A Shortcut from Metabolic-Associated Fatty Liver Disease (MAFLD) to Hepatocellular Carcinoma (HCC): c-MYC a Promising Target for Preventative Strategies and Individualized Therapy

Abstract

Background: Metabolic-associated fatty liver disease (MAFLD) has risen as one of the leading etiologies for hepatocellular carcinoma (HCC). Oncogenes have been suggested to be responsible for the high risk of MAFLD-related HCC. We analyzed the impact of the proto-oncogene c-MYC in the development of human and murine MAFLD and MAFLD-associated HCC.

Methods: alb-myc^tg mice were studied at baseline conditions and after administration of Western diet (WD) in comparison to WT littermates. c-MYC expression was analyzed in biopsies of patients with MAFLD and MAFLD-associated HCC by immunohistochemistry.

Results: Mild obesity, spontaneous hyperlipidaemia, glucose intolerance and insulin resistance were characteristic of 36-week-old alb-myc^tg mice. Middle-aged alb-myc^tg exhibited liver steatosis and increased triglyceride content. Liver injury and inflammation were associated with elevated ALT, an upregulation of ER-stress response and increased ROS production, collagen deposition and compensatory proliferation. At 52 weeks, 20% of transgenic mice developed HCC. WD feeding exacerbated metabolic abnormalities, steatohepatitis, fibrogenesis and tumor prevalence. Therapeutic use of metformin partly attenuated the spontaneous MAFLD phenotype of alb-myc^tg mice. Importantly, upregulation and nuclear localization of c-MYC were characteristic of patients with MAFLD and MAFLD-related HCC.

Conclusions: A novel function of c-MYC in MAFLD progression was identified opening new avenues for preventative strategies.

Keywords: c-myc; metabolic-associated fatty liver disease (MAFLD); metformin; oncogene; tumorigenesis.

Figure 1

High expression of c-MYC in patients with MAFLD and MAFLD-related HCC. (A) c-MYC nuclear expression inside tumor nodules and tumor-adjacent tissues in patients with MAFLD-related HCC (20× magnification). Black arrows indicate cells with positive nuclear c-MYC expression (B) Quantification of % c-MYC positive cells inside tumor nodules and tumor-adjacent tissues in patients with MAFLD-related HCC (n = 4). (C) c-MYC protein expression in MAFLD patients with advanced liver fibrosis (F3–F4), (20× magnification). Red arrows indicate cells with positive nuclear c-MYC expression. Data are expressed as the mean ± SD, * = p < 0.05, inside tumor nodules vs. tumor-adjacent tissues.

Figure 2

Metabolic profile of chow diet-fed alb-myc^tg mice at the age of 36 weeks. (A) Left: Growth curve demonstrating body weight gain, assessed at 7 days’ interval for each group. Right: The histogram represents the incremental area under the respective grow curve (n = 8–9). (B) Body mass index (BMI) (g/m²) (n = 8–9). (C) Weight of epididymal white adipose tissue (eWAT) (n = 8). (D) Size of eWAT cells by H&E staining. Scale bar = 100 µm. Size of individual cells from both groups was measured and average cell size (µm²) was quantified (n = 8–9). Asterisks indicate CLS. The dotted red line marks the individual adipocyte. (E) Levels of cholesterol and TG in serum (n = 8–9). (F) Basal glucose level in blood after 6 h fasting; Blood glucose concentration curve during intraperitoneal glucose tolerance test (n = 3). Data are expressed as the mean ± SD, * = p < 0.05; ** = p < 0.01, *** = p < 0.001, alb-myc^tg mice vs. WT controls.

Figure 3

Steatotic changes in the liver of transgenic mice. (A) Representative liver H&E staining demonstrating distended hepatocytes with foamy appearing cytoplasm and small lipid vesicles. Scale bar = 100 µm (n = 8–9). (B) Illustrative ORO staining. Scale bar = 100 µm. (C) Quantitative analysis of ORO-stained area (n = 5). (D) Direct TG quantification (n = 4). (E) Histology score for steatosis, hepatocyte ballooning, NAFLD activity score (NAS) (n = 5–6). Data are expressed as the mean ± SD, * = p < 0.05; ** = p < 0.01, alb-myc^tg mice vs. WT controls.

