Induction of hepatocellular carcinoma by in vivo gene targeting

Abstract

The distinct phenotypic and prognostic subclasses of human hepatocellular carcinoma (HCC) are difficult to reproduce in animal experiments. Here we have used in vivo gene targeting to insert an enhancer-promoter element at an imprinted chromosome 12 locus in mice, thereby converting ∼1 in 20,000 normal hepatocytes into a focus of HCC with a single genetic modification. A 300-kb chromosomal domain containing multiple mRNAs, snoRNAs, and microRNAs was activated surrounding the integration site. An identical domain was activated at the syntenic locus in a specific molecular subclass of spontaneous human HCCs with a similar histological phenotype, which was associated with partial loss of DNA methylation. These findings demonstrate the accuracy of in vivo gene targeting in modeling human cancer and suggest future applications in studying various tumors in diverse animal species. In addition, similar insertion events produced by randomly integrating vectors could be a concern for liver-directed human gene therapy.

Fig. 1.

Rian gene targeting induces liver tumors. (A) Map of chromosome 12 containing Rian and Mirg transcripts (exons in thick boxes) showing the target locus, AAV-Rian-CMV targeting vector, snoRNAs, microRNAs, Southern blot probe, and Ase I sites. The locations of sleeping beauty transposon and nonhomologous AAV vector insertions found previously in tumors are indicated. (B and C) The number of tumors and liver weights are shown at 6 mo (n = 4 per group), 10 mo (n = 4 per group), and experimental endpoints (11–18 mo; n ≥ 7 per group) for male and female mice (means ± SDs; *P < 0.05 by two-tailed t test). (D) Survival of injected and control mice over time (*P < 0.01 by Gehan-Breslow-Wilcoxon test). (E) Copy numbers of targeted alleles and vector genomes as determined by qPCR (means ± SEs). (F) Representative Southern blot of Ase I-digested genomic DNAs from seven tumors and adjacent normal tissues from vector-injected mice.

Fig. 2.

Appearance and histology of liver tumors. (A) Photographs of gross liver specimens from 10-mo-old mice. (B) H&E staining of livers with HCC nodules from vector-treated mice. (C) Staining of liver sections with specific antibodies for Pik3ca, Epcam, or BrdU. Serial sections stained with H&E are shown below. T, tumor nodules.

Fig. 3.

Small Afp⁺ foci transform into HCC. (A) Examples of Afp⁺ tumors detected by anti-Afp antibody staining shown below serial H&E sections. (B) Small foci of Afp⁺ hepatocytes within the normal liver tissue of mice injected with AAV-Rian-CMV. (C) The sizes of Afp⁺ foci are shown over time for male and female mice injected with AAV-Rian-CMV (means ± SEs). The average liver surface area examined was 228 ± 60 mm² per mouse (n = 4 per group). No Afp⁺ cells were detected in control mice.

Fig. 4.

Transcriptional effects of Rian gene targeting. (A) The expression levels of mRNA transcripts near the insertion site were determined quantitatively in tumors and compared with adjacent normal-appearing tissue by qRT-PCR (n = 3). Red and blue genes are imprinted RefSeq genes expressed from the maternal or paternal chromosomes respectively, with transparent red regions representing areas of non-RefSeq transcripts. (B) The expression levels of 6 snoRNAs and 42 microRNAs transcribed from the insertion site locus were determined from the same samples as in A by qRT-PCR and microarray analysis, respectively. The four microRNAs with binding sites in down-regulated genes (Table S5) are indicated by asterisks (miR-369–5p, miR-376, miR-134, and miR-758). (C and D) Gene ontology analysis of dysregulated genes (twofold or greater change, P < 0.05), in comparison with all genes present in the mouse genome.

Fig. 5.

Transcription profile of subclass C3 human HCCs. (A) The expression levels of mRNA transcripts near the insertion site were determined quantitatively in C3 HCC samples (n = 5) and compared with normal liver samples (n = 10) by microarray analysis. (B) The expression levels of two snoRNAs and 26 microRNAs transcribed from the insertion site locus were determined from the same samples as in A by qRT-PCR and microarray analysis, respectively. Human homologs of the microRNAs highlighted in Fig. 4B are marked with asterisks. (C) Copy number analysis as measured by SNP array. Each dot represents the mean copy number in a genomic region including MEG3, RTL1, MEG8 and the snoRNA-microRNA cluster on human chromosome 14 in 103 human HCC samples plotted against standardized theoretical quantiles. Dashed lines show the range of control values from nonmalignant cirrhotic samples. Orange symbols indicate 4 C3 HCC samples. (D) Methylation status of 11 CpG sites present in a CpG-rich region is shown for five C3 HCC samples and two normal liver specimens. The location of the CpG Island in the RTL1 gene and its predicted transcription start site (arrow) are indicated. CpG dinucleotides are represented as circles (black, methylated; white, unmethylated; gray, partial methylation). Missing circles were excluded because of aberrant base calls.