β-catenin signaling pathway in colorectal cancer

Biomedicine & Pharmacotherapy 110 (2019) 473–481

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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Therapeutic potential of targeting the Wnt/β-catenin signaling pathway in colorectal cancer Xiaofei Chenga,b, Xiangming Xua, Dong Chena,b, Feng Zhaoc, Weilin Wangb,

T

⁎

a

Department of Colorectal Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310003, China Key Laboratory of Precision Diagnosis and Treatment for Hepatobiliary and Pancreatic Tumor of Zhejiang Province, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China c Department of Radiation Oncology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Colorectal cancer (CRC) Therapeutics Wnt β-catenin Adenomatous polyposis coli (APC)

Aberrant Wnt/β-catenin signaling has often been reported in different cancers, particularly colorectal cancer (CRC), and this signaling cascade is central to carcinogenesis. Approximately 80% of CRC cases harbor mutations in the adenomatous polyposis coli gene, and half of the remaining cases feature mutations in the β-catenin gene that affect the Wnt/β-catenin signaling pathway. Unsurprisingly, the Wnt/β-catenin signaling pathway has potential value as a therapeutic target in the treatment of CRC. Several inhibitors of the Wnt/β-catenin signaling pathway have been developed for CRC treatment, but so far no molecular therapeutic targeting this pathway has been incorporated into oncological practice. In this review, we discuss the role of Wnt/β-catenin signaling in CRC and its potential as a target of innovative therapeutic approaches for CRC.

1. Introduction Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide [1]. Furthermore, the incidence of CRC continues to increase, probably as a result of a diet rich in red meat but low in fruits and vegetables [2]. The prognosis of CRC globally has slowly improved over the past decades. The 5-year relative survival rate has reached almost 70% in highincome countries but remains lower than 50% in low-income countries [3,4]. Early CRC can be readily cured by surgical resection, whereas the treatment of patients with distant metastasis or postsurgical recurrence remains challenging [5]. Recent advances in combination chemotherapy regimens, including FOLFIRI, XELOX/CAPOX, and FOLFOX, have significantly prolonged the survival of patients with stage III–IV CRC [6]. Cetuximab, an epidermal growth factor receptor antibody, and bevacizumab, a vascular endothelial growth factor antibody, in combination with chemotherapies can prolong survival compared with standard chemotherapies, demonstrating that targeted therapy for CRC has significant potential. However, it is important to recognize that the increased survival times afforded by targeted therapy are modest at present. It is therefore necessary to identify molecules and signaling pathways that are fundamentally essential for CRC and to develop new therapeutics targeting them. Since CRC patients have mutations in at

least one Wingless-type (Wnt) signaling pathway, Wnt signaling has emerged as a promising therapeutic target in CRC. 2. Wnt and the Wnt/β-catenin signaling pathway The Wnt gene is synonymous with the Drosophila segment polarity gene Wingless and the murine proto-oncogene integration 1, molecularly characterized from mouse tumor cells in 1982 [7]. Wnt ligands are a large family of 19 secreted glycoproteins produced in the endoplasmic reticulum that relay signals from the extracellular environment to the cell via cell surface receptors [8]. The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that participates in multiple developmental events during embryonic development and tissue homeostasis, including cell proliferation, stem cell self-renewal, and cellular differentiation [9]. The Wnt signaling pathway has historically been divided into two main categories: the canonical and noncanonical pathways. The canonical pathway is typically referred to as the β-catenin-dependent pathway, whereas the non-canonical pathway does not rely on β-catenin and is responsible for controlling cell movement during morphogenesis [10]. Both pathways have been implicated in cancer development [11,12], but the canonical pathway is most commonly recognized for its implications in CRC and will be addressed exclusively hereafter in this review.

⁎ Corresponding author at: Key Laboratory of Precision Diagnosis and Treatment for Hepatobiliary and Pancreatic Tumor of Zhejiang Province, First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou 310003, China. E-mail address: [email protected] (W. Wang).

https://doi.org/10.1016/j.biopha.2018.11.082 Received 9 August 2018; Received in revised form 5 November 2018; Accepted 19 November 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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Fig. 1. Schematic illustration of the Wnt/β-catenin signaling pathway. Left: Inactive Wnt/β-catenin pathway. In the absence of Wnt ligands, “destruction complex” phosphylates β-catenin for ubiquitination and proteolytic degradation; Right: Active β-catenin pathway and inhibitors. In the presence of Wnt ligands, formation of destruction complex is not accomplished, resulting in nuclear translocation of β-catenin. APC: Adenomatous polyposis coli; Axin: Axis inhibitor; CBP: cAMP response element-binding protein; CK1: Casein kinase 1; Dvl: Disheveled; Fzd: Frizzled; GSK3β: Glycogen synthase kinase-3β; LRP: Low density lipoprotein receptor-related protein; TCF: Transcription factor

