Targeting Wnt Signaling for Gastrointestinal Cancer Therapy: Present and Evolving Views

Simple Summary Therapeutic targeting of Wnt has long been suggested for gastrointestinal (GI) cancer treatment because deregulation of Wnt signaling is associated with GI cancers. However, therapeutic targeting of Wnt is still challenging because of the pleiotropic roles of Wnt signaling in the human body. Thus, targeting strategies of Wnt signaling are continuously evolving. The current flows of targeting Wnt signaling for cancer treatment are focused on increasing the specificity of drugs and combinatory treatment with other cancer drugs that minimize side effects and increase efficacy. Additionally, increased knowledge about the β-catenin paradox has expanded the cases that can be treated with Wnt targeting therapy, not strictly considering Wnt upstream and downstream mutations. Here, we discuss these evolving views of targeting Wnt signaling and describe examples of current clinical trials. Abstract Wnt signaling governs tissue development, homeostasis, and regeneration. However, aberrant activation of Wnt promotes tumorigenesis. Despite the ongoing efforts to manipulate Wnt signaling, therapeutic targeting of Wnt signaling remains challenging. In this review, we provide an overview of current clinical trials to target Wnt signaling, with a major focus on gastrointestinal cancers. In addition, we discuss the caveats and alternative strategies for therapeutically targeting Wnt signaling for cancer treatment.


Introduction
Evolutionarily conserved Wnt signaling was initially identified in Drosophila (Wingless) and the mammalian system (Int-1) [1,2]. Wnt signaling has been extensively studied, revealing its pivotal roles in orchestrating embryonic development, tissue homeostasis, and regeneration [3][4][5]. Notably, the deregulation of Wnt signaling is associated with many human diseases, including cancers [6]. Therefore, the manipulation of Wnt signaling has gained attention as a means of disease treatment and prevention [7,8].
Although it has been confirmed in in vitro and in vivo cancer studies that targeting Wnt signaling has drastic tumor-suppressing effects, no targeted drugs have been successively advanced to clinical applications to date [7][8][9]. This is mainly because Wnt signaling plays essential roles in maintaining a Figure 1. General view of canonical and non-canonical Wnt signaling. The switch of the canonical Wnt/β-catenin signaling pathway depends on the subcellular location of β-catenin. The stability of β-catenin is controlled by the destruction complex, consisting of AXIN, APC, CK1, and GSK3. In the absence of WNT ligands, cytoplasmic β-catenin is first phosphorylated by CK1 at Ser45 residue, followed by GSK3 phosphorylation at the Thr41, Ser37, and Ser33 residues. Next, the phosphorylated motif of β-catenin acts as a docking site for βTrCP, which induces the final ubiquitin-mediated degradation of β-catenin (Wnt off). When WNT ligands bind to Frizzled receptors (FZDs) and low density lipoprotein receptor-related protein co-receptor 5/6 (LRP 5/6), the destruction complex is recruited to the plasma membrane, triggering the translocation of β-catenin into the nucleus and activating its downstream target genes via binding directly to the TCF/LEF transcription factor family (Wnt on). Wnt/PCP signaling involves the triggering of a cascade that contains small GTPases RHOA (transforming protein RhoA) and Ras-related C3 botulinum toxin substrate 1 (RAC1), activating Rho-associated protein kinases (ROCKs) and JUN N-terminal kinases, respectively. Wnt/Ca 2+ signaling involves the activation of phospholipase C, which in turn triggers the release of Ca 2+ from intracellular stores and the activation of effectors such as calcium-or calmodulin-dependent protein kinase II, protein kinase C, and calcineurin (CaN). Next, CaN activates the nuclear factor of activated T cells, activating the transcription of downstream target genes.
In the Wnt/PCP pathway, the binding of WNT-FZDs triggers a cascade involving small GTPases RHOA (transforming protein RhoA) and RAC1 (Ras-related C3 botulinum toxin substrate 1), which in turn activates ROCKs (Rho-associated protein kinases) and JUN-N-terminal kinases, respectively [10,24,25]. It mainly regulates cell polarity, cell motility, and morphogenetic movements [10,24,25]. In the Wnt/Ca 2+ signaling pathway, the binding of WNT-FZDs activates phospholipase C (PLC), which in turn triggers the release of Ca 2+ from intracellular stores and the activation of effectors such as calcium-and calmodulin-dependent protein kinase II (CAMKII), protein kinase C (PKC), and calcineurin (CaN) [10,26]. CaN activates the nuclear factor of activated T cells, which regulates the transcription of the genes that control cell fate and cell migration [10,26]. Although both β-catenin-dependent and -independent Wnt signaling are involved in tumorigenesis, β-catenin-dependent Wnt signaling is relatively well defined in various cancer models. In line with this, current pharmacological trials targeting Wnt signaling have mainly focused on β-catenin-dependent Wnt signaling.

