Inhibitors of beta-Catenin Signaling

ChemDiv’s Inhibitors of beta-Catenin Signaling Library contains 8,000 compounds.

β-Catenin is a fascinating protein with many important cellular and developmental functions. The roles of b-catenin are ‘classically’ defined: as an adhesion protein and as a signaling protein, transducing extracellular signals to the nucleus to modify gene expression. β -catenin has many binding partners that mediate a diverse set of cellular functions, and the protein probably acts as a ‘hub’ on which many cellular signaling networks impinge. Wnt/β-catenin signaling is a branch of a functional network that is involved in a broad range of biological systems including stem cells, embryonic development and adult organs. Deregulation of components involved in Wnt/β-catenin signaling has been implicated in a wide spectrum of diseases including a number of cancers and degenerative diseases.

ChemDiv proposes the new library of beta-catenin inhibitors/modulators. This library represents a selection of drug-like compounds aimed at modulating protein-protein interaction (PPI) of β-catenin with different proteins involved in significant physiological processes. Library has been assembled using in house structural biology insight, molecular stimulation-modeling, virtual screening of ChemDiv’s novel chemistries and medicinal chemistry filtering/ranking of the resulting hits. A representative example of a ‘druggable’ ‘hot spots’ included specific topological features of the β -catenin interaction.

1. ß-catenin: structure and functions.

β-catenin is a multipurpose and evolutionarily conserved molecule that plays a critical role by metazoans in a variety of processes in development and homeostasis. Speaking more specifically, β-catenin is an integral structural component of cadherin-based adhesive junctions as well as it a key nuclear effector of canonical in nucleus Wnt signaling.

β-catenin belongs to the catenin molecule family. These proteins were firstly discovered in 1989. The molecular weight of proteins were 102, 88 and 80 kDa, so they were named α, β and γ-catenin. The primary structure of beta-catenin consists of an NH2-terminal domain (NDT), a central region represented by 12 Arm-repeats (R1–12), and a COOH-terminal end (CTD).

Vertebrate catenins contain a large number of Arm-domains including 3 subfamilies: β-tencains, p120 and plakophilins 1 and 3. All the diversity of catenins forms a complex functional network.

An imbalance in the structural and signaling properties of β-catenin often leads to disease and unregulated growth associated with cancer and metastasis.

β-catenin have functions which are involved in:

• adhesion mechanisms
• signal transmission
• anti-inflammatory effect

2. WNT/beta-catenin passway: components, mechanism and role

Wnt/β-catenin signaling, a highly conserved evolutionary pathway, regulates natural cellular functions including proliferation, differentiation, migration, genetic stability, apoptosis, and stem cell renewal. The Wnt pathway mediates the biological processes of the canonical or non-canonical pathway, depending on the involvement of β-catenin in signal transduction. β-catenin is a component of the complex cadherin complex whose stabilization is required to activate Wnt/β-catenin signaling. The multiple aberrations in the path that occur with many WNT- directed therapies represent an area having importance for developmental therapy. The recently described role of the Wnt/β-catenin pathway in the regulation of immune cell infiltration of microenvironment tumor resumes interest because of its potential impact to rection of immune therapy. This article presents the roles of the Wnt/β-catenin pathway in cancer and current therapeutic strategies involving this pathway.

Nussе and Varmus identified components of the Wnt/β-catenin pathway in 1982 in a study of oncogenic breast tumor viral diseases (MMTV) [1]. Proviral infection at the "site of the highest degree" was thought to be the mechanism of carcinogenesis, giving the first name to the gene found along this pathway as INT1. Simultaneous work in the field of developmental biology and work with Drosophila has established the INT1 gene is a homologue of the Drosophila segment polarity gene [2]. Human INT1 has been found to be very similar to mouse INT1, thus most revealing the highly conserved nature of this pathway across species [3]. Additional screens for MMTV provirus entry sites in tumors revealed several other upregulated genes that are associated with other gene development pathways such as INT2, INT3, and INT4 [4,5,6]. For example, INT2 is a member of the fibroblast growth factor family (FGF; INT2 is the same FGF-3 protein), but INT3 is associated with the NOTCH gene family (the INT3 protein is the same homologue of the notch 4/NOTCH4 neurogenic locus) [7, 8]. Identifying the "INT" nomenclature proved inadequate and confusing, a consensus was reached on the elective name "WNT" (wingless evaluation site) for gene designations belonging to the INT1/wingless family. INT1 - now called WNT1 - became a founding member [9].

