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Aop: 298

AOP Title
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Wnt ligand stimulation and Wnt signalling activation lead to cancer malignancy

Short name:
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Wnt activation leading to cancer malignancy

Graphical Representation
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Click to download graphical representation template

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Authors
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Shihori Tanabe, Akihiko Hirose, Takashi Yamada

Point of Contact
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Shihori Tanabe (email point of contact)

Contributors

Shihori Tanabe

Status
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Author status	OECD status	OECD project	SAAOP status
Under development: Not open for comment. Do not cite

This AOP was last modified on January 19, 2020 20:21
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Revision dates for related pages

Page	Revision Date/Time
Wnt ligand stimulation	May 29, 2019 21:13
Frizzled activation	May 29, 2019 21:17
GSK3beta inactivation	May 29, 2019 21:28
β-catenin activation	May 29, 2019 21:39
Snail, Zeb, Twist activation	May 29, 2019 21:44
Epithelial-mesenchymal transition	September 03, 2019 03:00
Cancer Malignancy	May 29, 2019 22:01
Increased, Reactive oxygen species	November 27, 2017 13:15
Oxidative Stress	December 06, 2018 11:35
Wnt ligand stimulation leads to Frizzled activation	May 29, 2019 22:07
Frizzled activation leads to GSK3beta inactivation	May 29, 2019 22:12
Increased, Reactive oxygen species leads to β-catenin activation	January 19, 2020 21:12
GSK3beta inactivation leads to β-catenin activation	May 29, 2019 22:18
β-catenin activation leads to Snail, Zeb, Twist activation	May 29, 2019 22:23
Snail, Zeb, Twist activation leads to Epithelial-mesenchymal transition	November 19, 2019 20:27
Epithelial-mesenchymal transition leads to Cancer Malignancy	October 08, 2019 02:06
Increased, Reactive oxygen species leads to Oxidative Stress	December 03, 2019 20:49
Wnt	May 29, 2019 03:59
WNT2	May 29, 2019 03:59

Abstract
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Wnt (Wingless and INT-1) ligands stimulate Frizzled receptors and activate Wnt signaling leading to cancer malignancy. This AOP workplan entitled “Wnt ligand stimulation and Wnt signaling activation lead to cancer malignancy” is suitable for the AOP programme in terms of revealing cancer signaling with the molecular signaling cascades induced by Wnt ligands. The current AOP includes MIE as Wnt ligand stimulation, KE1 as Frizzled activation, KE2 as GSK3beta inactivation, KE3 as beta-catenin activation, KE4 as Snail, Zeb, Twist1 activation, KE5 as epithelial-mesenchymal transition (EMT) and AO as cancer malignancy. The current AOP would be associated to the prediction of the cancer malignancy, which would be the regulatory toxicological endpoint, by chemicals or molecules activating Wnt signaling. The Wnt/beta-catenin signaling is well understood in terms of development and cancer. The relationship between EMT and cancer malignancy has recently been investigated in many research fields such as molecular signatures of cells.

Background (optional)
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Summary of the AOP
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Events: Molecular Initiating Events (MIE)
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Key Events (KE)
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Adverse Outcomes (AO)
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Sequence	Type	Event ID	Title	Short name

MIE	1115	Increased, Reactive oxygen species	Increased, Reactive oxygen species
MIE	1645	Wnt ligand stimulation	Wnt ligand stimulation
MIE	1392	Oxidative Stress	Oxidative Stress

KE	1646	Frizzled activation	Frizzled activation
KE	1647	GSK3beta inactivation	GSK3beta inactivation
KE	1648	β-catenin activation	β-catenin activation
KE	1649	Snail, Zeb, Twist activation	Snail, Zeb, Twist activation
KE	1650	Epithelial-mesenchymal transition	Epithelial-mesenchymal transition

	AO	1651	Cancer Malignancy	Cancer Malignancy

Relationships Between Two Key Events
(Including MIEs and AOs)
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Title	Adjacency	Evidence	Quantitative Understanding