Therapeutically Targeting Wnt Signaling in GI Cancer
Targeting Wnt signaling for cancer treatment normalizes the hyperactivated Wnt signaling that promotes cancer progression. For this purpose, many targeting strategies have been evaluated, including the inhibition of Wnt ligands and receptors or coreceptors, restoration of the destructive complex, and inhibition of β-catenin/β-catenin-dependent transcriptional machinery. Although these approaches have not been studied in phase III clinical trials or used clinically, dozens of Wnt-targeting agents are currently being evaluated in phase II clinical trials (Table 1). These important phase II clinical trials include LGK974, genistein, Foxy-5, DKN-01, niclosamide, PRI-724, and chloroquine/hydroxychloroquine.
In the next section, we provide an overview of the known and potential agents that target Wnt signaling, especially for GI cancers; we also describe their mechanisms of action and related clinical trials ( Table 2). All potential agents that inhibit Wnt signaling are listed in Table 3. In addition, the molecular targets of representative Wnt inhibitors on WNT signaling are illustrated in Figure 2.

Inhibiting WNT Ligands
Ipafricept (OMP-54F28) is a recombinant receptor that is comprised of the cysteine-rich domain of FZD8 fused to the human IgG1 Fc domain; it inhibits Wnt signaling by neutralizing WNT ligands [54]. Three trials evaluated ipafricept and its combination therapies (Table 2). A phase I trial evaluated the best dosage of ipafricept and revealed grade 1-2 adverse events (AEs), including dysgeusia, decreased appetite, fatigue, and muscle spasms [36]. Another phase I trial evaluated ipafricept combined with nab-paclitaxel and gemcitabine in metastatic pancreatic cancer and revealed grade ≥ 3 AEs, including increased aspartate aminotransferase, nausea, maculopapular rash, vomiting, and decreased white blood cells [37].
Secreted frizzled-related proteins (SFRPs) bound directly to WNTs via the cysteine-rich domain, preventing the WNT-FZD interaction [55][56][57]. SFRPs also form dimers with FZDs via the respective cysteine-rich domain to activate or inhibit WNT3A/β-catenin signaling, depending on their concentration [58]. In the nucleus, SFRPs act as biphasic modulators of β-catenin-mediated transcription, which promotes TCF7L2 recruitment and transactivation of cancer stem cell-related genes by binding to the β-catenin's C-terminus; however, they suppress transcriptional activities by binding to the N-terminus [59]. The phase II trial evaluated genistein, an SFRP2 silencer inhibitor, in combination with FOLFOX and bevacizumab in metastatic CRC; the study revealed mild AEs, including headaches, nausea, and hot flashes ( Table 2) [35]. In addition, Wnt inhibitory factor 1 directly binds to WNTs through the Wnt inhibitory factor domain and prevents WNTs from transducing Wnt signaling [60]. Cerberus also binds to and inhibits WNT8, inhibiting Wnt signaling [61]. However, no agents mimicking Wnt inhibitory factor 1 and Cerberus have been identified.

