aldrich), and cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), according to the manufacturer's instruction. Real-time PCR was performed using an Eppendorf Realplex PCR system with specific primers described in Table 1 and SYBR Green PCR Master Mix (Thermo Fisher Scientific). Expression of a target gene of interest relative to an internal control gene peptidylprolyl isomerase A (Ppia) was analyzed using the 2−ΔΔCT method.

Table 1.

PCR Primer Sequences

Gene symbol Species Primer sequences (forward and reverse) Application
Aldh2 Mouse 5′-TGATCAAGGAGGCAGGCTTT-3′
5′-CCACTTTGTCCACACCCTCA-3′		 qPCR
Ctnnb1 Mouse 5′-TCAGTGCAGGAGGCCGA-3′
5′-CAGGTCAGCTTGAGTAGCCAT-3′		 qPCR
Wnt2 Mouse 5′-GTCTGACCTGATGTAGACGCA-3′
5′-CTGTAGCTCTCATGTACCACCAT-3′		 qPCR
Wnt4 Mouse 5′-CAGAGCCACATGCTCCTAGA-3′
5′-TCACGTCCTGATAGGCACAG-3′		 qPCR
Wnt5a Mouse 5′-CAAGGAGTTCGTGGACGCTA-3′
5′-CAGGCTACATCTGCCAGGTT-3′		 qPCR
Wnt5b Mouse 5′-GTGCCAACACCAGTTTCGAC-3′
5′-GAAGGCAGTCTCTCGGCTAC-3′		 qPCR
Cyr61 Mouse 5′-AGAGGCTTCCTGTCTTTGGC-3′
5′-CCAAGACGTGGTCTGAACGA-3′		 qPCR
Ctgf Mouse 5′-TGCAGACTGGAGAAGCAGAG-3′
5′-GGCTTGGCGATTTTAGGTGT-3′		 qPCR
Adh1 Mouse 5′-GGAGCTTCACCACTGGACAA-3′
5′-GGTCACCTTGGCGACTTTGA-3′		 qPCR
Adh4 Mouse 5′-GCCAGAGTCGATGATGAGGC-3′
5′-CAGGCCAAAGACAGCACAAG-3′		 qPCR
Tet1 Mouse 5′-CTGCTGTCAGGGAGCTCATG-3′
5′-GAGCTCTTCCCTTCCTTCCC-3′		 qPCR
Tet2 Mouse 5′-GGCAAGAGCTCTCAGGGATG-3′
5′-AGGTCGCACTCGTACCAAAC-3′		 qPCR
Tet3 Mouse 5′-TACCCTCCGGAAGTATGGCA-3′
5′-TACATGCTCCAGGAACAGCC-3′		 qPCR
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qPCR, real-time quantitative PCR.

Statistical Analysis

Data are presented as means ± SEM. Comparisons between two groups were analyzed using nonparametric U-tests. Comparisons among multiple groups were performed using nonparametric Kruskal-Wallis tests (Prism version 10.2.1; GraphPad, La Jolla, CA).

Results

The PNPLA3148M Variant Exacerbates Alcohol-Related HCC Development

As reported previously,12 transgenic mice carrying human PNPLA3148I and PNPLA3148M genomic sequences were generated. Expression of human PNPLA3 protein in the transgenic mouse livers was verified by Western blot analysis of hepatic lipid droplet protein lysates (Figure 1A). The line #1 PNPLA3148I and line #2 PNPLA3148M transgenic mice were used for this study as they had comparable overexpression. To develop an ethanol-related HCC mouse model, WT, PNPLA3148I, and PNPLA3148M mice were treated with a liquid diet containing 5% ethanol (v/v) for 12 weeks plus weekly CCl4 injections for 10 weeks (Figure 1B). At the end of the experiment, body weights of the PNPLA3148M mice were not significantly different from those of the WT mice but were significantly less than those of the PNPLA3148I mice. There was no significant difference in liver weights among the three groups of mice (Figure 1, C–E). However, liver cancer phenotype was strikingly different (Figure 1F). Liver cancer incidence rates were 33.3%, 0%, and 85.7% in WT, PNPLA3148I, and PNPLA3148M mice, respectively (Figure 1G). Liver tumors were greater in number and larger in the PNPLA3148M mice than those in the WT mice (Figure 1, H and I). As expected, hepatic steatosis was significantly elevated in the PNPLA3148M mice compared to that in WT and PNPLA3148I mice (Figure 2, A and B). Tumor marker analysis confirmed worse HCC in the PNPLA3148M mice as AFP, ARG1, CD44, and CD133 protein levels were significantly higher in the liver sections of the PNPLA3148M mice than those in the WT or PNPLA3148I mice (Figure 2, C and D). Cell proliferation rates were also significantly increased in the PNPLA3148M mouse livers compared with those in the WT and PNPLA3148I mouse livers (Figure 3).