WNTs (translated products of the WNT gene) are cysteine-rich glycoproteins secreted by cells into the extracellular matrix that activate receptor- mediated signaling to cells in close proximity [10]. The WNT protein family consists of at least 19 secreted glycoproteins (with 350–400 amino acids long) that are highly conserved across species – from invertebrates to mammals [11]. WNT is

bound to the N-terminal extracellular cysteine-rich domain of the Frizzled family receptor, a member of the G protein-coupled receptor superfamily. It disrupts the β-catenin destruction complex (a tertiary complex formed by axin, adenomatous polyposis coli (APC), CK1a and GSK3β) and triggers cytoplasmic accumulation of β-catenin.

T-cell factor/lymphoid enhancer factor-1 (TCF/Lef1) is a transcription complex that mediates canonical gene transcription triggered by WNT [12, 13]. β- catenin translocates to the nucleus, where it interacts with TCF/Lef1 and activates the TCF/Lef1 transcription complex [14,15,16]. β-catenin is also localized in many subcellular locations, including the cytoplasm, where its levels are tightly controlled. β-catenin also promotes intercellular adhesion by accumulating at the sites of intercellular contact, namely, in adhesive junctions [17, 18]. Figure 1 illustrates the canonical Wnt/β-catenin signaling pathway. In addition to the classical canonical WNT-induced activation of β-catenin–TCF/Lef1 transcriptional complexes, WNT can elicit alternative responses through β-catenin-independent mechanisms, which are collectively known as non-canonical pathways [19]. In an alternative concept known as the integrated Wnt pathway, canonical and non- canonical pathways are combined and multiple inputs are integrated both at the level of binding to the Wnt receptor and at the level of the subsequent intracellular response [20].

The Wnt/β-catenin pathway is intricately involved in the pathogenesis of several types of cancer. Recent discoveries about its role in the regulation of immunomodulation have revived enthusiasm in this area.

3. Possible mutations in WNT-passway components and their influence on tumorigenesis.

According to modern data, mutations play a key role, leading to an abnormal interaction of β-catenin with the destruction complex, which in turn leads to the development of various tumors [4]. A number of studies have shown that β-catenin is a key modulator of tumor cell proliferation and survival [5]. It was also found this protein is able to support tumor growth by stimulating angiogenesis by participating in the regulation of expression of vascular endothelial growth factor [9]. β-catenin is involved in the mechanisms of tumor metastasis, enhancing the ability of cells to migrate and invade. In particular, it regulates the expression of matrix metalloproteinase genes, which products play a role in tissue remodeling, angiogenesis, proliferation, cell migration and differentiation, and apoptosis. In addition, by changing the expression of target genes in fibroblasts, macrophages, mesenchymal stem cells, endothelial cells. β-catenin influences the tumor microenvironment, which is of direct importance for the growth and progression of a malignant tumor. Impaired signaling and adhesive function β-catenin has been found in colorectal cancer, hepatocellular cancer, prostate cancer, thyroid cancer, and some other neoplasms. Involvement of the Wnt/β-catenin pathway in several malignancies:

• Colorector cancer (CRC)

The role of the Wnt/β-catenin pathway in carcinogenesis was first described in the context of APC gene mutation. APC mutations, which are typically acquired early in the pathogenesis of most colon cancers (over 80%), lead to cytosolic accumulation of β-catenin, which, in combination with TCF/Lef1, is transported to the nucleus functioning as a transcription factor and promoting cell proliferation. [21, 22]. Nuclear expression of β-catenin has been associated with more aggressive cancer biology. A study presents nuclear expression of β-catenin in 18 of 25 (72%) cases of ulcerative CRC, while it was present in only 7 of 26 (26.9%) cases of polypoid CRC (P < 0.001). This finding was independent of APC mutation and E- cadherin expression [23]. The Wnt/β-catenin pathway is also involved in cross-talk with the Hippo/YAP pathway. Consavage (et al.) showed that β-catenin/TCF4 complexes bind the DNA enhancer element in the first intron of the YAP gene, stimulating YAP expression in CRC cells, promoting carcinogenesis [24]. The Hippo–YAP signaling pathway may be an effector pathway downstream of APC, regardless of its involvement in the β-catenin degradation complex [25].