small intestine and colon, forming invaginations, termed crypts. The intestinal stem cells (ISCs) drive a massive renewal process to replenish the loss of differentiated intestinal epithelial cells at the crypt base [19]. Wnt/β-catenin pathway activity is highest at the base of the crypt [20–22]. Suppression of Wnt signaling leads to both suspended proliferation and ISC deficiency, resulting in ablation of the intestinal epithelium [23–25]. On the contrary, the number of ISCs is increased by the potentiation of Wnt signaling [26,27]. This shows that Wnt signaling plays a crucial role in ISC self-renewal and proliferation during homeostasis. The progression of CRC from normal colonic epithelium to a malignant phenotype often develops over a period of more than 10 years [28], accompanied by numerous genetic changes closely related to the Wnt/β-catenin signaling pathway. The Wnt/β-catenin signaling pathway is an evolutionarily conserved and unique signaling pathway that regulates gene expression and cell invasion, migration, proliferation, and differentiation in the initiation and progression of CRC [29]. The APC gene, as the main cause of familial adenomatous polyposis (FAP) syndrome, has been found to be up to 80% mutated in sporadic CRCs [30]. In the majority of cases, these mutations occur in the APC gene, but additional mutations may occur in genes such as β-catenin or Axin [31,32]. Of the known Wnt signaling cascades, at least one protein of the Wnt/β-catenin signaling pathway is mutated in more than 94% of CRC cases [29]. The development of these mutations is thought to be an early event and the primary driving force in early-stage CRC. When APC is absent or dysregulated, β-catenin accumulates to high levels, translocates to the nucleus, and associates with TCF/LEF, leading to its

The canonical Wnt pathway, also called the Wnt/β-catenin signaling pathway, is complex and functions by regulating the level of β-catenin available to regulate the expression of key developmental genes; β-catenin acts as an intracellular signal transducer in transcriptional regulation and chromatin interactions [13]. In the absence of Wnt signaling, β-catenin cannot accumulate in the cytoplasm as it is degraded by a multimeric protein complex consisting of adenomatous polyposis coli (APC), axis inhibitor (Axin), protein phosphatase 2 A, glycogen synthase kinase-3 beta (GSK3β), and casein kinase 1 (CK1). Receptor occupancy inhibits the kinase activity of the “destruction complex”, involving the direct interaction of Axin with low-density lipoprotein receptor-related protein 5 or 6 and/or the actions of the Axin-binding molecule Dishevelled (DVL). Consequently, due to stabilization of βcatenin by disrupted Axin-mediated phosphorylation, β-catenin accumulates and travels to the nucleus where it engages the N-termini of DNA-binding proteins of the T-cell factor (TCF)/lymphoid enhancerbinding factor (LEF) family to mediate transactivation of target genes [14–17]. A schematic diagram of this pathway is presented in Fig. 1. A large number of Wnt target genes that affect stemness, proliferation, and differentiation have been identified. Recent studies have shown that such mutations cause aberrant expression of Wnt ligands, secreted Frizzled-related proteins (sFRP), β-catenin, and APC, which are involved in various types of human cancers, particularly CRC [18]. 3. Wnt/β-catenin signaling in CRC A single layer of epithelial cells lines the lumen of the mammalian 474

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Table 1 Wnt/β-catenin Pathway Inhibitors of CRC in Clinical Development. Therapeutics

Molecular target

Development phase

Interventions

Trial identifier

Ref.

Natural compouds Vitamin D

β-catenin

Curcumin

Tcf/β-catenin

Genistein Resveratrol

GSK3β PDE4

Clinical I I I III II I/II I I

Cholecalciferol XELOX/mFOLFOX 5-fluorouracil Irinotecan Celecoxib preoperative neoadjuvant mFOLFOX/ mFOLFOX + Avastin SRT501 grapes

NCT02603757 NCT02172651 NCT02724202 NCT01859858 NCT00295035 NCT00745134 NCT01985763 NCT00920803 NCT00578396

[57] * [58] [59] [60] * * [61] [62]

β-catenin TCF β-catenin

III Clinical Clinical

Placebo – –

NCT02607072 –

[63] [64] [64]

Porcupine Porcupine Porcupine Dvl Dvl Axin Axin Axin Axin CK1 Tcf/β-catenin Tcf/β-catenin Tcf/β-catenin Tcf/β-catenin Tcf/β-catenin Tcf/β-catenin Tcf/β-catenin Tcf/β-catenin Tcf/β-catenin β-catenin? Stat3

CBP/β-catenin CBP/β-catenin CBP/β-catenin

Discovery Discovery I Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery Discovery III II II II Discovery Discovery II