Figure 1.

Figure 1

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PNPLA3148M promotes liver cancer in an ethanol-fed mouse model. A: Verification of human PNPLA3 transgenic mice by Western blot analysis. B: Schematic diagram of the mouse model used in this study. Wild-type (WT), PNPLA3148I, and PNPLA3148M mice were fed with a 5% (v/v) ethanol-containing diet for 12 weeks plus weekly CCl4 injections (0.32 μg/g, intraperitoneally) for 10 weeks. C–E: Body and liver weight measurements. F: Mouse liver gross images. G–I: Liver cancer incidence rates, tumor counts, and tumor sizes, respectively. Data are expressed as means ± SEM (C–E and G–I). n = 6 to 12 (C–E and G–I). #P < 0.05 for WT versus other groups; ∗∗P < 0.01 for PNPLA3148M versus PNPLA3148I. Plin2, perilipin 2.

Figure 2.

Figure 2

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Characterization of hepatocellular carcinoma. A: Representative hematoxylin and eosin staining of liver sections. B: Lipid droplet analysis by BODIPY staining. C and D: Immunofluorescence and quantification analysis of α fetoprotein (AFP), arginase 1 (ARG1), CD44, and CD133 in liver sections. Total magnification of the images was labeled as such. Data are expressed as means ± SEM (B and D). n = 6 (B); n = 4 (D). #P < 0.05, ##P < 0.01 for wild type (WT) versus other groups; ∗P < 0.05, ∗∗P < 0.01 for PNPLA3148M versus PNPLA3148I. Original magnification: ×100 (A); ×400 (B); ×200 (C).

Figure 3.

Figure 3

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Cell proliferation is increased in the liver of PNPLA3148M mice. A–C: Immunofluorescence and quantification analysis of proliferating cell nuclear antigen (PCNA) and Ki-67 in liver sections. Data are expressed as means ± SEM (B and C). n = 3 (B and C). ##P < 0.01, ###P < 0.001 for PNPLA3148M versus wild type (WT); ∗∗P < 0.01, ∗∗∗P < 0.001 for PNPLA3148M versus PNPLA3148I. Original magnification, ×400 (A).

PNPLA3148M Impairs Ethanol Detoxification and Exacerbates Oxidative Stress

To examine how the PNPLA3148M variant affects ethanol-induced liver injury, a key ethanol-detoxification enzyme, ALDH2, was first analyzed at both protein and mRNA levels. ALDH2 expression was increased in the liver of PNPLA3148I mice but decreased in the liver of PNPLA3148M mice (Figure 4, A–C). As a result, hepatic acetaldehyde levels were significantly elevated in the PNPLA3148M mice (Figure 4D). In addition, Adh1 and Adh4 mRNA levels were also significantly decreased in the liver of PNPLA3148M mice (Figure 4E). Next, reactive oxygen species and DNA damage were analyzed in the liver of ethanol-treated mice. Fluorescence intensities of dichlorodihydrofluorescein diacetate (a probe for hydrogen peroxide), dihydroethidium (a probe for superoxide and hydrogen peroxide), MDA, pH2A.X at Ser139, and TP53BP1 were significantly elevated in the liver of the PNPLA3148M mice compared with those in WT and PNPLA3148I mice (Figure 5, A and B). Biochemical analysis confirmed an increase in hydrogen peroxide, nitric oxide, and MDA in the liver of the PNPLA3148M mice compared with those in WT and PNPLA3148I mice, whereas hepatic total glutathione levels were not significantly different among the three groups of mice (Figure 5, C–F).

Figure 4.

Figure 4

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PNPLA3148M impairs ethanol detoxification. A and B: Hepatic aldehyde dehydrogenase 2 (ALDH2) protein analysis. C: Aldh2 mRNA analysis by real-time PCR. D: Hepatic acetaldehyde measurements. E: Alcohol dehydrogenase 1 (Adh1) and Adh4 mRNA analysis by real-time PCR. Data are expressed means ± SEM (B–E). n = 3 (A and B); n = 4 (C); n = 6 to 11 (D). #P < 0.05, ###P < 0.001 for wild type (WT) versus other groups; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 for PNPLA3148I versus PNPLA3148M. ACTN, actinin α.

Figure 5.

Figure 5

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PNPLA3148M exacerbates ethanol-induced oxidative stress and DNA damage. A and B: Fluorescence imaging analysis of dichlorodihydrofluorescein diacetate (DCFDA), dihydroethidium (DHE), malondialdehyde (MDA), phospho-histone H2A.X (pH2A.X), and tumor protein P53 binding protein 1 (TP53BP1) in liver sections. C–F: Hepatic hydrogen peroxide, nitric oxide (NO), MDA, and total glutathione (GSH) measurements, respectively. Data are expressed means ± SEM (B–F). n = 6 to 12 (C–F). #P < 0.05, ##P < 0.01, and ###P < 0.001 for wild type (WT) versus other groups; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 for PNPLA3148I versus PNPLA3148M. Original magnification, ×200 (A).

