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Armengol et al. Hepatoma Res 2021;7:50 https://dx.doi.org/10.20517/2394-5079.2021.19 Page 7 of 12

reduce toxic side effects of current treatments.

The neurokinin-1 receptor (NK1R) has been described as an integral part of cancer cells that can be targeted
by its antagonist aprepitant, a clinically approved drug used for chemotherapy-induced nausea and
[56]
vomiting . In high doses, aprepitant has been reported to suppress growth of HB cells propagated on
plastic as well as in vivo . As high expression of NK1R, especially of the truncated splice variant, can be
[57]
[57]
found in almost all HB cases , NK1R may serve as a novel therapeutic target in HB that warrants further
exploration.

Genetic permutation analysis of transcriptomic data revealed breast cancer 1 (BRCA1) and Fanconi anemia
(FA) complex genes (FANCI and FANCD2) to be upregulated in the HUH6 and HepG2 models, which
belong to the C2A group of the 4-gene signature . Although well-known FA/BRCA pathway inhibitors
[32]
such as GW7647, ML323, pimozide, and MLN4924 had very high effect concentrations in viability assays,
the proteasome inhibitor bortezomib was effective in the low nanomolar range, rendering it compatible for
[32]
clinical applications . Consequently, testing bortezomib in the preclinical HUH6 xenograft model
significantly reduced tumor volume.

Another gene with a significantly high expression in C2 tumors of the 16-gene signature is MYCN, which
maps to 2p24.1, a chromosomal region known to be frequently duplicated in HB . Eberherr et al. found
[58]
[11]
that MYCN is generally upregulated in pediatric liver tumors, and that treating HB and HCC cell lines with
the known MYCN inhibitors MLN8237 and JQ1 induced dose-dependent growth arrest by trapping cells in
the cell cycle.

Recent transcriptomic data reveal choline kinase alpha (CHKA), a key enzyme of the biosynthesis of
phosphatidylcholine via the CDP/choline pathway, to be the most widely overexpressed coding gene in
high-risk and intermediate-risk tumors . Treatment of the “classical” models HUH6 and HepG2 with the
[27]
CHKA inhibitors MN58b and TCD-717 showed a dose-dependent reduction of cell viability and colony
formation, and knockdown of CHKA expression in HUH6 cells phenocopied these effects .
[27]

Integrative analysis of transcriptomic and methylomic data of metastasized and non-metastasized HB
samples identified the Sp8 transcription factor (SP8) as a highly upregulated gene in metastatic tumors .
[59]
Interestingly, HB patients with high SP8 expression levels were also prognostic for poor survival. SP8-
mediated aggressive traits such as cell motility and clonogenic growth could be abrogated in SP8-silenced
cell models using RNA interference. Of clinical relevance, application of the FDA-approved pan SP
transcription factor mithramycin A abolished SP8-induced effects .
[59]

PATIENT-DERIVED XENOGRAFT MODELS
Several drugs have been identified as potentially effective against HB by cell line screening. Drug efficacy
validation in preclinical studies with animal models is usually required prior to clinical trials. Several
genetically engineered mouse models have been described that recapitulate HB features , including the
[60]
Cited1-Ctnnb1 mouse, which provided evidence that activating Ctnnb1 mutation is sufficient to drive
tumorigenesis in the liver for the first time . While these models are very useful tools to shed light on HB
[61]
biology, the lack of the human genetic background limits the use of transgenic mouse models for the
validation of new anticancer treatments. Patient-derived xenografts (PDXs) have recently become reference
models for the preclinical evaluation of anti-cancer therapy and are gaining a central tool in preclinical
pediatric oncology programs worldwide (http://www.ncipptc.org, https://www.itccp4.eu). During the last
years, several HB PDX models have been generated, by either orthotopic or heterotopic (interscapular fat