β-catenin cancer-enhancing genomic regions axis is involved in the development of fibrolamellar hepatocellular carcinoma

Abstract

Fibrolamellar hepatocellular carcinoma (FLC) is a disease that occurs in children and young adults. The development of FLC is associated with creation of a fusion oncoprotein DNAJB1-PKAc kinase, which activates multiple cancer-associated pathways. The aim of this study was to examine the role of human genomic regions, called cancer-enhancing genomic regions or aggressive liver cancer domains (CEGRs/ALCDs), in the development of FLC. Previous studies revealed that CEGRs/ALCDs are located in multiple oncogenes and cancer-associated genes, regularly silenced in normal tissues. Using the regulatory element locus intersection (RELI) algorithm, we searched a large compendium of chromatin immunoprecipitation-sequencing (ChIP) data sets and found that CEGRs/ALCDs contain regulatory elements in several human cancers outside of pediatric hepatic neoplasms. The RELI algorithm further identified components of the β-catenin-TCF7L2/TCF4 pathway, which interacts with CEGRs/ALCDs in several human cancers. Particularly, the RELI algorithm found interactions of transcription factors and chromatin remodelers with many genes that are activated in patients with FLC. We found that these FLC-specific genes contain CEGRs/ALCDs, and that the driver of FLC, fusion oncoprotein DNAJB1-PKAc, phosphorylates β-catenin at Ser675, resulting in an increase of β-catenin-TCF7L2/TCF4 complexes. These complexes increase a large family of CEGR/ALCD-dependent collagens and oncogenes. The DNAJB1-PKAc-β-catenin-CEGR/ALCD pathway is preserved in lung metastasis. The inhibition of β-catenin in FLC organoids inhibited the expression of CEGRs/ALCDs-dependent collagens and oncogenes, preventing the formation of the organoid's structure. Conclusion: This study provides a rationale for the development of β-catenin-based therapy for patients with FLC.

FIGURE 1

Summary of regulatory element locus intersection (RELI) algorithm search for cancer‐enhancing genomic region or aggressive liver cancer domain (CEGR/ALCD) sequences in a large collection of chromatin immunoprecipitation–sequencing (ChIP‐seq) data sets. (A) Example of CEGR/ALCD from the nuclear erythroid 2 p45‐related factor 2 (NRF2) gene used for the search and RELI algorithm strategy. Colors show 100% homological regions. Bottom part shows the schematic locations of these sequences within each CEGR/ALCD. (B) Diagram summarizing previous publications. (C) Diagram showing a strategy for the RELI algorithm. Right image shows the list of human cancers with activated CEGRs/ALCDs. (D) List of proteins that pulled down CEGRs/ALCDs with high scores. These proteins are detected in several cancers, and pulled down more than five CEGRs/ALCDs. (E) The RELI algorithm–based hypothesis for the activation of CEGR/ALCD‐dependent oncogenes in human cancers. HBL, hepatoblastoma.

FIGURE 2

Experimental data that provide a rationale for the studies of the DNAJB1‐PKAc‐β‐catenin‐TCF4 axis as the main cause of fibrolamellar hepatocellular carcinoma (FLC). (A) Identity of genes detected by RELI algorithm and their locations on human chromosomes. (B) Identification of the fusion DNAJB1‐PKAc transcript in patients with FLC. (C) Western blot shows expression of DNAJB1‐PKAc, total levels of β‐catenin, and levels of phosphorylated forms of β‐catenin. Antibodies (Abs) to total β‐catenin, ph‐Ser675‐β‐catenin, and ph‐Ser33/37/Thr41‐β‐catenin were used. β‐actin served as a loading control. (D) Expression of β‐catenin messenger RNA (mRNA) in patients with FLC2 and FLC4 was determined by quantitative real‐time polymerase chain reaction (PCR). (E) Staining of background and tumor sections with Abs to total β‐catenin. (D) Hypothesis for the role of the DNAJB1‐PKAc‐ph‐S675‐β‐catenin‐TCF4‐CEGRs/ALCDs axis in FLC. HCC, hepatocellular carcinoma.

FIGURE 3

DNAJB1‐PKAc‐ph‐S675‐β‐catenin pathway is active in patients with FLC. (A) Sirius red staining and ki67 staining of background and tumor sections of patients with FLC. (B) Immunostaining of FLC livers with antibodies to PKAc. (C) High‐magnification images show PKAc‐positive cells with nuclear (red arrows) and cytoplasmic signals (black arrows). (D) Immunostaining of background and tumor sections of FLC livers with antibodies to ph‐S675‐β‐catenin. Internal images: Cells with positive nuclear staining for ph‐S675‐β‐catenin. (E) An example of ph‐S675‐β‐catenin staining of a patient's sample that had predominantly nuclear localization of the ph‐S675‐β‐catenin. Bottom: Nuclear localization of ph‐S675‐β‐catenin and mitotic figures observed in ph‐S675‐β‐catenin positive cells. (F) Western blotting and Co‐IP analyses of background (B) and tumor (T) sections of the patient with FLC. IgGs are signals of immunoglobulins G observed in Co‐IP studies. Two repeats of western blot and Co‐IP are shown.

