Accelerated liver regeneration and hepatocarcinogenesis in mice overexpressing serine-45 mutant beta-catenin

Abstract

The Wnt/beta-catenin pathway is implicated in the pathogenesis of hepatocellular cancer (HCC). We developed a transgenic mouse (TG) in the FVB strain that overexpresses Ser45-mutated-beta-catenin in hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated beta-catenin at hepatocyte membrane with no increase in the Wnt pathway targets cyclin-D1 or glutamine synthetase. However, TG hepatocytes upon culture exhibit a 2-fold increase in thymidine incorporation at day 5 (D5) when compared to hepatocytes from wildtype FVB mice (WT). When subjected to partial hepatectomy (PH), dramatic increases in the number of hepatocytes in S-phase are evident in TG at 40 and WT at 72 hours. Coincident with the earlier onset of proliferation, we observed nuclear translocation of beta-catenin along with an increase in total and nuclear cyclin-D1 protein at 40 hours in TG livers. To test if stimulation of beta-catenin induces regeneration, we used hydrodynamic delivery of Wnt-1 naked DNA to control mice, which prompted an increase in Wnt-1, beta-catenin, and known targets, glutamine synthetase (GS) and cyclin-D1, along with a concomitant increase in cell proliferation. beta-Catenin-overexpressing TG mice, when followed up to 12 months, showed no signs of spontaneous tumorigenesis. However, intraperitoneal delivery of diethylnitrosamine (DEN), a known carcinogen, induced HCC at 6 months in TG mice only. Tumors in TG livers showed up-regulation of beta-catenin, cyclin-D1, and unique genetic aberrations, whereas other canonical targets were unremarkable.

Conclusion: beta-Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, beta-catenin overexpression makes TG mice susceptible to DEN-induced HCC.

Figure 1. Creation and characterization of TG mice. (*p<0.05)

(A) Topflash reporter assay showing transcriptional response in HEK293 cells following transfection with various β-catenin mutant expression vectors. (B) Full length human Ctnnb1 containing a point mutation (S45D) was inserted downstream of an albumin promoter/enhancer and injected into FVB mouse eggs to create transgenic mice. Genomic DNA was subjected to PCR using indicated primers (X and Y) whose position is indicated. Representative PCR show the presence of transgene in TG mice and not WT mice. (C) Liver weight in TG mice and age-matched WT (n≥3) (upper panel). Liver weight to body weight ratio (LW/BW) in TG and age-matched WT (n≥3) (lower panel). (D) Increase in the number of Ki-67 positive hepatocytes in TG liver at 1 month compared to WT. (E) WB showing expression levels of total cyclin-D1 and GS in TG and WT liver at 1 and 2 months. An increase in cyclin-D1 is observed in TG at 1 month. Actin was used as a loading control. IP studies from three representative livers shows enhanced association of β-catenin and E-cadherin in 6 months old TG livers as compared to WT. (F) IHC for β-catenin reveals excess membranous localization in TG.

Figure 2. TG hepatocytes have a growth advantage over WT cells in culture. (*p<0.05)

(A) WT and TG hepatocytes in culture are morphologically distinct at 120H. (B) Increased [³H] thymidine uptake by TG hepatocytes as compared to WT cells. The counts were normalized to values at 40H after culture for each group and presented as fold change. (C) While numbers of PCNA-positive hepatocytes remained constant between 40H and 120H in WT hepatocyte culture, a net and significant gain in numbers of PCNA-positive cells was evident between 40H and 120H in the TG hepatocytes. (D) A significant increase in cell viability as determined by MTT assay was observed in TG versus WT hepatocytes at 120 hours after culture.

Figure 3. Accelerated liver regeneration in TG mice after PH. (*p<0.05)

(A) At 40H, WT livers are quiescent; however, TG livers display several cells in mitosis (arrow). Both WT and TG livers have several mitotic figures (arrow) at 72H. (B) Quantification of the number of PCNA-positive cells in representative 10× fields (n=5) from three TG and WT livers (p<0.001). (C) PCNA staining at 20H is low and approximately the same in both WT and TG samples; however, at 40H TG livers show increased PCNA-positive hepatocytes in S-phase, while WT livers are PCNA-negative. WT livers show several PCNA-positive hepatocytes at 72H post-PH. (D) Increase in Cyclin-D1 in TG livers at 40H after PH is seen in whole cell extracts while levels of GS and c-myc remain unchanged. Actin is a loading control. (E) Nuclear extracts from regenerating livers show an increase in both nuclear β-catenin and Cyclin-D1 that begins at 40H in TG and 72H in WT livers. Lamin B1 verifies equal loading. (F) After PH, at 40H, a clear increase in cytoplasmic (arrow) and nuclear (arrowhead) staining of β-catenin is observed in the TG and not in WT livers.

Figure 4. Hydrodynamic delivery of Wnt-1 plasmid and not pcDNA3 through tail vein induces Wnt/β-catenin activation in the liver. (*p<0.05)

(A) WB showing increased protein levels of Wnt-1 and GS in the Wnt-1 injected group vs. controls at 30H after PH (B) WB shows increased nuclear β-catenin in Wnt-1 injected livers vs. pcDNA3-injected controls at 30H after PH. (C) GS and Cyclin-D1 are increased in Wnt-1 injected animals at 30H after PH, as shown by IHC. A concomitant increase in PCNA staining is evident in the livers of Wnt-1 injected animals vs. control at 30H after PH. (D) Quantification of the number of PCNA-positive cells in five representative 10× fields from Wnt-1 or pcDNA3-injected livers (n=3, p<0.05).

Figure 5. Activation of Wnt pathway in TG tumors at 6 months after exposure to DEN. (All images at 100×, unless indicated)

(A) H&E staining shows tumor formation in TG and not WT at 6 months after exposure to DEN. Consecutive sections show IHC for β-catenin, seen localizing to cytoplasm and nuclei in TG tumors while mostly membranous localization is evident in WT. (B) IHC for cyclin-D1, GS and PCNA in consecutive sections from TG and WT livers at 6-months after DEN-exposure. Increased cyclin-D1 is evident in TG tumor, while GS shows typical peri-central localization as compared to WT. Increase PCNA-positive cells are evident in TG tumors only.

Figure 6. Activation of Wnt pathway in TG tumors at 9 months after exposure to DEN. (All images at 100×, unless indicated)

(A) H&E staining shows tumor formation in both WT and TG mouse liver 9 months after DEN exposure. Consecutive sections show IHC for β-catenin, which localizes predominantly to membrane in WT tumors and in addition in cytoplasm and nuclei in TG tumors. (B) IHC on consecutive sections show greater numbers of cyclin-D1 and PCNA-positive cells in TG tumors as compared to WT, while GS localization continued to be pericentral and unaltered in tumors. Inset for cyclin-D1 is at 400× magnification. (C)IHC for β-catenin and cyclin-D1 show activation of Wnt pathway in a minority of WT tumors as shown in a representative group. Consecutive sections show histology by H&E and nuclear/cytoplasmic β-catenin and increased numbers of cyclin-D1 and PCNA-positive cells by IHC. These tumors were negative for GS, which showed peri-central staining. Inset for cyclin-D1 is at 400× magnification.