Wnt signaling pathway is a highly conserved signaling pathway in eukaryotes and commonly divided into canonical (β-catenin dependent) and non-canonical (β-catenin independent) pathways (Polakis, 2012). Originally, many components of the Wnt signaling were identified as key mediators of patterning decisions during embryonic development by genetic screening (Mazzotta et al., 2016). In the last decade, aberrant Wnt/β-catenin pathway activation in carcinogenesis has most prominently been described for CRC. Data from the Cancer Genome Atlas (TCGA) suggests that Wnt/β-catenin pathway is activated in 93% of nonhypermutated CRC and 97% of hypermutated CRC (Li et al., 2012; Sebio et al., 2014; Voorneveld et al., 2015). The status of Wnt/β-catenin pathway is mainly related to the stability of ß-catenin controlled by the ß-catenin destruction complex that is comprised of scaffolding proteins APC, Axin and the kinases casein kinase 1 (CK1) and Glycose synthase kinase 3β (GSK3β). Absence of Wnt ligands stimuli, the cytosolic ß-catenin is phosphorylated by GSK3β, ubiquitinated by ß-TrCP²⁰⁰ and targeted for proteasomal degradation. The ligand Wnt binds to the cell surface receptor Frizzled and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to form a trimer, which recruits the Dishevelled (Dvl) protein to the plasma membrane, leading to dissociation of the destruction complex followed by cytosolic accumulation of ß-catenin. Consequently, the ß-catenin translocates to the nucleus where nuclear ß-catenin cooperates with TCF/LEF family transcription factors to active target genes such as c-myc, MMP-7, SNAIL and EGFR (Zhan et al., 2017). The activation of Wnt/β-catenin signaling is indispensable for the progression of CRC (Figure 1).

The best-known mutation of APC is the major driver of Wnt pathway in colorectal tumorigenesis which functions as a negative regulator and its importance was further highlighted by several recent studies (Hankey et al., 2018). By using the CRISPR/Cas9 technique to introduce APC mutation into human intestinal organoids, the tumorigenesis of CRC could be modeled in vivo (Drost et al., 2015; Matano et al., 2015). Moreover, these studies in human and mouse models indicated that the genotypes of APC mutations are consistent with the distinct levels of canonical Wnt pathway and these alterations are associated with characteristic tumor locations within the large intestine (Buchert et al., 2010; Christie et al., 2013). Besides APC, ring finger protein 43 (RNF43) mutations and R-spondin translocations are noted in over 18 and 9% patients with CRC respectively by preventing removal of Wnt receptor. Both RNF43 and R-spondin fusion are completely opposite to APC mutations (Schatoff et al., 2017). In addition to the well-established function of Wnt/β-catenin in CRC, there is accumulating evidence indicating that the KRAS is also an important and frequently mutant gene during colorectal cancinogenesis. Up to 40% of KRAS mutations occur in patients with CRC (Arrington et al., 2012). The discovery of small-molecule RAS inhibitors or a siRNA targeting RAS displayed anti-proliferative activity on xenografts of human CRC cell line SW480 (Song et al., 2020). The mutations of KRAS result in the hyper-activation of RAS-extracellular signal-regulated kinase (ERK) pathway involving transformation of cells and tumorigenesis. Series of studies confirmed the regulation of the RAS-ERK pathway by Wnt/β-catenin signaling and its roles, such as Axin, APC, and GSK3β, and so on (Vincan and Barker, 2008). The crosstalk of RAS and Wnt/β-catenin pathways relies on the phosphorylation of RAS mediated by GSK3β. GSK3β, a key component of the ß-catenin destruction complex, is identified as a kinase inducing phosphorylations of ß-catenin and RAS at the different sites of the threonine, and subsequently recruits the ß-TrCP E3 linker for the proteasomal degradation. Inactivation of GSK3β caused by Wnt stimuli or APC loss further leads to high concentration of cytoplasmic ß-catenin and KRAS (Lee et al., 2018a). Therefore, both mutations of APC and KRAS have a positive connection with the Wnt/β-catenin pathway in colorectal tumorigenesis (Figure 1).

