Discussion
While GEMMs are essential for advancing the cellular and molecular
understanding of liver cancer (17), the combinatorial character of AP-1
homo- and hetero-dimers complicates the identification of dimer-specific
functions, when using conventional monomer-based gain- or
loss-of-function GEMMs. Using a single-chain, forced dimer strategy
approach, the present study dissects for the first time the contribution
of a specific c-Jun/Fra-2 AP-1 dimer to HCC pathogenesis in vivo .
Hepatic expression of c-Jun~Fra-2 leads to hepatocyte
proliferation, decreased hepatic fat content, moderate liver
inflammation and limited fibrosis, with the subsequent development of
liver tumors that have HCC characteristics. We identify a crucial
pathogenic interaction between c-Jun/Fra-2 and c-Myc (Figure 6H) as an
important initiating event and identify the consequences of switching
off the c-Jun~Fra-2 oncogenic driver or therapeutically
targeting c-Myc activation in established liver tumors.
Mice expressing Fra-1, Fra-2 or c-Jun~Fra-2 in the liver
express lower levels of Pparγ and are protected from steatohepatitis
(22, 24). Repression of Pparγ is maintained during liver carcinogenesis
in c-Jun~Fra-2hep mice, while a
c-Jun~Fra-1 dimer has apparently no effect. As Pparγ
expression across Fra-1/2hep and
c-Jun~Fra-1/2hep mice is not
correlated with the occurrence of liver tumors, decreased hepatic Pparγ
is likely not causally involved in the early
c-Jun~Fra-2-driven oncogenic events. However, it might
potentiate transformation, as Pparγ+/- mice are more
susceptible to DEN-induced HCC (52). Strikingly, signs of mild
inflammation, fibrosis and even ER stress, are observed in
c-Jun~Fra-2hep livers despite a
dyslipidemic, low hepatic fat context. Future experiments will clarify
if any or all of these events are essential for tumorigenesis and how
they occur independently of steatosis. The
c-Jun~Fra-2hep GEMM constitutes a
convenient model to dissect the interactions between pre-neoplastic or
fully transformed hepatocytes and their non-parenchymal environment.
c-Myc and Myc pathway activation is a major oncogenic event in many
tumor types including HCC (53, 54). A modest but consistent increase in
c-Myc mRNA and protein expression was measured in livers of
c-Jun~Fra-2hep mice already before
tumors were observed. Importantly, and consistent with a crucial role
for increased c-Myc in c-Jun~Fra-2–driven hepatocyte
transformation, tumors that escaped switching off
c-Jun~Fra-2 maintained c-Myc expression, while it was
decreased to control levels in the adjacent non-tumoral areas. In
addition, the tumor-static effect of JQ-1, which decreased Myc
expression and activity in c-Jun~Fra-2 tumors, is in
line with the idea that these tumors are addicted to increased c-Myc
expression that is initiated by the c-Jun/Fra-2 AP-1 dimer.
Several signalling pathways, such as IL6/JAK/Stat3 and PI3K/AKT/GSK3β
(42, 43), both elevated in
c-Jun~Fra-2hep livers and tumors, can
increase c-Myc expression. These pathways might also contribute to
maintain c-Myc expression in tumors escaping switching off
c-Jun~Fra-2, together or along with increased Fos
expression that is observed in some escaping tumors. Using
Jun~Fra-2hep mice, mouse and human
liver cell lines and publicly available human cell lines and human liver
cancer datasets, we demonstrate that c-Jun/Fra-2 activates c-myctranscription by binding a conserved 3’ enhancer in the c-mycgene. Importantly, hepatocyte-specific expression of the closely related
c-Jun~Fra-1 dimer, or freely dimerizing Fra-1 or Fra-2
monomers, had no impact on c-myc expression and did not lead to
spontaneous tumors. Conversely, pre-neoplastic livers expressing Fos
(19) had elevated c-myc mRNA, while increased fos mRNA was
observed in three out of five tumor escapers that maintained c-Myc
expression. These data indicate that only specific AP-1 complexes, such
as c-Jun/Fra-2 and Fos-containing dimers can activate c-myctranscription in hepatocytes. Ongoing work using a similar forced dimer
strategy will certainly shed light on the identity of the Fos-containing
dimers modulating c-myc in hepatocytes. A likely consequence of
this functional dimer redundancy, supported by loss-of-function
experiments, is that no Jun or Fos protein is essential for c-mycexpression. In HepG2 cells, where ChIP experiments indicate that c-Jun
and Fra-2 form a functional dimer on the MYC 3’ enhancer,
knock-down of either JUN or JUNB moderately decreased MYC mRNA,
while a significant reduction in c-myc mRNA and protein was
observed during HBV-driven carcinogenesis in mice lacking hepatic c-Jun.