– – PDR001 – – – – – – – – – – – – – – – – Placebo Panitumumab + Cet Pembrolizumab Nivolumab – – mFOLFOX6+ Bev

– – NCT01351103 – – – – – – – – – – – – – – – – NCT01830621 NCT01776307 NCT02851004 NCT03647839 – – NCT02413853

[65] [66] [67] [68] [69] [70] [71] [72] [72] [73] [74] [74] [74] [75] [76] [77] [78] [79] [80] [81] * * * [82] [83] *

Fzd Fzd

Discovery Discovery

– –

– –

[84] [84]

Existing drugs Aspirin Celecoxib Sulindac Small molecules IWPs ETC-159 LGK 974 LMO2 NSC668036 XAV939 IWR G007-LK G244-LM Pyrvinium PKF115-584 CGP049090 PKF222-815

iCRT3/5/14 HI-B1 MSAB PNU-74654 LF3 CWP232228 BBI608

ICG-001 IC-2 PRI-724 biological agents OMP-18R5 OMP-54F28 *

https://clinicaltrials.gov.

role in the development of CRC [41]. Matrix metalloproteinase-7, another target of β-catenin/TCF signaling, is expressed in up to 90% of CRCs and is associated with unfavorable outcomes in CRC [42,43]. In recent years, Musashi1 (Msi1) was confirmed to be a Wnt target gene that regulates APC translation in both mouse intestinal epithelium and human colon cells [44]. Wnt signaling also plays an important role in promoting epithelial–mesenchymal transition (EMT) by inducing the expression of EMT-related transcription factors. EMT contributes to invasion as well as the metastatic dissemination of CRC and is associated with chemotherapeutic resistance [45]. Wnt signaling plays a critical role in CRC stem cell (CSC) self-renewal in the intestinal crypt [46]. CSCs can self-renew and give rise to the cellular bulk of the tumor. Depending on whether the recruited coactivator of β-catenin is CREB-binding protein or p300, Wnt activity will favor either differentiation or proliferation of CSCs. p300/β-catenin binding promotes CSC differentiation, whereas CREB-binding protein/β-catenin binding favors the maintenance of CSC potency [47]. In addition, the Wnt/β-catenin signaling target gene Lgr5 is a primary marker of CSCs in the intestinal crypt [48]. Other putative CSC markers, including CD44 [49], CD24 [50], CD133 [51], ABC cassette genes [52], and EpCAM [53] are in fact direct Wnt target genes. CSCs are thought to be resistant to standard chemotherapeutic agents and, therefore, therapies targeting CSCs may provide an effective means of combating

binding to DNA and subsequent transcription of genes associated with CRC development [33]. Activation can be further enhanced by tumor suppressor proteins, such as cytoplasmic Dickkopf [34] and sFRP [35], and by modifying the expression of non-canonical Wnt signaling members [36]. As negatively regulated proteins of canonical Wnt signaling, sFRPs bind to Wnt in the extracellular matrix and prevent binding to bona fide Frizzled (FZD) receptors; Dickkopf can bind to Wnt-activated lipoprotein receptor-related protein and promote its internalization. β-catenin, the key mediator of Wnt signaling, is found in multiple subcellular localizations, including adherens junctions, where it helps stabilize cell–cell contacts; the cytoplasm, where its levels are controlled by processes regulating protein stability; and the nucleus, where it is involved in transcriptional regulation and chromatin interactions. Approximately 1% of CRCs have activating mutations in the β-catenin protein [37,38], and high levels of β-catenin in the nucleus are associated with a poor prognosis in CRC patients [39]. Furthermore, nuclear β-catenin cooperates with TCF/LEF to activate the expression of Wnt/βcatenin signaling target genes. For example, the c-MYC oncogene, identified as a target gene in the Wnt signaling pathway, is overexpressed in CRC and thus plays a potential role in the development of CRC [40]. Increased transcription of cyclin D1 (a target gene in the Wnt signaling pathway) by RAS and β-catenin is likely to play an important 475

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inactivates Wnt signaling by upregulating the expression of GSK3β and E-cadherin, plays a role in reversing resistance to fluoropyrimidines and platinum compounds [91,92]. This agent has been tested in a phase I–II clinical trial in combination with FOLFOX or FOLFOX-Avastin to reduce chemotherapy resistance and improve response rates for stage IV colon or rectal neoplasms (NCT01985763). Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a phytoalexin produced by certain plants when damaged by pathogens such as bacteria or fungi. Resveratrol inhibits Wnt signaling to repress the growth of human CRC cells [93]. A clinical phase I study (NCT00920803) to assess the safety and pharmacodynamics of micronized resveratrol SRT501 in subjects with CRC and hepatic metastases showed that SRT501 was well tolerated and significantly increased cleaved caspase3, by 39%, in malignant hepatic tissue compared to tissue from placebotreated patients [94]. Another clinical phase I study (NCT00578396) to investigate the dietary influence of resveratrol-rich fresh red grapes in CRC prevention is underway. Other naturally occurring compounds that may inhibit Wnt include quercetin, green tea polyphenol epigallocathechin-3-gallate, 3,3-diindolylmethane, magnolol, indole-3carbinol, and lycopene, among others [95–99].