Hepatic Fibrosis Is Comparable Between the PNPLA3148I and PNPLA3148M Mice

As PNPLA3148M has been previously implicated in hepatic fibrosis,14, 15, 16, 17, 18, 19, 20 several fibrosis markers were also analyzed in the liver by immunofluorescence imaging and immunoblotting. Expression of COL1, COL3, ACTA2, and TIMP1 was higher in the liver of WT mice than that in the PNPLA3148I and PNPLA3148M mice (Figure 6). This suggests that hepatic fibrosis might not be a major driver for the ethanol-induced HCC in the PNPLA3148M mice.

Figure 6.

Figure 6

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Extracellular matrix (ECM) protein analysis in the PNPLA3148M mouse livers. A and B: Immunofluorescence analysis of collagen 1 (COL1), collagen 3 (COL3), smooth muscle actin α 2 (ACTA2), and TIMP metallopeptidase inhibitor 1 (TIMP1) in liver sections. C and D: Western blot analysis of ECM-related proteins in liver tissues. Data are expressed as means ± SEM (B and D). n = 4 (A and B); n = 3 (C and D). #P < 0.05 for wild type (WT) versus other groups. Original magnification, ×100 (A). ACTB, actin β.

Wnt and β-Catenin Are Elevated in the PNPLA3148M Mouse Liver

As β-catenin is often involved in HCC development, expression of β-catenin and Wnt ligands was analyzed. Ctnnb1 and Wnt family members 4 and 5b (Wnt4 and Wnt5b) mRNAs were elevated in the liver of PNPLA3148M mice compared with those in WT and PNPLA3148I mice (Figure 7, A and B). Immunoblot analysis also showed a trend of increase in β-catenin protein levels (Figure 7, C and D). Immunofluorescence analysis confirmed an increase in WNT4 and β-catenin proteins in the PNPLA3148M mouse livers (Figure 7, E and F).

Figure 7.

Figure 7

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The Wnt/β-catenin pathway is elevated in the PNPLA3148M mouse livers. A and B: Real-time PCR analysis of β-catenin (Ctnnb1) and Wnt gene expression. C and D: Western blot analysis of β-catenin in mouse liver lysates. E and F: Immunofluorescence imaging analysis of Wnt4 and β-catenin in the liver sections. Data are expressed as means ± SEM (A, B, D, and F). n = 4 (A, B, E, and F); n = 3 (C and D). #P < 0.05 for wild type (WT) versus other groups; ∗P < 0.05 for PNPLA3148I versus PNPLA3148M. Original magnification: ×630 (E, top panels); ×200 (E, bottom panels). ACTN, actinin α; Ppia, peptidylprolyl isomerase A.

The Hippo Signaling Pathway Is Dysregulated in the PNPLA3148M Mouse Liver

Next, key factors were analyzed in the downstream of the Hippo signaling pathway. Immunoblot analysis showed that phosphorylated YAP (Ser127) levels trended down, whereas TAZ and TEAD1 protein levels trended up (Figure 8, A and B). Immunofluorescence analysis also showed that YAP and TAZ had increased nuclear translocation in the PNPLA3148M mouse livers (Figure 8, C–E). As a result, expression of the Hippo downstream target genes cysteine-rich angiogenic inducer 61 (Cyr61; alias Ccn1) and connective tissue growth factor (Ctgf; alias Ccn2) trended up in the liver of the PNPLA3148M mice (Figure 8, F and G). In addition, expression of the ten-eleven translocation (Tet) methylcytosine dioxygenase genes was also increased (Figure 8H), which is consistent with a previous report suggesting that Tet1 is a YAP target gene.21

Figure 8.

Figure 8

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Yes-associated protein (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ) are activated in the PNPLA3148M mouse livers. A and B: Western blot analysis of phosphorylated YAP (p-YAP), YAP, TAZ, and TEA domain transcription factor 1 (TEAD1) in liver lysates. C–E: Immunofluorescence imaging analysis of YAP and TAZ in liver sections. F–H: Real-time PCR analysis of cysteine-rich angiogenic inducer 61 (Cyr61), connective tissue growth factor (Ctgf), and ten-eleven translocation (Tet) methylcytosine dioxygenase 1/2/3 mRNA levels. Data are expressed as means ± SEM (B and D–H). n = 3 (A and B); n = 4 (C–H). #P < 0.05, ##P < 0.01 for wild type (WT) versus other groups; ∗P < 0.05, ∗∗P < 0.01 for PNPLA3148I versus PNPLA3148M. Original magnification, ×630 (C). ACTN, actinin α; Ppia, peptidylprolyl isomerase A.

