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Kwee et al. Hepatoma Res 2021;7:8 I http://dx.doi.org/10.20517/2394-5079.2020.124 Page 3 of 13

In a genetically-engineered mouse model of melanoma that exhibits stabilized b-catenin expression, it was
first discovered that aberrant activation of this pathway could lead to the disappearance of immune cells
from the tumor microenvironment (TME) and the development of tumor resistance to anti-PD-L1/anti-
CTLA-4 monoclonal antibody therapy [13,20] . b-catenin expression in this model was also associated with
decreases in the expression of immune-oncology relevant chemokines such as CCL4 that are important
for recruiting dendritic cells and consequently T-cells into the TME. The mechanism by which b-catenin
reduces CCL4 expression was tied to the induction of ATF3, a transcriptional repressor, and its binding
to the CCL4 promoter [13,20,21] . This immune evasion mechanism has since been recapitulated in a similarly
engineered model of b-catenin activated HCC, where it was observed that aberrant b-catenin activation led
to tumor resistance to anti-PD1 therapy and that reinstating the expression of CCL5, a chemokine that was
[22]
downregulated in the b-catenin driven tumors, could restore tumor immune surveillance .

TME infiltration by antigen presenting dendritic cells and T-cells, leading to a so-called inflamed TME,
is now recognized as a critical factor that allows anti-PD1 and anti-PD-L1 antibodies to exert their
therapeutic effects [21,22] . Across numerous cancer types, analysis of TCGA data has revealed a significant
difference in the percentage of Wnt/b-catenin activating mutations between immune inflamed and non-
[12]
inflamed tumors, with the largest differences observed in HCC and esophageal cancer . Histologically, the
TME of b-catenin activated HCC also displays a paucity of immune cells . Hence, one may speculate that
[23]
it may be possible to infer the status of anti-tumor immunity in HCC by analyzing tumor biomarkers that
capture the status of b-catenin activation.

WNT/b-CATENIN SIGNALING
The Wnt/b-catenin signaling pathway is highly conserved across species and constitutively involved in
[24]
embryonic development, cell migration, and tissue homeostasis . This pathway has been shown to play
multiple roles in tumorigenesis and tumor immune-evasion [12,25-29] . In the canonical Wnt/b-catenin pathway,
the nuclear transcription co-activator protein b-catenin undergoes continual degradation in the cytoplasm
by a destruction complex comprised of the scaffolding protein Axin (AXIN1) along with casein kinase 1
(CK1), glycogen synthase kinase 3 (GSK3), and the adenomatous polyposis coli (APC) gene product
[Figure 1A] . Wnt binding at the cell membrane by Frizzled protein and low-density lipoprotein receptor-
[30]
related proteins 5 and 6 (LRP5/6) promotes recruitment of this Axin complex to the cell membrane via
dishevelled (DVL) proteins. Membrane bound, this b-catenin destruction complex is no longer able to
[31]
mediate the phosphorylation, ubiquitination, and subsequent proteasomal degradation of b-catenin . As
a consequence, the stable form of b-catenin accumulates in the nucleus where it can interact with DNA-
bound T cell factor/lymphoid enhancer factor (TCF/LEF) proteins to promote Wnt target gene expression
[Figure 1B] .
[32]

Aberrant activation of this pathway in cancer can be caused by a number of different molecular
mechanisms, including gene mutations, epigenetic alterations, and abnormal regulation of other pathways.
Mutations involving catenin-beta-1 (CTNNB1), the gene that encodes for b-catenin, are among the
most common causes in HCC, as they are found in approximately one-third of HCC tumors [33-36] . The
specific CTNNB1 mutations implicated in tumorigenesis and immune evasion have been noted to
disproportionately affect exon 3 at amino acid positions 29-49, corresponding to sites on the b-catenin
protein that undergo phosphorylation by GSK3 to initiate its degradation . Protected from normal break-
[34]
down, mutated b-catenin remains stable and is free to accumulate in the nucleus where it can activate multiple
transcriptional programs that can promote tumor progression and immune avoidance [Figure 1C] [12,13] .

Mutations involving other Wnt/b-catenin pathway-related genes can also lead to aberrant b-catenin
signaling [12,33,35,36] . For example, the AXIN1 gene is mutationally inactivated in 5%-19% of HCC tumors,
making it the second most frequently mutated gene leading to aberrant Wnt/b-catenin activation in