Wnt signaling in cancer: therapeutic targeting of Wnt signaling beyond β-catenin and the destruction complex

Wnt/β-catenin signaling is implicated in many physiological processes, including development, tissue homeostasis, and tissue regeneration. In human cancers, Wnt/β-catenin signaling is highly activated, which has led to the development of various Wnt signaling inhibitors for cancer therapies. Nonetheless, the blockade of Wnt signaling causes side effects such as impairment of tissue homeostasis and regeneration. Recently, several studies have identified cancer-specific Wnt signaling regulators. In this review, we discuss the Wnt inhibitors currently being used in clinical trials and suggest how additional cancer-specific regulators could be utilized to treat Wnt signaling-associated cancer.


Introduction
Wnt signaling orchestrates various biological processes, such as cell proliferation, differentiation, organogenesis, tissue regeneration, and tumorigenesis [1][2][3][4][5] . Classically, Wnt signaling is divided into β-catenin-dependent (canonical, Wnt/β-catenin pathway) and β-catenin-independent (noncanonical, Wnt/planar cell polarity [PCP] and calcium pathway) signaling 6,7 . Canonical Wnt signaling mainly regulates cell proliferation, and noncanonical Wnt signaling controls cell polarity and movement. However, this terminological distinction is unclear, and has been questions by studies proposing the involvement of both β-catenin-dependent and β-cateninindependent Wnt signaling in tumorigenesis 8 . For instance, APC and β-catenin are not only involved in cell proliferation but have also been linked to cell-to-cell adhesion 9 . In this review, we will discuss an ongoing effort to inhibit Wnt signaling and suggest potential approaches to target Wnt signaling for cancer therapies proposed from recent studies.

WNT ligands and receptors
Under physiological conditions, Wnt signaling is activated by the binding of secreted WNT ligands to LRP5/6 coreceptors and frizzled (FZD) receptors 54 , which induces the recruitment of the protein destruction complex to the LRP receptors and the subsequent phosphorylation of the Ser/Pro-rich motif of the LRP cytoplasmic domain via GSK3 15,55,56 . This event activates dishevelled (DVL) and inhibits GSK3, resulting in the inhibition of the phosphorylation-mediated β-catenin protein degradation and the stabilization/accumulation of β-catenin. Then, β-catenin undergoes nuclear translocation and transactivates Wnt target genes 57 . The secretion of WNT ligands mainly depends on acylation by Porcupine (PORCN) 58,59 . PORCN is a membrane-bound O-acyltransferase that mediates the palmitoylation of WNT ligands to induce their secretion. In line with this observation, PORCN shows increased genetic alterations in various human cancers, including esophageal, ovarian, uterine, lung, and cervical cancers 60 .

Therapeutic targeting of Wnt/β-catenin signaling
To suppress WNT ligands or receptors for cancer treatment, PORCN inhibitors, WNT ligand antagonists, and FZD antagonists/monoclonal antibodies have been examined in clinical trials of various Wnt signalingassociated human cancers (Table 1 and Fig. 1).
(ii) CK1 agonist Pyrvinium is an FDA-approved anti-helminthic drug. Pyrvinium binds to CK1 family members in vitro and promotes CK1 kinase activity 96 .
(ii) Inhibitor of Wnt target genes SM08502 (NIH clinical trial number NCT03355066) is a small molecule that inhibits serine and arginine-rich splicing factor (SRSF) phosphorylation and disrupts spliceosome activity. Upon oral administration, SM08502 was shown to downregulate Wnt signaling-controlled gene expression.

Additional layers of Wnt/β-catenin signaling activation
The β-catenin paradox Wnt signaling hyperactivation by mutations in β-catenin destruction complex components or β-catenin itself contributes to tumorigenesis. In addition to APC mutations, β-catenin can be further activated by additional layers of regulation 39,40,[111][112][113][114][115][116][117] , which demonstrated the complexity of Wnt signaling deregulation in cancer. Accumulating evidence supports the notion that additional regulatory processes contribute to Wnt signaling hyperactivation in cancer, as demonstrated in the following examples. (a) Mutant APC is still able to downregulate β-catenin 39,40 . (b) Even in the presence of APC mutations, blockade of WNT ligands triggers apoptosis or growth inhibition 40,113,118 . (c) β-Catenin fold induction is essential for the activation of β-catenin target genes [119][120][121] . (d) Increased AXIN1 by Tankyrase inhibitor suppresses cell proliferation of cancer cells where Wnt/β-catenin signaling is genetically hyperactive 43,90,93,95,122 . (e) Mutations in RNF43 and ZNRF3 E3 ligases that degrade Wnt receptors contribute to tumor development 111,115 . (f) Ras/ MAPK signaling is also required for Wnt signaling activation 112,123 . These reports suggest that additional layers further enhance Wnt signaling activation in cancer.