Discussion

In this work, a significant role of the PNPLA3148M variant was illustrated in the development of alcohol-related HCC. Previously, the PNPLA3148M variant was associated with alcohol-related cirrhosis and HCC in multiple human cohort studies.8, 9, 10 The present data have provided some potential molecular links to HCC development. First, PNPLA3148M hepatocytes have attenuated alcohol detoxification function as ALDH2 is decreased and acetaldehyde is increased in the PNPLA3148M mouse liver. This is consistent with the previous reports suggesting that ALDH2 deficiency increases the risk of HCC development in human patients with cirrhosis and alcohol-associated liver disease and an alcohol-associated liver disease plus CCl4 mouse model as well.22,23 Second, reactive oxygen species are elevated in the PNPLA3148M mouse liver. As a result, biomolecules, including lipids, proteins, and DNAs, may be damaged by free radicals. It is likely that DNA damage could lead to oncogenic mutations and/or dysregulation. Third, key oncogenic pathways, including Wnt/β-catenin and the Hippo pathway, are dysregulated in the PNPLA3148M mouse liver. Those alterations could promote HCC initiation and progression. Collectively, those alterations significantly increase the risk of HCC development through the interaction between the PNPLA3148M variant and alcohol metabolism. Mutation of the corresponding 148I to 148M in the mouse Pnpla3 gene does not lead to HCC development in mice under either chow or Western diet plus 10% (v/v) ethanol and 23.1 g/L fructose and 18.9 g/L glucose in the drinking water for up to 50 weeks.24 The differential phenotypes are attributed to multiple factors, including human versus mouse Pnpla3 gene,11 experimental design, and animal housing facility.

As PNPLA3 is a lipid droplet-associated protein, lipid droplet metabolism could play a role in the PNPLA3148M variant-associated HCC.11 This seems consistent with the histology of the liver tumors in the PNPLA3148M mice. PNPLA3148M sequesters abhydrolase-domain containing 5 (ABHD5 or commonly known as CGI-58) from PNPLA2 (commonly known as ATGL for adipose triglyceride lipase) and thus decreases lipolysis.25 Intriguingly, alcohol metabolism, especially accumulation of acetaldehyde, is also impaired by the PNPLA3148M variant. This could partially explain the elevated risk of HCC in the PNPLA3148M mice. However, hepatic fibrosis is not exacerbated by the PNPLA3148M variant under the alcohol feeding and tetrachloride treatment conditions, even though PNPLA3148M promotes hepatic stellate cell activation in vitro.14

Somatic mutations of the CTNNB1 gene, coding for β-catenin, are found in nearly 50% of patients with alcohol-related HCC.5 A genome-wide association analysis identified a protective WNT3A-WNT9A rs708113 [T] allele in alcohol-related HCC cohorts. Interestingly, this protective allele is associated with HCC tumors with a lower rate of CTNNB1 somatic mutations. Further analysis suggests that the rs708113 [T] allele might enhance tumor immune response in the nontumor liver tissue.26 In this study, PNPLA3148M activates the Wnt/β-catenin pathway partly by increasing both Wnt ligands and nuclear β-catenin. But the specific mechanism is still elusive. As PNPLA3 is normally localized on lipid droplet, it is reasonable to believe that the Wnt/β-catenin pathway is indirectly regulated by PNPLA3148M.

The Hippo pathway is implicated in liver cancer development27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43; however, whether it is also involved in alcohol-related HCC is unclear. Current data showed that YAP, TAZ, and TEAD1 in the downstream of the Hippo pathway are elevated in the PNPLA3148M mouse livers, suggesting that the Hippo pathway is involved in the alcohol-related HCC. YAP is activated by PNPLA3148M in human hepatic stellate cells.44 How the Hippo pathway is regulated by PNPLA3148M and whether this occurs in the early or late stage of the HCC development is unclear. Additional studies are needed to address these questions.

HCC originates from dedifferentiated hepatocytes that may express cancer stem cell markers, such as CD44 and CD133.45,46 The PNPLA3148M mouse livers exhibited elevated levels of these markers. Lineage tracing approaches would be useful to track whether those CD44+ or CD133+ cells are the initiating cells for HCC. PNPLA3148M increased the number of those cells under the alcohol and CCl4 conditions. It would be interesting to illustrate the role of PNPLA3148M in the generation of those potential HCC initiating cells.

In summary, current data showed an elevated risk for alcohol-related HCC in PNPLA3148M variant carriers. PNPLA3148M impaired alcohol detoxification and increased hepatic oxidative stress and DNA damage. The Wnt/β-catenin and YAP/TAZ pathways were activated in the PNPLA3148M livers. These findings suggest that PNPLA3 can be targeted for the prevention or treatment of alcohol-associated HCC in PNPLA3148M variant carriers.