Figure 4

Transgenic overexpression of c-MYC in the livers leads to induction of ER stress, apoptosis, inflammation and fibrosis. (A) qPCR analysis of hepatic mRNA expression of Acadm and Cpt1 genes (n = 6). (B) 4-HNE expression (brown) of representative livers was evaluated by immunohistochemistry. Scale bar = 100 µm. The histogram represents the 4-HNE staining intensity (% square microns) (n = 4). (C) Immunoblot analysis of c-MYC, CYP2E1, CHOP (n = 3). Uncropped blots are presented in Figure S13. (D) Levels of ALT in serum (n = 8–9). (E) Protein expression of CC3 and PCNA (n = 3). GAPDH was used as a control for protein loading). Uncropped blots are presented in Figure S14. (F) Representative IF images of hepatic inflammation (CD45 and F4/80 positive cells are stained in green). (G) Sirius red (SR) staining of liver paraffin sections showing accelerated onset of fibrosis formation in alb-myc^tg. * = p < 0.05; ** = p < 0.01.

Figure 5

WD feeding accelerates liver injury in alb-myct^g mice. WT (n = 6) and alb-myc^tg (n = 7) mice fed for 24 weeks with WD. (A) Body mass index (BMI) (g/m²) of mice was calculated at the age of 36 weeks (n = 3–4). (B) H&E staining of epididymal white adipose tissue (eWAT). The dotted red line marks the individual adipocyte. Scale bar = 100 µm. (C) Fasting blood glucose, levels of cholesterol and TG in serum (n = 6–8). (D) Representative liver sections stained with H&E and ORO are shown. Scale bar = 100 µm. Histogram showing TG quantification of mice fed WD (n = 4). (E) Representative images of hepatic inflammation (CD45 positive cells stained in green). (F,G) Liver fibrosis (SR and αSMA IHC staining). (H) Hepatic proliferation (Ki-67 positive cells in green). Scale bar = 100 µm. (I) Protein expression of cleaved caspase 3, GAPDH used as loading control (n = 3). Uncropped blots are presented in Figure S15. Data are expressed as the mean ± SD, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, alb-myc^tg mice fed with WD compared to WT fed with WD.

Figure 6

WD feeding accelerates the initiation of MAFLD associated tumorigenesis in alb-myc^tg mice. WT (n = 3) and alb-myc^tg (n = 5) mice fed 40 weeks with WD. (A) Gross liver. Numbers represent tumor incidence. (B) Representative liver sections stained with H&E. (C) Representative liver sections stained with SR. Quantification of % SR positive area (n = 3–5) Scale bar = 100 µm. (D) Ki-67 immunostaining of paraffin sections showing increased cell proliferation (brown) of hepatocytes in alb-myc^tg mice liver. Quantification of Ki-67 positive cells (n = 3–5). Scale bar = 100 µm. (E) c-MYC IHC staining of paraffin sections. Quantification of c-MYC positive cells (n = 3–5). Scale bar = 100 µm. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 7

Improved hepatic phenotype in alb-myc^tg mice after metformin treatment. alb-myc^tg mice (n = 6) and WT mice (n = 6) fed 20 weeks received metformin enriched chow diet, alb-myc^tg (n = 8) received chow diet; all animals were sacrificed at the age of 36 weeks. (A) Body mass index (BMI) (g/m²) of mice at the age of 36 weeks (n = 3–6). (B) Basal glucose level after 6 h fasting. Blood glucose concentration curve during intraperitoneal GTT. (n = 3–6). (C) eWAT weight (n = 6–8). (D) Serum levels of cholesterol and TG in serum (n = 6–8). Representative liver sections stained with H&E (E), ORO (F), SR (G) and Ki-67 (H) stainings. Scale bar = 100 µm. (I) Protein expression of SREBP-1, GAPDH used as loading control (n = 3). Uncropped blots are presented in Figure S16. Data are expressed as the mean ± SD, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, alb-myc^tg mice fed metformin enriched chow diet vs. alb-myc^tg mice fed chow diet; ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, WT mice fed metformin enriched chow diet vs. alb-myc^tg mice fed chow diet.

Figure 7

Improved hepatic phenotype in alb-myc^tg mice after metformin treatment. alb-myc^tg mice (n = 6) and WT mice (n = 6) fed 20 weeks received metformin enriched chow diet, alb-myc^tg (n = 8) received chow diet; all animals were sacrificed at the age of 36 weeks. (A) Body mass index (BMI) (g/m²) of mice at the age of 36 weeks (n = 3–6). (B) Basal glucose level after 6 h fasting. Blood glucose concentration curve during intraperitoneal GTT. (n = 3–6). (C) eWAT weight (n = 6–8). (D) Serum levels of cholesterol and TG in serum (n = 6–8). Representative liver sections stained with H&E (E), ORO (F), SR (G) and Ki-67 (H) stainings. Scale bar = 100 µm. (I) Protein expression of SREBP-1, GAPDH used as loading control (n = 3). Uncropped blots are presented in Figure S16. Data are expressed as the mean ± SD, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, alb-myc^tg mice fed metformin enriched chow diet vs. alb-myc^tg mice fed chow diet; ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, WT mice fed metformin enriched chow diet vs. alb-myc^tg mice fed chow diet.