Involvement in pathogenesis
The Wnt/β-catenin protein signaling pathway plays a significant role in the development of HCC and is essential for regulating several physiological and pathological liver activities. There is approximately a 40% genetic variation between its key components, and aberrant expression and mutations have been demonstrated to be crucial for the development and dedifferentiation of HCC.55,56 EVs in HCC maintain a certain level of invasiveness of HCC cells by regulating Wnt signaling and delivering contents between cells, which in turn mediates the biological behavior of the cells and promotes tumor progression.57 It has been confirmed that a large amount of abnormally activated Wnt and its contents exist in HCC tissues, further activating downstream target genes through its signaling pathway, which maintains the high invasiveness of HCC.58,59
The most common gene mutation in the Wnt/β-catenin signaling pathway occurs in exon 3 of the β-catenin-encoding CTNNB1 gene.60 According to one study, about 30% of the CTNNB1 gene, which codes for β-catenin proteins, is mutated in HCC cells. This mutation interacts with T cell factor (TCF) 4, a transcription factor specific to Wnt cells, to initiate metabolism and proliferation before Wnt response elements are recruited. This results in blocked phosphorylation of β-catenin proteins, which, in combination with mutant histone modifying factors and chromatin remodeling factors, prevents their degradation by the proteasome, allowing the translocation of β-catenin proteins to the nucleus. The findings indicate that a compound containing APC, AXIN1, GSK3β, and CK1 is responsible for this process.61–63 Two of the most well-studied mechanisms of aberrant activation of the Wnt/β-catenin signaling pathway are cellular mutations and epigenetic modifications. Promoter methylation and the aberrant synthesis of non-coding RNAs are among the other factors. Regional and ethnic factors affect CTNNB1 mutations, with significantly more CTNNB1 mutations found in patients from Asia and Europe compared to HCC patients from Africa.64 In addition, etiological factors of HCC also play a role. Research indicates that CTNNB1 mutations are more common in HCC caused by HCV than in HCC caused by HBV, and alcohol-associated HCC has the highest correlation with the frequency of CTNNB1 mutations, approximately 42%.65,66 Mutations affecting AXIN, GSK-3β, and APC, or their lack of function as AXIN complex constituents, can also trigger the pathway and cause cancer. APC gene inactivation results in cancer through the combined effects of GSK-3β phosphorylation, hepatic β-catenin overexpression, and AXIN mutations.67,68 Zhang et al. found in earlier research that the Wnt3/FZD7-mediated β-catenin signaling pathway was more highly expressed in HCC. Subsequent analysis of the effects of Wnt3 binding to the N-glycosylation site-directed mutation of FZD7 on HCC cell development showed that Wnt3/β-catenin signaling pathway-related protein levels were negatively impacted by downregulating Wnt3’s ability to bind to FZD7, which in turn affected cell invasion, migration, and proliferation.69 Similarly, related studies have demonstrated that a variety of Wnts act in conjunction with various FZD receptors, overexpressing and decreasing the levels of secreted frizzled-associated proteins in tissues and leading to their methylation in HCC and its surrounding tumor microenvironment. Initially, secreted frizzled-associated proteins functioned to oppose the Wnt/β-catenin signaling pathway; however, as their quantity decreased and inactivation occurred, the ability of the pathway to be activated increased, gradually accumulating and promoting HCC tumorigenesis.70–72 This study has validated two important factors that lead to aberrant activation of the Wnt/β-catenin protein signaling pathway in HCC tumor formation: the previously reported CTNNB1 gene mutations and the loss of function or mutations in AXIN, GSK-3β, and APC complexes.73,74 Among these, missense mutations or deletions account for most loss-of-function mutations of GSK-3β and APC axis protein complexes, resulting in a decrease in the negative regulatory function of the pathway. These also facilitate the translocation of β-catenin