Chronic jet lag induces spontaneous hepatocellular carcinoma (HCC) in wild-type mice following a mechanism very similar to that observed in obese humans. The process initiates with non-alcoholic fatty liver disease (NAFLD) that progresses to steatohepatitis and fibrosis before HCC detection. This pathophysiological pathway is driven by jet-lag-induced genome-wide gene deregulation and global liver metabolic dysfunction, with nuclear receptor-controlled cholesterol/bile acid and xenobiotic metabolism among the top deregulated pathways. Ablation of farnesoid X receptor dramatically increases enterohepatic bile acid levels and jet-lag-induced HCC, while loss of constitutive androstane receptor (CAR), a well-known liver tumor promoter that mediates toxic bile acid signaling, inhibits NAFLD-induced hepatocarcinogenesis. Circadian disruption activates CAR by promoting cholestasis, peripheral clock disruption, and sympathetic dysfunction.

Non-alcoholic fatty liver disease (NAFLD) is predicted to become the leading cause of hepatocellular carcinoma (HCC) due to the prevalence of obesity and chronic circadian disruption, but the underlying mechanisms are poorly understood. We found that circadian dysfunction promotes NAFLD-induced hepatocarcinogenesis by maintaining persistent liver gene deregulation and metabolic disruption, closely mimicking that observed in obese humans. We demonstrate that the profound circadian dysregulation of nuclear receptor-controlled hepatoprotective pathways, especially those controlled by the bile acid receptor FXR and xenobiotic receptor CAR, plays an essential role in NAFLD-induced HCC. Thus, circadian dysfunction is an independent risk factor of HCC, and restoration of bile acid homeostasis and inhibition of CAR are promising complementary strategies for prevention of metabolic syndrome-induced HCC.

Based on the discoveries described above, we hypothesize that chronic circadian disruption is sufficient to induce spontaneous hepatocarcinogenesis by driving persistent liver metabolic dysfunction and oncogenic activation.

Intrahepatic cholestasis can also activate the constitutive androstane receptor (CAR, NR1I3) (), a central regulator of xenobiotic metabolism. CAR activation is the basis for phenobarbital-promoted non-genotoxic or mutagen-induced hepatocarcinogenesis in mice (), which is strongly linked to β-catenin activation or mutation (), a common molecular lesion in human HCC (). We have recently shown that the combined pharmacologic activation of CAR and the genetic activation of β-catenin in mice is sufficient to induce liver tumors that share an extensive gene expression signature with a subset of human HCC displaying β-catenin activation ().

Overexpression of glutamine synthetase is associated with beta-catenin-mutations in mouse liver tumors during promotion of hepatocarcinogenesis by phenobarbital.

Bile acids are synthesized from cholesterol in the liver and function to promote dietary fat and lipid absorption. As detergents, bile acid intrahepatic levels are tightly controlled by a classic endocrine negative-feedback loop in which bile acids act as ligands for the farnesoid X receptor (FXR, NR1H4), which then indirectly suppresses Cyp7A1, the rate-limiting enzyme in bile acid biosynthesis (). FXR also plays a broad role in liver function by inhibiting steatosis and inflammatory genes (). Ablation of Nr1h4 (hereafter referred to as Fxr) not only promotes cholestasis but also NAFLD, NASH, and HCC in mice ().

The liver is the focus of circadian regulation of metabolic pathways, especially those controlling the synthesis and metabolism of glucose, lipid, cholesterol, and bile acid (). We have previously reported that mutations in both Per1 and Per2 or phase-shifts of the liver clock by restricted feeding both promote uncontrolled intrahepatic bile acid accumulation (cholestasis) and disrupt xenobiotic metabolism in mice (), and that HCC is the second most commonly observed malignancy in circadian gene mutant mouse models ().

