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Liu et al. Hepatoma Res 2020;6:7 I http://dx.doi.org/10.20517/2394-5079.2019.39 Page 11 of 16

exposure to DEN. Alb-Cre/Fbxl5 flox/flox mice were injected with DEN (25 mg/kg) intraperitoneally at Day 15
and tumours were significantly increased in number and size compared to DEN-injected control mice at
36 weeks in both males and females. The study demonstrated FBXL5 deficiency led to a sequence of events
(iron overload, oxidative stress, liver damage and regenerative proliferation), which, with the addition of
DEN, gave rise to liver tumours with high mutational load. Previously, hepatocyte-specific FBXL5 deletion
without the addition of DEN was shown to cause liver inflammation but not tumours. The authors went
on to analyse FBXL5 mRNA expression in five different human HCC cohorts and found that low FBXL5
expression level was indeed strongly associated with poorer prognosis in human HCC. Finally, the impact
of iron on hepatocarcinogenesis has also been evaluated using a xenograft model. In this study, 3-4-week-
old female BALB/c athymic mice (nu/nu) were injected subcutaneously with human HCC cell lines (Hep3B
[23]
or HepG2) and followed for 21 days . The authors showed that TSC24 (a potent iron chelator) suppressed
tumour growth in a dose-dependent manner by reducing available iron, and triggering cell-cycle arrest and
apoptosis.

THE ROLE OF THE GUT MICROBIOME
Increasingly, the role of the gut microbiome has been implicated in alcoholic and metabolic liver diseases
and HCC via the gut-liver axis, which refers to bidirectional communication between the gut (and its
[78]
microbiome) and the liver . In one direction, the liver secretes bile acids and antibodies into the intestine,
which influences the gut microbiome composition. Reciprocally, the microbiome and its metabolites
translocate the gut to reach the liver via the portal vein (the enterohepatic circulation) and regulate
metabolic functions. This gut-liver axis exists in a homeostasis, which becomes disrupted in metabolic liver
diseases.

Bacterial dysbiosis has been consistently demonstrated in the gut microbiomes of patients and mice with
[79]
metabolic liver diseases and HCC . Mouse model studies have already revealed several mechanisms by
which the gut microbiome contributes to HCC development.

Bacterial metabolism of compounds
[80]
In a model of NASH-associated HCC, Yoshimoto et al. induced HCC by treatment with a chemical
carcinogen [dimethylbenz (a)anthracene] and HFD. The authors found a strong increase in Gram-positive
bacteria (particularly Clostridium spp.) as well as levels of deoxycholic acid (DCA), a secondary bile acid
whose production relies on metabolism of primary bile acids by bacteria such as Clostridium. Significantly,
DCA was shown to promote a senescence-associated secretory phenotype in hepatic stellate cells, which
leads to hepatocarcinogenesis via activation of the TLR2 pathway [79,80] .

Leaky gut
Increased levels of lipopolysaccharide in the systemic circulation (due to increased intestinal permeability)
and its interaction with TLR4 have been demonstrated to promote HCC formation in a CD-HFD-fed
NASH model as well as a chemotoxin model with combination DEN and CCl 4 [81,82] . This process can be
abrogated by gut sterilisation with oral antibiotics, especially in late-stage disease.

Immunosuppressive microenvironment
The gut microbiome also modulates tumoural adaptive immune responses. The aforementioned study by
Shalapour et al. showed that manipulating the gut microbiome in mice with NASH-driven HCC either
[72]
by knocking out their polyimmunoglobulin receptor (which regulates IgA transport into the gut lumen and
maintains microbial homeostasis) or giving them broad-spectrum antibiotics (which reduces gut bacterial
load) promoted and inhibited HCC development, respectively. Both these interventions modulate liver and
circulating IgA levels and hence anti-tumour cytotoxic T cell activation, as discussed above.