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.