Circadian homeostasis in mammals is maintained by a central clock located in the hypothalamic suprachiasmatic nucleus that constantly synchronizes to external solar light cues and controls subordinate clocks in peripheral tissues via circadian output pathways. Among these pathways, the sympathetic nervous system (SNS) targets peripheral organs via adrenergic receptor-mediated intracellular signaling (). SNS dysfunction is closely associated with metabolic syndrome, oncogenic activation, neoplastic growth, and tumor initiation (). The clock is operated by circadian genes. The current model of the molecular clock is based on the cyclic expression of the bHLH-Periodic Acid Schiff transcription factors BMAL1 and CLOCK and their downstream targets Cryptochrome (Cry1 and Cry2) and Period (Per1, Per2, and Per3), which encode the repressors of BMAL1/CLOCK heterodimer. The molecular clock also targets clock-controlled genes (CCGs) to couple diverse functions of peripheral organs with daily physical activity (). Recent studies have revealed that germline or tissue-specific ablation of core circadian genes promotes genomic instability and accelerates both tumorigenesis and cancer progression in mice ().

The prevalence of obesity and NAFLD in our society is coupled with the epidemic of chronic circadian disruption, also termed “social jet lag” (). Circadian dysfunction among night-shift workers and individuals suffering from sleep dyspnea has been identified as a common risk factor of obesity, metabolic disorders, NAFLD, and cancer, including HCC ().

As in HCC caused by other risk factors, NAFLD-induced HCC is predominantly diagnosed in males, but at older ages and earlier disease stages (). However, no significant difference in overall 5-year survival is found among HCC patients with or without NAFLD-related etiologies (). The lack of proper mouse models that develop NAFLD in response to chronic metabolic stress and progress to NAFLD-initiated liver injury, inflammation, fibrosis, and HCC as observed in obese human subjects, has significantly impaired the study on the mechanism of metabolic syndrome-induced hepatocarcinogenesis (). Despite the impending burden of NAFLD-induced HCC, no oncogene addiction loops specific to this disease have been identified ().

NAFLD is found in 30%–40% of the general population and in up to 95% of those with morbid obesity (). Excessive fat accumulation induces liver injury, inflammation, and regeneration, which progresses to non-alcoholic steatohepatitis (NASH) in a fraction of patients. This further stimulates the formation of scar tissue (fibrosis) and cirrhosis, both of which predispose to HCC (). Although severe NAFLD can lead to hepatocarcinogenesis without NASH and fibrosis (), NASH and advanced fibrosis increase HCC risk by 15- and 25-fold in humans, respectively (). The co-existence of NASH with other metabolic disorders such as leptin resistance and cholestasis significantly accelerates liver fibrosis ().

Hepatocellular carcinoma (HCC), the most common liver malignancy, was previously considered a rare cancer in the US, but a nearly 3-fold increase in incidence since the 1980s has made it the fastest rising cause of cancer-related death (). Unlike the situation in developing countries, where hepatitis viral infection and aflatoxin contamination are major HCC risk factors, up to 30%–50% of HCC diagnosed in the US results from liver metabolic diseases. Among these diseases, non-alcoholic fatty liver disease (NAFLD) is predicted to become the leading cause of HCC in the 21st century as a secondary consequence of the obesity pandemic ().

We found that under entrained conditions, pCREB and BMAL1 displayed a coupled and robust rhythmic interaction with both Car and Per1 promoters, which peaked at ZT2 for pCREB and ZT6 for BMAL1. Although c-FOS binding to the Per1 promoter displayed a strong circadian rhythm and peaked at ZT2, its binding to the Car promoter was constitutively low. Chronic jet lag abolished BMAL1 binding to both Car and Per1 promoters, significantly dampened c-FOS and pCREB interaction with the Per1 promoter, but induced a coupled and dramatically increased binding of c-FOS and pCREB to the Car promoter at all times studied ( Figure 7 H), which, as shown by promoter functional analysis, strongly activated Car transcription ( Figure 7 B). As expected, the binding of BMAL1 or c-FOS and pCREB to Per1 and Car promoters was not detected in livers of Alb;Bmal1or β-less mice, respectively ( Figure 7 H). Thus, jet-lag-induced SNS dysfunction and liver clock disruption are sufficient to promote Car dysregulation.