Wnt ligand stimulation leads to Frizzled activation	adjacent	High	Moderate
Frizzled activation leads to GSK3beta inactivation	adjacent	High	Moderate
GSK3beta inactivation leads to β-catenin activation	adjacent	High	Moderate
β-catenin activation leads to Snail, Zeb, Twist activation	adjacent	High	Moderate
Snail, Zeb, Twist activation leads to Epithelial-mesenchymal transition	adjacent	High	Moderate
Epithelial-mesenchymal transition leads to Cancer Malignancy	adjacent	High	Low
Increased, Reactive oxygen species leads to Oxidative Stress	adjacent	High	Moderate

Increased, Reactive oxygen species leads to β-catenin activation	non-adjacent	Moderate	Moderate

Network View
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Stressors
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Name	Evidence Term
Wnt	High
WNT2	High

Life Stage Applicability
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Life stage	Evidence
All life stages	High

Taxonomic Applicability
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Term	Scientific Term	Evidence	Link
Homo sapiens	Homo sapiens	High	NCBI

Sex Applicability
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Sex	Evidence
Unspecific	High

Overall Assessment of the AOP
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Attached file:

1. Support for Biological Plausibility of KERs
MIE => KE1: Wnt ligand stimulation leads to Frizzled activation	Biological Plausibility of the MIE => KE1 is high. Ratioinale: Upon the stimulation with Wnt ligand, Wnt ligand binds to FZD and form the complex with LRP5/6 (MacDonald et al., 2009).
KE1 => KE2: Frizzled activation leads to GSK3beta inactivation	Biological Plausibility of the KE1 => KE2 is high. Ratioinale: Upon Wnt ligand stimulation, FZD is activated and Axin is recruited to the phosphorylated tail of LRP dimerized with the activated FZD, the seven-transmembrane receptor, followed by GSK3beta inactivation to prevent beta-catenin degradation (Aberle, Bauer, Stappert, Kispert, & Kemler, 1997) (Clevers & Nusse, 2012).
KE2 => KE3: GSK3beta inactivation leads to β-catenin activation	Biological Plausibility of the KE2 => KE3 is high. Ratioinale: GSK3beta recruitment to LRP6 leads to form un-phosphorylated beta-catenin inducing the stabilization and translocation of the beta-catenin (MacDonald, Tamai, & He, 2009). Stabilized beta-catenin accumulates in cytosol and translocates into the nucleus leading to beta-catenin activation (MacDonald et al., 2009).
KE3 => KE4: β-catenin activation leads to Snail, Zeb, Twist activation	Biological Plausibility of the KE3 => KE4 is high. Ratioinale: The treatment of human gastric cancer cells with INC280, which inhibits c-MET overexpressed in diffuse-type gastric cancer with poor prognosis, shows downregulation in beta-catenin and Snail expression, (Sohn et al., 2019). The treatment with garcinol, a polyisoprenylated benzophenone derivative that is obtained from Garcinia indica extract, induced ZEB1 and ZEB2 down-regulation, increase in phosphorylated beta-catenin and decrease in nuclear beta-catenin in human breast cancer cells (Ahmad et al., 2012). Sortilin, a member of the Vps10p sorting receptor family which is highly expressed in high-glade malignant glioma, positively regulates GSK-3beta/beta-catenin/Twist signaling pathway in glioblastoma (W. Yang et al., 2019).
KE4 => KE5: Snail, Zeb, Twist activation leads to Epithelial-mesenchymal transition, induced	Biological Plausibility of the KE4 => KE5 is high. Ratioinale: The transcription factors such as Snail, Zeb and Twist inhibit the CDH1 expression through their binding towards the promoter of CDH1, which leads to inhibition of cell adhesion and EMT (Diaz et al., 2014).
KE5 => AO: Epithelial-mesenchymal transition, induced leads to Cancer Malignancy	Biological Plausibility of the KE5 => AO is high. Ratioinale: The morphological and physiological changes associated with EMT are involved in invasiveness and drug resistance (Shibue & Weinberg, 2017). The EMT-activated particular carcinoma cells in primary tumors invade the surrounding stroma (Shibue & Weinberg, 2017). The EMT –activated carcinoma cells interact with the surrounding extracellular matrix protein to induce focal adhesion kinase and extracellular signal-related kinase activation, followed by the transforming growth factor beta (TGFbeta) and canonical and/or noncanonical Wnt pathways to induce cancer stem cell (CSC) properties which contribute to the drug resistance (Shibue & Weinberg, 2017). EMT-associated down-regulation of multiple apoptotic signaling pathways induce drug efflux and slow cell proliferation to induce the general resistance of carcinoma cells to anti-cancer drugs (Shibue & Weinberg, 2017). Snail, an EMT-related transcription factor, induces the expression of the AXL receptor tyrosine kinase, which enables the cancer cells to survive by the activation of AXL signaling triggered by the binding of its ligand growth arrest-specific protein 6 (GAS6)(Shibue & Weinberg, 2017). The EMT-activated cells evade the lethal effect of cytotoxic T cells, which include the elevated expression of programmed cell death 1 ligand (PD-L1) which binds to the programmed cell death protein 1 (PD-1) inhibitory immune-checkpoint receptor on the cell surface of cytotoxic T cells (Shibue & Weinberg, 2017).