CRC [54]. The high frequency (> 90%) of classical gene mutations (i.e., APC truncations, β-catenin mutations) associated with Wnt/β-catenin signaling in CRC is different from other types of malignancy. Based on the properties of the above Wnt/β-catenin signaling pathways in CRC, modulators that target upstream factors may fail to suppress the aberrant gene transcription caused by these mutations. Almost all mutations in the Wnt/β-catenin signaling pathway in CRC eventually cause the accumulation of β-catenin. Therefore, inhibitors of β-catenin/TCF interactions or antagonists of transcriptional co-activators may be a potential option for CRC therapy. Recent studies showed that restoration of APC function can in turn restore crypt homeostasis and normal levels of Wnt signaling in mice, even in the presence of Tp53 and KRAS gene mutations [55]. The progression of CRC induced by the loss of APC could be effectively slowed through the elimination of β-catenin/TCF activity [56]. Not surprisingly, the relationship between aberrant regulation Wnt/β-catenin signaling and CRC suggests that Wnt/β-catenin inhibitors are a promising targeted therapy for CRC. 4. Targeting Wnt/β-catenin signaling in CRC therapy

4.2. Existing drugs

As with many solid tumors, the only fully curative treatment for CRC is surgery. However, many CRC patients are asymptomatic until the disease has progressed to a stage that precludes curative surgery. These patients may be treated with a combination of chemotherapy, radiotherapy, and biotherapy. The use of the therapeutic antibodies cetuximab or bevacizumab in combination chemotherapy, such as FOLFIRI, XELOX/CAPOX, FOLFOX, and FOLFOXIRI, has been shown to increase survival. Preliminary results indicate that combination therapy will be required to effectively treat most malignancies, including CRC. Several phase I–II trials are underway in which a Wnt antagonist or modulator is used in combination with chemotherapy agents (https:// clinicaltrials.gov/). CRC therapeutics currently under investigation that involve Wnt/β-catenin signaling include natural compounds, existing drugs, small molecules, and biological agents, as summarized in Fig. 1 and Table 1.

Non-steroidal anti-inflammatory drugs (NSAIDs) can effectively inhibit CRC recurrence in patients by approximately 40–50% [100]. A mass of evidence obtained from a range of CRC cell lines suggests that NSAIDs inhibit cancer by preventing activation of the Wnt/β-catenin signaling pathway via suppression of β-catenin-mediated transcription, leading to reduced expression of Wnt/β-catenin target genes [64,101]. However, the precise mechanism of Wnt inhibition by NSAIDs is not fully understood. Sulindac and celecoxib have been demonstrated to reduce adenomas in patients with FAP. A recent prospective study suggested that the long-term administration of aspirin specifically reduces the risk of developing CRC [102] but, at the same time, causes a dramatic increase in cardiovascular events and gastrointestinal ulcers. A multi-center randomized phase IV trial (NCT02607072) to investigate the role of aspirin in the prevention of postsurgical recurrence and metastasis in Asian CRC patients is currently in progress.

4.1. Natural compounds The active form of vitamin D, 1,25(OH)2D3, promotes binding of βcatenin to the vitamin D receptor and increases expression of E-cadherin, thus decreasing the available β-catenin molecules that can bind to TCF/LEF transcription factors [85]. Higher plasma 1,25(OH)2D3 levels are associated with a decreased risk of CRC and improved survival [86,87]. Furthermore, vitamin D deficiency is highly prevalent in patients with stage IV CRC, particularly black and female patients [88]. The Legacy Health system produced by Dr Eric Anderson is being used as a pilot study to test whether there is an association between baseline vitamin D levels, vitamin D supplementation, and survival in patients with stage III colon and stage II/III rectal cancer receiving chemotherapy (NCT02603757). A randomized, multicenter, doubleblinded phase III study (NCT03389659), exploring the effect of vitamin D3 in combination with XELOX/mFOLFOX or XELOX/mFOLFOX as first-line chemotherapy in previously untreated advanced or metastatic CRC (mCRC), is currently in progress. Curcumin (diferuloylmethane) is a natural compound derived from the rhizome of Curcuma longa, an East Indian plant, commonly called turmeric. It has been shown to possess a potent anti-proliferative effect against a variety of cancer cell lines in vitro, which stem from its ability to suppress Wnt-inhibiting activity [89,90]. The safety and effectiveness of curcumin in combination with 5-fluorouracil (NCT02724202), irinotecan (NCT01859858), celecoxib (NCT00295035), and preoperative neoadjuvant standard radiation therapy and chemotherapy (NCT00745134) in treating patients with CRC is currently being tested in phase I–III clinical trials. Genistein, a soy-derived isoflavone and phytoestrogen that