proteins from the cytoplasm to the nucleus.75,76 Mutations in CTNNB1 are more likely to occur in both ways. Missense mutations or deletions of APC hypermethylate the WNT inhibitory factor-1 promoter, preventing the WNT inhibitory factor-1 from being expressed, causing cells to lose their usual control and encouraging the growth of HCC cells.77 Activation of receptor tyrosine kinases, high levels of R-spondins, and TGF-β-dependent activation in HCC are just a few of the ways that the Wnt/β-catenin signaling pathway is activated. The interaction of multiple HCC-related high-risk factors, either together or separately, leads to HCC tumorigenesis, which is extremely complex. These mechanisms are still under investigation.78–80
Promote malignant biological behavior
Proliferation
When the Wnt signaling pathway is activated, the amount of β-catenin protein increases. This protein can enter the nucleus and bind to TCF or lymphatic factor (LEF) to regulate the production of downstream target genes such as c-Myc and Cyclin D1.85,86 These genes are upregulated, which promotes cell cycle progression and boosts cell proliferation.87 In HCC cells, the Wnt signaling pathway is often aberrantly activated, releasing β-catenin from the GSK-3β degradation complex and causing it to accumulate in the cytoplasm.88 Subsequently, β-catenin penetrates the nucleus and associates with TCF/LEF family members to form a transcriptional complex.89 A cell’s Frizzled and LRP5/6 receptors are the sites where Wnt ligands bind to activate the Wnt signaling pathway, which in turn inhibits GSK-3β activity.90 GSK-3β is typically responsible for phosphorylating β-catenin, which accelerates its breakdown.91 GSK-3β inhibition prevents β-catenin from being phosphorylated and eliminated, causing it to accumulate in the cytoplasm.92 The accumulating β-catenin is then translocated to the nucleus, where it binds to TCF/LEF transcription factors.93 Once the transcriptional complexes are formed, downstream genes are activated to express themselves.94 These β-catenin-induced genes promote rapid cell division by facilitating the transition from the G1 phase to the S phase of the cell cycle.95 The G1/S phase transition requires CDK4/6 activity, which can be increased by upregulating β-catenin.96 Additionally, β-catenin can prevent the production of genes linked to apoptosis, such as Bax and Bim, thereby inhibiting apoptosis.97 Studies have demonstrated that increased β-catenin accelerates HCC cell proliferation, invasion, and metastasis, and encourages tumor development.98
Activation of the Wnt signaling pathway can sustain liver stem cells’ capacity for self-renewal, allowing them to proliferate without differentiating into fully developed hepatocytes.99 HCC may originate from these undifferentiated stem cells.100 Several genes linked to stem cell properties can also be activated by the accumulation of β-catenin in the nucleus.101 Studies have indicated that HCC cells driven by the Wnt signaling pathway exhibit stem cell-like characteristics, including the ability to differentiate in multiple directions and self-renew.102 By upregulating the expression of genes related to the cell cycle, inhibiting apoptosis, and promoting HCC cell proliferation, the Wnt pathway enables HCC cells to survive and grow in harsh conditions.103 The Wnt signaling pathway also promotes cell division by upregulating the production of cyclins and kinases, thereby maintaining the stem cell pool, activating DNA damage repair mechanisms, and helping HCC cells repair DNA damage, which supports their self-renewal ability.104,105
Invasion and metastasis