2. Support for essentiality of KEs
KE5: Epithelial-mesenchymal transition	Essentiality of the KE5 is moderate. Rationale for Essentiality of KEs in the AOP:
3. Empirical support for KERs
MIE => KE1: Wnt ligand stimulation leads to Frizzled activation	Empirical Support of the MIE => KE1 is high. Ratioinale: Dishevelled (DVL), a positive regulator of Wnt signaling, form the complex with FZD and lead to trigger the Wnt signaling together with Wnt coreceptor low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (Clevers & Nusse, 2012; X. Jiang et al., 2015). Wnt binds to FZD and activate the Wnt signaling (Clevers & Nusse, 2012; Janda et al., 2012; Nile et al., 2017). Wnt binding towards FZD induce the formation of the protein complex with LRP5/6 and DVL, leading to the down-stream signaling activation (Clevers & Nusse, 2012).
KE1 => KE2: Frizzled activation leads to GSK3beta inactivation	Empirical Support of the KE1 => KE2 is high. Ratioinale: ・ The ligand-stimulated FZD induces the regulation of the phosphorylation by GSK3beta to inactivate GSK3beta which phosphorylates beta-catenin (Clevers & Nusse, 2012). ・ The binding of Axin to the cytoplasmic tail of LRP5 bound to Wnt is crucial for the Wnt signaling pathway regulation and GSK3 beta inactivation in Wnt/beta-catenin signaling (Mao et al., 2001). ・ Axin-LRP6 binding is the important step for the phosphorylation of the LRP5/6 tail by GSK3 beta which phosphorylates the serine in the PPPSP motif found in beta-catenin, Axin, APC (He, Semenov, Tamai, & Zeng, 2004; Tamai et al., 2004; Zeng et al., 2005). ・ Wnt3a induces phosphorylation of LRP6 leading to beta-catenin activation, while beta-catenin is not activated in FZD-inhibited cells (Zeng et al., 2008).
KE2 => KE3: GSK3beta inactivation leads to β-catenin activation	Empirical Support of the KE2 => KE3 is high. Ratioinale: GSK3beta inactivation induces the beta-catenin stabilization (Pez et al., 2013). GSK3beta inactivation induces beta-catenin translocation into the nucleus (MacDonald et al., 2009; Pez et al., 2013). WNT2 knockdown induces the accumulation of GSK3beta in the cytoplasm and reduced the expression of beta-catenin, which WNT2 overexpression reduces the expression of GSK3beta in the cytoplasm and induces beta-catenin translocation into the nucleus (Wang, Li, & Kidder, 2010). WNT2 siRNA knockdown increases the GSK3beta expression and decreases beta-catenin expression, and WNT2 overexpression reduces the GSK3beta and increases beta-catenin in granulosa cells in Mus musculus (Wang et al., 2010).
KE3 => KE4: β-catenin activation leads to Snail, Zeb, Twist activation	Empirical Support of the KE3 => KE4 is high. Ratioinale: The inhibition of c-MET, which is overexpressed in diffuse-type gastric cancer, induced increase in phosphorylated beta-catenin, decrease in beta-catenin and Snail (Sohn et al., 2019). The garcinol, that has anti-cancer effect, increases phosphorylated beta-catenin, decreases beta-catenin and ZEB1/ZEB2, and inhibit EMT (Ahmad et al., 2012). The inhibition of sortilin by AF38469 (a sortilin inhibitor) or small interference RNA (siRNA) results in decrease in beta-catenin and Twist expression in human glioblastoma cells (W. Yang et al., 2019).
KE4 => KE5: Snail, Zeb, Twist activation leads to Epithelial-mesenchymal transition, induced	Empirical Support of the KE4 => KE5 is high. Ratioinale: Histone deacetylase inhibitors affect on EMT-related transcription factors including ZEB, Twist and Snail (Wawruszak et al., 2019). Snail and Zeb induces EMT and suppress E-cadherin (CDH1) (Batlle et al., 2000; Diaz et al., 2014; Peinado, Olmeda, & Cano, 2007).
KE5 => AO: Epithelial-mesenchymal transition, induced leads to Cancer Malignancy	Empirical Support of the KE5 => AO is high. Ratioinale: Slug/Snai2, a ces-1-related zinc finger transcription factor gene, confers resistance to p53-mediated apoptosis of hematopoietic progenitors by repressing PUMA (also known as BBC3, encoding Bcl-2-binding component 3) (Inukai et al., 1999; Shibue & Weinberg, 2017; W.-S. Wu et al., 2005). EMT activation induces the expression of multiple members of the ATP-binding cassette (ABC) transporter family, which results in the resistant to doxorubicin (Saxena, Stephens, Pathak, & Rangarajan, 2011; Shibue & Weinberg, 2017) TGFbeta-1 induced EMT results in the acquisition of cancer stem cell (CSC) like properties (Pirozzi et al., 2011; Shibue & Weinberg, 2017). Snail-induced EMT induces the cancer metastasis and resistance to dendritic cell-mediated immunotherapy (Kudo-Saito, Shirako, Takeuchi, & Kawakami, 2009). Zinc finger E-box-binding homeobox (ZEB1)-induced EMT results in the relief of miR-200-mediated repression of programmed cell death 1 ligand (PD-L1) expression, a major inhibitory ligand for the programmed cell death protein (PD-1) immune-checkpoint protein on CD8+ cytotoxic T lymphocyte (CTL), subsequently the CD8+ T cell immunosuppression and metastasis (Chen et al., 2014).

