c-Jun~Fra2hep tumors are reversible, but addicted to c-myc expression
We next investigated whether c-Jun~Fra-2 is necessary to maintain the tumor phenotype by utilizing the “tetracycline switch” in c-Jun~Fra-2hep mice. c-Jun~Fra-2 was induced for 9 months, and the mice subsequently put back on Dox to halt c-Jun~Fra-2 expression and sacrificed 6 months later (Figure 5A). At this OFF endpoint, approximately 2/3 of the c-Jun~Fra-2hep mice, hereafter termed “reverted”, had no visible liver nodule at necropsy, while the rest of the c-Jun~Fra-2hep mice, hereafter “escapers”, presented at least one visible surface nodule (Figure 5B). Liver to body weight ratio (Suppl. Figure 5A) and serum AFP (Figure 5C) were comparable to controls in the reverted mice, while half (liver/body) to most (AFP) escapers had higher values and were similar to c-Jun~Fra-2hep mice sacrificed after 9 months of c-Jun~Fra-2 expression (ON). Serum ALT at end point was more heterogeneous, but escapers were still in the higher ranges (Suppl. Figure 5A).
Six c-Jun~Fra-2hep mice, hereafter termed mutant-1 to -6, were next followed longitudinally by liver ultrasonography (US) and serum monitoring along the reversion protocol. These mice had 1 to 3 tumors of variable size and roughly similar AFP, ALT, and ALP values (Suppl. Table 1A). While all 6 c-Jun~Fra-2hep mice displayed a sharp drop in serum AFP, approaching control levels after 8 weeks, AFP increased again in mutant-5 and even more in mutant-6 (Suppl. Figure 5B). High ALT concentrations were also measured at end point in these 2 mice, whereas ALP was comparable to control values in all 6 c-Jun~Fra-2hep mice (Suppl. Table 1A). US monitoring revealed that the fate of the individual tumors was heterogeneous and rather independent of their initial size or mouse of origin (Figure 5D, Suppl. Table 1A). Some tumors regressed to very small (T3 in mutant-4 and T1 in mutant-6) or below detection limit (T1 and T2 in mutant-4, all tumors in mutant-1 and mutant -2), while other tumors initially regressed, but resumed growing after a variable period (T1 in mutant-5 and T2 in mutant-6). We also observed the emergence of new tumors, with different sizes, latencies and growth kinetics, such as T3* in mutants 5 and 6 (Figure 5D, Suppl. Table 1A). Although we cannot rule out that these tumors were overlooked at start due to US limitations, it remains striking that the timing of the AFP “rebound” in mutants 5 and 6 roughly corresponds to the regrowth of pre-existing tumors and/or detection of new tumors in these mice (Figure 5D, Suppl. Figure 5B).
Liver tumors were next dissected from a group of escapers, together with non-tumoral (NT) areas and compared to (tumor-free) livers from reverted and control littermates, as well as non-tumoral and tumoral areas from mice sacrificed after 9 months of c-Jun~Fra-2 expression (ON). qRT-PCR for fra-2 (Figure 5E) andc-Jun~Fra-2 (Suppl. Figure 5C) confirmed that c-Jun~Fra-2 was barely detectable in the samples collected at the OFF endpoint. However, while c-myc mRNA was decreased to control levels in reverted livers and in escapers’ NT areas, escaping tumors had high c-myc expression (Figure 5F) and detectable c-Myc-positive cells by IHC (Suppl. Figure 5D). qRT-PCR analyses revealed high expression of oncofetal, cancer cell stemness, HCC and replicative senescence markers in escaping tumors (Figure 5G), as well as foxm1 , p21 and a panel of c-Myc target genes (Suppl. Figure 5E), while the corresponding NT areas displayed an expression profile more similar to controls (Figure 5G, Suppl. Figure 5E). These data are consistent with c-Myc being an essential molecular determinant of tumor formation and maintenance in c-Jun~Fra-2hep mice. Interestingly, some of the escaping tumors displayed increased Fos mRNA (Suppl. Figure 5F) and protein (Suppl. Figure 5D). As c-myc and c-Myc target gene expression is increased in the pre-neoplastic livers of Foshep mice (Suppl. Figure 5G), Fos-containing AP-1 dimers likely substitute for c-Jun~Fra-2 to maintainc-myc expression, at least in a subset of escaping tumors.