To test this hypothesis, we performed in vivo chromatin immunoprecipitation (ChIP) using anti-BMAL1, c-FOS, and pCREB antibodies ( Figures S8 B–S8D), primers flanking the AP1, CRE, or E-box 8 sequences in the Car promoter, E-box 3, AP1, or CRE motifs in the Per1 promoter, and liver nuclear extracts from 12 week old WT, Alb;Bmal1, and β-less mice at ZT2, 6, and 18. In the livers of WT mice, BMAL1/CLOCK transcriptional activity is low at ZT2 and 18 but peaks at ZT6 (), while nuclear accumulation of AP1 and pCREB is lowest at ZT6, slightly elevated at ZT2, and peaks at ZT18 ( Figures 7 D and 7E). Thus, ZT2, 6, and 18 are the best times in a circadian cycle to study the interplay between SNS-controlled cell signaling and the liver clock in controlling Car expression.

Previous studies have suggested that BMAL1/CLOCK and the PAR-domain basic leucine zipper transcription factor DBP may co-activate Car over a 24-hr period in the liver (). However, Bmal1 and Dbp were both suppressed by circadian disruption ( Figures 1 C, 1D, and 4 C). Thus, jet-lag-induced CAR overexpression in WT mice appeared to be controlled by a previously unknown mechanism. We found that apart from multiple D- and E-boxes potentially recognized by DBP and BMAL1/CLOCK, human and mouse CAR promoters contain conserved AP1 and CRE motifs potentially activated by SNS signaling ( Figure 7 A ). In vitro transfection assays showed that the Car promoter was moderately stimulated by low levels but suppressed by higher levels of AP1 or CREB expression. However, co-expression of AP1 and CREB, even at a moderate level, dramatically stimulated Car promoter activity ( Figure 7 B). We also found that chronic jet lag significantly increased SNS tone in the sleep phase. This was coupled with a constitutive AP1 and Ser133 phospho-CREB (pCREB) nuclear accumulation in mouse livers, which is a well-known consequence of cellular response to SNS-ADRβ-c-AMP-PKA signaling ( Figures 7 C–7E). Ablation of all three β-adrenergic receptors in mice (β-less) completely inhibited AP1 and pCREB nuclear accumulation and also Car, Fos, Myc, and Cyp2b10 expression in mouse livers under both entrained and jet lag conditions ( Figures 7 D–7G), which was associated with strong HCC resistance ( Figure S8 A). Thus, jet-lag-induced SNS dysfunction may directly deregulate Car expression via activation of AP1 and CREB.

(H) BMAL1, c-FOS, and pCREB ChIP signals on Car and Per1 promoters in the livers of WT, β-less, and Alb;Bmal1mice at 12 weeks of age, with ChIP signals for each transcription factor detected in control WT mice at ZT2 as the a.u. 1. Negative control immunoglobulin G and qPCR primers are explained in Supplemental Experimental Procedures

(G) The summary of three independent RT-PCR studies on hepatic expression of Fos, Myc, Car, and Cyp2b10 mRNAs in control and jet-lagged WT and β-less mice at 12 weeks of age.

(E) The summary of three independent Western blots studying the hepatic expression of c-FOS, pCREB, and T-CREB in WT and β-less mice at 12 weeks of age.

(D) Western blots show nuclear expression of c-FOS and S133 phopho-CREB (pCREB) (top panel), and total CREB (T-CREB) levels in total liver protein extracts (bottom panel) of WT and β-less mice at 12 weeks of age.

(B) The summary of four to five independent co-transfection assays studying the role of AP1 and CREB in Car promoter activation.

(A) The schematic illustration of conserved E-boxes, AP1 (A1 and A2) and CRE (C1 and C2) binding motifs and ChIP qPCR primers in the Car promoter.

