The role of NFE2L2 mutations and the epigenetic regulator UHRF1 in hepatoblastoma

Hepatoblastoma is a malignant disease of the liver. It accounts for
about 1 % of all childhood cancers and is the most common malignant
liver tumor in infancy. Hepatoblastoma is assumed to arise from
immature liver progenitor cells by aberrant activation of genes
important in the embryonic development. Based on its early
manifestation it is generally assumed that hepatoblastoma displays
a relatively normal genomic background. Whole-exome sequencing
performed in our group identified hepatoblastoma as one of the
genetically simplest tumors ever described, with recurrent
mutations in beta-catenin (CTNNB1) and nuclear factor
(erythroid-derived 2)-like 2 (NFE2L2). Based on this finding we
performed targeted genotyping of a large cohort of primary
hepatoblastomas, hepatoblastoma cell lines and transitional liver
cell tumors and identified CTNNB1 and NFE2L2 to be mutated in 72.5
% and 9.8 % of cases, respectively. CTNNB1 is a key effector
molecule of canonical WNT signaling pathway, a pathway that is
essential in organogenesis and cellular processes such as cell
proliferation, differentiation, survival and apoptosis. However,
NFE2L2 is involved in the activation of the cellular antioxidant
response to combat the harmful effects such as xenobiotics and
oxidative stress. Interestingly, all NFE2L2 mutations were located
in or adjacent to the DLG and ETGE motifs of the NFE2L2 protein
that are needed to get recognized by the KEAP1/CUL3 complex for
proteasomal degradation. Functional analysis showed that cells
transfected with mutant NFE2L2 were insensitive to KEAP1-mediated
downregulation of NFE2L2 signaling and that depletion of the NFE2L2
via siRNA downregulates the NAD(P)H dehydrogenase (quinine) 1
(NQO1), a target gene of NFE2L2, and inhibits proliferation. In the
clinical setting, NQO1 overexpression in tumors was significantly
associated with metastasis, vascular invasion, the adverse
prognostic C2 gene signature as well as poor outcome. RNA
sequencing in our group identified the ubiquitin-like with PHD and
ring finger domains 1 (UHRF1), a protein known to preferentially
bind to hemi-methylated DNA, to be highly overexpressed in
hepatoblastoma tumors. UHRF1 is as a key regulator in the
epigenetic crosstalk, by controlling DNA methylation and histone
modification. Using immunoprecipitation, we were able to show that
UHRF1 binds in concert with DNA methyltransferase 1 (DNMT1) and
ubiquitin specific peptidase 7 (USP7) as a trimeric complex to
promoter regions of tumor suppressor genes (TSG) relevant in
hepatoblastoma, such as hedgehog interacting protein (HHIP),
insulin-like growth factor binding protein 3 (IGFBP3), and secreted
frizzled-related protein 1 (SFRP1). These genes are epigenetically
silenced in hepatoblastoma, as evidenced by heavy DNA methylation
and enrichment of the repressive H3K27me3 and H3K9me2 chromatin
mark. Interestingly, knockdown of UHRF1 expression via RNA
interference resulted in promoter demethylation, but no
reactivation of TSG gene expression. Additionally, no effect on
tumor cell proliferation was observed after UHRF1 knockdown.
Chromatin immunoprecipitation experiments revealed a decrease of
the repressive chromatin marks H3K27me3 and H3K9me2 after UHRF1
depletion, but neither a clear shift towards the active H3K4me2
chromatin mark nor enrichment of RNA Polymerase at the TSG loci was
observed. Statistical analyses revealed that a high expression of
UHRF1 was associated with advanced disease state and a worse
overall survival. Taken together our study demonstrates that
activation of WNT signaling in concert with activation of the
NFE2L2-KEAP1 pathway might be the driving force in the development
of liver cancers. Moreover, we defined aberrant NQO1 expression as
a marker for adverse course of disease and poor outcome. In
addition, we showed that an aberrant expression of the epigenetic
regulator UHRF1 and its excessive binding on promoter regions
results in methylation of TSGs. This may represent an important
mechanism in the initial phases of embryonal tumorigenesis.
However, UHRF1 depletion alone was not sufficient to re-induce TSG
expression. Therefore, UHRF1 might be more useful as a biomarker
for the prognosis of hepatoblastoma than a direct anti-cancer
target for hepatoblastoma therapy.