The increased malignant biological behavior of HCC patients is a major cause of their high mortality rate. Wnt signaling pathway activation and the development of EMT are closely related.106 EMT enables epithelial cells, such as liver cells, to shed their epithelial characteristics and acquire mesenchymal characteristics, such as the ability to migrate and invade. EMT is considered a key indicator of tumor cells’ propensity to infiltrate.107 The Wnt signaling pathway regulates the expression of N- and E-cadherin, which can further this process.108 E-cadherin is a protein responsible for the adhesion of epithelial cells. The Wnt signaling pathway prevents the expression of E-cadherin by accumulating β-catenin, and its decreased expression leads to reduced intercellular adhesion, promoting EMT.109 The β-catenin-TCF/LEF complex can activate mesenchymal markers such as vimentin, fibronectin, and N-cadherin. The upregulation of these proteins imparts interstitial properties to cells.110,111 The mesenchymal characteristics acquired during EMT facilitate the migration of HCC cells to surrounding tissues. Mesenchymal cells also exhibit enhanced invasion ability, allowing them to enter the blood circulation by passing through the vascular wall and basement membrane, thereby increasing the metastatic potential of HCC.112 Additionally, HCC cells with mesenchymal characteristics enable epithelial cells to survive even in the absence of adhesion, inhibiting tumor cell apoptosis.113 HCC cells’ ability to upregulate angiogenesis-related factors during EMT, such as vascular endothelial growth factor (VEGF), helps supply nutrients and oxygen for tumor development, potentially promoting the formation of tumor blood vessels.114 The Wnt signaling pathway can increase the expression of matrix metalloproteinase (MMP) genes, such as MMP-2 and MMP-9, and facilitate β-catenin’s nuclear translocation and binding to the transcription factor TCF/LEF. These enzymes promote tumor cell motility and invasion by breaking down collagen and elastin, two components of the extracellular matrix (ECM). While MMP activity is negatively regulated by tissue inhibitors of metalloproteinases (TIMPs), MMPs are crucial for matrix disintegration.115 By promoting the breakdown of TIMPs, the Wnt signaling pathway can increase MMP activity and reduce ECM inhibition. In some cases, the Wnt signaling pathway may directly increase TIMP expression, but this typically occurs in conjunction with the degradation of MMPs to maintain proper ECM degradation.116
The Wnt signaling pathway can also increase integrin expression. Integrins bind to ECM components, enhancing cell migration and invasion, and activating Rho family GTPases, including Rac1 and RhoA. These GTPases affect cell morphology and movement by regulating the polymerization and recombination of cytoskeletal proteins (such as actin and tubulin). These functions are primarily achieved by upregulating cell migration-related genes, such as MMPs, TIMPs, and cytoskeleton-related proteins.117,118 Moreover, Wnt signaling pathway activation may aid in modifying the HCC tumor microenvironment. In addition to promoting VEGF production, it can also support immunosuppression by inhibiting T cell activation and function, and by triggering the release of various cytokines and growth factors from tumor cells, such as platelet-derived growth factor, TGF-β, and fibroblast growth factor, all of which promote tumor invasion and metastasis.119,120 Furthermore, it can regulate the expression of cell adhesion molecules and chemokines, influencing immune cell recruitment and tumor cell infiltration. This suggests that tumor cells have an increased propensity to penetrate blood vessels and the lymphatic system, facilitating their entry into the blood circulation, infiltration, and metastasis.121–123
Therapy
Chemotherapy is the mainstay of treatment for patients with advanced HCC. However, the emergence of targeted therapy has brought new hope for these patients. Compared with traditional chemotherapy methods, targeted therapy is more specific. It has less impact on the normal internal environment of the human body, fewer side effects, better drug compliance, and more obvious efficacy.124 Because of the Wnt signaling pathway’s crucial role in HCC, targeting it has become a feasible therapeutic strategy.125 The related studies include Wnt signaling pathway inhibitors, downstream Wnt signaling pathway inhibitors, combination therapies targeting cell adhesion and migration, etc. (Table 1).126–134
Table 1Drugs acting on the Wnt/β-catenin signaling pathway in HCC
| Medicines | Action mechanism | Clinical stage | References |
|---|
| LGK974 | Inhibition of Porcupine reduces the secretion of Wnt ligands | Clinical phase I | 126 |
| PRI-724 | Inhibit the interaction between β-catenin and CBP, block the signal transduction | Clinical phase I | 127 |