Under entrained conditions, Carmice had an intact liver clock and maintained a circadian profile of FXR and Cyp7A1 expression ( Figures 6 A and 6B ). However, they lacked β-catenin nuclear activation and showed dramatically dampened c-Myc and Cyp2B10 protein expression in the liver ( Figures 6 B–6D). CAR mRNA and protein levels also displayed a robust circadian rhythm in 24-hr LD cycles, but were arrhythmic and elevated at most times in the livers of jet-lagged WT mice, coupled with constitutive overexpression of Cyp2b10 mRNA, a direct consequence of CAR-directed transcriptional activation ( Figure 6 E). Chronic jet lag disrupted the liver clock in both Carand WT mice but failed to fully activate oncogenic, steatotic, and inflammatory genes in Carmice compared with jet-lagged WT controls. This, again, was coupled with the lack of direct CAR target Cyp2b10 mRNA expression in jet-lagged Car mutants, which confirmed the ability of jet lag to activate CAR ( Figures 6 A and 6E). Thus, Car is a CCG. Its circadian dysregulation stimulates the pathophysiological progression from NAFLD to HCC.

(E) The summary of three to six independent RT-PCR studies on the expression of Car, Cyp2b10, Fxr, Cyp7a1, Myc, Fos, Nr1c3, Srebp1c, Tnf, and Il6 in the livers of 12 week old WT, Car −/− , and Alb cre ;Bmal1 fl/fl mice, with the expression level in control WT mice at ZT2 as the a.u. 1.

(D) The summary of three independent western blots on hepatic expression of FXR, CAR, c-Myc, pβ-catenin, p53, Cyp7A1, and Cyp2B10 in 12 week old WT and Car −/− mice, with the expression level in control WT mice at ZT2 as the a.u. 1.

(B and C) Western blots show the expression of BMAL1, CRY1, PER2, FXR, Cyp7A1, and Cyp2B10 (B), and CAR, p53, c-Myc, phospho-β-catenin (pβ-catenin), and total β-catenin (T-β-catenin) (C) in WT and Car −/− mouse livers at 12 weeks of age.

(A) The summary of three to six independent RT-PCR studies on core circadian gene expression in WT and Car −/− mouse livers at 12 weeks of age, with the expression level in control WT mice at ZT2 as the a.u. 1.

We and others have previously demonstrated that elevated bile acids, as observed in mice lacking circadian homeostasis, activates CAR (), which promotes hepatocarcinogenesis independent of exogenous mutagens (). To define the role of cholestasis and CAR in NAFLD-induced HCC, we studied HCC risk in Fxrand Nr1i3(hereafter Car) C57BL/6J mice. Under entrained conditions, Fxrmice displayed the highest levels of intrahepatic bile acids and triglycerides throughout a 24-hr period among all mouse models studied, and also the highest risk of NAFLD and HCC, as expected. Chronic jet lag further accelerated NAFLD development and led to a greater than 2-fold increase in HCC incidence in Fxrmice by 90 weeks of age ( Figure 5 A and Table 1 ). In contrast, although Carmice were also prone to NAFLD and circadian disruption-induced cholestasis and glycogen storage disease, they showed a dramatically decreased risk of jet-lag-induced hepatomegaly, inflammation, hepatocyte proliferation and necrosis, and fibrosis, and were completely resistant to spontaneous HCC ( Figures 5 and S7 and Table 1 ).

, Increase; #, decrease, compared with WT control samples at the same age or time,p < 0.05,p < 0.01,p < 0.001, ±SEM. See also Figure S7

(G) Gross image (top) and histological diagnosis of HCC by H&E staining (bottom) show NAFLD in jet-lagged WT, Car −/− , and Fxr −/− mice and HCCs found in jet-lagged WT and Fxr −/− mice. Kidneys that do not show significant changes in size among different mouse models are included in gross images. Tumors in H&E slides are circled by dashed green lines. Arrows show representative fat droplets in H&E slides. Scale bars represent 200 μm.

(F) The level of bile duct and hepatocyte proliferation (Student’s t test), hepatocyte necrosis (Student’s t test), and liver fibrosis (ImageJ) in 30 week old control and jet-lagged WT and mutant mice. All analyzed at 10× magnification with CK19, Ki67, and TUNEL signals detected in the livers of control WT mice as the a.u. 1.

(E) TUNEL and Sirius Red staining shows incidence of hepatocyte death (TUNEL) and fibrosis (Sirius Red) in the livers of WT, Car −/− , and Fxr −/− mice. Arrows show representative TUNEL + hepatocytes (TUNEL) and areas of liver fibrosis (Sirius Red). Scale bars represent 50 and 100 μm in the TUNEL and Sirius Red slides, respectively.