FIGURE 4

DNAJB1‐PKAc‐ph‐S675‐β‐catenin pathway activates expression of CEGR/ALCD‐containing genes in patients with FLC. (A) Immunostaining of the same areas of the liver with antibodies to ph‐S675‐β‐catenin and PGAP1. Arrows show co‐localizations of staining. (B) Western blotting shows an increase of expression of post GPI attachment to proteins 1 (PGAP1), HACE1, and RUNDC1 in tumor sections of patients with FLC. (C) Western blotting shows expression of the components of ph‐S675‐β‐catenin‐TCF4‐p300 complexes. (D) Co‐IP studies. ph‐S675‐β‐catenin was immunoprecipitated, and PKAc, TCF4, p300, and ph‐S675‐β‐catenin were detected by western blot. € Pull‐down assay. Cytoplasmic and nuclear extracts from FLC samples were incubated with streptavidin‐linked TCF4 oligomer, and the interacting proteins were examined by western blot. β‐actin is a negative control. (F) ChIP assay with CEGRs/ALCDs from three oncogenes shown on the left.

FIGURE 5

Identification of CEGR/ALCD‐containing genes that are elevated in patients with FLC. (A) RNA‐sequencing (RNA‐seq) results for patients with FLC who have elevated DNAJB1‐PKAc‐β‐catenin‐CEGRs/ALCDs pathways. Red colors show mRNAs coding for components of the DNAJB1‐PKAc‐β‐catenin‐TCF4 axis. (B) Sequences of CEGRs/ALCDs in the COL4A1, SPARC, and VCAN genes. (C) Confirmation of RNA‐seq results by quantitative real‐time PCR. (D) Expression of up‐regulated proteins detected by western blotting. (E) ChIP assay performed with CEGRs/ALCDs for HDAC1, DNAJB1, and SPARC genes.

FIGURE 6

DNAJB1‐PKAc‐β‐catenin‐TCF4‐CEGRs/ALCDs pathway is preserved in lung metastasis of a patient with FLC. (A) Expression of the fusion DNAJB1‐PKAc transcript in liver tumor and lung metastasis. (B) Hematoxylin and eosin (H&E) staining, sirius red staining, and immunostaining of primary hepatic tumor (upper) and lung metastasis (bottom) of the patient with FLC with antibodies to PKAc and ph‐S675‐β‐catenin. The ×20 insert shows nuclear staining of cells with ph‐S675‐β‐catenin. Cells with nuclear (red arrows) and cytoplasmic (black arrows) staining are shown. (C) Examples of ph‐S675‐β‐catenin‐positive cells with mitotic figures are observed in lung metastasis of a patient with FLC. (D) Western blotting shows the expression of proteins of the β‐catenin‐TCF4‐p300 complexes in lung metastasis. Right: Co‐IP studies. (E) Western blotting shows the expression of CEGR/ALCD‐dependent genes in lung metastasis. (F) ChIP assay of the CEGRs/ALCDs from DNAJB1, HDAC1, and SPARC genes. (G) Hypothesis that is based on the studies of primary tumors and a lung metastasis of patients with FLC.

FIGURE 7

Inhibition of β‐catenin in FLC organoids reduces expression of FLC‐specific CEGR/ALCD‐containing genes and inhibits the development of FLC. (A) Western blot shows levels of DNAJB1‐PKAc in transfected cells. Bar graphs show luciferase activity of CEGR/ALCD‐luciferase constructs with CEGRs/ALCDs from p53, β‐catenin, and NRF2 genes in untreated cells, and in cells transfected with DNAJB1‐PKAc, untreated and treated with the inhibitor of β‐catenin PRI‐724 (5 μm). (B) Left: Treatment of FLC organoids with PRI‐724 (5 μm) for 24 h inhibits cyclin D1 and β‐catenin, but elevates p21 and Rb. Co‐IP study shows that PRI‐724 destroys β‐catenin‐TCF4‐p300 complexes in FLC organoids. Images on the right show levels of proteins, the genes of which contain CEGRs/ALCDs. (C) Treatment of FLC organoids with PRI‐724 (5 μm) for 96 h inhibits the proliferation of organoids and the formation of organoid structures. (D) List of top up‐regulated mRNAs that were determined by RNA‐seq in FLC organoids after treatments with PRI‐724 for 96 h (5 μm, n = 4/group). (E) List of genes reduced in FLC organoids with inhibited β‐catenin activity. (F) Western blot confirms changes of expression of proteins, the mRNAs of which were found by RNA‐seq.

FIGURE 8

The reduction of DNAJB1‐PKAc by inhibition of β‐catenin identified an auto‐regulatory loop that supports high levels of the fusion oncoprotein in patients with FLC. (A) Diagram showing fusion points for DNAJB1‐PKAc isoforms detected in FLC organoids. (B,C) Reduction of transcripts of DNAJB1‐PKAc isoforms in FLC organoids, treated with PRI‐724 (5 μm, n = 4/group; *adjusted p < 0.05). (D) Western blotting of proteins from FLC organoids with antibodies to the N‐Terminal part of DNAJB1 and the C‐terminal part of PKAc. (E) Hypothesis for auto‐regulatory loop. Bottom image shows an additional part of the loop, in which DNAJB1‐PKAc‐β‐catenin‐TCF4 increases levels of TCF4. A sequence of the CEGR/ALCD in the TCF4 gene is shown.