| ICG-001 | Inhibition of β-catenin binding to CBP blocks Wnt/β-catenin pathway | Preclinical study/clinical phase I | 128 |
| FH535 | Inhibition of Wnt/β-catenin and PPAR pathways | Preclinical study | 129 |
| XAV939 | Inhibit Tankyrase, increase AXIN level and reduce the accumulation of β-catenin | Preclinical study | 130 |
| Vantictumab | Antibody drugs, targeting Frizzled receptors, block Wnt signaling | Preclinical study/clinical phase I | 131 |
| DKK1 | Blocking Wnt binding to Frizzled receptor and inhibiting Wnt pathway | Preclinical study | 132 |
| Niclosamide | Inhibition of Wnt/β-catenin pathway reduces cell invasion ability. | Preclinical study | 133 |
| Salinomycin | By inhibiting the Wnt/β-catenin pathway, it reduces the migration and invasion of cancer cells. | Preclinical study | 134 |
β-catenin inhibitors, which are further subdivided into GSK-3β inhibitors and small molecule β-catenin inhibitors, are the most significant class of Wnt signaling pathway inhibitors.135 β-catenin inhibitors in small molecules function in two ways. First, they bind directly to β-catenin, blocking its entry into the nucleus and activation of downstream genes. The second mechanism limits the activity of ubiquitinating enzymes (such as MMP7) to reduce β-catenin breakdown, stabilizing it and lowering its activity.136 Importantly, GSK-3β inhibits the Wnt signaling pathway. Inhibiting its activity stabilizes β-catenin and leads to its accumulation in the nucleus. This indirect suppression of the Wnt signaling pathway has led to the development of GSK-3β inhibitors.137 In addition, Wnt competitive inhibitors are substances that competitively bind to Wnt receptors, preventing the activation of the Wnt signaling pathway. Currently, clinical trials for small molecule β-catenin inhibitors are underway, represented by Inducible Cytotoxic GPC3-001, which has entered the clinical trial stage. In patients with HCC, phase I clinical research has shown some anti-tumor activity and good tolerability; the phase II clinical trial is currently in progress to assess its safety and efficacy in more detail.138 Wnt response inhibitors (IWR) are the main type of GSK-3β inhibitor. Research on IWR-1 has attracted widespread attention and has been clinically tested in various cancer types, showing certain anti-tumor activity and potential for the treatment of HCC.139 Representative Wnt competitive inhibitors include κBα (IκBα) analog inhibitors. IκBα analogs inhibit the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway by mimicking IκBα, which decreases IκB kinase activity and increases the expression of anti-apoptotic proteins (such as Bcl-2) while decreasing pro-apoptotic proteins (such as Bax), promoting apoptosis of HCC tumor cells.140,141 Clinical studies of related competitive inhibitors and antibodies in HCC are ongoing, and these drugs may provide new treatment options for HCC patients.
The downstream components of the Wnt signaling pathway are primarily responsible for obtaining, transmitting, and amplifying Wnt signals, including related receptors, TCF/LEF transcription factors, and effector molecules. An essential part of the Wnt signaling pathway is Frizzled (Fzd). Fzd receptors initiate signal transduction by attaching to Wnt ligands on the cell membrane. Fzd inhibitors interact with Frizzled receptors to prevent Wnt protein from binding, thus interrupting Wnt signal transduction, reducing downstream target gene transcription, lowering HCC cell survival and proliferation, and inhibiting tumor growth (Table 2).142–144 Inhibition of Fzd3 has been shown to suppress HCC cell proliferation and invasion while promoting tumor growth in animal models, providing a strong basis for further human studies.145,146 TCF/LEF transcription factors are crucial in the downstream Wnt signaling cascade. Inhibitors of TCF/LEF have the potential to restrict the expression of Wnt target genes by blocking the interaction between β-catenin and TCF/LEF, disrupting the Wnt signaling pathway and decreasing tumor cell proliferation and survival.147 Specific inhibitors of TCF/LEF include BAY 11-7082. Recent research has demonstrated that BAY 11-7082, in conjunction with CDK4/6 inhibitors, is beneficial against a range of malignancies, including HCC, in animal trials.148 Furthermore, studies have shown a negative correlation between tumor growth and the production of WISP1, a downstream effector of the Wnt signaling pathway. WISP1 stops HCC progression by negatively regulating β-catenin/TCF/LEF signaling, suggesting a potential therapeutic avenue for targeting Wnt downstream effector molecules to halt HCC progression.149