The WNT pathway has been implicated in maintaining cancer stem cell (CSC) production in colorectal cancer. In vitro data suggest that chronic “ stemness” caused by chemotherapeutic stress is associated with attenuated WNT signaling [26]. Vermeulen et al. showed that high activity of the WNT pathway was observed predominantly in tumor cells located close to stromal myofibroblasts, which are believed to secrete factors such as hepatocyte growth factor (HGF), which activate β-catenin-dependent transcription. This maintains CSC clonogenicity and restores the CSC phenotype in more differentiated tumor cells both in vitro and in vivo [27]. HGF additionally induces nuclear translocation of β-catenin via Met/β-catenin dissociation in a Wnt-independent pathway [28].

• Noncolorectal gastrointestinal cancer

β-catenin mutations have been implicated in the early stages of carcinogenesis by activating the WNT pathway in gastric and non-hepatitis- associated hepatocellular cancer [29, 30]. Cholangiocarcinoma has also been shown to be a WNT-dependent cancer that thrives on canonical activation of the WNT pathway. Activation of Wnt/β-catenin signaling by pluripotent mesenchymal stem cells recruited to the tumor site appears to play a central role in modulating the tumor microenvironment, promoting metastasis growth and resistance to chemotherapy in cholangiocarcinoma [31]. M2-polarized tumor-associated macrophages (TAMs) in the surrounding stroma are known to maintain a highly activated WNT pathway in the tumor [32].

• Desmoid tumors

Crago et al. demonstrated that mutations affecting APC and CTNNB1 (the gene encoding β-catenin) are common in desmoid tumors (111 of 117; 95%). Even true wild-type CTNNB1 tumors (determined by next-generation sequencing) can have genomic changes associated with WNT activation, such as a 6-loss/BMI1 chromosome mutation maintaining Wnt/β-catenin activation as an important pathway regulating desmoid initiation [33] .

• Breast cancer

WNT/β-catenin signaling regulates self-renewal and migration of CSCs, thereby promoting tumor growth and metastasis in breast cancer [34]. TAMS in the invasive anterior portion of primary breast tumors have a higher expression of molecules involved in WNT signaling, indicating their role in tumor cell migration, invasiveness, and metastasis [35]. In triple-negative breast cancer, β-catenin expression has been associated with poor overall survival and disease-specific survival [36]. Wnt/β-catenin upregulation has been shown to be a mechanism for resistance to PI3K inhibitors, and the use of β-catenin inhibitors can increase the sensitivity of PIK3CA mutant breast cancer to PI3K inhibition [37].

• Tumor of the adrenal cortex

Wnt/β-catenin activation has been shown to be an independent predictor of overall and disease-free survival in patients with resected primary adrenal cortical carcinoma. The presence of nuclear staining with β-catenin was significantly associated with higher tumor stage and risk score, frequent necrosis, mitosis, and other associated mutations [38]. The results obtained in animal models have shown that β-catenin can function as an adrenal oncogene, causing progressive hyperplasia of subcapsular cells, ectopic expansion of spongiocytes and subcapsular cells with subsequent dysplasia, as well as pronounced differentiation defects leading to primary hyperaldosteronism and the development of malignant features, such as uncontrolled neovascularization and local invasion when there is prolonged activation [39].

Melanoma. Wnt/β-catenin activation may play a controversial role in the metastatic spread of melanoma. β-catenin signaling reduced the migration of melanocytes and melanoma cell lines in vitro, but promoted lung metastasis in an NRAS-driven mouse model of melanoma. β-catenin appears to be a major factor in the spread of melanoma to the lymph nodes and lungs in a mouse model based on melanocyte-specific PTEN loss and BRAF (V600E) mutation. The level of β- catenin also controls tumor differentiation and regulates both MAPK/Erk and PI3K/Akt signaling. In fact, activation of the Wnt/β-catenin and AKT pathways mediates chemoresistance and increased invasion in melanoma cell lines [32].

Glioblastoma multiforme. Components of the WNT pathway are commonly overexpressed in glioblastoma multiforme (GBM) tumors. Overexpression of PLAG2 may play a role in inducing upregulation of WNT6, FZD9, and FZD2, which ultimately leads to the maintenance of stemness in GBM stem cells. β-catenin also increases the expression of the DNA repair enzyme O6- methylguanine-DNA-methyltransferase (MGMT) via Tcf/Lef binding located in the 5'-flanking regulatory region of hmMGMT. Genetic or pharmacological inhibition of Wnt/β-catenin signaling reduces MGMT expression and enhances the cytotoxic effects of temozolomide [15].