Domain of Applicability
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Homo sapiens

Essentiality of the Key Events
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Wnt signaling is involved in cancer malignancy (Tanabe, 2018).

Key Events Frizzled activation, GSK3beta, beta-catenin activation and Zeb, Twist and Snail transcription factors are essential to this AOP.

Upon stimulation with Wnt ligand to Frizzled receptor, Wnt/beta-catenin signaling is activated. Wnt/beta-catenin consists of GSK3 beta inactivation, beta-catenin activation and up-regulation of transcription factors such as Zeb, Twist and Snail. The transcription factors Zeb, Twist and Snail relate to the activation of EMT-related genes. EMT is regulated with various gene networks (S. Tanabe, 2015).

Evidence Assessment
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The Wnt signaling promotes EMT and cancer malignancy in colorectal cancer (Lazarova & Bordonaro, 2017). Although the potential pathways other than Wnt signaling exist in EMT induction and the mechanism underlaid cancer malignancy, Wnt signaling is one of the main pathways to induce EMT and cancer malignancy (Polakis, 2012).

Quantitative Understanding
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Wnt signaling activates the CSCs to promote cancer malignancy (Reya & Clevers, 2005). The responses between each KEs from Frizzled activation and GSK3beta inactivation, beta-catenin activation, Snail, Zeb, Twist activation are dose-dependently related. The quantification of EMT and cancer malignancy would require the further investigation.

Considerations for Potential Applications of the AOP (optional)
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AOP entitled “Wnt ligand stimulation and Wnt signaling activation lead to cancer malignancy” might be utilized for the development and risk assessment of anti-cancer drugs. EMT is involved in the acquisition of drug resistance, which is one of the critical features of cancer malignancy. The assessment of EMT would be the potential prediction of the adverse effects of anti-cancer drugs.