DLK1/DIO3 locus upregulation by a β-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis

Abstract

The CTNNB1 gene, encoding β-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained β-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (Apc^Δhep) or Ctnnb1-exon 3 (β-catenin^ΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/β-catenin complexes in an open conformation upon sustained β-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in β-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, Apc^Δhep HB and β-catenin^ΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during β-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.

Keywords: enhancer site; in vivo CRISPR-Cas9; non-coding RNAs; primary liver cancers; targeted therapies; transgenic mice; β-catenin.

Graphical abstract

Figure 1

The Dlk1/Dio3 locus is induced in mouse HCC- and HB-like tumors driven by β-catenin (A) Schematic representation of the DLK1/DIO3 locus. (B) In situ hybridization of Meg3 and miR-127 with staining of glutamine synthetase (GS) or active β-catenin in WT and Apc^Δhep livers and in Apc^Δhep and β-catenin^ΔExon3 HCC- or HB-like tumors. CV, central vein; PV, portal vein. (C–F) Expression of Rian, Mirg, and miR-127 with error bars representing SEM by RT-qPCR in Apc^Δhep tumors (TUM) compared to adjacent non-tumor (NT) tissue (C); in Apc^Δhep HCC and HB-like tumors (D); in β-catenin^ΔExon3 tumors (E); and in DEN tumors without β-catenin activation (F). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0001; ns, non-significant (Mann-Whitney).

Figure 2

β-Catenin binding at the DLK1-WRE site opens chromatin and exerts enhancer activity in Apc^Δhep hepatocytes (A) ChIP-seq targeting TCF-4 in WT, Apc^Δhep, and β-cat^Δhep hepatocytes and ATAC-seq data in WT and Apc^Δhep hepatocytes. TCF-4 binding site is framed in the blue box (DLK1-WRE) and sites common with 3C in pink. (B, D, F, and G) ChIP-qPCR analysis at the DLK1-WRE site for TCF-4, H3K4me1, and H3K27ac relative to isotype control in Apc^Δhep hepatocytes with error bars representing SEM compared to WT (B and D) and compared to Apc^Δhep-DLK1/DIO3^ΔWRE hepatocytes (F and G). (C and H) ATAC-qPCR analysis at the DLK1-WRE site compared to WT (C) and to Apc^Δhep-DLK1/DIO3^ΔWRE hepatocytes (H). (E) Relative contact frequencies in arbitrary units (a.u.) between the DLK1-WRE site (blue vertical bar) and 19 genomic sites (small vertical black bars on the map below) measured in 3C experiments performed on WT, Apc^Δhep-Rosa26, and Apc^Δhep-DLK1/DIO3^ΔWRE liver nuclei with error bars representing SEM of six, five, and three biological replicates, respectively. Regions of interest (highlighted in pink) are numbered from 1 to 6. The lower panel illustrates the different chromatin loops distributed into six interaction zones: the darker the pink, the stronger the interaction. Figure made with BioRender. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0001; ns, non-significant (Mann-Whitney).

Figure 3

The DLK1/DIO3 locus is induced in human HB in correlation with CTNNB1 mutations and DLK1-WRE opening (A) Pseudo bulk snATAC-seq aggregated with Cellranger-atac of three human HBs (T) and their adjacent NT tissue (N) at the DLK1-WRE site; the pink panel represents ChIP-seq data targeting TCF-4 in the human HB HepG2 cell line (Gsm782122). (B) RNA-seq expression data for the entire DLK1/DIO3 locus in HB normalized to their adjacent NT tissue (N = 22, cohort 1). A white square in the β-catenin lane indicates HB with intact CTNNB1, a yellow square HB with point mutation in CTNNB1, an orange square HB with CTNNB1 exon 3 deletion. (C) Correlation between RIAN, MIRG, DIO3OS, DIO3, MEG3, miR-411, and miR-136 expressions in cohort 1. (D) RIAN, RTL-1, and MEG3 expression determined by RT-qPCR in primary (N = 83) and recurrent HB (N = 17) versus non-tumor liver (NTL) (N = 100, cohort 2). (E) Expression of DIO3OS, MEG3, and RIAN determined by RT-qPCR in the different subgroups of HB: embryonal (green), fetal hepatocytic 1 (pink), fetal hepatocytic 2 (yellow), and mesenchymal (purple). (F) Expression of RIAN, DIO3OS, and MEG3 determined by RT-qPCR in primary HB with CTNNB1 mutations (CTNNB1^mut, N = 76) or with intact CTNNB1 (CTNNB1^WT, N = 7). ∗p < 0.05, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001; ns, non-significant (Kruskal-Wallis),with error bars representing SEM.

Figure 4

DLK1-WRE editing affects Apc^Δhep hepatocyte proliferation through inhibition of mitosis and cytokinesis regulators (A) Percentage of Ki-67+ hepatocytes in WT, Apc^Δhep-Rosa26, and Apc^Δhep-DLK1/DIO3^ΔWRE livers. (B) Percentage of liver to body weight ratio in WT, Apc^Δhep-Rosa26, and Apc^Δhep-DLK1/DIO3^ΔWRE livers. (C) Expression of Top2a, Kif20b, Nuf2, and Nusap1 in Apc^Δhep-DLK1/DIO3^ΔWRE hepatocytes relative to Apc^Δhep-Rosa26 hepatocytes. (D–F) RNA-seq analysis on Apc^Δhep−DLK1/DIO3^ΔWRE and Apc^Δhep-Rosa26 hepatocytes. (D) The histograms summarize ratio obtained with gene set enrichment analysis (GSEA) between the number of genes in the intersection of the query set with a set from MSigDB (k/K), with p value and FDR q-values for each item. (E) Schematic representation of the most significantly deregulated RNAs. (F) Main hub obtained by STRING analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.001; ns, non-significant (Mann-Whitney), with error bars representing SEM.