Table 2Drugs targeting downstream effector molecules of Wnt signaling in HCC
| Medicines | Action mechanism | Clinical stage | References |
|---|
| PNU-74654 | It directly binds to β-catenin, prevents its formation with TCF complex, and reduces gene transcription | Preclinical study | 142 |
| MSAB | Inhibition of β-catenin-mediated transcriptional activity reduces Wnt signaling downstream gene expression | Preclinical study | 143 |
| PBP(Pyrvinium pamoate) | By interfering with the Wnt signaling pathway and inhibiting the activity of β-catenin, the transcription of downstream genes is inhibited | Preclinical study | 144 |
MMPs are important players in the Wnt signaling pathway, stimulating HCC tumor cells’ adhesion, motility, and invasion. Inhibition of MMPs can reduce these processes and prevent premature metastasis of tumor cells.150 MMP inhibitors directly block the activity of MMPs through chemical structures or biological pathways, reducing ECM degradation and regulating MMP gene expression or protein synthesis to lower MMP production.151 The current research on MMP inhibitors focuses on Batimastat (BB-94) and Marimastat (BB-2516).152 MMP inhibitors have been demonstrated in several clinical trials to reduce HCC tumor invasion and improve prognosis. Studies have shown that BB-94 can prevent HCC cells from migrating and inhibit angiogenesis in the tumor vascular system. Furthermore, the tumor blood vessels that are prevented from growing cannot supply enough oxygen and nutrients for the remaining HCC cells.153 MMP inhibitors target MMPs overexpressed in the tissues and cells of hepatocellular carcinoma. After years of research, many clinical drugs have been produced, such as Bamalabaster, Novartis, BAY12-9566, AG-3340, OPB-3206, KBR07785, and KBR-8301.154–156 Cell adhesion molecules (CAMs) are important for mediating interactions between cells or between cells and the extracellular matrix.157 CAM modulators can affect CAM content and activation pathways, influencing tumor cell adhesion, migration, and invasion (Table 3).158–175 Various types of CAM modulators include E-cadherin agonists, which can enhance intercellular adhesion and reduce tumor cell invasion by promoting E-cadherin expression and function.176 SMADS agonists can initiate SMADS phosphorylation and nuclear translocation through the TGF-β/SMAD signaling pathway. By binding to the E-cadherin promoter region and promoting E-cadherin production, SMADS interacts with β-catenin to improve cell adhesion and Wnt signaling. Studies have indicated that interferon-α and TGF-β1 may effectively block the development of the β-catenin/TCF4/Smads complex, suggesting that these molecules could be employed as potential therapeutics for HCC patients.177,178 Integrin modulators inhibit integrins from binding to ligands, reducing cell adhesion and migration. Examples include antibody-coupled drugs including ramucirumab and atezolizumab, which can bind integrins and target tumor cells.179,180 Cetuximab, a small molecule integrin blocker, may be particularly effective in preventing advanced HCC by obstructing the interaction between integrins and EGFR.181
Table 3Drugs targeting cell adhesion and migration in the Wnt signaling pathway in HCC
| Medicines | Action mechanism | Clinical stage | References |
|---|
| Batimastat (BB-94) | Broad-spectrum MMP inhibitors inhibit MMP-1, MMP-2, MMP-9, etc., and reduce tumor invasion and migration | Clinical stage I/II | 158 |
| Marimastat (BB-2516) | Broad-spectrum MMP inhibitors inhibit MMP-1, MMP-2, MMP-9, etc., and reduce tumor invasion and migration | Clinical Phase II | 159 |
| Tanomastat (BAY 12-9566) | MMP inhibitors, targeting MMP-2 and MMP-9, inhibit the degradation of extracellular matrix and prevent tumor metastasis | Clinical Phase II | 160 |
| TIMP-2 | Endogenous MMP inhibitors directly inhibit MMP-2 activity and reduce matrix degradation and tumor invasion | Preclinical study | 161 |