Metastasis is a hallmark of advanced cancer and a major challenge to clinic treatment. Epithelial-mesenchymal transition (EMT) is a crucial process by which epithelial cells lose cell polarity and cell-cell adhesion, and closely associate with invasion and metastasis in many types of malignancies including CRC (Spaderna et al., 2006; Vu and Datta, 2017). There is a complicated network involved in the regulation of EMT, containing different signaling pathways. Many investigations indicated that aberrant activation of the canonical Wnt pathway promotes EMT-associated dedifferentiation located at the invasive front of colorectal tumors. Enhanced Wnt/β-catenin signaling in CRC cells induces the action of E-cadherin repressors SNAIL and upregulation of matrix metalloproteinases (MMP) involving CRC invasion and metastasis (Gu et al., 2016). However, inactivating mutations of APC and AXIN2 can up-regulate the canonical Wnt pathway, thereby promoting EMT. Furthermore, in vitro and in vivo experiments showed that WNT3a overexpression induces SNAIL expression and promotes invasion (Qi et al., 2014).

In addition, increasing evidences suggest that cancer stem cells (CSCs) theory underlies tumor proliferation, differentiation and metastasis. Although there is still no consensus on the concept of cancer stemness, the vital role of the Wnt pathway for the function of normal and cancer stem cells is commonly accepted (Reya and Clevers, 2005). In the intestinal crypt, Wnt/β-catenin pathway exerts a crucial role in the self-renewal of CSCs in CRC (Yan K. S. et al., 2017). R-spondin receptor Lgr5, one putative mark of intestinal stem cells, is a direct target gene of the canonical Wnt signaling cascade and able to promote tumor proliferation after APC is deleted in these cells. The experiments in mouse models showed that the Lgr5⁺ stem cells can increase additionally the population of Lgr5-positive cells and drive adenoma expanding in colon (Barker et al., 2009). CD44v6, as another CSC marker in colorectal cancer, is promoted by Wnt/β-catenin signaling and cytokines secreted from tumor-associated cells (Todaro et al., 2014). Moreover, the tumor environment has an important effect on maintenance of cancer stemness in some studies, such as hepatocyte growth factor, which is secreted by myofibroblasts in tumor micro-environment and can induce stemness features in colorectal cancer cells by improving Wnt activity (Clara et al., 2020). Recently, several studies uncovered potential relations between Wnt pathway and non-coding microRNAs in CSCs. Scientists have discovered miR-142 can inhibit stem cell-like traits by targeting APC gene whose mutations are linked to colon cancer (Isobe et al., 2014). Taken together, these findings indicate that canonical WNT signaling plays a vital role in the maintenance and expansion of CSCs in CRC.

Due to the importance of canonical Wnt/β-catenin signaling in human carcinogenic development, small molecule inhibitors targeting Wnt signaling have been developed for the treatment of CRC (Table 1). Activation of Wnt signaling through ß-catenin is a critical event in CRC progression. Porcupine (PORCN) is a membrane-bound O-acyltransferase protein which regulates Wnt ligands secretion outside the cell membrane through palmitoylation. In recent years, PORCN has emerged as a molecular target for treating Wnt-driven cancers. ETC-159, WNT974 (LGK974) and Rxc004 has been identified as potent inhibitors of Wnt secretion inhibiting ß-catenin activity in preclinical studies. ETC-159 has been proven to be remarkably efficacious in treating CRCs with R-spondin translocation in vivo and in vitro experiments (Soo and Keller, 2015). During in vitro studies in RNF43 mutant and R-spondin fusion CRC cell lines, Rxc004 could potently repress the cell proliferation by arresting cell cycle at G1/S and G2/M phase (Shah et al., 2021). IWP-2 is another inhibitor of PORCN. Experiments on organoid derived from CRC patients unveiled that IWP-2 is sensitive to the cancers with loss of function RNF43 mutations (Masaru, 2017). Pyrvinium, a FDA-approved drug, has been shown to bind to CK1α and form a degradation complex with GSK-3, APC, and Axin, resulting in the inhibition of Wnt signaling. Moreover, Pyrvinium suppresses the proliferation of CRC with mutations of APC or ß-catenin in HCT116 and SW480 cell lines (Momtazi-Borojeni et al., 2018). ICG-001, a selective inhibitor of Wnt/β-catenin pathway, binds to the CREB-binding protein (CBP) and down-regulates ß-catenin/Tcf transcription. As a consequence, ICG-001 selectively induces apoptosis in colon carcinoma cells but not in normal colonic epithelial cells, which is effective in mouse with APC mutations or nude mouse xenograft models of colon cancer. PRI-724, the second generation specific CBP/catenin antagonist for oncology, has been proved to have an acceptable safety profile in early clinical trials and is now under further clinical investigation (Bahrami et al., 2017). Windorphen (WD) is an inhibitor of Wnt/β-catenin signaling by directly targeting p300 to disrupt the association of ß-catenin with p300. These findings suggest that WD can selectively kill cancer cells with aberrant activation of Wnt signaling (Hao et al., 2013). Other small molecules, such as NSC668036 and Pen-N3, block the Wnt signaling pathway through binding to the Dishevelled (Dvl) PDZ domain and interrupting the receptor Frizzled (Fz)-Dvl interaction in colon cells (Shan et al., 2005; Zhang et al., 2009).