(D) H&E and Ki67 staining detects liver inflammation and hepatocyte proliferation in control and jet-lagged WT, Car −/− , and Fxr −/− mice. Arrows show representative areas of hematopoietic cell infiltration in H&E and Ki67 + hepatocytes in Ki67 slides. Scale bars represent 50 and 60 μm in the H&E and Ki67 slides, respectively.

(C) The ratios of liver versus body weight of WT, Car −/− , and Fxr −/− mice at 30 weeks of age (Student’s t test).

(B) Daily hepatic glycogen storage in control and jet-lagged WT and Car −/− mice detected by PAS staining. Values indicate average levels of hepatic glycogen storage detected at ZT2, 10, and 18 for each mouse model, compared with those in control WT mice as the a.u. 1 (ImageJ/Color Deconvolution plugin quantification).

(A) Circadian profiles of serum and hepatic bile acids and triglyceride (TG) in 12 week old control and jet-lagged WT, Car −/− , and Fxr −/− mice (Student’s t test).

Further analysis revealed that all HCC-prone mouse models in our study displayed significantly elevated serum and hepatic bile acid levels, characteristic of intrahepatic cholestasis, regardless of their molecular mechanisms of liver clock disruption and overall metabolic phenotypes ( Figures 1 C, 1D, 4 G, and S6 E). Thus, the co-existence of intrahepatic cholestasis with NAFLD may play a key role in spontaneous hepatocarcinogenesis.

Strikingly, nuclear receptor-controlled cholesterol, bile acid, and xenobiotic metabolism were among the top deregulated pathways in the livers of jet-lagged WT mice at all ages studied ( Table S6 ). Among these nuclear receptors, the suppression of FXR and induction of CAR, as reported for human HCC (), were found in jet-lagged WT and circadian gene mutant mice, coupled with upregulation of transcription factors stimulating cell proliferation and steatosis such as β-catenin, c-Myc, SREBP1, and PPARγ (encoded by Nr1c3), as well as Cyp2B10, a direct target of CAR activation, and Cyp7A1, the rate-limiting enzyme for bile acid synthesis negatively regulated by FXR ( Figures 4 D–4F and S6 A–S6D).

Dysregulation of homologs of molecular markers of human HCCs, such as Ctnnb1 (encoding β-catenin), Myc, and Trp53 (), was also found at the mRNA and/or protein levels in the livers of jet-lagged WT and circadian gene mutant mice ( Figures 4 C, 4D, S6 A, and S6B). However, unlike in mouse thymus where circadian disruption is sufficient to suppress p53 (), chronic jet lag induced a coupled activation of p53 and c-Myc in the livers of WT mice at a young age. Thus, the p53 response to Myc oncogenic activation was still intact in the liver at the initial stage of circadian disruption ( Figure 4 D). This finding agrees with previous reports that the complete loss of p53 function is associated with the progression but not initiation of hepatitis B virus or aflatoxin B1-induced HCC in humans ().

The deregulation of key genes, including circadian genes Bmal1, Clock, Per1, Per2, Cry1, and Nr1d1 (encoding REV-ERBα), was validated at the mRNA level by RT-PCR ( Figure 4 C). Together with the results of protein expression studies shown in Figures 1 C and 1D, these results demonstrated that chronic jet lag is sufficient to disrupt the liver clock independent of circadian gene mutations. Surprisingly, in the livers of Alb;Bmal1mice, Per2 mRNA still displayed a shifted and dampened circadian expression profile, while Cry1 mRNA was constitutively overexpressed. These findings were consistent with a previous report on Cry1 mRNA hepatic overexpression in Alb;Bmal1mice (), and with our finding that the negative loop proteins PER2 and CRY1 still display a robust circadian expression in the livers of these mice under entrained condition ( Figures 1 C, 1D, and 4 C).

Importantly, the jet-lag-induced gene deregulation signature significantly overlaps with that found in human HCC ( Figure 4 B), and also includes pathways frequently deregulated in other human cancers, such as pancreatic adenocarcinoma, and bladder cancer, small-cell lung cancer, prostate cancer, and estrogen-dependent and hereditary breast cancers ( Tables S4 and S5 ) ().

