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Page 10 of 16 Liu et al. Hepatoma Res 2020;6:7 I http://dx.doi.org/10.20517/2394-5079.2019.39

Genetically obese mice with metabolic syndrome such as ob/ob (leptin deficient) db/db (leptin-receptor
deficient), and foz/foz (mutated Alms1 gene) promote tumourigenesis in the presence of a secondary insult
(e.g., DEN) but do not otherwise develop HCC spontaneously [62,67,69] . Furthermore, ob/ob and db/db mice
fail to develop significant liver fibrosis or NASH histology without the addition of one of the dietary models
[62]
[21]
above . Park et al. utilised a dietary (HFD) and genetic (ob/ob) obesity model in combination with
DEN to show that obesity (no matter how it was achieved) promoted the development of DEN-induced
HCC in C57BL/6 mice by enhanced production of the pro-inflammatory cytokines IL-6 and TNF. Many
other genetic models have been developed to study NAFLD-associated HCC including PTEN knockout,
PPARα knockout, AOX knockout, KK-Ay/a (agouti gene mutation) and MAT1A knockout mice. While
they all reliably form HCC, they fail to recapitulate NASH itself (in KK-Ay/a mice) or its associated aspects
such as obesity and metabolic syndrome (in PTEN, PPARα, AOX and MAT1A knockout mice) [21,62,67] . As an
example, PTEN knockout mice (which develop tumours between 40 and 78 weeks) are hyper-responsive
to insulin instead of being insulin resistant. Unsurprisingly, gene expression signatures from PTEN
knockout mice are markedly different from that of other NASH mouse models [28,70] . One promising genetic
[71]
model of NASH-driven HCC is the MUP-uPA transgenic mouse combined with HFD . MUP-uPA mice
express high amounts of urokinase plasminogen activator in hepatocytes leading to hepatocyte-specific
endoplasmic reticulum stress and liver damage. These mice exhibited weight gain, insulin resistance, classic
signs of NASH (steatosis, inflammation, ballooning), fibrosis and, importantly, spontaneous HCC in 80%
[71]
at 40 weeks via processes dependent on TNF produced by inflammatory liver macrophages . As expected,
HFD-fed wild type mice developed simple steatosis and no HCC over the same period. Furthermore,
transcriptomic data from MUP-uPA mice and human NASH datasets showed signalling similarities,
especially in the regulation of the immune system, innate immune response and the response to cytokine
[67]
[72]
gene sets . Recently, Shalapour et al. used both MUP-uPA and STAM mice fed with HFD to make
a landmark discovery that hepatocarcinogenesis in NASH was facilitated by immunosuppressive liver-
+
resident IgA plasma cells, which directly inhibit anti-tumour cytotoxic CD8+ T lymphocyte activation.
Of the models mentioned above, it seems the MUP-uPA and DIAMOND mice (which require a
combination of genetic modification and dietary manipulation) best replicate NASH-associated
HCC. However, tumour formation in these models requires lengthy periods and there is considerable
heterogeneity in their mutational landscapes which may limit utility and reliability in some settings, e.g.,
[67]
drug development studies . Although STAM mice can develop tumours more quickly than these models
(20 weeks vs. 40 weeks), they are physiologically less similar to human NASH (lacking insulin resistance).

HH-associated HCC models
Hepatocarcinogenesis arising from iron accumulation is thought to be secondary to oxidative DNA
damage from ROS generated by free hepatic iron. This leads to a cycle of cell death, and compensatory
proliferation, which favours the accumulation of mutations in hepatocytes and ultimately malignant
transformation [13,73] . Recreating this in an animal model is difficult. The most common form of HH is
caused by mutations in the HFE gene. Although HFE gene knockout produces the phenotype of HH in
mice, spontaneous liver tumours do not develop . In a dietary model where BALB/cJ male mice were
[74]
fed ad libitum with chow supplemented with 3% carbonyl-iron, hepatic iron concentrations at 12 months
were 13-fold that of normal chow-fed controls . No liver tumours developed; however, hepatocyte nuclei
[75]
changes were observed (iron-containing ferritin inclusions, enlarged nucleus, increased mitotic index
and abnormal mitotic figures), which may have represented preneoplastic changes. Rothenberg et al.
[76]
created a model of HH by knocking out β2-microglobulin (the chaperone protein for HFE) in C57BL/6
mice and reported that spontaneous HCCs developed in only a minority (31%) of mice. Because tumour
development was not predictable and time-consuming (taking up to two years), this model has not been
widely used to study HH-related HCC. Recently, Muto et al. developed a novel model of HCC induced
[77]
by iron overload by deleting the iron-sensing ubiquitin ligase FBXL5 specifically in hepatocytes and