4.3. Small molecules The small-molecule inhibitors of CRC can be divided into three categories according to the Wnt/β-catenin signaling pathway: (1) Molecules that bind to the PDZ domain of DVL or affect stabilization of the “destruction complex”, (2) inhibitors of β-catenin/TCF interactions, and (3) antagonists of transcriptional co-activators (CBP, p300, etc.). 4.3.1. Molecules that bind to the PDZ domain of DVL or affect stabilization of the “destruction complex” Porcupine (PORCN), a membrane-bound O-acyltransferase, is essential for proper secretion of Wnt ligands [103]. Inhibition of PORCN can block Wnt/β-catenin signaling activities. Inhibitors of Wnt production, a novel type of PORCN-targeted Wnt antagonist, were discovered by using a cell-based Wnt/β-catenin reporter assay in the LWnt-STF cell line [65]. A recent study showed that a novel PORCN inhibitor, ETC-159, is highly effective toward genetically defined human CRCs with RSPO2/3 translocations [66]. LGK974 is a potent PORCN inhibitor that inhibits the secretion of Wnt3A and effectively suppresses the growth of murine tumors induced by mouse mammary tumor virus-driven ectopic Wnt1 expression. A phase I clinical trial (NCT01351103) of LGK974 in combination with PDR001, for treating patients with BRAF mutant CRC, is currently underway [104]. Recently, salinomycin, a novel small molecule inhibitor of LRP6, is detected to target CSCs via suppression of Wnt signaling by LRP6 degradation [105]. Formation of the DVL protein-FZD complex is triggered by Wnt 476

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vitro and in mouse cancer models [77]. Furthermore, PNU-74654 and 2,4-diamino-quinazoline were also identified as inhibitors of the TCF/βcatenin pathway [78,118]. LF3, a 4-thioureido-benzenesulfonamide derivative, inhibited TCF/β-catenin interaction robustly in a mouse xenograft model of colon cancer and blocked the self-renewal capacity of colon and head and neck cancer stem cells in a concentration-dependent manner [79]. CWP232228, a novel inhibitor of TCF/β-catenin interaction, inhibits Wnt/β-catenin signaling and depletes CD133+/ ALDH + liver cancer stem cells, thus ultimately diminishing the selfrenewal capacity of liver cancer stem cells in vitro and in vivo [80]. However, none of these small molecules have yet been identified as having true drug potential, and further preclinical studies should be performed to evaluate their efficacy in vivo and their side effects. Napabucasin (BBI608), which is currently in clinical development, is a first-in-class cancer stemness inhibitor that inhibits signal transducer and activator of transcription-3 (STAT3), kills CSCs by STAT3 inhibition, and blocks cancer metastasis and relapse [119,120]. STAT3 regulates the β-catenin expression in CSC self-renewal [121,122]. The potent and broad-spectrum anti-cancer activity of BBI-608, alone and in combination with other agents, was observed in vitro and in vivo [123]. A randomized phase III trial (NCT01830621) aimed to test BBI608 versus placebo in advanced CRC. The median overall survival was 4.4 months (95% confidence interval [CI]: 3.7–4.9) in the BBI608 group and 4.8 months (4.0–5.3) in the placebo group (adjusted hazard ratio, 1.13, 95% CI: 0.88–1·46, p = 0.34) [81]. However, in a prespecified biomarker analysis of pSTAT3-positive patients, overall survival was longer in the BBI608 group than in the placebo group. This implies that STAT3 might be an important target for the treatment of CRC with elevated pSTAT3 expression [81]. A phase Ib/II multi-center study in mCRC patients was performed to confirm RP2D and signs of anti-cancer activity of BBI608 in combination with FOLFIRI +/- bevacizumab. Disease control (CR + PR + SD) was observed in 55 out of 66 patients who underwent the RECIST evaluation (83%), with 1 CR (1.5%), 13 PR (20%), and 41 SD, and with 27 (66%) of these patients experiencing tumor regression [124]. The results suggested that BBI608 can be safely combined with FOLFIRI +/- bevacizumab, and shows encouraging signs of efficacy in pretreated mCRC patients, including those previously treated with FOLFIRI +/- bevacizumab [124]. BBI608 is under consideration for mCRC in several phase IeII clinical trials in combination with other agents, such as panitumumab/cetuximab (NCT01776307), pembrolizumab (NCT02851004), and nivolumab (NCT03647839).