The lysosome and Wnt signaling
The lysosome contains 40 types of hydrolytic enzymes, including cathepsins, which become active under acidic conditions 124 . Lysosomal hydrolytic enzymes mediate the degradation of phagocytosed material and proteolysis of cytosolic proteins through fusion with the multivesicular body (MVB). Luminal acidification of the lysosome is required for lysosomal protein degradation, which is mainly controlled by vacuolar H + transporters in the lysosomal membrane 125 .
Recently, this classical view of lysosomal functions has evolved into new perspectives highlighting the roles of lysosomes in transcriptional regulation and metabolic homeostasis 126 . In human cancer, lysosomal dysfunction is involved in the generation of building blocks, cell proliferation, metastasis, angiogenesis, and tumor suppressor degradation 39,127 .
It has been reported that Wnt signaling is involved in the endocytosis-mediated formation of the LRP signalosome into the MVB 123,128 . GSK3 in the LRP signalosome is sequestered into the MVB, which leads to an increase in the level of cytosolic β-catenin and inhibition of Wnt signaling 129 . However, decreased GSK3 kinase activity by MVB sequestration lasts approximately 1 h 129,130 . Moreover, it is unclear how sequestrated APC, GSK3, AXIN, and CK1 in MVB are processed. A recent study showed that clathrin-mediated endocytosis is required for Wnt signaling activation, which is inhibited by APC 131 . These studies suggest that vesicular acidification and trafficking also play crucial roles in controlling Wnt/β-catenin signaling through modulation of the protein destruction complex. Next, we discuss how APC is deregulated for Wnt signaling hyperactivation in cancer cells.
Wnt signaling activation requires v-ATPase (vacuolar H + -ATPase; an electrogenic H + transporter) 125,132,133 . Previous studies imply that in cancer cells, the upregulation of v-ATPase activity might trigger abnormal Wnt/ β-catenin signaling and contribute to Wnt signalingdependent tumorigenesis. Growing evidence has demonstrated the effect of v-ATPase on various oncogenic processes, including cellular signaling, survival, drug resistance, and metastasis 125,134 . Moreover, the v-ATPase subunits are highly expressed in colorectal, breast, prostate, liver, ovarian, and pancreatic cancer cells [135][136][137][138] . The v-ATPase complex is composed of the V1 domain (in the cytosol) and V0 domain (on the membrane) 139,140 . The V1 domain shows reversible disassociation from the V0 domain under physiological conditions, including glucose concentration, starvation of amino acids, and infection of cells by influenza virus [141][142][143][144] . Recently, TMEM9 (transmembrane protein 9) was identified as an activator of v-ATPase and is highly expressed in cancer 39 . TMEM9 amplifies Wnt signaling through the v-ATPase-mediated lysosomal protein degradation of APC 39 . Given that TMEM9 is highly expressed in CRC cells and that Tmem9 knockout mice are also viable 39 , molecular targeting of TMEM9 may selectively suppress Wnt signaling activity in cancer cells.

Novel therapeutic target: v-ATPase
Conventional approaches targeting Wnt/β-catenin have led to various side effects, as mentioned above. Therefore, cancer-specific Wnt signaling regulators such as v-ATPase may be attractive molecular targets for Wnt signaling blockade. Chloroquine (CQ) and hydroxychloroquine (HCQ), inhibitors of lysosomes and autophagy, are clinically used for the treatment of diseases such as malaria and rheumatoid arthritis 145 . While the mechanism of action of CQ and HCQ is somewhat unclear, other v-ATPase inhibitors, such as bafilomycin (BAF) and concanamycin (CON), directly bind to and inhibit v-ATPase 146,147 . Compared with CQ and HCQ, BAF and CON showed marked inhibition of Wnt/ β-catenin signaling in CRC. In addition, BAF and CON displayed an antiproliferative effect in CRC patient-driven xenograft and animal models without toxicity to normal cells and animals 39 . In addition, BAF and CON also strongly inhibit Wnt signaling activity in CRC cells, regardless of APC mutations. Thus, further research may lead to the development of not only safer but also more potent anti-v-ATPase drugs as cancer-specific Wnt/ β-catenin inhibitors (Fig. 2).

Conclusion
Genetic and epigenetic deregulation of Wnt/β-catenin signaling contributes to human cancer, which has led to the development of extensive approaches targeting Wnt/ β-catenin signaling as cancer therapies. Nonetheless, the blockade of Wnt signaling impairs tissue homeostasis and regeneration, which needs to be resolved. Recent studies have identified several Wnt signaling regulators whose expression is specific to cancer cells. These cancer-specific regulatory processes of Wnt signaling may be druggable vulnerabilities of Wnt signaling-associated cancer.
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