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.