Renal cell cancer (RCC). Higher levels of β-catenin expression are associated with poor prognosis, higher staging, nodal involvement, vascular invasion, and sarcomatoid differentiation in RCC. Multilayered omics analysis, including methylome and transcriptome assays, also demonstrated a significant role for the Wnt/β-catenin signaling pathway in the pathogenesis of RCC. Mutations in GCN1L1, MED12, and CCNC, members of the CDK8 mediator complex that directly regulate β-catenin-driven transcription, were identified in 16% of RCCs [16].

Osteosarcoma. Expression of the Wnt LRP-5 receptor correlates with worse event-free survival in patients with osteosarcoma [17]. Targeting LRP5 receptor signaling via a dominant-negative form of the receptor inhibited tumor growth and metastasis and reduced the expression of markers associated with cancer cell invasiveness in animal models of osteosarcoma.

4. Regulation of WNT/beta-catenin passway: activators and inhibitors.

Interestingly, some of the natural agents exhibit antitumor activity through regulation of the canonical Wnt signaling pathway. Curcumin, isolated from the rhizome of Curcuma longa, modulates the Wnt signaling pathway and exhibits antitumor activity in melanoma, lung cancer, breast cancer, colon cancer, endothelial carcinoma, gastric carcinoma, and hepatocellular carcinoma. 3,3'- diindolylmethane (DIM), a natural compound derived from cruciferous vegetables, inhibits colon and colorectal cancer cell proliferation via the Wnt/β-catenin pathway, emerging as a promising chemopreventive or chemo-radiosensitizer to prevent tumor recurrence in cancer therapy. Formononetin isolated from red clover showed antitumor activity in breast cancer and glioma cells with high IC50 values. To achieve high potency, formononetin was modified with a coumarin unit to give derivative 10 using a molecular hybridization strategy. Analog 10 showed antiproliferative effects through the Wnt/β-catenin pathway in gastric cancer [3]. In addition, Wogonin, a major flavonoid compound isolated from Scutellaria radix, reduced intracellular levels of Wnt proteins and activated β-catenin degradation for proteasomal degradation. Gigantol, a bibenzyl compound from orchid species, has also been reported to inhibit Wnt/β-catenin signaling through down-regulation of phosphorylated LRP6 and cytosolic β-catenin in breast cancer cells. Moreover, treatment with echinacoside, a phenylethanoid glycoside from Tibetan herbs, significantly reduced tumor growth and regulation of Wnt/β-catenin signaling. In addition, nimbolide, a limonoid present in the leaves of the neem tree, simultaneously suppressed canonical Wnt signaling and induced intrinsic apoptosis in hepatocarcinoma cells. Further, isoquercitrin, a natural flavonol compound, had an inhibitory effect on Wnt/β-catenin, where the flavonoid regulated translocation of β-catenin to the downstream nucleus. It has also been noted that tryptonide, a diterpenoid epoxide present in Tripterygium wilfordii, can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or the β-catenin-related nuclear component and inducing apoptosis of Wnt-dependent cancer cells. Moreover, there is information that the fungus Exobasidium vexans and its subcomponent atranorin inhibit lung cancer cell motility and tumorigenesis by affecting β-catenin nuclear import and downstream β-catenin/LEF target genes.

5. WNT/beta-catenin passway inhibitors used in clinical trials

PORCN inhibitors. PORCN, a member of the membrane-bound O- acyltransferase (MBOAT) family, is key for the secretion of Wnt ligands [30]. Several PORCN-targeting inhibitors prevent palmitoylation of Wnt proteins in the endoplasmic reticulum, which subsequently prevents their secretion [13, 24]. Blocking WNT acylation with a PORCN inhibitor to stop WNT secretion becomes an effective treatment strategy. WNT974 (LGK974) is an orally available small molecule inhibitor that reduces the viability of epithelial ovarian cancer (EOC) cells in vitro and inhibits tumor growth in vivo. In preclinical mouse models of EOC, WNT974 shows enhanced antitumor effects when combined with paclitaxel.

Wnt/FZD antagonists. With antagonism of Wnt ligands and FZD receptors, the canonical Wnt signaling pathway was suppressed and indicated a potential strategy in cancer therapy. Ipafricept (OMP54F28; IPA) is a recombinant fusion protein comprising a cysteine-rich FZD8 domain fused to a human IgG1 Fc fragment [34]. This structure can bind directly to Wnt ligands, competing for the binding of Wnt ligands to the FZD8 receptor, thereby inhibiting Wnt-regulated processes [33].