| Doxycycline | Antibiotics, non-specific MMP inhibitors, inhibit MMP-2 and MMP-9, reduce tumor invasion and metastasis | Preclinical study/clinical phase I | 162 |
| Ilomastat (GM6001) | Broad-spectrum MMP inhibitors, inhibit MMP-1, MMP-2, MMP-9, reduce extracellular matrix degradation | Preclinical study | 163 |
| AG3340 (Prinomastat) | Selective MMP inhibitors, mainly inhibit MMP-2 and MMP-9, reduce extracellular matrix degradation and invasion | Clinical phase I | 164 |
| ONO-4817 | Selective inhibition of MMP-2 and MMP-9 reduced tumor cell invasion and matrix degradation | Preclinical study | 165 |
| Minocycline | Inhibit the activity of MMP-2 and MMP-9, reduce matrix degradation and tumor cell migration | Preclinical study | 166 |
| E7820 | Inhibit the expression of integrin α2, reduce tumor cell adhesion and angiogenesis | Clinical stage I / II | 167 |
| Volociximab | Targeting integrin α5β1 prevents the binding of tumor cells to the extracellular matrix and inhibits tumor invasion | Clinical Phase II | 168 |
| Etaracizumab (Abegrin) | Targeting integrin αvβ3 reduces tumor cell adhesion and angiogenesis, and inhibits tumor growth and metastasis | Clinical Phase II | 169 |
| Cilengitide | Inhibition of integrin αvβ3 and αvβ5 inhibits tumor angiogenesis and tumor cell invasion. | Clinical Phase II | 170 |
| Simtuzumab (GS-6624) | Anti-LOXL2 (lysyl oxidase-like protein 2) antibody prevents extracellular matrix remodeling and reduces tumor invasion | Clinical Phase II | 171 |
| Disulfiram | It inhibits the expression of cell adhesion molecules (such as N-cadherin) and prevents cancer cell migration and invasion | Preclinical study | 172 |
| ADH-1 | Targeting N-cadherin inhibits intercellular adhesion and reduces invasion and metastasis of tumor cells | Preclinical study | 173 |
| Galunisertib (LY2157299) | TGF-β inhibitors inhibit the expression of adhesion molecules and reduce tumor cell invasion and metastasis | Clinical Phase II | 174 |
| Metformin | Indirectly inhibit the expression of cell adhesion molecules, reduce the invasion ability of tumor cells | Preclinical study | 175 |
Several therapeutic modalities related to the Wnt signaling pathway, including immune checkpoint inhibitors and CAR-T cell therapy, are now in the preclinical or animal experimental stages, in addition to the previously mentioned treatment strategies.182,183 The combination of Wnt signaling pathway-related treatments with other therapeutic measures (such as immunotherapy, VEGF inhibitor therapy, etc.) can improve efficacy, eliminate the side effects of monotherapy, and reduce drug resistance.184 Targeting the Wnt signaling pathway holds great potential in clinical practice, but it also faces several challenges. Therapeutic approaches targeting the Wnt pathway may lead to non-specific off-target effects, affecting the function of normal cells and tissues, particularly in the liver, intestine, and bones.185 Although targeting the Wnt pathway has shown promising efficacy in vitro and in animal models, drug resistance is a common issue in clinical applications. Activation of the Wnt signaling pathway involves multiple regulatory mechanisms, including the stability of β-catenin, upstream Frizzled receptors, and downstream transcription factors. The complexity of these regulatory processes allows tumor cells to bypass targeted therapies in various ways.186 Additionally, abnormal stability or nuclear translocation of β-catenin may be maintained through mutations or other pathways, reducing the effectiveness of drugs.187 The regulation of the Wnt pathway is highly complex, involving multiple key molecules, and different types of tumors may depend on different Wnt signaling subtypes, which complicates targeted Wnt therapy and introduces issues of tumor heterogeneity.188 The Wnt signaling pathway’s high degree of intricacy and variety in regulation is a result of its numerous interactions with other signaling pathways. Accurate targeting is extremely challenging, which highlights the significance of combination therapy. Due to the Wnt pathway’s intricate role in the human body, therapeutic techniques for it are currently being developed slowly and require further research.