ligands binding to the FZD-LRP receptor complex in the canonical Wnt signaling pathway. Upregulation of the level of DVL was reported to activate Wnt signaling in CRC [106,107]. The proto-oncogene LIMdomain only 2 (LMO2) was traditionally considered to be a pivotal transcriptional regulator in hematopoiesis and leukemia [108]. The current study demonstrated that LMO2 binds to DVL-1/2 proteins at the extracellular level to attenuate breast and colorectal tumor growth [68]. NSC668036 was found to prevent interaction of DVL with FZD through the formation of a complex with DVL by binding to its PDZ domain [69]. At present, this type of inhibitor has yet to enter the clinical trial stage. Axin binds to APC/ GSK3β/ CK1 to form a “destruction complex” and plays a pivotal role in β-catenin degradation. Loss-of-function mutations of Axin or decreased Axin expression have been found to increase the expression of the Wnt downstream targets [109]. Therefore, mutations of the Axin gene are deemed to be linked to numerous neoplasms, especially CRC [110]. In addition, it has been suggested that increased Axin protein levels can compensate for the loss of APC tumor suppressor function [111,112]. This is particularly important in CRC because approximately 80% of CRC cases harbor mutations in APC. XAV939, initially identified as selectively inhibited β-catenin-mediated transcription via Axin stabilization, stabilized Axin by inhibiting tankyrase 1 and tankyrase 2 [70,113]. SW480 CRC cells treated with XAV939 increased the level of the Axin-GSK3β complex to block the abnormally activated Wnt signaling pathway [70]. Chen et al. identified a novel type of inhibitor of the Wnt response that not only inhibits the activity of PORCN, but also abrogates the destruction of Axin proteins, which are suppressors of Wnt/β-catenin pathway activity [65,71]. Novel tankyrase small-molecule inhibitors, G007-LK and G244-LM, that reduce Wnt/β-catenin signaling by preventing poly (ADP-ribosylation)-dependent Axin degradation, display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines [72]. Pyrvinium selectively potentiates the CK1α kinase activator, which is used to treat colon cancer HCT116 and SW480 cell lines with mutation of the APC gene or β-catenin, and inhibits both Wnt signaling and proliferation [73]. GSK-3β is an important part of the “destruction complex” in Wnt/βcatenin signaling and was initially identified as a phosphorylating and inactivating agent of glycogen synthase. Small molecules of GSK-3β, such as SB-216763 [114], CHIR99021 [115], BIO(6-bromoindirubin-3′oxime) [116], or LY2090314 [117], have been identified. Unfortunately, although these small molecules of GSK-3β work well for melanoma, they have not been found to be effective for CRC.

4.3.3. Antagonists of transcriptional co-activators The region of β-catenin binding to its cofactors CBP and BCL9 is distinct from the region binding to TCF [125,126]. Therefore, small molecules that disrupt the interaction between β-catenin and cofactors (CBP and BCL9) might serve as an alternative therapeutic approach. ICG-001, an antagonist of the interaction of β-catenin with CBP, has been shown to decrease the growth of CRC cells in mice having a mutation in one allele of the APC or nude mouse SW620 of CRC [82,127,128]. Interestingly, ICG-001, by specifically binding to CBP without interacting with the highly homologous coactivator p300, can avoid triggering the switch from β-catenin/CBP to β-catenin/p300, which controls fundamental stem and/or progenitor cell-switching points [82,129]. In combination with 5-FU, IC-2, a derivative of ICG001, can stimulate tumor-suppressive effects in CRC [83]. Therapeutic agent PRI-724, a second-generation CBP/catenin-specific antagonist, was shown to increase p300/β-catenin binding and promote stem cell differentiation in a similar manner to ICG-001 [127]. PRI-724 showed an acceptable toxicity profile in treating patients with advanced pancreatic cancer in a phase I clinical trial (NCT01764477). Unfortunately, a phase II trial (NCT02413853) of combination chemotherapy comprising mFOLFOX6 and bevacizumab, with or without PRI-724, for treating patients with metastatic CRC has now been withdrawn due to supply issues with the study drug.