Some studies indicate that tankyrases (TNKS) are novel targets for Wnt inhibition by regulating stabilization of Axin and hence leading to increased ß-catenin degradation. XAV939 and JW55 have been shown to target Wnt/β-catenin pathway through inhibiting the poly-ADP-ribose polymerase (PARP) domains of TNKS in DLD-1 and SW480 cell lines in vitro (Huang et al., 2009). JW55 also reduces the growth of tumor in conditional APC mutation mice (Waaler et al., 2012). G007-LK and G244-LM are two other types of small-molecule tankyrase inhibitors (Lau et al., 2013). In particular, G007-LK has greater stability and displays favorable pharmacokinetic properties to inhibit Wnt/β-catenin signaling in APC-mutant CRC xenograft tumors (Tanaka et al., 2017; Katoh, 2018). IWR-1 is another tankyrase inhibitor which interacts with PARP enzyme (Mashima et al., 2017).

β-catenin is a key mediator of Wnt signaling, regulating the stabilization of the destruction complex and consequently intracellular ß-catenin levels. Ewan K et al. revealed that three small molecule inhibitors including CCT031374, CCT036477, and CCT070535 can block the Wnt/β-catenin signaling through reducing the level of ß-catenin without altering its stability, which is different from drugs involving inhibition of TCF-dependent transcription in SW480 cells (Ewan et al., 2010). Interaction of ß-catenin with TCF binding proteins is a crucial step in the activation of target genes in response to the activation of Wnt/β-catenin pathway. A cohort of Wnt antagonists including iCRT3, iCRT5, iCRT14, PKF115-584, PKF222-815, CGP049090, and BC21 have been demonstrated to suppress the Wnt/β-catenin signaling by breaking the association between Tcf4 and ß-catenin (Gonsalves et al., 2011; Tian et al., 2012; Yan M. et al., 2017). NC043 is an inhibitor of ß-catenin/TCF4, which decreases ß-catenin/TCF4 association without affecting the cytosol-nuclear distribution of soluble ß-catenin in vivo and in vitro (He et al., 2017).

In recent years, a small molecular KYA1797K has been identified to suppress the formation of CRCs along with the mutations of APC and KRAS via activating GSK3β and subsequently reducing the level of both ß-catenin and Ras as showed both in vitro and in vivo studies. Moreover, KYA1797K can alleviate the resistant to the EGFR-targeting therapies because of KRAS mutations (Lee et al., 2018b). Whereas, KY1022 destabilizes both ß-catenin and Ras by targeting the Wnt/β-catenin signaling in the process of metastasis involving EMT, which is different from the action of KY1797K (Cho et al., 2016). As indicated above, small molecule inhibitors targeting Wnt/β-catenin pathway exhibit promising therapeutic effects on CRC. However, to the best of our knowledge, few of these small molecules has gone into clinical trials. In the future, many scientists will make great efforts to identify more small molecules targeting Wnt/β-catenin and convert them into effective therapies.