To define the mechanism of jet-lag-induced global disruption of liver metabolism and pathology, we carried out a large-scale microarray analysis using total liver RNA prepared from 12 and 30 week old control and jet-lagged WT mice at ZT2, 10, and 18. As expected, chronic jet lag induced persistent and genome-wide gene deregulation in mouse livers ( Figure 4 A and Tables S1 and S2 ). This includes overexpression of genes promoting lipid, amino acid, and nucleotide biosynthesis and storage, cytoplasmic glycolysis, glycogenolysis, oxidative stress, hepatocyte proliferation and death, cholestasis and fibrosis, suppression of genes stimulating fatty acid mitochondria transportation and β-oxidation, glycogen synthesis and tumor suppression, deregulation of genes controlling cell cycle checkpoints, DNA damage repair, and both innate and adaptive inflammatory responses ( Table S3 ).

, Increase; #, decrease, compared with WT controls at the same time,p < 0.05,p < 0.01,p < 0.001, Student’s t test, ±SEM. See also Figure S6 and Tables S1 S5 , and S6

(G) Serum and hepatic bile acid levels over a 24-hr period in circadian gene mutant and WT mice at 7, 12, and 30 weeks of age.

(D–F) Western blots show the expression of Ser552 phospho-β-catenin (pβ-catenin), total β-catenin (T-β-catenin), c-Myc, and p53 in (D), FXR, CAR, Cyp2B10 and Cyp7A1 in (E), and SREBP1 and PPARγ in (F) in the livers of 12 week old WT mice.

(C) The summary of three independent RT-PCR studies on the expression of core circadian genes, Myc and Fxr in the livers of 12 week old circadian gene mutant and WT mice, with levels detected in control WT mice at ZT2 as the a.u. 1.

(B) Venn diagrams showing persistent overlap of deregulated liver transcriptomic signatures in jet-lagged WT mice with that of human HCC from 12 to 30 weeks of age (hypergeometric test).

Thus, chronic jet lag induces a global shift in liver metabolism to promote lipid synthesis and storage via accelerating cytoplasmic glycolysis. This is also coupled with elevated intracellular oxidative stress that induces liver damage and increased biosynthetic intermediates that support rapid cell division ( Figures 1 E, 1F, 2 A, 2B, S1 C, S2 S4 , and S5 ), a pattern of metabolic adaptation closely reminiscent of that well-characterized for most types of cancer cells ().

To study the mechanism of circadian disruption-induced NAFLD, we conducted a large-scale circadian metabolomics study on serum and hepatic carnitines, lipids and prostaglandins, coenzyme A (CoAs), and tricarboxylic acid cycle (TCA) metabolites in WT mice at 12 and 30 weeks of age. The majority of metabolites studied displayed robust circadian rhythms in control WT mice, but were deregulated in jet-lagged WT mice. Jet-lagged WT mice in particular displayed increased serum and hepatic levels of lactate and pyruvate, coupled with elevated levels of essential bioprecursors for amino acids, nucleotides, triglyceride, and cholesterol synthesis, such as α-ketoglutarate, glycerol 3-phosphate, glutamine, ribose 5-phosphate, acetyl CoA, malate, fumarate, etc. These were associated with deregulation of fatty acid transporting carnitines and accumulation of TCA cycle intermediates, such as isocitrate, succinate, fumarate, and malate, an indication of mitochondrial electron transport chain disruption and redox deregulation ( Figures 3 S4 , and S5 ).

Hierarchical clustering heatmaps show serum (left panel) and hepatic (right panel) carnitines, lipids and prostaglandins, CoAs, and TCA metabolites in control WT mice and the persistent deregulation of these metabolites in jet-lagged WT mice. See also Figures S4 and S5

The development of metabolic syndrome in jet-lagged WT mice is coupled with persistent liver damage as indicated by elevated and deregulated plasma liver parameters, such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, and total bilirubin ( Figures 2 A and 2C). This is accompanied by hepatomegaly ( Figure 2 D), increased bile duct proliferation and chronic liver inflammation ( Figure 2 E), accelerated hepatocyte proliferation and death, and the appearance of fibrosis long before HCC detection ( Figure 2 F). Thus, chronic circadian disruption not only induces NAFLD but also persistent liver damage and fibrosis long before spontaneous hepatocarcinogenesis. Per1;Per2and Alb;Bmal1mice also displayed similar deregulation of serum and hepatic metabolic parameters and liver pathological changes in addition to NAFLD prior to HCC detection ( Figure S3 ).