Targeting Lipid Modification of WNT Ligands
The palmitoylation of WNT ligands by the protein-serine O-palmitoleoyltransferase porcupine in the endoplasmic reticulum [62] is essential for the maturation and extracellular secretion of WNT ligands. The palmitoylated WNT ligands bind to Wntless homolog in the Golgi and are ferried to the plasma membrane via secretory exosomes [63]. Porcupine inhibitors (CGX1321, ETC-159, and LGK974 [WNT794]), which suppress Wnt signaling by blocking the secretion of WNT ligands, are currently being evaluated in clinical trials (Table 2). A phase I trial evaluated the best dosage of ETC-159 and revealed well-tolerated AEs, including vomiting, anorexia and fatigue, dysgeusia, and constipation [34]. The lipid modification of WNTs can be enzymatically removed by the palmitoleoyl-protein carboxylesterase NOTUM, thereby inhibiting Wnt signaling [64]. The NOTUM inhibitor, ABC99, is effective in the treatment of benefiting osteopenia and osteoporosis by enhancing Wnt signaling (Table 3) [64,65]. However, no agents have been identified that mimic NOTUM to inhibit GI cancers. Alternatively, metalloprotease TIKI1 (Trabd2a) acts as a protease to cleave eight amino acid residues of WNTs, resulting in oxidized WNT oligomers with minimized receptor binding capability in frogs [66,67]. However, no agents have been identified that mimic the impact of TRABD on Wnt signaling in humans.

Antibodies against FZDs
Vantictumab (OMP-18R5) is a monoclonal antibody that binds to FZD 1, 2, 5, 7, and 8 and inhibits Wnt signal transduction [54]. A phase I trial evaluating the best dosage of vantictumab combined with nab-paclitaxel and gemcitabine in metastatic pancreatic cancer was terminated because of the increased risk of bone fracture [39]. Moreover, FZD5 has been identified as a dominant FZD receptor in RNF43-mutant pancreatic cancer cells and may be a therapeutic index [68]. However, no agents targeting FZD5 have been introduced.

Mimetic Agents Binding to FZDs
Initially, WNT5A was classified as a non-canonical Wnt family member. It activates Wnt/Ca 2+ signaling by stimulating intracellular Ca 2+ flux in zebrafish and frogs [69][70][71][72]. In 2006, Mikels et al. found that WNT5A also activates canonical Wnt signaling via FZD4 and LRP5 [73]. Intriguingly, WNT5A additionally inhibits WNT3A-induced canonical Wnt signaling via FZD2 and tyrosine-protein kinase transmembrane receptor ROR2 [73,74]. Therefore, the function of WNT5A is considered not limited to the field of Wnt signaling and is more dependent on the context of receptors. Foxy-5, a WNT5A peptide mimic, reduces the metastatic capacity of invasive breast cancer via epithelial discoidin domain-containing receptor 1 (DDR1), which decreases the motility and the invasive potential of breast epithelial cells [75][76][77]. However, whether these mechanisms are also true in GI cancers remains unknown. Foxy-5 is being evaluated in phase I-II clinical trials of metastatic CRC, but no results have been published [38] (Table 2).
On the basis of the tumorigenic role of DKK1, DKN-01, a DKK1 monoclonal antibody, was developed for cancer therapy. Four trials evaluating DKN-01 and its combination therapies are ongoing ( Table 2). A phase I trial assessing DKN-01 combined with paclitaxel in advanced esophageal and gastroesophageal junction cancer revealed that 35% of patients experienced a partial response [40,89]. Another phase I trial of the best dosage of DKN-01 combined with gemcitabine and cisplatin in advanced biliary cancer revealed that 33.3% of patients experienced a partial response [41]. Sclerostin domain-containing protein 1 can activate or inhibit Wnt signaling by mimicking WNT ligands or by competing with WNT8 for binding to LRP6, respectively [90,91]. However, no agents simulating sclerostin domain-containing protein 1 have been identified.
A phase I trial evaluated the best dosage of rosmantuzumab (OMP-131R10), a monoclonal antibody against RSPO3, for metastatic CRC; no results have been published (Table 2). BNC101, a monoclonal antibody against LGR5, demonstrated antitumor activity in multiple CRC patient-derived xenografts, but the clinical trial was terminated (Table 2) [97]. Niclosamide, a teniacide in the anthelmintic family, promotes FZD1 endocytosis, inhibiting WNT3A/β-catenin signaling in CRC and osteosarcoma and inducing LRP6 degradation in prostate and breast cancer [98][99][100]. The NIKOLO trial and NCT02687009 have been evaluating niclosamide in CRC ( Table 2). The NIKOLO trial has revealed no drug-related AEs [43].