However, c-Myc protein expression was unaltered in DEN-induced
c-JunΔli liver tumors, while hepatic c-Myc decreased
upon genetic inactivation of the AP-1-upstream kinase JNK1 (14). Bulk
RNAseq analyses of Fos-expressing and Fos-deficient livers (19)
indicated that hepatic c-myc expression was increased in
c-Foshep, but unchanged in DEN-treated
c-FosΔli mice. c-Myc transcription is also not
affected by the single inactivation of Fra-2 in hepatocytes and
Fra-2Δli mice, subjected here for the first time to an
HCC paradigm, display unaltered DEN-induced tumorigenesis. While the
consequences of inactivating other AP-1 monomers, such as JunB, JunD and
Fra-1 on hepatic c-myc expression and tumorigenesis remain to be
formally tested, these experiments indicate that the requirement for
AP-1-forming proteins to modulate c-myc expression during liver
carcinogenesis is dimer- but also context- specific. Targeting one or
multiple AP-1 dimers might not be a straightforward therapeutic option,
although our in silico analysis of the
TCGA-LIHC dataset, as well as
preliminary immune-histochemical analyses of a set of HCC tumors,
indicates that patient stratification according to JUN/FRA2 and MYC
expression might help identifying patients likely to respond to such
AP-1 and/or Myc-targeted therapies.
Despite being heterogeneous in size, molecular profiles and growth
kinetics, liver tumors arising in
Jun~Fra-2hep mice regressed upon
switching off c-Jun~Fra-2 expression. However, a
fraction of tumors relapsed and new tumors arose within few weeks,
possibly in a c-Myc-dependent manner as indicated by the analyses of
tumors collected 6 months later. The cellular and molecular events
occurring immediately after turning OFF c-Jun~Fra-2, the
involvement of Fos-containing dimers, and their connection to the
various pro-tumorigenic functions of c-Myc certainly warrant further
evaluation. Unbiased, possibly single-cell, RNA and proteome profiling
of a large number of tumors in different ON and OFF settings and
subsequent comparison with the OMIC data generated using
c-Myc-switchable liver mice (55, 56) will help narrowing down the
essential molecular and cellular players.
Several therapeutic strategies targeting Myc, mostly indirectly, have
been evaluated (57, 58). The early tool compound JQ-1 (48) and other
BET-family inhibitors (BETi) have preclinical benefits in several
cancers, often through Myc/Myc target suppression (59). While a
Myc-independent anti-tumorigenic decrease in Fra-1 transcription has
been reported after BETi (60), JQ-1 does not impact
c-Jun~Fra-2 mRNA or protein expression and its positive
effects in c-Jun~Fra-2 hep tumors
appears to be Myc-dependent. This is also in line with the idea that
Myc-dependent tumorigenesis is reversible even when Myc is not the
initiating oncogenic lesion, as shown in lung adenocarcinoma induced by
oncogenic Ras (61), an upstream activator of AP-1.
Despite a wealth of studies, there is no effective therapy for HCC due
to limited mechanistic knowledge of this heterogeneous disease and the
lack of biomarkers to select clinical trial patients most likely to
benefit from a specific therapy. HCC prevention by limiting viral
hepatitis, currently accounting for 75% of liver cancer deaths, remains
the key strategy, while Sorafenib is still a standard of care for HCC in
low income countries, despite limited efficacy. The increased relevance
of non-viral risk factors is a major concern aggravated by the poor
prognosis of HCC patients, even in high income countries with the widest
portfolio of therapeutic options and where immunotherapies have become
first-line treatment for advanced HCC. While BETi have shown mixed
results as single agents (49, 59), immunotherapies are costly and have
yet to fulfil their promises (2, 3). Combination therapies involving BET
inhibitors, for example flight tested in this preclinical model, may
enhance treatment effectiveness for selected patients with high AP-1/Myc
expression and might help achieve widespread access to affordable and
more efficient HCC treatment.