Figure 5

DLK1-WRE editing impairs FoxM1 binding at Ccna2, Kif20a, and Cdc2 promoters (A) RT-qPCR analysis of Foxm1 expression in Apc^Δhep-Rosa26 and Apc^Δhep-DLK1/DIO3^ΔWRE hepatocytes compared to WT hepatocytes. (B) Number of Foxm1+ nuclei in IHC. (C) Representative images of ChIP-PCR targeting FoxM1 at Ccna2, Kif20a, Cdc2, and Cenpf promoters compared to isotype control in Apc^Δhep-Rosa26 and Apc^Δhep-DLK1/DIO3^ΔWRE hepatocytes and inputs (#1 and 2 for Apc^Δhep-Rosa26 hepatocytes, #3 and 4 for Apc^Δhep-DLK1/DIO3^ΔWREhepatocytes); the lower panel represents the PCR band quantification with ImageJ of all ChIP experiments against FoxM1 relative to isotype control. For the cdc2 promoter in Apc^Δhep-DLK1/DIO3^ΔWRE hepatocytes, the cropped images are for two different mice analyzed on two gels with the same conditions of exposure. (D) Quantification of Meg3 RNA co-immunoprecipitated with FoxM1 in RIP-qPCR in Apc^Δhep-DLK1/DIO3^ΔWRE (n = 4) compared to Apc^Δhep-Rosa26 hepatocytes (n = 2); data are represented as the relative binding compared to 18S. Figure made with BioRender. ∗p < 0.05, ∗∗p < 0.01; ns, non-significant (Kruskal-Wallis or Mann-Whitney), with error bars representing SEM.

Figure 6

DLK1-WRE site editing slows tumor growth of Apc^Δhep HB through decreased expression of mitotic entry regulators (A) Examples of GS staining of Apc^Δhep HB showing a heterogeneous staining with many stromal cells; HB cells losing several metabolic features of mature hepatocytes express low level of GS compared to Apc^Δhep HCC cells. (B) Analysis of tumor editing by PCR band quantification with ImageJ in Apc^Δhep HB. The upper panel is a representative image obtained from NT tissue and tumors (T). (C) Progression of cumulative tumor areas in Apc^Δhep-DLK1/DIO3^ΔWRE HB and Apc^Δhep-DLK1/DIO3^WT HB compared to Apc^Δhep-Rosa26 HB with cumulative area at sacrifice indicated in the right panel. (D) Representative images of Ki-67 staining on Apc^Δhep HB with high or low proliferation rate (left panel) and quantification of Ki-67+ hepatocytes in percentage for all tumors (right panel). (E) RT-qPCR analysis of Rian and Mirg in Apc^Δhep HB compared to their NT tissues. (F) RT-qPCR analysis of Mki67, Ccna2, Nuf2, Top2a, Axin2, Kif20b, Nusap1, Cenpf, and Ckap2 relative to their NT tissues. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001; ns, non-significant (Kruskal-Wallis), with error bars representing SEM.

Figure 7

DLK1-WRE site editing impairs the pro-tumorigenic capacities of murine hepatoma Hepa1-6 cells mutated for Ctnnb1 (A–D) Analysis of DLK1/DIO3^ΔWRE Hepa1-6 clones versus Rosa26 control clones. Proliferation rate at 48 h (A); percentage of cells in G2/M phase determined by flow cytometry (B); cyclin B1 and A2 protein level determined by western blot (C); representative quantification of Meg3 RNA co-immunoprecipitated with FoxM1 in RIP-qPCR (n = 2) with data represented as the relative binding compared to 18S (D). (E–K) Analysis of DLK1/DIO3^ΔWRE Hepa1-6 tumors versus Rosa26 tumors. Tumor volumes measured every 2 days (E) and tumor weights at sacrifice (F); percentage of Ki67+ tumor cells (G); percentage of tumor cells with cleaved caspase-3 (H); Fadd level determined by RT-qPCR (I); FADD protein level determined by western blot (J); Glul expression determined by RT-qPCR (K). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0001 (Mann-Whitney),with error bars representing SEM.

Figure 8

DLK1-WRE site editing impairs the pro-tumorigenic capacities of human Huh6 HB cells mutated for CTNNB1 (A–C) Analysis of DLK1/DIO3^ΔWRE Huh6 clones versus non-edited clones. Proliferation rate at 48 h (A); percentage of cells in G2/M phase determined by flow cytometry (B); number of spheroids (C). (D–L) Analysis of DLK1/DIO3^ΔWRE Huh6 tumors versus non-edited tumors. Tumor volumes measured every 2 days (D); tumor weights at sacrifice (E); percentage of Ki67+ tumor cells (F); MKI67 level determined by RT-qPCR (G); representative FOXM1 binding at CCNA2, KIF20A, and CDC2 promoters normalized to isotype control in ChIP-qPCR experiments (n = 2) (H); percentage of tumor cells with cleaved caspase-3 (I); FADD mRNA level determined by RT-qPCR (J); FADD protein level determined by western blot (K); GLUL expression determined by RT-qPCR (L). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0001 (Mann-Whitney)with error bars representing SEM