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.
