Enhanced proliferation and moderate inflammation in
c-Jun~Fra-2hep livers
Genome-wide transcription profiling by RNAseq was performed on 2- and
9-month liver samples. Unsupervised principal-component analysis (PCA)
clearly separated the samples along PC1 and PC2 according to genotype
and age, respectively (Figure 2A). Interestingly, while tumoral samples
also separated from non-tumoral (NT) along PC2, the two tumors isolated
from the same mouse appeared more distant from each-other than the two
tumors isolated from different mice, consistent with inter-tumoral
heterogeneity (Figure 2A). Gene set enrichment analysis (GSEA (30))
revealed enrichment in MSigDB Hallmarks gene sets related to cell cycle,
p53 pathway, cell death and hypoxia in the 3 mutant groups, when
compared to their respective control littermates (Suppl. Figure 2A).
CIBERSORTx (31) computational deconvolution at 2 months using a murine
hepatocyte matrix (32) indicated perturbed liver zonation, with
increased Zone 2 and undetectable Zone 3 hepatocytes (Suppl. Figure 2B),
which was confirmed by diffuse peri-central Glutamine synthetase IHC
positivity in mutants (Suppl. Figure 2C). The mean expression profile of
the 4 tumors relative to control livers was next compared by GSEA with a
collection of human and murine liver cancer signatures. A significant
correlation was observed with HCC gene signatures, in particular those
associated with poor outcome, such as Hoshida subclass S1 (33), Boyault
subclass G3 (34), Woo cancer recurrence (35), the hepatoblast subtype of
human HCC with prominent AP-1 (36) and paediatric hepatoblastoma with
upregulated Myc signalling (37) (Figure 2B). These gene signatures are
all characteristic of dedifferentiation, fetal liver–like gene
expression, high proliferation, and aggressiveness. There was also a
good correlation with murine liver cancer signatures (38), in particular
those arising in mice expressing a Myc transgene (Figure 2B). Increased
proliferation and altered cell cycle was confirmed by Ki67 and Cyclin D1
IHC (Figure 2C-D) as well as immunoblot and qRT-PCR for a panel of
cyclins and Cdks (Suppl. Figure 2D-E). Increased Cyclin A is consistent
with ccna2 (encoding Cyclin A2) being a direct target of the
c-Jun/Fra-2 dimer in cultured cells (25). Increased Ki67-positivity was
also observed in non-parenchymal, likely immune cells as early as 2
months (Figure 2D), along with increased interleukin 6 (il6 ) mRNA
(Suppl. Figure 2E). Therefore, the immune and inflammatory profile of
Jun~Fra-2hep livers was examined in
more detail. A moderate but consistent increase in immune cell-related
marker expression was observed by IHC (Figure 2E, Suppl. Figure 2F) and
qRT-PCR (Figure 2F, Suppl. Figure 2G). Furthermore, GSEA using human
MSigDB C8 liver cell gene sets (39) revealed that Kupffer cell
signatures were among the top enriched in mutant
Jun~Fra-2hep livers (Suppl. Figure
2H). Elevated myeloid cell abundance in mutant livers was confirmed by
CIBERSORTx deconvolution using a murine matrix (32), and TREM2-positive
macrophages, that are high in HCC and associate with poor prognosis (40)
were notably increased (Figure 2G).
We next evaluated signalling pathways that could connect inflammation
and proliferation. The relative phosphorylation of ERK, JNK and p38 was
not noticeably changed at 9 months, while PTEN, AKT and GSK3β
phosphorylation was increased to variable extents (Suppl. Figure 2I).
The MSigDB Hallmarks gene sets: Inflammatory response, TNF/NF-κB and
IL6/JAK/STAT3 were enriched in the
Jun~Fra-2hep mutant groups (Figure
2H). This is in line with increased relative STAT3 phosphorylation and
increased p-STAT3-positive cells at 2 and 9 months, although the
phosphorylation of the p65 NF-κB subunit was not changed (Figure 2I,
Suppl. Figure 2J-K). These results imply that hepatic
Jun~Fra-2 expression leads to cellular and molecular
characteristics of malignant transformation in a context of moderate
inflammation, even before visible tumors are detected.