Disclosure Statement

None declared.

Acknowledgments

We thank Dr. Xiongbin Lu for the scientific discussion and useful advice; and Rachel Schweiger and Drs. Jiazhi Xu and Lu Wang for the excellent technical assistance.

Author Contributions

J.-H.C. and H.-G.K. performed experiments and wrote the manuscript; M.H., S.W., A.L., Y.Z., and Z.F. performed experiments; S.L. and K.M. analyzed data; J. Wang and J. Wan analyzed and interpreted data; W.L. developed the animal model; and X.C.D. designed the experiments, analyzed the data, and wrote the manuscript.

Footnotes

Supported in part by the National Institute of Diabetes and Digestive and Kidney DiseasesR01DK121925 (X.C.D.), R01DK120689 (X.C.D.), R01DK124612 (W.L. and X.C.D.), and P30DK097512 (M.H.); the National Institute on Alcohol Abuse and AlcoholismR01AA028506 (X.C.D.) and UT2AA031151 (W.L. and X.C.D.); the National Institute on AgingR21AG072288 (X.C.D.); and the Heartland Children’s Nutrition Collaborative Fund at Riley Children’s Foundation (M.H.).

J.-H.C. and H.-G.K. contributed equally to this work.

References

  • 1.Huang D.Q., Mathurin P., Cortez-Pinto H., Loomba R. Global epidemiology of alcohol-associated cirrhosis and HCC: trends, projections and risk factors. Nat Rev Gastroenterol Hepatol. 2023;20:37–49. doi: 10.1038/s41575-022-00688-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Chang B., Tian H., Huang A., Zhai X., Wang Q., Han L., Jin X., Gao L., Liang Q., Li B., Lu Y., Xie H., Ji D., Zou Z. Prevalence and prediction of hepatocellular carcinoma in alcohol-associated liver disease: a retrospective study of 136 571 patients with chronic liver diseases. eGastroenterology. 2024;2 doi: 10.1136/egastro-2023-100036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Gao H., Jiang Y., Zeng G., Huda N., Thoudam T., Yang Z., Liangpunsakul S., Ma J. Cell-to-cell and organ-to-organ crosstalk in the pathogenesis of alcohol-associated liver disease. eGastroenterology. 2024;2 doi: 10.1136/egastro-2024-100104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Rodriguez F.D., Covenas R. Biochemical mechanisms associating alcohol use disorders with cancers. Cancers (Basel) 2021;13:3548. doi: 10.3390/cancers13143548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Xu C., Xu Z., Zhang Y., Evert M., Calvisi D.F., Chen X. beta-Catenin signaling in hepatocellular carcinoma. J Clin Invest. 2022;132 doi: 10.1172/JCI154515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Driskill J.H., Pan D. The Hippo pathway in liver homeostasis and pathophysiology. Annu Rev Pathol. 2021;16:299–322. doi: 10.1146/annurev-pathol-030420-105050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Patel S.H., Camargo F.D., Yimlamai D. Hippo signaling in the liver regulates organ size, cell fate, and carcinogenesis. Gastroenterology. 2017;152:533–545. doi: 10.1053/j.gastro.2016.10.047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Buch S., Stickel F., Trepo E., Way M., Herrmann A., Nischalke H.D., et al. A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis. Nat Genet. 2015;47:1443–1448. doi: 10.1038/ng.3417. [DOI] [PubMed] [Google Scholar]
  • 9.Falleti E., Cussigh A., Cmet S., Fabris C., Toniutto P. PNPLA3 rs738409 and TM6SF2 rs58542926 variants increase the risk of hepatocellular carcinoma in alcoholic cirrhosis. Dig Liver Dis. 2016;48:69–75. doi: 10.1016/j.dld.2015.09.009. [DOI] [PubMed] [Google Scholar]
  • 10.Trepo E., Guyot E., Ganne-Carrie N., Degre D., Gustot T., Franchimont D., Sutton A., Nahon P., Moreno C. PNPLA3 (rs738409 C>G) is a common risk variant associated with hepatocellular carcinoma in alcoholic cirrhosis. Hepatology. 2012;55:1307–1308. doi: 10.1002/hep.25518. [DOI] [PubMed] [Google Scholar]
