WNT/β-catenin pathway activation in hepatocellular carcinoma: a clinical perspective

Figures & data

Table 1 Clinical evidence of altered the WNT/β-catenin signaling pathway components in HCC

Components of the WNT/ β-catenin signaling pathway	Expression levels in HCC versus paratumor/normal liver tissues	Method of detection	References
Ligands
WNT1	↑	Western blot	125,126
WNT3	↑	qRT-PCR	128
WNT5A	↓	IHC	129
WNT11	↓	qRT-PCR and western blot	131
Antagonists
DKK1	↑	ELISA (serum)	133
DKK4	↓	qRT-PCR	136
DKK3	↓	qRT-PCR	134
WIF-1	↓	qRT-PCR	135
sFRP1,2,5	↓	Methylation	81
Receptors
FZD3	↑	IHC	138
FZD6	↑	IHC	138
FZD7	↑	qRT-PCR, IHC	77,138
LRP6	↑	qRT-PCR	140
“Destruction complex” components
β-catenin (CTNNBI)	↑	Sequencing/IHC	57,58,100,101
AXINI (AXINI)	↓	Sequencing/IHC	48
APC (APC)	↓	Promoter methylation	121

Abbreviations: APC, adenomatous polyposis coli; DKK, dickkopf; ELISA, enzyme-linked immunosorbent assay; FZD, frizzled receptors; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; LRP, low density lipoprotein receptor-related protein; qRT-PCR, quantitative real time – polymerase chain reaction; sFRP, secreted frizzled related protein; WIF, WNT inhibitory factor.

Table 2 Mutations of key components involved in the WNT/β-catenin pathway in HCC

Gene	Mutations and truncations	Effect of functional alteration	References
CTNNBI	S33C, S33Y, S33P, S37Y, S37C, S37F, T41A, T41F, T41P S45P, S45A, S45F, S45Y L30Q, D32G, D32C, D32V, G34V, 135S del A5-A80, del S35-G38, del V22-Y64, del w25-1140	Inhibit β-catenin phosphorylation by GSK3β, and protect β-catenin from subsequent ubiquitin-mediated degradation. Inhibit β-catenin phosphorylation by CK1, and the subsequent phosphorylation by GSK3β, protect β-catenin from subsequent ubiquitin-mediated degradation. Alter the ubiquitin DSG amino acid targeting sequence, and protect β-catenin from ubiquitin-mediated degradation. Keep β-catenin activity, and inhibit β-catenin phosphorylation by GSK3β, diminish β-catenin ubiquitin-mediated degradation.	100–102,105–110
AXINI	D94A, L106R, F201C G425S, G651S, P849T 1bp deletion (codon 302) 8bp deletion (codon 603-606) 13bp deletion (codon 522-526) 25bp deletion (codon 481-489) codon 443; GAG-TAG codon 284; TGG-TGA codon 443; GAG-TAG	Lack the binding motifs APC, and inhibit β-catenin phosphorylation and degradation. Lack the binding motifs of GSK3β, β-catenin and other Axins, and inhibit β-catenin phosphorylation and degradation. Result in frameshift, lack the binding motifs of GSK3p and β-catenin, and inhibit β-catenin phosphorylation and degradation. Result in premature termination, interrupt its binding to GSK3 and β-catenin, and inhibit β-catenin phosphorylation and degradation.	48,105,116
APC	codon 682; GGA-TGA	Result in premature termination that lacks the β-catenin-binding motif, and inhibit β-catenin phosphorylation and degradation.	120

Abbreviations: APC, adenomatous polyposis coli; CK, casein kinase; DSG, disuccinimidyl glutarate; GSK, glycogen synthase kinase; HCC, hepatocellular carcinoma.

Figure 1 Targeting the WNT/β-catenin pathway in HCC.

Notes: In normal liver cells (“WNT OFF” state), β-catenin is sequestered by the multiprotein “destruction complex” in the cytoplasm (which consists of APC, AXIN, GSK3 and CK1). The “destruction complex” sequentially phosphorylates β-catenin, which is then recognized by the E3 ubiquitin ligase complex-gamma-TrCP and is further degraded by proteasomes. In HCC cells (activated or “WNT ON” state), the protein dishevelled inhibits GSK3 phosphorylation activity and the “destruction complex” disintegrates. As a result, β-catenin accumulates in the cytoplasm and translocates to the nucleus, where it interacts with TCF4/LEF to transcriptionally activate downstream target genes. Investigational agents targeting various components that have been tested in HCC are indicated in red boxes.
Abbreviations: APC, adenomatous polyposis coli; AXIN, ; CK1, casein kinase; EpCAM, epithelial cell adhesion molecule; FZD, frizzled receptors; GSK3, glycogen synthase kinase-3; TCF, T-cell factor; GSK, glycogen synthase kinase; HCC, hepatocellular carcinoma; LRP, low density lipoprotein receptor-related protein; SiRNA, small interfering RNA; TCF4/LEF, T-cell factor 4/lymphoid enhancer-binding factor.

Table 3 Experimental approaches in targeting the WNT/β-catenin signaling pathway in HCC

Target(s)	Drug	Cell line/animal model	Findings	References
WNT1	Anti-WNT1 antibody	HCC cell lines, xenograft mouse model	Suppressed cell proliferation and reduced tumor growth.	125
FZD7	sFZD7	HCC cell lines	sFZD7 dose-dependently decreased viability of three HCC cell lines.	148
FZD9	siRNA of FZD9	HCC cell lines	Reduced FZD9 significantly suppressed cell proliferation and cell motility in HCC cell lines.	150
FZD7	Small interfering peptides interfere with FZD7-DVL protein-protein interaction	HCC cell lines and transgenic mouse model of HCC	Induced cell viability via apoptosis in vitro and in vivo.	149
β-catenin	β-catenin siRNA	HCC cell lines	Decreased growth and survival.	152
β-catenin and TCF4/LEF complex	PKF118-310, PKF115-584 and CGP049090	HCC cell lines, xenograft model	Decreased tumor growth.	151

Abbreviations: DVL, dishevelled; FZD, frizzled receptors; HCC, hepatocellular carcinoma; Refs, references; sFZD7, soluble ectodomain of the FZD7 receptor; siRNA, small interfering RNA; TCF4/LEF, T-cell factor 4/lymphoid enhancer-binding factor.