OMP-18R5 (vantictumab) is a monoclonal antibody that targets FZD1, FZD2, FZD5, FZD7 and FZD8. OMP-18R5 blocks in mouse models the tumor growth of breast, pancreatic, colon, lung, and head and neck cancers and is being evaluated in a number of phase I trials for these tumor types.

LRP5/6 inhibitors. As a Wnt co-receptor, LRP5/6 phosphorylation promotes activation of the Wnt/β-catenin signaling pathway. The molecular complex Wnt-FZD-LRP5/6-DVL forms a structural region for interaction with axin, which disrupts the degradation of β-catenin. BMD4503-2, a fragment of quinoxaline, has been identified as a novel small molecule inhibitor of LRP5/6 interaction with sclerostin using pharmacophore-based virtual screening and in vitro assays. Compound BMD4503-2 can restore reduced activity of the Wnt/β- catenin signaling pathway by competitive binding to the LRP5/6-sclerostin complex.

DVL inhibitors. DVL is important for Wnt signaling by attracting components of the β-catenin destruction complex to the cell membrane. DVL binds to the cytoplasmic carboxyl terminus of FZD proteins via its domain. NSC668036, FJ9 and 3289-8625 are some agents that block DVL-PDZ interaction resulting in subsequent inhibition of the signal transduction pathway.

6. Conducted studies, the effectiveness of the use of inhibitors in the treatment of cancer, safety

The paper represents several therapeutic strategies which have been developed to inhibit the WNT pathway, and many drugs are in early clinical trials. Porcupine membrane-bound O-acyltransferase (PORCN) has become a molecular target of interest for the treatment of cancer caused by WNT [3]. The addition of palmitoyl groups to WNT proteins is catalyzed by PORCN, a biochemical process known as palmitoylation or S-acylation in the endoplasmic reticulum that enhances the secretion of WNT into the cytoplasm. WNT974, a PORCN inhibitor, had a cytostatic effect on ovarian cancer cells in vitro and reduced tumor growth and metastasis in in vivo models of head and neck squamous cell carcinoma [19].

A phase I/II study is ongoing evaluating WNT974 in combination with LGX818, a specific BRAF inhibitor, and cetuximab in patients with metastatic colorectal cancer carrying both WNT and BRAF mutations (NCT02278133). Another study is evaluating WNT974 in patients with metastatic head and neck squamous cell carcinoma (NCT02649530).

WNT agonists, in particular WNT-5a activation, have been shown to inhibit metastasis. Increased WNT-5a signaling suppressed endothelial tumor cell migration and invasion and inhibited metastasis in an in vivo model of breast cancer. Foxy-5 is a WNT-5a mimic hexapeptide that binds and activates the WNT-5a, Frizzled-2, and Frizzled-5 receptors [6]. Two Phase I trials evaluate Foxy-5 in advanced solid tumors (breast, colon, and prostate) with loss or decrease in WNT-5a protein expression (NCT02020291 and NCT02655952).

CWP232291 is a novel small molecule that binds Src associated with the mitosis protein 68K (Sam68). Preclinical data showed selective inhibitory activity against the WNT gene reporter and decreased expression of the target genes β- catenin, cyclin D1 and survivin. CWP232291 is being tested in a phase I clinical trial in patients with acute myeloid leukemia (NCT01398462) and relapsed or refractory myeloma (NCT02426723) [6].

Genistein, a dietary compound present in soy-based foods, is thought to mediate antitumor activity through pleiotropic mechanisms that may include inhibition of the WNT pathway [2]. Phase I and II studies investigating the clinical activity of genistein and other soy isoflavone compounds in cancer treatment and chemoprevention have been published with disappointing results.

OMP-54F28 (Ipafricept) is a fusion protein that combines the Fc domain of an immunoglobulin with the cysteine-rich domain of the frizzled family receptor 8 (Fzd8), which competes with the native Fzd8 receptor for its ligands and counteracts WNT signaling. Phase I dose escalation has shown that OMP-54F28 is well tolerated, and dose escalation groups with standard therapy are currently underway for advanced hepatocellular carcinoma (NCT02069145), pancreatic cancer (NCT02050178), and recurrent platinum-sensitive ovarian cancer (NCT02092363) [7]. Another monoclonal antibody against curled receptors, OMP-18R5, is in phase 1 clinical trials for patients with metastatic breast cancer in combination with paclitaxel. DKN-01 is a humanized monoclonal antibody (Mab) with neutralizing activity against Dickkopf-1 (Dkk-1). Convincing preclinical evidence of synergistic activity with chemotherapeutic agents is the basis for clinical trials of these agents in combination.

