Liver Cancer Checks in When Bile Acid Clocks Out

Abstract

In this issue of Cancer Cell, Kettner et al. identify the disruption of normal circadian rhythmicity as an independent risk factor for hepatocellular carcinoma (HCC) in experimental animals and reveal opposing roles for the nuclear receptors FXR and CAR in disease progression from non-alcoholic fatty liver disease (NAFLD) to HCC.

With the advent of readily accessible air travel, the connection between our internal biological clocks and the external day/night cycle is increasingly being disrupted. Of note, these disruptions in our normal circadian rhythms are being linked to disease states. Indeed, a large body of evidence indicates that metabolic disorders are direct consequences of a dysregulated molecular clock. Furthermore, the correlations between disrupted circadian clocks and tumor initiation and progression are such that the International Agency for Research on Cancer (IARC) has categorized “shift work that involves circadian disruption” as a probable carcinogen to humans (Fu and Kettner, 2013).

The circadian clock is a cell-autonomous timekeeper that controls virtually all aspects of physiology in humans and other animals. While the master pacemaker is located in the suprachiasmatic nucleus (SCN), peripheral clocks are believed to be present in every type of tissue. Both central and peripheral clocks are controlled by interconnecting transcriptional and translational feedback loops (Reppert and Weaver, 2002), such that transcriptional activators (BMAL1/CLOCK) and repressors (REV-ERBs) temporally regulate the expression of a host of clock-controlled genes (CCGs) (Bugge et al., 2012; Cho et al., 2012). The temporal control of these CCGs shapes the transcriptional landscape of core circadian clock system and its output genes important to many physiological processes.

A mechanistic link between circadian clocks and cancer is supported by several recent studies (Fu and Kettner, 2013), but the connection is complicated. For example, animals harboring mutations in Per2, a transcriptional repressor of the BMAL1/CLOCK complex, show spontaneous and radiation-induced tumor formation and are insensitive to radiationinduced cell death. In contrast, loss of Cry1 and Cry2, Per2’s partner transcriptional repressors, does not lead to a cancer phenotype and actually reduces cancer susceptibility in a Trp53 knockout background (Fu and Kettner, 2013). However, the recent explosion in studies linking cancer to alterations in circadian regulation reflects a growing appreciation for a fundamental connection between these processes. These reports further clarify the direct link between misaligned clocks and different types of malignancies, including lung adenocarcinoma, acute myeloid leukemia, and neuroblastoma, and also uncovered the mechanistic basis of cancer development under these conditions (Papagiannakopoulos et al., 2016; Puram et al., 2016).

Hepatocellular carcinoma (HCC) is one of the most aggressive types of cancer with no effective treatment. However, a definitive model to elucidate the relationship between disrupted clocks and HCC remains elusive. In a tour de force, Kettner and colleagues now demonstrate the progression to HCC in a chronic jet lag mouse model that closely resembles disease progression in man (Kettner et al., 2016).

By employing a chronic jet lag paradigm, Kettner et al. uncover a clear increase in the incidence, as well as an acceleration in the progression, of HCC (as well as other types of tumors) in both wild-type mice and mice lacking core components of the clock (Per1^−/−; Per2^−/−, Cry1^−/−;Cry2^−/−, and Alb^cre; Bmal1^fl/fl). Through the analysis of liver pathology and metabolic parameters, the authors chronicle the initial induction of NAFLD, its progression to NASH and fibrosis, and ultimately its progression to HCC. Based on extensive serum and hepatic metabolomics studies, the authors propose a model in which chronic jet lag induces a global shift in liver metabolism. Notably, the increases in lipid synthesis and storage, oxidative stress and associated liver damage, and increased biosynthetic intermediates supporting rapid cell division induced in the jet lag model are reminiscent of those observed in cancer cells. The authors link this metabolic shift to the dysregulation of hepatic cholesterol and bile acid metabolism and implicate the nuclear receptors farnesoid X receptor (FXR) and constitutive androstane receptor (CAR) in this progression. In particular, Cyp7A1, the rate-limiting enzyme for bile acid synthesis negatively regulated by FXR, and Cyp2B10, a direct target of CAR activation, as well as genes stimulating cell proliferation and steatosis including Ctnnb1, Myc, Srebf1, and Pparg, are aberrantly regulated in jet-lagged mice.

Supporting a protective role for FXR in HCC, Fxr^−/− mice developed NAFLD at an accelerated rate and had a >2-fold increase in the incidence of spontaneous HCC under the jet lag condition. In contrast, loss of Car expression dramatically decreased the risk of jet-lag-induced hepatomegaly, inflammation, and fibrosis, and Car^−/− mice were resistant to HCC. Furthermore, the authors categorize Car as a novel clock-controlled gene (CCG). In their jet lag model, sympathetic neuron dysfunction leads to arrhythmic Car expression and higher transcript and protein levels, leading to the conclusion that Car is an important driver for HCC formation by stimulating NAFLD to NASH transition.

The study presented by Kettner et al. provides insight into the potentially damaging effects of our modern lifestyles, including “social jet lag,” in which global liver metabolic alterations facilitate cancer initiation and progression. Indeed, because jet lag leads to spontaneous tumor formation in animal models, it too should be considered to be an independent risk factor for HCC. This study highlights the importance of deregulated bile acid metabolism as an early event during HCC formation, with all mouse models displaying cholestasis in addition to NAFLD prior to HCC detection. Fxr^−/− mice have extremely high intrahepatic bile acid levels, which correlate with elevated HCC incidence. This suggests that loss of bile acid homeostasis due to circadian disruption may be an early initiating event for the liver metabolic shift, as observed in obese humans. Persistent circadian dysregulation would then further drive the transition from NAFLD to HCC by altering the function of CAR (Figure 1).

Figure 1. Induction of HCC by Chronic Jet Lag Results from Global Gene Dysregulation Mediated through FXR and CAR.

Disruption of the peripheral clock by chronic jet lag leads to a sequential progression from NAFLD to NASH, cirrhosis, and ultimately HCC due to global metabolic dysfunction and gene dysregulation mediated through FXR and CAR.

It should be noted that the increased HCC risk in this jet lag model could also be due to additional mechanisms, including increased stress, increased hepatic toxic bile acid species, and alteration to the gut microbiota (Leone et al., 2015). Recent studies indicate that the actions of FXR in the intestine are protective against HCC. Indeed, re-expressing a constitutively active FXR in the intestine prevents the development of HCC in aged Fxr^−/− mice (Degirolamo et al., 2015). Moreover, intestinal activation of FXR ameliorates NAFLD (Fang et al., 2015). These effects may be mediated by circulating bile acids, reshaped by intestinal FXR and the microbiota. Furthermore, circadian disruption has also been reported to modulate the gut microbiota and bile acid pool. All of these factors may contribute to the jet-lag-modelinduced HCC. Given these points, the current study sheds new light on the relationship between a disrupted circadian clock and cancer development and defines new players important in HCC development.