Activating CK1 and GSK3
CK1 and GSK3 sequentially phosphorylate β-catenin to induce the ubiquitination and proteasomal degradation of β-catenin [16]. Therefore, CK1 and GSK3 activators likely reduce the level of β-catenin that translocates into the nucleus, consequently inactivating Wnt signaling. pyrvinium, a CK1 activator that binds to the C-terminal regulatory domain of its isoform CK1A1, has been introduced, but it has not been evaluated in clinical trials (Table 3) [117]. In addition, no GSK3 activators have been introduced.

Targeting Core Components of Wnt Signaling
The major caveat in Wnt targeting strategies is their detrimental side effects on normal cells in which Wnt signaling plays pivotal roles in tissue homeostasis and regeneration [3][4][5]. For example, intestinal stem cells replenish the intestinal epithelium every 3 to 4 days; this is tightly regulated by constitutively active Wnt signaling in the crypt bottom [139,140]. Inhibiting Wnt signaling disrupts intestinal homeostasis and induces the severe loss of the crypt-villi structure. Similarly, upon Wnt blockade, tissue homeostasis disruption also takes place in hair follicles, the stomach, and the hematopoietic system, where Wnt signaling is indispensable for the maintenance of stem cells and their niches [141][142][143]. Indeed, the treatment of the FZD inhibitor (vanctumab) and antagonist (ipafricept) leads to side effects, including tiredness, diarrhea, vomiting, constipation, bone metabolism disorders, and abdominal pain [36,54]. Wnt signaling is also required for tissue homeostasis and regeneration in the lungs, liver, skin, and pancreas [3][4][5]. Therefore, Wnt signaling targeting strategies need to be meticulously designed and evaluated on the basis of their specificity and efficacy, which is discussed in the next section.

Targeting Upstream vs. Downstream
Targeting the downstream effectors of Wnt signaling, e.g., β-catenin and TCF/LEF, might maximize Wnt signaling inhibition on the basis of signaling convergence into downstream gene regulation. However, targeting downstream Wnt signaling might also generate severe side effects by disrupting Wnt signaling in normal tissues. Conversely, targeting the upstream molecules of Wnt signaling, e.g., ligands and receptors, was initially considered ineffectual in cancer cells carrying mutations in Wnt signaling downstream (i.e., APC and β-catenin/CTNNB1) [93]. Intriguingly, accumulating evidence suggests that targeting Wnt signaling upstream is also effective independent of Wnt signaling downstream mutations. This evolving concept, the "β-catenin paradox", is discussed below.

Cancer-and Tissue-Specific Wnt Signaling Targeting
Targeting cancer type-or tissue-specific Wnt signaling components or modulators may overcome the side effects of Wnt signaling blockade on normal tissues. For instance, specifically targeting the constitutively active form of β-catenin mutants may be ideal. A recent study found that small-molecule enhancers of mutant β-catenin and its E3 ligase (β-TrCP) interaction potentiate the ubiquitination-mediated degradation of mutant β-catenin [119], suggesting one possible approach to targeting the mutant form of β-catenin.
There are also several promising preclinical and clinical studies evaluating antibodies against RSPOs and LGRs, Wnt signaling amplifiers [42]. Since RSPOs and LGRs are differently expressed in different tissues and cancers [144,145], targeting them might diminish normal tissue damage. LGR5 has been suggested as a cancer stem cell marker [146,147], and targeting LGR5+ cells with anti-LGR5 antibody-drug conjugates suppressed tumor growth and metastasis in a preclinical model [145,148]. Anti-LGR5 therapy and anti-RSPO3 (rosmantuzumab) are currently being evaluated in phase I trials for the treatment of metastatic CRC (NCT02726334 and NCT02005315) ( Table 2). RSPO3-LGR4-maintained Wnt signaling is essential for the stemness of acute myeloid leukemia, and the clinical-grade anti-RSPO3 antibody eradicated leukemia stem cells [149], which might be effective in GI cancer. The results of these studies indicate that blockage of cancer-or tissue-specific Wnt signaling components or regulators are viable options for GI cancer treatment.