4.3.2. Inhibitors of β-catenin/TCF interactions There are different extracellular and intracellular abnormal Wnt signaling pathways in CRC, and these eventually affect cellular selfregulation through the TCF/β-catenin interaction. Disruption of the TCF/β-catenin interaction has been shown to effectively block target gene activation and inhibit colon cancer cell growth in vitro [21,41]. Therefore, small-molecule drugs disrupting the TCF/β-catenin interaction would have great potential for CRC treatment. Three inhibitors, PKF115-584, CGP049090, and PKF222-815, from fungal organisms were found to consistently act as potent inhibitors of TCF/β-catenin binding [74]. Interestingly, these three inhibitors might disrupt the TCF/β-catenin complex in similar ways because they share a common core chemical structure [74]. Another study showed that catenin responsive transcription inhibitor 3 (iCRT3), iCRT5, and iCRT14 also disrupted the TCF/β-catenin interaction and inhibited CRC cell Wnttargeted gene expression [75]. A recent study showed that small-molecule inhibitor HI-B1 (patent number: US9616047), rationally designed by cyclization of resveratrol, suppressed CRC growth in vitro and in vivo through disrupting the β-catenin-TCF4 interaction [76]. MSAB (methyl 3-benzoate), as a selective inhibitor of Wnt/β-catenin signaling, showed a potent and selective Wnt-dependent anti-tumor effect, specifically downregulating Wnt/β-catenin target gene CRC cells in 477

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2013 and 2018 were reported at the ASCO meeting. Studies on inhibitors of Wnt/β-catenin signaling in CRC stem cells are at very early stages and still have a long way to go. In summary, advances in understanding Wnt/β-catenin signaling mechanisms and the development of new technologies have facilitated the discovery of drugs that may provide a foundation for innovative therapeutic approaches to the treatment of CRC. Although most drugs are still at a very early stage of development, the significance of this pathway in CRC provides the ability to benefit from these new therapies.

4.4. Biological agents The monoclonal antibodies OMP-18R5 and OMP-54F28 interact with FZD receptors and block the canonical Wnt signaling induced by multiple Wnt family members [84]. As the efficacy of most of these biological agents in CRC treatment is equivocal, future studies using combination therapies are likely to result in more potent and durable inhibition of CRC. 5. Challenges in targeting the Wnt/β-catenin signaling pathway in CRC

Competing interests The authors declare that they have no competing interests.

More than 30 years after the milestone discovery of Wnt/β-catenin signaling and identification of the importance of aberrant Wnt/β-catenin in CRC, it remains unclear whether we will be able to successfully target the Wnt/β-catenin signaling cascade for CRC therapeutic purposes. Three factors have thwarted progress in this field: (1) The Wnt signaling cascade is bewilderingly complex. At least 15 receptors and 19 Wnt ligands are distributed over seven protein families in mammals [130]. In addition, crosstalk from non-canonical Wnt signaling has also been reported to modulate nuclear β-catenin accumulation. Even the simple notion that targeting aberrantly high Wnt/β-catenin signaling in cancer would be universally beneficial has been called into question. For instance, active Wnt/β-catenin signaling is associated with a lower proliferative index and a more favorable prognosis in patients with melanoma [131,132], in contrast to those with CRC. (2) The relevance of Wnt/β-catenin signaling to physiological homeostasis in the normal intestinal epithelia cannot be ignored. The binding site between β-catenin and TCF4 overlaps with the binding sites between β-catenin and APC/E-cadherin [133,134]. Binding β-catenin with E-cadherin is essential for cell adhesion and normal stem cell functions, while binding β-catenin with APC is necessary for β-catenin degradation. Given that the β-catenin binding sites among TCF4, APC, and E-cadherin are rather similar, such high selectivity is difficult to achieve. The binding sites of CBP with β-catenin and p300 with β-catenin have a similar problem. (3) A high frequency (> 90%) of classical gene mutations (i.e., APC truncations, β-catenin mutations) is associated with Wnt/β-catenin signaling in CRC. Some mutations of APC, Axin, and β-catenin occurring in CRC cause constitutively active β-catenin, which is difficult to inhibit by inhibitors of the upstream proteins of Wnt/β-catenin signaling. Together, these are the main challenges and complexities associated with developing safe and effective therapeutic agents targeting the Wnt/βcatenin signaling cascade in CRC.