A number of other strategies to counteract Wnt/β-catenin-mediated carcinogenesis have been considered in preclinical studies, although they have not yet been tested in clinical trials. Inducing or stabilizing the “destruction complex” of β-catenin, thereby reducing its intracellular levels and excluding its transcriptional activity, is a promising strategy. Axin is a concentration-limiting component of the β-catenin degradation complex, and its stability is regulated by tankyrase, a key regulatory enzyme that is responsible for poly(ADP-ribosylation) anionation (parsylation) of axin and induces its proteasomal degradation [9]. Waler et al. demonstrated the antitumor effect of tankyrase inhibition in models of colorectal adenoma and adenocarcinoma. Potential side effects of this therapy include diarrhea and intestinal toxicity. Tankyrase inhibition has been shown to restore resistance to PI3K and AKT inhibitors in sphere cultures obtained from colorectal cancer patients and mouse tumor xenografts. High expression of nuclear β-catenin predicted resistance to PI3K and AKT inhibitors. Combination treatment with a WNT/tankyrase inhibitor reduced nuclear β-catenin levels, restored resistance to PI3K and AKT inhibitors, and suppressed tumor growth.

Another possible strategy to reduce signaling along the canonical Wnt/β- catenin pathway is to antagonize β-catenin/TCF mediated transcription. Since the regulation of transcription of the β-catenin/TCF complex requires several coactivators, such as cAMP response element binding protein (CREB)-binding protein (CBP), this molecule was considered a potential target for inhibition.

INT-001, a specific CBP antagonist, exhibited antitumor activity in preclinical models of tamoxifen-resistant pancreatic, colon, and breast cancer [17].

Numerous observational studies and randomized controlled trials have demonstrated a chemopreventive role for aspirin, especially in the development of colorectal neoplasia. Given the critical importance of WNT dysregulation in colorectal carcinogenesis, the interaction between aspirin and canonical WNT signaling has become the subject of research [7]. By inhibiting cyclooxygenase-2 (COX-2), aspirin reduces the availability of prostaglandin E2 (PGE2), thereby reducing WNT levels. Interestingly, the effect of aspirin on cancer cells in vitro led to the inhibition of the WNT pathway due to an increase in β-catenin ubiquitination and subsequent degradation, also independent of COX inhibition [9].

Findings

The Wnt/β-catenin pathway is increasingly recognized as a potentially important target for anticancer therapy, with several relevant inhibitors in various stages of clinical development. The growing role of immunotherapy in cancer and the recent understanding of the role of the Wnt pathway in cancer-related immune regulation may give a new dimension to this area of developmental therapy. Only tumors with enhanced Wnt/β-catenin signaling, such as colorectal cancer, have been investigated as targets for Wnt inhibition. However, due to their role in immunomodulation, Wnt inhibitors may play a wider role in cancers such as melanoma, lung and kidney cancer, where immunotherapy has come to the fore. There are some nuances that need to be considered in the exact role of Wnt signaling in immunomodulation before it can be applied in clinical practice.

