c-Jun~Fra-2 binds a c-myc 3’ enhancer and increases c-Myc and Myc target gene expression
c-Myc is central to HCC pathogenesis and is connected to the IL6/JAK/Stat3 and PI3K/AKT/GSK3β pathways (42, 43). Consistent with the prominent enrichment in Myc-related murine and human liver cancer signatures (Figure 2B) and the increased IL6/JAK/Stat3 and PI3K/AKT/GSK3β pathway activities (Figure 2H-I, Suppl. Figure 2I-K), c-Myc protein expression was increased in c-Jun~Fra-2hep livers at 2 and 9 months (Figure 4A, Suppl. Figure 4A). c-myc mRNA was also increased at 2 months, but not in age-matched Jun~Fra-1hep or Frahep mice (Suppl. Figure 4B). foxm1 , an HCC-relevant protein often connected to Myc (44), was also increased (Suppl. Figure 4C), along with a panel of c-Myc target genes (Figure 4B).
A 3’ enhancer, 1.4 kb downstream of the MYC transcriptional stop, is bound and activated by JUN-containing dimers in human colorectal cancer cells, cooperatively with β-catenin/TCF4 (45). This Wnt-responsive enhancer (WRE) is conserved in the mouse, including the AP-1 consensus motif TGACTCA, and a similar motif was identified in the c-mycpromoter (Figure 4C). Chromatin immunoprecipitation using hepatic chromatin from c-Jun~Fra-2hep mice at 2 months and Fra-2 (Figure 4D) or Flag (Figure 4E-F) antibodies followed by quantitative PCR (ChIP-qPCR) revealed that c-Jun~Fra-2 efficiently bound the c-myc -WRE and the AP-1-responsive Dusp1 promoter used as a positive control, but not the c-myc promoter. The enrichment in WRE ChIP-qPCR fragments was negligible, when hepatic chromatin from Fra-2hep mice was employed (Figure 4F), consistent with unaltered c-mycexpression in these samples (Suppl. Figure 4B). Transient transfection experiments using the murine AML12 liver cell line revealed that c-Jun~Fra-2 expression increased endogenous c-mycmRNA along with the activity of a c-myc -WRE luciferase reporter, while Fra-2 had little to no effect (Figure 4G).
c-myc expression was next evaluated in experimental HCC models with Fra-2 or c-Jun deficiency. Hepatic c-myc and c-junexpression was unchanged upon injection of the chemical carcinogen diethylnitrosamine (DEN) to adult Fra-2Δli mice lacking Fra-2 in hepatocytes (Suppl. Figure 4D). Furthermore, DEN-induced tumorigenesis was similar between Fra-2Δliand Fra-2-proficient littermates, as were serum AFP and ALT (Suppl. Figure 4E, F) and c-myc and c-jun expression in isolated tumors (Suppl. Figure 4G). Mice lacking hepatic c-Jun are resistant to experimental HCC paradigms (13, 15, 17, 18) and a significant reduction in c-myc mRNA (Figure 4H) and protein (Figure 4I) expression are observed in c-JunΔli livers during HBV-driven tumorigenesis (18). These data indicate that Fra-2 is dispensable, while c-Jun is needed to modulate c-myc expression, at least in the context of DEN- and HBV-induced tumorigenesis, respectively.
We next explored the connection between JUN, FRA2 and MYC in human liver cancer. Datamining of genome-wide ChIPseq of HepG2 hepatoma cells (46) revealed JUN- and FRA2- ChIP peaks in a transcriptionally active genomic area consistent with the MYC WRE (Suppl. Figure 4H). Furthermore,MYC mRNA expression was abrogated in HepG2 cells upon CRISPR/cas9 deletion of the MYC WRE, while it was increased after transient expression of c-Jun~Fra-2 in the parental cell line (Suppl. Figure 4I) and decreased upon siRNA knock-down of JUN or JUNB (Suppl. Figure 4J). HCC RNAseq data from treatment-naïve patients (TCGA-LIHC) generated by The Cancer Genome Atlas (47) were next explored. MYC expression, reported as the average number of ‘Fragments Per Kilobase of exon per Million reads’ (FPKM), correlated with each of JUN and FRA2 independently (Suppl. Figure 4K). Cohorts with high (HH) or low (LL) JUN and FRA2 expression where next defined, corresponding to 37% and 27% of the samples, respectively (Suppl. Figure 4K, right panel). Strikingly, MYC, FOXM1 and Cyclin D1 (CCND1) expression was found higher (Suppl. Figure 4L) and overall survival lower (Suppl. Figure 4M) in the HH group. Taken together these data indicate that the modulation of c-myc expression by c-Jun/Fra-2 is likely also occurring in human hepatocytes and could be relevant to HCC progression.