Circadian dysfunction induces NAFLD-related human liver cancer in a mouse model

Abstract

Background & aims: Chronic circadian dysfunction increases the risk of non-alcoholic fatty liver disease (NAFLD)-related hepatocellular carcinoma (HCC), but the underlying mechanisms and direct relevance to human HCC have not been established. In this study, we aimed to determine whether chronic circadian dysregulation can drive NAFLD-related carcinogenesis from human hepatocytes and human HCC progression.

Methods: Chronic jet lag of mice with humanized livers induces spontaneous NAFLD-related HCCs from human hepatocytes. The clinical relevance of this model was analysed by biomarker, pathological/histological, genetic, RNA sequencing, metabolomic, and integrated bioinformatic analyses.

Results: Circadian dysfunction induces glucose intolerance, NAFLD-associated human HCCs, and human HCC metastasis independent of diet in a humanized mouse model. The deregulated transcriptomes in necrotic-inflammatory humanized livers and HCCs bear a striking resemblance to those of human non-alcoholic steatohepatitis (NASH), cirrhosis, and HCC. Stable circadian entrainment of hosts rhythmically paces NASH and HCC transcriptomes to decrease HCC incidence and prevent HCC metastasis. Circadian disruption directly reprogrammes NASH and HCC transcriptomes to drive a rapid progression from hepatocarcinogenesis to HCC metastasis. Human hepatocyte and tumour transcripts are clearly distinguishable from mouse transcripts in non-parenchymal cells and tumour stroma, and display dynamic changes in metabolism, inflammation, angiogenesis, and oncogenic signalling in NASH, progressing to hepatocyte malignant transformation and immunosuppressive tumour stroma in HCCs. Metabolomic analysis defines specific bile acids as prognostic biomarkers that change dynamically during hepatocarcinogenesis and in response to circadian disruption at all disease stages.

Conclusion: Chronic circadian dysfunction is independently carcinogenic to human hepatocytes. Mice with humanized livers provide a powerful preclinical model for studying the impact of the necrotic-inflammatory liver environment and neuroendocrine circadian dysfunction on hepatocarcinogenesis and anti-HCC therapy.

Impact and implications: Human epidemiological studies have linked chronic circadian dysfunction to increased hepatocellular carcinoma (HCC) risk, but direct evidence that circadian dysfunction is a human carcinogen has not been established. Here we show that circadian dysfunction induces non-alcoholic steatohepatitis (NASH)-related carcinogenesis from human hepatocytes in a murine humanized liver model, following the same molecular and pathologic pathways observed in human patients. The gene expression signatures of humanized HCC transcriptomes from circadian-disrupted mice closely match those of human HCC with the poorest prognostic outcomes, while those from stably circadian entrained mice match those from human HCC with the best prognostic outcomes. Our studies establish a new model for defining the mechanism of NASH-related HCC and highlight the importance of circadian biology in HCC prevention and treatment.

Keywords: circadian disruption; circadian transcriptomes; cirrhosis; hepatocellular carcinoma; non-alcoholic steatohepatitis (NASH).

Fig. 1.. Chronic jet lag increases HCC incidence in humanized TIFR mice.

(A) A schematic representation of humanized TIRF mice. Control (Ctrl): maintained in steady 24-hour LD cycles. Chronically jet-lagged (CJ): chronically treated with a human 8-hour shiftwork schedule. CJ/CA: chronically jet-lagged and fed with a chow diet containing 2% cholic acid. (B) The summary of human hepatocyte donors. (C) A H&E-stained section of healthy h-liver (circled by dashed blue lines) shows less eosinophilic human hepatocytes readily distinguishable from more acidic mouse cells in a natural liver microenvironment (scalebar: 100 μm). (D) The survival of humanized TIRF mice in each group. p1: Ctrl vs. CJ; p2: CJ vs. CJ/CA, Kaplan-Meier Statistics, *p<0.05. (E) Representative images of HCCs found in TIRF mice. h-liver, humanized liver; HCC, hepatocellular carcinoma; TIRF mice, transgene-free II2rg^−/−;Rag2^−/−;Fah^−/− mice.

Fig. 2.. Circadian dysfunction induces humanized HCCs.

(A) A representative image of PCR genotyping of HCCs (293: human embryonic kidney cells; MCF-7: human breast cancer cells; C57BL/6J: C57BL/6J mouse tails). (B) The summary of the sizes of HCCs in each group. (C-D) The incidence of h-HCC in each group. (E) A gross image shows a h-HCC metastasized from the liver to the lumbar vertebrae in a jet-lagged TIRF mouse. (F) Gross images (left panel) of a primary h-HCC (top) metastasized to the lumbar bone (bottom) in a jet-lagged TIRF mouse. H&E-stained sections (central panel) show a primary (top) and a metastasized (bottom) h-HCC. IHC sections (right panel) show strong human MYC expression in both primary (top) and metastasized (bottom) h-HCCs circled by dashed blue lines (scalebars: 50 μm). (G) The rate of human hepatocyte repopulation in control and jet-lagged mice. (H) The h-HCC risk among TIRF mice generated by each human donor. (I) Serum levels of glucose, insulin, ALT, TNFα, and IL-6 in control mice with healthy livers (Ctrl h-Liver) and jet-lagged and h-HCC-free mice with NASH and/or cirrhosis (CJ h-NASH/Cirrhosis). Student’s t test, ANCOVA, and chi-square test, **p <0.01, ***p <0.001 (±SEM). ALT, alanine aminotransferase; h-, humanized-; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; TIRF mice, transgene-free II2rg^−/−;Rag2^−/−;Fah^−/− mice.