THE LIST OF REFERENCES

• Cai J, Maitra A, Anders RA, Taketo MM, Pan D. Beta-catenin destruction complex - independent regulation of Hippo-YAP signaling by APC in intestinal tumorigenesis. Gene Dev. 2015;29:1493-506.
• Damsky WE, Curley DP, Santhanakrishnan M, Rosenbaum LE, Platt JT, Gould Rothberg BE, Taketo MM, Dankort D, Rimm DL, McMahon M, Bosenberg M. Beta-catenin signaling controls metastasis in Braf-activated Pten-deficient melanoma. Cancer cell. 2011; 20:741-54.
• Konsavage Jr WM, Kyler SL, Rennoll SA, Jin G, Yochum GS. Wnt/beta-catenin signaling regulates Yes-associated protein (YAP) gene expression in colorectal carcinoma cells. J Biol Chem. 2012;287:11730-9.
• Merino VF, Cho S, Liang X, Park S, Jin K, Chen Q, Pan D, Zahnow CA, Rein AR, Sukumar S. STAT3, beta-catenin, and IGF-1R inhibitors sensitize murine PIK3CA mutant breast cancer to PI3K inhibitors. Mol Oncol. 2017;11(5):552-66.
• Nusse R, Brown A, Papkoff J, Scambler P, Shackleford G, McMahon A, Moon R, Varmus H. New nomenclature for int-1 and related genes: the Wnt gene family. Cell. 1991;64:231.
• Riese J, Yu X, Munnerlyn A, Eresh S, Hsu SC, Grosschedl R, Bienz M. LEF-1, nuclear factor coordinating signaling from wingless and decapentaplegic. Cell. 1997;88:777-87.
• Roelink H, Wagenaar E, Lopes da Silva S, Nusse R. Wnt-3, a gene activated by provirus injection into mouse mammary tumors, is homologous to int-1/Wnt-1 and is commonly expressed in mouse embryos and adult human brain . Proc Natl Academy Sci U S A. 1990;87:4519-23.
• Ayadi M, Bouygues A, Ouare D, Ferrand N, Shuaib S, Thierry J.P., Muchardt S, Sabbah M, Larsen AK. Chronic chemotherapy stress promotes stem cell development and WNT/beta- catenin signaling in colorectal cancer cells: implications for the clinical use of inhibitors of WNT signaling. oncotarget. 2015;6:18518-33.
• Behrens J., Dzherkhov B.A., Wurtele M., Grimm J., Asbrand S., Wirtz R., Kuhl M., Wedlich D., Birchmeier V. Functional interaction of the axin homologue, conductin, with beta- catenin , APC and GSK3beta. The science. 1998;280: 596-9.
• Behrens J, von Kris JP, Kuhl M, Brun L, Wedlich D, Grossheadl R, Birchmeier W. Functional interaction of beta-catenin with transcription factor LEF-1. Nature. 1996; 382:638-42.
• Burton A, Sahut-Barnola I, Lambert-Lenglet C, de Jussino C, Damon-Subeyran C, Louise E, Taketo Mm, Tissier F, Bertera J., Lefrancois-Martinez A.M. and others. Constitutive activation of beta-catenin causes adrenal hyperplasia and promotes the development of adrenal cancer. Hum Mol Genet. 2010;19:1561-76.
• Boulter L., Guest R.W., Kendall T.J., Wilson D.H., Wojtacha D., Robson A.J., Ridgway R.A., Samuel K., Van Rooyen N., Barry ST. and others. WNT signaling stimulates the growth of cholangiocarcinoma and can be pharmacologically suppressed. J Clin Invest. 2015;125:1269-85.
• Brunner E, Peter O, Schweiser L, Basler K. Pangolin encodes a Lef-1 homologue that acts downstream of the armadillo to transmit the wingless signal in Drosophila. Nature. 1997;385:829–33.
• Wang V, Zhong V, Yuan Jay, Yan S, Hu S, Tong Wu, Mao Wu, Hu T, Zhang B, Song G: Involvement of WNT/beta-catenin signaling in mesenchymal stem cells promotes metastatic growth and chemoresistance of cholangiocarcinoma . Oncotarget 2015.
• van Ooyen A, Qui V, Nusse R. Nucleotide sequence of the human mammary int-1 oncogene; evolutionary conservation of coding and non-coding sequences. Embo J. 1985; 4:2905-9.
• Veeman M.T., Axelrod J.D., Moon R.T. Second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. dev cell. 2003;5:367-77.
• Vermeulen L., De Souza E.F., van der Heyden M., Cameron K., de Jong J.H., Borowski T., Tuynman J.B., Todaro M., Merz S., Rodermond H. et al. Wnt activity determines colon cancer stem cells and is regulated by the microenvironment. Natural Cell Biol. 2010;12:468– 76.
• Gallahan D, Callahan R. The mouse mammary tumor associated INT3 gene is a unique member of the NOTCH (NOTCH4) gene family. Oncogene. 1997;14:1883–90.
• Gallahan D, Callahan R. Mammary tumorigenesis in wild mice: identification of a novel int locus in mammary tumors induced by mouse mammary tumor virus (Czech II). J Virol. 1987;61:66-74.
• Gallagher CJ, Rambow F, Kumasaka M, Champeval D, Bellakosa A, Delmas W, Larue L. Beta-catenin inhibits melanocyte migration but causes melanoma metastasis. Oncogene. 2013;32:2230–8.

