Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas

Abstract

Although inappropriate activation of the Wnt/beta-catenin pathway has been implicated in the development of hepatocellular carcinoma (HCC), the role of this signaling in liver carcinogenesis remains unclear. To investigate this issue, we constructed a mutant mouse strain, Apc(lox/lox), in which exon 14 of the tumor-suppressor gene adenomatous polyposis coli (Apc) is flanked by loxP sequences. i.v. injection of adenovirus encoding Cre recombinase (AdCre) at high multiplicity [10(9) plaque-forming units (pfu) per mouse] inactivated the Apc gene in the liver and resulted in marked hepatomegaly, hepatocyte hyperplasia, and rapid mortality. beta-Catenin signaling activation was demonstrated by nuclear and cytoplasmic accumulation of beta-catenin in the hepatocytes and by the induction of beta-catenin target genes (glutamine synthetase, glutamate transporter 1, ornithine aminotransferase, and leukocyte cell-derived chemotaxin 2) in the liver. To test a long-term oncogenic effect, we inoculated mice with lower doses of AdCre (0.5 x 10(9) pfu per mouse), compatible with both survival and persistence of beta-catenin-activated cells. In these conditions, 67% of mice developed HCC. beta-Catenin signaling was strongly activated in these Apc-inactivated HCCs. The HCCs were well, moderately, or poorly differentiated. Indeed, their histological and molecular features mimicked human HCC. Thus, deletion of Apc in the liver provides a valuable model of human HCC, and, in this model, activation of the Wnt/beta-catenin pathway by invalidation of Apc is required for liver tumorigenesis.

Fig. 1.

Activation of β-catenin signaling, hepatomegaly, and altered survival in Apc-inactivated mice infected with AdCre at high multiplicity. (A) Various alleles of Apc in floxed mice. Restriction sites: E, EcoRI; B, BamHI; X, XbaI; EV, EcoRV. Exons 11–15 are represented by black bars; primers and the lengths of the PCR fragments generated are indicated by arrows. (B) Cre delivery strategy. (C) Survival curve for Apc^lox/lox and Apc^+/lox mice after injection of various doses of AdCre. (D) Liver as a percentage of body weight and proliferation quantified by Ki67 scores in Apc^lox/lox compared with Apc^+/lox Cre-infected livers. *, P < 0.001; **, P = 0.0028. (E) PCR of DNA extracted from livers of Apc floxed mice showing the accumulation of the Apc^Δex14 band in AdCre-treated livers compared with noninjected (NI) mouse livers. (F) β-Catenin immunostaining of membranes in Apc^+/lox injected mice (Apc^+/–). (G) Cytosolic and nuclear accumulation of β-catenin staining in injected, Apc^lox/lox mice (Apc^–/–). (Scale bars in F and G:20 μm.)

Fig. 2.

Dose-dependent Apc inactivation and cell-autonomous induction of liver-specific β-catenin target genes. (A) β-Catenin and GS immunostaining of Apc^+/– and Apc^–/– mice injected with various doses of AdCre. (B and C) Coimmunostaining of β-catenin and its liver-specific targets (GS, GLT1, and LECT2, in blue) in perivenous (B) and nonperivenous (C) areas. β-Catenin stains red-brown at the membrane of every hepatocyte and accumulates in the cytosol and nuclei of Apc^–/– hepatocytes. CV, centrolobular vein; PS, portal space. (Scale bars: A, 100 μm; B and C, 20 μm.) (D) Percentage of hepatocytes that are GS⁺ in nonperivenous areas of Apc^–/– mice, according to dose of AdCre. Filled ovals represent mice with a hepatomegaly (liver > 8.5% of body weight), and open ovals represent mice without hepatomegaly.

Fig. 3.

Hepatocarcinogenesis from Apc^–/– hepatocytes. (A and B) HCC1 and HCC2 developed 8 and 9 months, respectively, after AdCre infection. (C) PCR analysis from tumoral DNA shows the loss of the Apc^lox allele and the gain of the Apc^Δex14 allele. (D–F) MD HCCs. (G–I) PD HCCs. Hematoxylin/eosin staining shows atypical hepatocytes organized in a trabecular (D) or pseudoglandular (G) pattern; β-catenin mislocalizes in the nucleus (E and H), and GS is overexpressed (F and I). Nontumoral (NT) and tumoral (T) tissues are delineated by a dashed line. (J and K) GS immunostaining in macronodular HCCs (J) and in a micronodular preneoplastic lesion (K), indicated by arrows. (Scale bars: A and B, 1 cm; D–I, 50 μm; J and K, 1 mm.)

Fig. 4.

Comparative expressions of β-catenin-dependent target genes in Apc^–/– hyperplastic livers and in β-catenin-activated HCC. (A) Northern blot analysis of livers from adult (3–5 months old) Apc floxed mice, not injected (0) or on D7 after injection of 10⁹ pfu of AdCre (10⁹). Apc^–/– and PK/c-Myc tumoral (T) or nontumoral (NT) livers are also shown. HCC1 and HCC2 are the WD and MD HCCs shown in Fig. 3 A and B, respectively. Note for c-Myc the presence of two bands in PK/c-Myc livers: The lower band is the c-Myc transgene (tg) mRNA, and the upper band is the endogenous c-Myc mRNA. OAT, ornithine aminotransferase; 28S and 18S are the rRNAs. (B) Real-time quantitative RT-PCR analysis of cyclin D1 (CycD1) and c-Myc mRNAs extracted from D7 infected livers, HCC1 and HCC2. For analyses at D7, three 1-month-old and four 5-month-old animals were analyzed in each group. No significant differences were observed, except those marked by an asterisk, where P =0.016.

Fig. 5.

p53 immunostaining of Apc^–/– HCC. (A) p53 accumulation in a hyperplastic liver of large T antigen-expressing ATIII-SV40 transgenic mice (37), used as a positive control for nuclear staining. (B) No p53 staining was seen in an Apc^–/– MD HCC. (C) Intense p53 staining was seen in the nuclei of tumoral cells from an Apc^–/– PD HCC. (Scale bars: 50 μm; 20 μmin Insets.)