It is well documented that uncontrolled cell proliferation is a typical feature in many types tumor development, especially in CRC. The complex balance between proliferation and apoptosis is intimately connected with tissue homeostasis (Diwanji and Bergmann, 2018) and in general, increased cell proliferation along with reduced apoptosis, drives tumor formation. It has been found that many compounds or extracts from TCMs could inhibit colorectal tumorigenesis by targeting different molecules in Wnt/β-catenin pathway. Therefore, we summarized the single-herb and formula of TCM against the different stages of CRC via Wnt/β-catenin pathway (Table 2).

CRC is often diagnosed at an advanced stage when tumor cell dissemination has already taken place and chemotherapy was one of the major methods for the treatment of CRC in the past few decades. In clinic, it is obviously clear that fluoropyrimidines, irinotecan, and oxaliplatin have been widely applied to chemotherapeutic regimens for tumors (Gustavsson et al., 2015). The recent introduction of small molecular target agents, such as anti-EGFR (cetuximab, panitumumab) and antiangiogenic molecules (bevacizumab) have led to profound improvements in the life expectancy of patients with advanced CRC (Franke et al., 2019), but with potential lethal adverse drug events and drug resistance. Therefore, it is necessary to develop new and neo-adjuvant therapies in combination with other chemotherapeutics. TCMs and their active compounds with multi-targets was reported to prevent and treat CRC patients as promising candidates, which is distinct from small molecular inhibitors that depend on single target (Yeh et al., 2020). In addition, because of relatively lower toxicity and cheaper price, TCMs can be more accepted by patients with CRC physically and psychologically.

On account of the significance of Wnt/β-catenin pathway in CRC development and metastasis, some native components of TCMs was developed as novel drugs specifically targeting this signaling pathway and are already in clinical trials (Table 3). Resveratrol is a naturally occurring polyphenol with antioxidant, which has been used in many diseases involving cancers. Recently, in vitro studies suggest that resveratrol exhibited preventative colon cancer effects and this was associated with Wnt signaling (93). In this clinical trial, patients with colon cancer were randomly provided a treatment with resveratrol, and relevant studies tested its effects directly on colon cancer and normal colonic mucosa. These results showed that resveratrol could inhibit Wnt/β-catenin signaling in the normal colonic mucosa, but not in colon cancer (Nguyen et al., 2009). Thus, resveratrol represented a potential colon cancer preventive strategy in this phase I study. Genistein is also identified to block Wnt/β-catenin signaling and has a cooperative effect with chemotherapeutic agents in lab. According to pre-clinical data, investigators found that combining genistein with standard chemotherapeutic regimens could reduce chemotherapy resistance and improve patient’s response rates (Authors Anonymous, 2021b).

Besides, small molecular weight Wnt 974, a potential inhibitor of Wnt/β-catenin signaling, has been used to assess its safety and antitumor activity in combination with chemotherapeutic agents in patients with BRAF-mutant metastatic CRC and Wnt pathway mutations (Authors Anonymous, 2021c). Nevertheless, so far, the study results have not been published. ABT-888 (veliparib) has also been used in combination with chemotherapeutic drugs to inhibit the growth of metastatic CRC in phaseⅠandⅡclinical trials (Authors Anonymous, 2021d). But it has not yet been approved by the FDA for use in this cancer. Foxy5, identified by WntResearch, can prevent migration of epithelial cancer cells by mimicking the functions of Wnt-5a and thereby play the anti-metastatic role. The safety and tolerability of Foxy-5 were established and early signs of anti-metastatic activity were evaluated in subjects with resectable colon cancer. Further, researchers have already examined the maximum tolerated dose and dose-limiting toxicity of this drug (Authors Anonymous, 2021e). Interestingly, another small molecule niclosamide, an anti-helminthic drug, has been proved to have an obviously suppressive effect on colorectal tumorigenesis by attenuating Wnt/β-catenin signaling lately. In this experiment, investigators devised a double-blind randomized controlled trial to evaluate the effect of niclosamide on patients with FAP. Unfortunately, to date, this project is still in the recruitment stage (Authors Anonymous, 2021f).