• Gojou S, Grabar S, Fassnacht M, Ragazon B, Lone P, Liebe R, Shokri I, Odeburg A, Royer B, Sbiera S, et al. Beta-catenin activation is associated with specific clinical and pathological characteristics and poor outcome in adrenocortical carcinoma. Clinic Cancer Res. 2011;17:328-36.
• Dixon S, Peters G. Potential oncogenic product associated with growth factors. Nature. 1987;326:833.
• Dixon S, Smith R, Brooks S, Peters G. Oncogenesis by mouse mammary tumor virus: proviral cellular gene activation in the common int-2 integration region. Cell. 1984;37:529-36.
• Clements V.M., Van J., Sarnaik A., Kim O.J., McDonald J., Fenoglio-Preizer S., Groden J., Lowy A.M. Beta-catenin mutation is a common cause of Wnt pathway activation in gastric cancer. Cancer Res. 2002;62:3503-6
• Crago A.M., Khmeletsky J., Rosenberg M., O'Connor R., Byrne S., Wilder F.G., Thorn K., Agius P., Cooke D., Sochchi N.D. et al. Near-Universal Detection of Alterations in CTNNB1 and Wnt Pathway Regulators in Desmoid-Type Fibromatosis Using Whole-Exome Sequencing and Genomic Analysis. Cancer genes of chromosomes. 2015;54:606-15.
• Laurent-Puig P, Legua P, Bluto O, Belgiti J, Franco D, Bino F, Monges G, Thomas G, Biulak-Sage P, Zucman-Rossi J. Genetic Changes, associated with hepatocellular carcinomas define different pathways of hepatocarcinogenesis. Gastroenterology. 2001;120:1763–73.
• Miller RK, Hong Ji, Munoz VA, McCree, police. Beta-catenin versus other armadillo catenins: an evaluation of our current view of canonical Wnt signaling. Prog Mol Biol Transl Sci. 2013;116:387-407.
• Molenaar M, van de Wetering M, Osterwegel M, Peterson-Maduro J, Godsave S, Korinek W, Rousse J, Destri O, Clevers H. Transcription factor XTcf-3 mediates beta-catenin- induced axis formation in Xenopus embryos. . Cell. 1996;86:391-9.
• Monga S.P., Mars V.M., Pediaditakis P., Bell A., Mule K., Bowen V.K., Van H, Zarnegar R., Michalopoulos G.K. Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after dissociation of Met-beta-catenin in hepatocytes. Cancer Res. 2002;62:2064-71.
• Maureen PJ. Beta-catenin signaling and cancer. Bioassays. 1999;21:1021-30.
• Niers S. The complex world of WNT receptor signaling. Nat Rev Mol Cell Biol. 2012; 13:767-79.
• Nusse R, van Ooyen A, Cox D, Fung YC, Varmus H. A method for proviral activation of putative mammary oncogene (int-1) on mouse chromosome 15. Nature. 1984; 307:131-6.
• Nusse R., Varmus ON. Many mouse mammary tumor virus-induced tumors contain a provirus integrated into the same region of the host genome. Cell. 1982;31:99-109.
• Ohalvo L.S., Whittaker K.A., Condelis J.S., Pollard J.W. Analysis of the expression of macrophage genes that promote tumor invasion confirms the role of Wnt signaling in mediating their activity in primary breast tumors. J Immunol. 2010;184:702-12.
• Fagotto F. Looking beyond the Wnt pathway to the deep nature of beta-catenin. Representative EMBO 2013;14:422-33.

• Huber O, Korn R, McLaughlin J, Osugi M, Hermann BG, Kemler R. Nuclear localization of beta-catenin by interaction with transcription factor LEF-1. Mechdev. 1996;59:3-10.
• Chang GB, Kim Ji, Cho SD, Park KS, Chung Ji, Lee HI, Hong YS, Nam Jay S. Blockade of Wnt/beta-catenin signaling inhibits breast cancer metastasis by inhibiting a CSC- like phenotype. sci rep. 2015;5:12465.
• Chiang J.M., Chow Yu.H., Chen T.K., Ng K.F., Lin J.L. Expression of nuclear beta- catenin is closely associated with ulcerative growth of colorectal carcinoma. Br J Cancer. 2002;86:1124–9.
• Shen T, Zhang K, Seagal GP, Wei S.: Predictive value of E-cadherin and beta-catenin in triple-negative breast cancer. Am J Clin Pathol 2016.