  • 11.Dong X.C. PNPLA3-a potential therapeutic target for personalized treatment of chronic liver disease. Front Med (Lausanne) 2019;6:304. doi: 10.3389/fmed.2019.00304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Liu Z., Zhang Y., Graham S., Wang X., Cai D., Huang M., Pique-Regi R., Dong X.C., Chen Y.E., Willer C., Liu W. Causal relationships between NAFLD, T2D and obesity have implications for disease subphenotyping. J Hepatol. 2020;73:263–276. doi: 10.1016/j.jhep.2020.03.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Fang Z., Kim H.G., Huang M., Chowdhury K., Li M.O., Liangpunsakul S., Dong X.C. Sestrin proteins protect against lipotoxicity-induced oxidative stress in the liver via suppression of C-Jun N-terminal kinases. Cell Mol Gastroenterol Hepatol. 2021;12:921–942. doi: 10.1016/j.jcmgh.2021.04.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Bruschi F.V., Claudel T., Tardelli M., Caligiuri A., Stulnig T.M., Marra F., Trauner M. The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells. Hepatology. 2017;65:1875–1890. doi: 10.1002/hep.29041. [DOI] [PubMed] [Google Scholar]
  • 15.Lee S.S., Byoun Y.S., Jeong S.H., Woo B.H., Jang E.S., Kim J.W., Kim H.Y. Role of the PNPLA3 I148M polymorphism in nonalcoholic fatty liver disease and fibrosis in Korea. Dig Dis Sci. 2014;59:2967–2974. doi: 10.1007/s10620-014-3279-z. [DOI] [PubMed] [Google Scholar]
  • 16.Trepo E., Pradat P., Potthoff A., Momozawa Y., Quertinmont E., Gustot T., Lemmers A., Berthillon P., Amininejad L., Chevallier M., Schlue J., Kreipe H., Deviere J., Manns M., Trepo C., Sninsky J., Wedemeyer H., Franchimont D., Moreno C. Impact of patatin-like phospholipase-3 (rs738409 C>G) polymorphism on fibrosis progression and steatosis in chronic hepatitis C. Hepatology. 2011;54:60–69. doi: 10.1002/hep.24350. [DOI] [PubMed] [Google Scholar]
  • 17.Valenti L., Al-Serri A., Daly A.K., Galmozzi E., Rametta R., Dongiovanni P., Nobili V., Mozzi E., Roviaro G., Vanni E., Bugianesi E., Maggioni M., Fracanzani A.L., Fargion S., Day C.P. Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease. Hepatology. 2010;51:1209–1217. doi: 10.1002/hep.23622. [DOI] [PubMed] [Google Scholar]
  • 18.Pingitore P., Dongiovanni P., Motta B.M., Meroni M., Lepore S.M., Mancina R.M., Pelusi S., Russo C., Caddeo A., Rossi G., Montalcini T., Pujia A., Wiklund O., Valenti L., Romeo S. PNPLA3 overexpression results in reduction of proteins predisposing to fibrosis. Hum Mol Genet. 2016;25:5212–5222. doi: 10.1093/hmg/ddw341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Linden D., Ahnmark A., Pingitore P., Ciociola E., Ahlstedt I., Andreasson A.C., Sasidharan K., Madeyski-Bengtson K., Zurek M., Mancina R.M., Lindblom A., Bjursell M., Bottcher G., Stahlman M., Bohlooly Y.M., Haynes W.G., Carlsson B., Graham M., Lee R., Murray S., Valenti L., Bhanot S., Akerblad P., Romeo S. Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice. Mol Metab. 2019;22:49–61. doi: 10.1016/j.molmet.2019.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Scheiner B., Mandorfer M., Schwabl P., Payer B.A., Bucsics T., Bota S., Aichelburg M.C., Grabmeier-Pfistershammer K., Stattermayer A., Ferenci P., Trauner M., Peck-Radosavljevic M., Reiberger T. The impact of PNPLA3 rs738409 SNP on liver fibrosis progression, portal hypertension and hepatic steatosis in HIV/HCV coinfection. PLoS One. 2015;10 doi: 10.1371/journal.pone.0143429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wu B.K., Mei S.C., Chen E.H., Zheng Y., Pan D. YAP induces an oncogenic transcriptional program through TET1-mediated epigenetic remodeling in liver growth and tumorigenesis. Nat Genet. 2022;54:1202–1213. doi: 10.1038/s41588-022-01119-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Seo W., Gao Y., He Y., Sun J., Xu H., Feng D., Park S.H., Cho Y.E., Guillot A., Ren T., Wu R., Wang J., Kim S.J., Hwang S., Liangpunsakul S., Yang Y., Niu J., Gao B. ALDH2 deficiency promotes alcohol-associated liver cancer by activating oncogenic pathways via oxidized DNA-enriched extracellular vesicles. J Hepatol. 2019;71:1000–1011. doi: 10.1016/j.jhep.2019.06.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Wen Y., Ma L., Ju C. Recent insights into the pathogenesis and therapeutic targets of chronic liver diseases. eGastroenterology. 2023;1 doi: 10.1136/egastro-2023-100020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Patsenker E., Thangapandi V.R., Knittelfelder O., Palladini A., Hefti M., Beil-Wagner J., Rogler G., Buch T., Shevchenko A., Hampe J., Stickel F. The PNPLA3 variant I148M reveals protective effects toward hepatocellular carcinoma in mice via restoration of omega-3 polyunsaturated fats. J Nutr Biochem. 2022;108 doi: 10.1016/j.jnutbio.2022.109081. [DOI] [PubMed] [Google Scholar]