Fig. 3.. Circadian dysfunction Induces NAFLD-related h-HCCs.

(A) ORO and FAH staining of consecutive cryostat sections show a healthy humanized liver (Ctrl h-liver) and a jet lag induced humanized NAFLD liver (CJ h-NAFLD). Note: only the FAH-positive humanized liver region shows fat accumulation. (B) H&E-, Sirius Red-, FAH-, and F4/80-stained consecutive sections of a Ctrl h-liver (top panel) and a jet lag induced humanized NASH liver (CJ h-NASH, lower panel). (C-D) The summary of ORO, F4/80, and Sirius Red (C) and FAH (D) signals in Ctrl h-liver, CJ h-NASH, and CJ h-HCC sections (10X magnification, Image J quantification, Student’s t test, *p <0.05). Signals in Ctrl h-Liver as arbitrary unit 1 (***p <0.001, ±SEM). (E) Representative h-GPC-3, h-CTNNB1, h-c-MYC, and FAH IHC-stained (top panel) and H&E-stained (lower panel) consecutive sections of CJ h-NASH and CJ h-HCCs, circled by dashed blue lines (Scalebars: 50 μm in A and B, and 100 μm in E). CJ, chronically jet-lagged; Ctrl, maintained in stable 24-hour LD cycles; DEGs, differentially expressed genes; h-, humanized-; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; ORO, Oil Red O.

Fig. 4.. Chronic jet lag activates hallmarks of cancer in humanized livers and HCCs.

(A) Venn diagrams show circadian expression of human genes in healthy h-livers and control and jet-lagged h-NASH and h-HCCs, defined by comparing DEGs between ZT10 and 2, ZT18 and 2, and ZT18 and 10 within each group. (B) The distribution of deregulated human genes at ZT2, 10, and 18 in control and jet-lagged h-NASH and h-HCCs, defined by comparing human genes expressed in h-NASH and h-HCCs to those in control healthy h-livers at same ZTs. (C–F) Top five deregulated human gene pathways in control and jet-lagged h-NASH and h-HCCs at ZT2, 10, and 18 defined by over-representation analysis (red: among top five deregulated pathways at all ZTs studied). The length of bar indicates the total number of DEGs. The blue colour in bars: the numbers of downregulated genes. The yellow (C), orange (D), red (E), and purple (F) colour in bars: the numbers of upregulated genes. The number right next to each bar: -log10 p value of the pathway (*p <0.05). CJ, chronically jet-lagged; Ctrl, under stably entrained condition; DEGs, differentially expressed genes; EMT, epithelial to mesenchymal transition; h-, humanized-; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; ZT, zeitgeber time. Venn Diagram and Over Representation Analysis (ORA, *p <0.05).

Fig. 5.. Jet lag disrupts the liver clock and deregulates serum and hepatic biomarkers.

(A) Reverse-transcription PCR study of human (on black boxes) and mouse (on blue boxes) gene expression in the same h-livers or h-HCCs. Signals of each human gene expression in control h-livers at ZT2 are arbitrary unit 1. (B) Circadian profiles of representative BAs in serum (upper panel) and h-livers or h-HCCs (lower panel) (Ctrl h-Liver: healthy humanized livers isoalted under entrained condition; Ctrl h-NASH: mice with h-NASH but h-HCC-free under the entrained condition. CJ h-NASH: jet-lagged mice with h-NASH but h-HCC-free). Signals of BAs detected in serum and h-livers of control healthy mice at ZT2 are arbitrary unit 1. Student’s t test and ANCOVA, *p <0.05, **p <0.01; ***p <0.001 (±SEM). BAs, bile acids; CJ, chronically jet-lagged; Ctrl, under entrained condition; h-, humanized-; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; ZT, zeitgeber time.

Fig. 6.. Humanized HCCs share the same molecular mechanism with human HCCs.

(A-B) Venn diagram analysis of similarities of control and jet-lagged h-NASH transcriptomes with human NASH (A) and cirrhosis (B) transcriptomes. (C) Venn diagram shows shared and unique DEGs in control and jet-lagged h-NASH (left) and the top 15 jet lag induced deregulated pathways in NASH, defined by over-representation analysis of the 1,085 DEGs specific to CJ h-NASH (right). (D) Venn diagrams show the similarities between the human HCC transcriptome with the combined h-HCC transcriptome (C/h-HCC: derived from both human and mouse DEGs in all h-HCCs studied), the tumour-specific transcriptome (H/h-HCC: derived from human DEGs in all h-HCCs studied), and the tumour stroma-specific transcriptome (M/hHCC: derived from mouse DEGs in all h-HCCs studied). (E) Three human HCC gene clusters associated with differences in patient survival. (F) Venn diagrams show shared and unique DEGs in control and jet-lagged h-HCCs. (G) Venn diagrams comparing the similarities of control and jet-lagged h-HCC-specific gene signatures with those unique to human HCC gene cluster 1 (best survival, left) and cluster 3 (worst prognosis, right). (H–I) The top 15 pathways unique to control (H) and jet-lagged (I) h-HCC transcriptomes (Red: cell proliferation, death, and oncogenic signalling. Blue: damage responses and repair. Black: immune responses. Green: metabolism and biosynthesis. Brown: epithelium-stroma interaction. Purple: sex disparity). Venn Diagram/phyper (*p <0.05) and Over Representation (ORA,*p <0.05) analyses. CJ, chronically jet-lagged; Ctrl, under entrained condition; DEGs, differentially expressed genes; h-, humanized-; HCC, hepatocellular carcinoma; NASH, nonalcoholic steatohepatitis.