Funding The study was Supported by Chinese traditional medicine Foundation of Zhejiang Province (2017ZA077), Zhejiang Province Natural Science Foundation of China (LY18H160014; LY17H160008) and Zhejiang education department scientific research project (Y201636334). Acknowledgements We gratefully acknowledge the contributions made by Jianjiang Lin and Wenbin Chen. References [1] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. Clin. (2018). [2] A.J. Cross, L.M. Ferrucci, A. Risch, B.I. Graubard, M.H. Ward, Y. Park, A.R. Hollenbeck, A. Schatzkin, R. Sinha, A large prospective study of meat consumption and colorectal cancer risk: an investigation of potential mechanisms underlying this association, Cancer Res. 70 (6) (2010) 2406–2414. [3] H. Brenner, A.M. Bouvier, R. Foschi, M. Hackl, I.K. Larsen, V. Lemmens, L. Mangone, S. Francisci, E.W. Group, Progress in colorectal cancer survival in Europe from the late 1980s to the early 21st century: the EUROCARE study, International journal of cancer, Int. J. Cancer Suppl. 131 (7) (2012) 1649–1658. [4] R.L. Siegel, K.D. Miller, S.A. Fedewa, D.J. Ahnen, R.G.S. Meester, A. Barzi, A. Jemal, Colorectal cancer statistics, 2017, CA Cancer J. Clin. 67 (3) (2017) 177–193. [5] G.J. Chang, M.A. Rodriguez-Bigas, J.M. Skibber, V.A. Moyer, Lymph node evaluation and survival after curative resection of colon cancer: systematic review, J. Natl. Cancer Inst. 99 (6) (2007) 433–441. [6] H.T. Arkenau, D. Arnold, J. Cassidy, E. Diaz-Rubio, J.Y. Douillard, H. Hochster, A. Martoni, A. Grothey, A. Hinke, W. Schmiegel, H.J. Schmoll, R. Porschen, Efficacy of oxaliplatin plus capecitabine or infusional fluorouracil/leucovorin in patients with metastatic colorectal cancer: a pooled analysis of randomized trials, J. Clin. Oncol. 26 (36) (2008) 5910–5917. [7] T. Reya, H. Clevers, Wnt signalling in stem cells and cancer, Nature 434 (7035) (2005) 843–850. [8] Y. Komiya, R. Habas, Wnt signal transduction pathways, Organogenesis 4 (2) (2008) 68–75. [9] H. Clevers, Wnt/beta-catenin signaling in development and disease, Cell 127 (3) (2006) 469–480. [10] M.T. Veeman, J.D. Axelrod, R.T. Moon, A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling, Dev. Cell 5 (3) (2003) 367–377. [11] R.H. Giles, J.H. van Es, H. Clevers, Caught up in a Wnt storm: wnt signaling in cancer, Biochim. Biophys. Acta 1653 (1) (2003) 1–24. [12] A. Kikuchi, H. Yamamoto, Tumor formation due to abnormalities in the beta-catenin-independent pathway of Wnt signaling, Cancer Sci. 99 (2) (2008) 202–208. [13] B.T. MacDonald, K. Tamai, X. He, Wnt/beta-catenin signaling: components, mechanisms, and diseases, Dev. Cell 17 (1) (2009) 9–26. [14] B. Rubinfeld, B. Souza, I. Albert, O. Muller, S.H. Chamberlain, F.R. Masiarz, S. Munemitsu, P. Polakis, Association of the APC gene product with beta-catenin, Science 262 (5140) (1993) 1731–1734. [15] L.K. Su, B. Vogelstein, K.W. Kinzler, Association of the APC tumor suppressor protein with catenins, Science 262 (5140) (1993) 1734–1737. [16] J. Behrens, J.P. von Kries, M. Kuhl, L. Bruhn, D. Wedlich, R. Grosschedl, W. Birchmeier, Functional interaction of beta-catenin with the transcription factor LEF-1, Nature 382 (6592) (1996) 638–642. [17] P. Bhanot, M. Brink, C.H. Samos, J.C. Hsieh, Y. Wang, J.P. Macke, D. Andrew, J. Nathans, R. Nusse, A new member of the frizzled family from Drosophila functions as a Wingless receptor, Nature 382 (6588) (1996) 225–230. [18] L. Vermeulen, E.M.F. De Sousa, M. van der Heijden, K. Cameron, J.H. de Jong,

6. Summary Despite increasing attention recently being paid to the Wnt/β-catenin signaling pathway involved in CRC, this pathway remains difficult to target safely and effectively. However, the spectra of mutations found in CRC would have provided sufficient clues as to how to assemble our current inventory of Wnt/β-catenin signaling components with the help of genomic sequencing. Future research regarding Wnt/βcatenin signaling in CRC should focus on (1) achieving a deeper understanding of crosstalk among these pathways (e.g., AKT/PI3K, NOTCH, mTOR pathways); (2) optimizing and evaluating Wnt/β-catenin inhibitors, while also being highly selective to avoid unnecessary side effects; (3) more rational selection of patients who might benefit from therapy; and (4) identifying additional inhibitors downstream of the Wnt/β-catenin signaling pathway. APC mutations in CRC are a long-standing challenge. CK1α kinase activator and TNKS enzyme inhibitors appear to selectively block βcatenin activity, even in APC mutation mice. These have great potential in CRC, although research into these types of inhibitors is still at an early stage. BBI608, another potential inhibitor, in combination with chemotherapy and other targeted drugs, showed great potential in advanced CRC. The results of a clinical trial of BBI608 conducted between 478

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