Efficacy and Combination Therapy
An alternative method of overcoming limitations in Wnt signaling targeting strategies is to identify a safe dose that is highly effective but does not disrupt normal physiologic processes. A specific dose of LGK794 had lower severity of side effects with effective pharmacologic outcomes in a phase I clinical trial [7]. It is also noteworthy that different tissues showed different levels of Wnt signaling threshold in vivo [150], supporting the theory that localizing treatment is an alternative strategy to avoid toxicity and side effects.
In general, combination therapy is considered to result in more AEs. However, it does not always induce more AEs than does monotherapy. The incidences and degrees of AEs depend on various factors, such as the doses of single drugs, the timing of administration, the period of treatment, the supportive treatment, and the heterogeneity of the patients themselves. Thus, certain drug dose combinations may be more effective, with fewer AEs. Furthermore, monotherapy targeting one pathway does not guarantee complete anticancer activity because of multiple crosstalks and compensations by other signaling pathways. Although its efficacy may be counterbalanced by correspondingly increased toxicity, combination therapy that simultaneously targets several pathways might be more efficient.
In addition, combination therapy is the most common approach to achieving survival benefits in clinical practice, and most promising phase III Wnt targeting trials use combination therapy.
ICG-001 and PRI-724 inhibit Wnt target gene expression by antagonizing CBP, a β-catenin coactivator [133,151]. PRI-724 was effective in a phase I clinical trial of PDAC when used in combination with gemcitabine (NCT01764477). Other cases include the combination of anti-FZD antibody with chemotherapy. Vantictumab (OMP-18R5) resulted in promising outcomes in the preclinical setting [152,153] and is currently being evaluated in phase I clinical trials for multiple cancers in combination with paclitaxel [154]. Ipafricept (OMP-54F28/FZD8-Fc) is being evaluated in a phase I clinical trial to treat advanced pancreatic cancer in combination with nab-paclitaxel and gemcitabine [36]. Although antibodies against pan-Wnts or pan-FZD were not tissue-specific, their combination in advanced solid tumors had promising effects [36,154]. In addition, as a neoadjuvant therapy, Foxy-5 is currently being evaluated in phase II trials for colon cancer, as described above (NCT03883802).

β-Catenin Paradox
The β-catenin paradox was introduced on the basis of heterogeneous Wnt signaling activity in CRC cells, carrying homogenous genetic alterations in APC or β-catenin/CTNNB1 [155]. This observation was followed by discoveries of several Wnt signaling regulators and multiple crosstalks of Wnt/β-catenin signaling with MAPK and PI3K pathways [156][157][158][159][160][161][162][163][164][165]. Additionally, accumulating evidence suggests that the blockade of Wnt signaling upstream molecules suppresses tumor growth despite the presence of oncogenic mutations in Wnt signaling components [96,108,116,152,166,167], demonstrating the existence of additional regulatory modules in Wnt signaling, independent of genetic alterations. Additionally, truncated mutant APC remains partially functional to induce β-catenin protein degradation [116,167]. Moreover, the blockade of WNTs/RSPOs inhibits the growth of tumor cells that harbor APC mutations [96,116]. In line with this, Tankyrase inhibitor-stabilized AXIN protein suppresses the proliferation of CRC cells that carry constitutively active mutations in β-catenin or APC [108,110]. A recent gastric cancer mouse model study also revealed that vantictumab, the pan-FZD inhibitor, inhibits gastric adenoma growth independently of APC mutations [152]. Therefore, molecular targeting of the upstream molecules of APC and β-catenin might be promising in Wnt/β-catenin signaling-associated cancer.