In addition to developing leptin resistance (), jet-lagged WT mice also displayed a decrease in serum triglycerides and free fatty acids coupled with an increase in hepatic level of triglycerides and free fatty acids. This was associated with persistent high plasma levels of glucose and insulin, characteristic of insulin resistance ( Figure 2 A ), and a dramatic decrease in hepatic glycogen storage throughout a 24-hr period ( Figures 2 B and S2 ).

Scale bars represent 60 μm (CK19), 50 μm (H&E), 60 μm (Ki67), 50 μm (TUNEL), and 150 μm (Sirius Red) in corresponding slides., Increase; #, decrease, compared with WT controls at the same time or agep < 0.05,p < 0.01,p < 0.001, ±SEM. See also Figures S2 and S3

(F) Ki67, TUNEL, and Sirius Red staining show incidence of hepatocyte proliferation and death, and liver fibrosis in control and jet-lagged WT mice. Arrows show representative Ki67 + or TUNEL + hepatocytes (Ki67 or TUNEL), and liver fibrosis (Sirius Red).

(E) Cytokeratin 19 (CK19) and H&E staining shows the incidence of intrahepatic bile duct proliferation and inflammation in control and jet-lagged WT mice. Arrows show representative proliferating bile ducts (CK19) and areas of hematopoietic cell infiltration (H&E).

(B) Daily hepatic glycogen storage detected by Periodic Acid Schiff (PAS) staining in control and jet-lagged WT mice. Values indicate average levels of glycogen storage detected at ZT2, 10, and 18 for each mouse model, with that in control WT mice as the a.u. 1 (ImageJ/Color Deconvolution plugin quantification).

Liver pathology in jet-lagged WT and mutant mice, including Cry mutants that are deficient in fat storage in adipose tissue (), had spontaneous onset of NAFLD at a young age. Jet lag also accelerated NAFLD progression in Alb;Bmal1mice ( Figures 1 E–1G and S1 C). Together, these findings demonstrate that chronic circadian misalignment is sufficient to disrupt the liver clock and induce NAFLD-related HCC in WT mice. Since ”social jet lag,” but not circadian gene germline or tissue-specific ablation, is the major type of circadian disruption in humans (), we then focused on studying the mechanism of NAFLD-induced HCC using jet-lagged WT mice, with Alb;Bmal1and Per1;Per2mice that display two distinct patterns of liver clock disruption and are easier to breed as controls to confirm the role of the clock in the pathologies observed ( Figures 1 C and 1D).

Despite the fact that C57BL/6J inbred WT mice are normally resistant to spontaneous and carcinogen-induced HCC (), chronically jet-lagged WT mice developed HCC after 78 weeks of age. This was also male dominant and at an age correspondent to the median age of spontaneous HCC diagnosis at 67–72 years in humans () ( Figures 1 B, S1 A, and S1B and Table 1 ).

The major cancer types diagnosed in mutant and jet-lagged mice include pancreatic and ovarian tumors, B-lymphoma, and HCC ( Figures 1 B and S1 A), with lymphoma and HCC as the leading causes of cancer-related early euthanization. Unlike γ-irradiated mice that are especially susceptible to lymphoma (), unirradiated jet-lagged WT and mutant mice displayed a similar risk of lymphoma and HCC regardless of the differences in the molecular signatures of liver clock disruption, with HCC incidence specific to each mouse model by 90 weeks of age ( Figures 1 C and 1D and Table 1 ). Both sexes of mutant mice displayed an increased risk of hepatocarcinogenesis, with males showing a greater risk of HCC than females. Under entrained conditions, Per or Cry mutants developed fewer but larger HCCs first detected at 50 weeks of age, while Alb;Bmal1mice developed a large number but small HCCs first detected after 70 weeks of age. Chronic jet lag increased both numbers and sizes of tumors in Per and Cry mutants and also the size of tumor in HCC-bearing Alb;Bmal1mice ( Figures 1 B, S1 A, and S1B and Table 1 ). Thus, chronic circadian disruption not only increases tumor incidence but also accelerates tumor progression.