  • 25.Wang Y., Kory N., BasuRay S., Cohen J.C., Hobbs H.H. PNPLA3, CGI-58, and inhibition of hepatic triglyceride hydrolysis in mice. Hepatology. 2019;69:2427–2441. doi: 10.1002/hep.30583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Trepo E., Caruso S., Yang J., Imbeaud S., Couchy G., Bayard Q., Letouze E., Ganne-Carrie N., Moreno C., Oussalah A., Feray C., Blanc J.F., Clement B., Hillon P., Boursier J., Paradis V., Calderaro J., Gnemmi V., Nault J.C., Gueant J.L., Deviere J., Archambeaud I., Vitellius C., Turlin B., Bronowicki J.P., Gustot T., Sutton A., Consortium G., Ziol M., Nahon P., Zucman-Rossi J. Common genetic variation in alcohol-related hepatocellular carcinoma: a case-control genome-wide association study. Lancet Oncol. 2022;23:161–171. doi: 10.1016/S1470-2045(21)00603-3. [DOI] [PubMed] [Google Scholar]
  • 27.Zhou D., Conrad C., Xia F., Park J.S., Payer B., Yin Y., Lauwers G.Y., Thasler W., Lee J.T., Avruch J., Bardeesy N. Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell. 2009;16:425–438. doi: 10.1016/j.ccr.2009.09.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lee K.P., Lee J.H., Kim T.S., Kim T.H., Park H.D., Byun J.S., Kim M.C., Jeong W.I., Calvisi D.F., Kim J.M., Lim D.S. The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis. Proc Natl Acad Sci U S A. 2010;107:8248–8253. doi: 10.1073/pnas.0912203107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lu L., Li Y., Kim S.M., Bossuyt W., Liu P., Qiu Q., Wang Y., Halder G., Finegold M.J., Lee J.S., Johnson R.L. Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver. Proc Natl Acad Sci U S A. 2010;107:1437–1442. doi: 10.1073/pnas.0911427107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Song H., Mak K.K., Topol L., Yun K., Hu J., Garrett L., Chen Y., Park O., Chang J., Simpson R.M., Wang C.Y., Gao B., Jiang J., Yang Y. Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression. Proc Natl Acad Sci U S A. 2010;107:1431–1436. doi: 10.1073/pnas.0911409107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kowalik M.A., Saliba C., Pibiri M., Perra A., Ledda-Columbano G.M., Sarotto I., Ghiso E., Giordano S., Columbano A. Yes-associated protein regulation of adaptive liver enlargement and hepatocellular carcinoma development in mice. Hepatology. 2011;53:2086–2096. doi: 10.1002/hep.24289. [DOI] [PubMed] [Google Scholar]
  • 32.Tschaharganeh D.F., Chen X., Latzko P., Malz M., Gaida M.M., Felix K., Ladu S., Singer S., Pinna F., Gretz N., Sticht C., Tomasi M.L., Delogu S., Evert M., Fan B., Ribback S., Jiang L., Brozzetti S., Bergmann F., Dombrowski F., Schirmacher P., Calvisi D.F., Breuhahn K. Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma. Gastroenterology. 2013;144:1530–1542.e12. doi: 10.1053/j.gastro.2013.02.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Wang J., Ma L., Weng W., Qiao Y., Zhang Y., He J., Wang H., Xiao W., Li L., Chu Q., Pan Q., Yu Y., Sun F. Mutual interaction between YAP and CREB promotes tumorigenesis in liver cancer. Hepatology. 2013;58:1011–1020. doi: 10.1002/hep.26420. [DOI] [PubMed] [Google Scholar]
  • 34.Perra A., Kowalik M.A., Ghiso E., Ledda-Columbano G.M., Di Tommaso L., Angioni M.M., Raschioni C., Testore E., Roncalli M., Giordano S., Columbano A. YAP activation is an early event and a potential therapeutic target in liver cancer development. J Hepatol. 2014;61:1088–1096. doi: 10.1016/j.jhep.2014.06.033. [DOI] [PubMed] [Google Scholar]
