Results
Spontaneous liver tumors in c-Jun~Fra-2hep mice
Jun~Fra-2hep mice had a shorter lifespan compared to controls, with a median survival of 45 weeks after switching on the transgene (Suppl. Figure 1A). Mutant and control littermates were sacrificed at different time points after transgene induction, which was always started at weaning by Doxycycline (Dox) removal. All time points indicated hereafter are post-transgene induction. Macroscopically visible liver tumors were observed at 9 months (Figure 1A), while increased liver to body weight ratio was already apparent at 2 months (Suppl. Figure 1B). Nodule number and size was variable (Suppl. Figure 1C), but the phenotype was highly penetrant, with almost 90% of Jun~Fra-2hep mice having at least one macroscopically visible tumor nodule at 9 months (Suppl. Figure 1D). Notably, Fra-1hep, Fra-2hep and Jun~Fra-1hep mice expressing Fra-1/2 monomers (23) or Jun~Fra-1 dimers, generated with a similar strategy (see methods and Suppl. Figure 7) and kept up to 15 months off Dox never or rarely developed liver tumors (Suppl. Figure 1D). Histologically, hyperplastic nodules, adenoma and HCC were identified in Jun~Fra-2hep liver sections (Figure 1A), while the HCC biomarker (26) ‘Protein-induced-by-vitamin-K-absence-or-antagonist-II’ (PIVKA), was increased in Jun~Fra-2hep sera at 9 months (Figure 1B). Alpha-fetoprotein (AFP), a more commonly used HCC biomarker, was elevated in the serum of Jun~Fra-2hep mice as early as 1 month (Figure 1C), but not in aged Fra-1hep, Fra-2hep of Jun~Fra-1hep mice (Suppl. Figure 1E). AFP was also detected by IHC in Jun~Fra-2hep liver tumors (Figure 1D). IHC-positivity for ‘minichromosome-maintenance-complex-component-2’ (Mcm2) and Sox9, which have been associated with HCC development and Sorafenib resistance (27-29), was also observed in the tumors (Figure 1D). Hypoalbuminemia (Suppl. Figure 1F), increased alanine (ALT) and aspartate (AST) aminotransferases as well as alkaline phosphatase (ALP) (Suppl. Figure 1G) were also observed in Jun~Fra-2hep mice, consistent with early onset liver dysfunction and damage.
Macroscopically visible liver tumors were dissected from Jun~Fra-2hep mice at 9 months, together with small liver pieces from areas that appeared macroscopically tumor-free, hereafter termed ‘non-tumoral’ (NT), and compared to livers of control littermates by RNA and protein analyses. Quantitative reverse transcription-PCR (qRT-PCR) revealed increased mRNA expression of oncofetal (h19 , nope , dlk1,bex1 ), cancer cell stemness (cd133, cd44, sox9 ), HCC (mcm2, gp73, ly6d) and replicative senescence (p16) markers in Jun~Fra-2hep tumors and non-tumoral (NT) areas (Figure 1E). Increased Gp73 and Bex was confirmed by immunoblotting (Figure 1F). Activation of endoplasmic reticulum (ER) stress with increased PERK and eIF2a phosphorylation and Bip protein expression was also evident in Jun~Fra-2hep tumoral and non-tumoral extracts compared to control livers (Suppl. Figure 1H). Increased p53, p21 and S139-phosphorylation of histone H2AX (γH2AX), a surrogate marker of DNA damage, as well as decreased p19 were consistent with aberrant cell cycle and replicative stress (Figure 1G, Suppl. Figure 1I). Overall, most of the molecular changes observed in the tumors were also seen in the non-tumoral areas indicating that these areas are likely pre-neoplastic. Consistently, p21-and γH2AX-positive hepatocytes and increased p21, p16 and p53 mRNA were already apparent in the livers of Jun~Fra-2hep mice at 2 months (Figure 1H, Suppl. Figure 1I-J).