Generalization of Wnt Targeting Therapy
Aberrant Wnt signaling is crucial for the potential clonal source of tumor cells and is considered an environmental and metastatic niche for tumor progression. Indeed, LGR5+ colon cancer cells are required for the formation of metastatic colonization in the liver [146]. A study using patient-derived pancreatic organoids revealed differing Wnt-niche dependency among organoids [168]. Furthermore, in a recent study of lung cancers that barely harbor Wnt mutations, Wnt signaling was shown to be required for lung cancer progression as a niche factor in a mouse lung adenocarcinoma model [169]. In that context, Wnt targeting by porcupine inhibitor, WNT794 (LGK794), revealed the suppression of lung tumor progression [169]. These results suggest that Wnt targeting therapy can be generalized to various types of non-Wnt-mutated cancers in which Wnt signaling has tumor-promoting or metastatic roles.

New Candidates for Targeting Wnt Signaling in GI Cancers
Several cancer-specific Wnt signaling regulators were identified in GI cancers. Amplification of USP21 deubiquitinase promotes pancreatic cancer cell growth and stemness via Wnt/β-catenin signaling [170]. RNF6, a CRC-upregulated E3 ligase, promotes CRC cell growth through the degradation of Tele3, a transcriptional repressor of the β-catenin/TCF4 complex [171]. Another deubiquitinase USP7 serves as a tumor-specific Wnt activator in APC-mutated CRC by promoting β-catenin deubiquitination [172]. Transcriptional coactivators of β-catenin, BCL9 and BCL9l, redundantly demonstrated CRC-specific upregulation, and their loss suppressed intestinal tumorigenesis in a mouse model [173]. BCL9 and BCL9l inhibitors were recently developed [135,174,175]. Targeting BCL9 and BCL9l has been suggested as a therapeutic approach to CRC-specific treatment. FZD5 mainly expressed in RNF43 mutated tumor cells was proposed as a molecular target for pancreatic cancer treatment [68]. Given that gut-specific knockout of FZD5 is feasible in the mouse models [176,177], it is likely that targeting of FZD5 can be used in RNF43 mutated intestinal or gastric tumors. In addition, CRC-upregulated PAF/KIAA0101 hyperactivates Wnt/β-catenin signaling and accelerates tumorigenesis in vitro and in vivo [178,179]. As an amplifier of Wnt signaling, TMEM9 hyperactivates β-catenin via APC degradation to promote intestinal and hepatic tumorigenesis [116,166]. Of note, germline deletion of Tmem9 or Paf did not display any discernible phenotypes, suggests that blockade of cancer-related Wnt signaling activators or amplifiers minimizes side effects in Wnt signaling targeting approaches.
Additionally, recent technological advances in organoids made it feasible to perform high-throughput chemical screening (clinical drugs or drug library) and genetic screening (gene knock-out or knock-down) of tumor organoids [180][181][182]. Moreover, patient-derived organoids become valuable resources to identify most effective drug(s) for precision medicine including pharmacogenomics [183][184][185]. Therefore, with the emergence of such new technology, it is anticipated that novel tumor-specific and druggable vulnerabilities related to Wnt signaling hyperactivation will be identified.

Conclusions
To date, many studies have reported the marked impact of molecular targeting of Wnt signaling on tumor suppression in preclinical settings. Despite the ongoing clinical trials, it is still imperative to overcome recurring pitfalls-catastrophic adverse effects on tissue homeostasis and regeneration. Like the sword of Damocles, targeting Wnt signaling poses a high risk but has significant potential in cancer therapy. With evolving concepts in Wnt signaling deregulation and manipulation, new and improved approaches, including molecular targeting of upstream signaling modules or cancer-specific regulators and combination therapy, are expected to open a new window of opportunity in the treatment of Wnt signaling-associated cancer.