  • 35.Tao J., Calvisi D.F., Ranganathan S., Cigliano A., Zhou L., Singh S., Jiang L., Fan B., Terracciano L., Armeanu-Ebinger S., Ribback S., Dombrowski F., Evert M., Chen X., Monga S.P.S. Activation of beta-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice. Gastroenterology. 2014;147:690–701. doi: 10.1053/j.gastro.2014.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Fitamant J., Kottakis F., Benhamouche S., Tian H.S., Chuvin N., Parachoniak C.A., Nagle J.M., Perera R.M., Lapouge M., Deshpande V., Zhu A.X., Lai A., Min B., Hoshida Y., Avruch J., Sia D., Camprecios G., McClatchey A.I., Llovet J.M., Morrissey D., Raj L., Bardeesy N. YAP inhibition restores hepatocyte differentiation in advanced HCC, leading to tumor regression. Cell Rep. 2015;10:1692–1707. doi: 10.1016/j.celrep.2015.02.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Hayashi H., Higashi T., Yokoyama N., Kaida T., Sakamoto K., Fukushima Y., Ishimoto T., Kuroki H., Nitta H., Hashimoto D., Chikamoto A., Oki E., Beppu T., Baba H. An imbalance in TAZ and YAP expression in hepatocellular carcinoma confers cancer stem cell-like behaviors contributing to disease progression. Cancer Res. 2015;75:4985–4997. doi: 10.1158/0008-5472.CAN-15-0291. [DOI] [PubMed] [Google Scholar]
  • 38.Xiao H., Jiang N., Zhou B., Liu Q., Du C. TAZ regulates cell proliferation and epithelial-mesenchymal transition of human hepatocellular carcinoma. Cancer Sci. 2015;106:151–159. doi: 10.1111/cas.12587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Nishio M., Sugimachi K., Goto H., Wang J., Morikawa T., Miyachi Y., Takano Y., Hikasa H., Itoh T., Suzuki S.O., Kurihara H., Aishima S., Leask A., Sasaki T., Nakano T., Nishina H., Nishikawa Y., Sekido Y., Nakao K., Shin-Ya K., Mimori K., Suzuki A. Dysregulated YAP1/TAZ and TGF-beta signaling mediate hepatocarcinogenesis in Mob1a/1b-deficient mice. Proc Natl Acad Sci U S A. 2016;113:E71–E80. doi: 10.1073/pnas.1517188113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Sohn B.H., Shim J.J., Kim S.B., Jang K.Y., Kim S.M., Kim J.H., Hwang J.E., Jang H.J., Lee H.S., Kim S.C., Jeong W., Kim S.S., Park E.S., Heo J., Kim Y.J., Kim D.G., Leem S.H., Kaseb A., Hassan M.M., Cha M., Chu I.S., Johnson R.L., Park Y.Y., Lee J.S. Inactivation of Hippo pathway is significantly associated with poor prognosis in hepatocellular carcinoma. Clin Cancer Res. 2016;22:1256–1264. doi: 10.1158/1078-0432.CCR-15-1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Lin C., Hu Z., Lei B., Tang B., Yu H., Qiu X., He S. Overexpression of Yes-associated protein and its association with clinicopathological features of hepatocellular carcinoma: a meta-analysis. Liver Int. 2017;37:1675–1681. doi: 10.1111/liv.13428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Chen R., Zhu S., Fan X.G., Wang H., Lotze M.T., Zeh H.J., 3rd, Billiar T.R., Kang R., Tang D. High mobility group protein B1 controls liver cancer initiation through yes-associated protein -dependent aerobic glycolysis. Hepatology. 2018;67:1823–1841. doi: 10.1002/hep.29663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Moya I.M., Castaldo S.A., Van den Mooter L., Soheily S., Sansores-Garcia L., Jacobs J., Mannaerts I., Xie J., Verboven E., Hillen H., Alguero-Nadal A., Karaman R., Van Haele M., Kowalczyk W., De Waegeneer M., Verhulst S., Karras P., van Huffel L., Zender L., Marine J.C., Roskams T., Johnson R., Aerts S., van Grunsven L.A., Halder G. Peritumoral activation of the Hippo pathway effectors YAP and TAZ suppresses liver cancer in mice. Science. 2019;366:1029–1034. doi: 10.1126/science.aaw9886. [DOI] [PubMed] [Google Scholar]
  • 44.Bruschi F.V., Tardelli M., Einwallner E., Claudel T., Trauner M. PNPLA3 I148M up-regulates Hedgehog and Yap signaling in human hepatic stellate cells. Int J Mol Sci. 2020;21:8711. doi: 10.3390/ijms21228711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Huang H., Tsui Y.M., Ng I.O. Fueling HCC dynamics: interplay between tumor microenvironment and tumor initiating Cells. Cell Mol Gastroenterol Hepatol. 2023;15:1105–1116. doi: 10.1016/j.jcmgh.2023.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Brouillet A., Lafdil F. Risk factors of primary liver cancer initiation associated with tumour initiating cell emergence: novel targets for promising preventive therapies. eGastroenterology. 2023;1 doi: 10.1136/egastro-2023-100010. [DOI] [PMC free article] [PubMed] [Google Scholar]

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