We have reported recently that chronic circadian disruption is sufficient to induce leptin resistance in mice independent of diet choice and the time or amount of food intake (). We further studied the role of prolonged circadian disruption in HCC risk by conducting a large-scale survival study with C57BL/6J inbred wild-type (WT) and congenic mice lacking Per1 and Per2 (Per1;Per2), Cry1 and Cry2 (Cry1;Cry2), or Bmal1 in the liver (Alb;Bmal1), under entrained or chronic jet-lagged conditions from 4 to 90 weeks of age. Compared with control littermates maintained in steady 24-hr light/dark (24-hr LD) cycles, jet-lagged mice showed significantly reduced lifespan with disease development, including neurodegeneration, severe ulcerative dermatitis, aging, cystic renal dysplasia, and cancer ( Table 1 ), leading to early euthanization ( Figure 1 A ).

(D) Summary of three independent Western blotting analyses on hepatic expression of BMAL1, CRY1, and PER2 (higher protein band only for PER2) with the expression level in control WT mice at ZT2 as the a.u. 1 (Student’s t test, ±SEM). ∗ , Increase; #, decrease, compared with WT controls at the same time, ∗/# p < 0.05, ∗∗/## p < 0.01, ∗∗∗/### p < 0.001.

(C) Western blots show BMAL1, CRY1, and PER2 expression in the livers of 12 week old WT and circadian gene mutant mice. ZT, zeitgeber time, with light on at ZT0 and off at ZT12.

c 32.1% early euthanization from 12 to 77 weeks of age due to overgrowth of teeth, severe ulcerative dermatitis, cystic renal dysplasia, or neurological disorders.

Discussion

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The complex liver pathology in jet-lagged WT mice is driven by global hepatic gene deregulation, which can be detected soon after the initiation of jet lag and displays a pattern overlapping significantly with a human HCC transcriptomic signature, including the induction of key human HCC molecular markers such as Trp53, Myc and β-catenin. Thus, jet-lagged WT mice likely also develop spontaneous HCC following a molecular mechanism very similar to that in obese humans.

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Buchmann A.

Schwarz M. Overexpression of glutamine synthetase is associated with beta-catenin-mutations in mouse liver tumors during promotion of hepatocarcinogenesis by phenobarbital. The tumor-promoting function of CAR is tightly associated with β-catenin activation or mutation (). Jet-lag activated CAR, as measured by constitutive overexpression of the CAR target gene Cyp2b10 over a 24-hr period, was coupled with β-catenin and c-Myc overexpression. Ablation of Car completely inhibited nuclear β-catenin activation and suppressed jet-lag-induced activation of oncogenic, steatotic, and inflammatory genes, leading to strong HCC resistance.

Lee et al., 2010 Lee S.

Donehower L.A.

Herron A.J.

Moore D.D.

Fu L. Disrupting circadian homeostasis of sympathetic signaling promotes tumor development in mice. Magnon et al., 2013 Magnon C.

Hall S.J.

Lin J.

Xue X.

Gerber L.

Freedland S.J.

Frenette P.S. Autonomic nerve development contributes to prostate cancer progression. In the hierarchical circadian system, peripheral organs rely on signaling from circadian output pathways to maintain synchrony with the central clock. We found that chronic jet lag promotes sympathetic dysfunction and peripheral clock disruption, both of which have been linked to oncogenic activation and increased risk of cancer (). We demonstrated that jet-lag-induced sympathetic dysfunction and peripheral clock suppression are sufficient to promote AP1 and CREB oncogenic activation independent of somatic mutations, which drives Car overexpression throughout a 24-hr period to mediate persistent toxic bile acid signaling in jet-lagged WT mice.