AOP ID and Title:

Short Title: Increase in ROS leading to human treatment-resistant gastric cancer

Authors

Shihori Tanabe¹⁾, Sabina Quader²⁾, Ryuichi Ono³⁾, Horacio Cabral⁴⁾, Ed Perkins⁵⁾

¹Division of Risk Assessment, Center for Biological Safety and Research, National Institute of Health Sciences, Japan

²Innovation Centre of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, Japan

³Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, National Institute of Health Sciences, Japan

⁴Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Japan

⁵Environmental Laboratory, US Army Engineer Research and Development Center, United States

Status

Abstract

This AOP entitled “Increase in reactive oxygen species (ROS) leading to human treatment-resistant gastric cancer” consists of MIE as KE1115 “Increase, ROS” followed by KE1 as KE1753 “Chronic ROS,” KE2 as KE1754 “porcupine-induced Wnt secretion and Wnt signaling activation,” KE3 as KE1755 “beta-catenin activation,” KE4 as KE1457 “epithelial-mesenchymal transition (EMT),” and AO as KE1651 “human treatment-resistant gastric cancer.” ROS has multiple roles in disease, such as the development and progression of cancer or apoptotic induction, causing anti-tumor effects. In this AOP, we focus on sustained levels of chronic reactive oxygen species (ROS) in inducing therapy resistance in human gastric cancer. Epithelial-mesenchymal transition (EMT), a cellular phenotypic change from epithelial to mesenchymal-like features, demonstrates cancer stem cell-like characteristics in human gastric cancer. EMT is induced by Wnt/beta-catenin signaling, providing the rationale to have Wnt secretion and beta-catenin activation as KE1 and KE2 on the AOP, respectively. The AOP might be useful for the development of anti-cancer drugs or the prediction of adverse effects of therapeutics, which are of possible regulatory relevance.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Wnt

WNT induces EMT (J. Zhang, Tian, & Xing, 2016). 

WNT2

WNT2 induces EMT in cervical cancer (Zhou et al., 2016).

Porcupine

Porcupine palmitoleates Wnt and facilitates the secretion of the Wnt ligand (Yu & Virshup, 2014) .

Wntless

Wntless binds to and transport Wnt to the plasma membrane leading to the secretion of Wnt ligand (Yu & Virshup, 2014).

ferric nitrilotriacetate

Carcinogenic iron(III)-nitrilotriacetate induces reactive oxygen species production via trasfer of an electron to molecular oxygen to form reactive oxygen species [Tsuchiya K, Akai K, Tokumura A, Abe S, Tamaki T, Takiguchi Y, Fukuzawa K. Biochim Biophys Acta. 2005 Aug 30;1725(1):111-9. doi: 10.1016/j.bbagen.2005.05.001, Akai K, Tsuchiya K, Tokumura A, Kogure K, Ueno S, Shibata A, Tamaki T, Fukuzawa K. Free Radic Res. 2004 Sep;38(9):951-62. doi: 10.1080/1071576042000261945.].

 

Overall Assessment of the AOP

1. Support for Biological Plausibility of KERs
MIE => KE1: Increase, ROS leads to porcupine-induced Wnt secretion and Wnt signaling activation	Biological Plausibility of the MIE => KE1 is moderate. Rationale: Sustained ROS caused by/causes DNA damage, which will alter several signaling pathways including Wnt signaling. ROS stimulate inflammatory factor production and Wnt/b-catenin signaling. Macrophages accumulate into injured tissue to recover the tissue damage, which may be followed by porcupine-induced Wnt secretion (Vallée & Lecarpentier, 2018).
KE1 => KE2: Porcupine-induced Wnt secretion and Wnt signaling activation leads to beta-catenin activation	Biological Plausibility of the KE1 => KE2 is moderate. Rationale: Secreted Wnt ligand stimulates Wnt/b-catenin signaling, in which b-catenin is activated. Wnt ligand binds to Frizzled receptor, which leads to GSK3b inactivation. GSK3b inactivation leads to b-catenin dephosphorylation, which avoids the ubiquitination of the b-catenin and stabilize the b-catenin (Clevers & Nusse, 2012).
KE2 => KE3: beta-catenin activation leads to Epithelial-mesenchymal transition (EMT)	Biological Plausibility of the KE2 => KE3 is moderate. Rationale: b-catenin activation, of which mechanism include the stabilization of the dephosphorylated b -catenin and translocation of b-catenin into the nucleus, induce the formation of b-catenin-TCF complex and transcription of transcription factors such as Snail, Zeb and Twist (Clevers & Nusse, 2012) (Ahmad et al., 2012; Pearlman et al., 2017; Sohn et al., 2019; Yang W et al., 2019). EMT-related transcription factors including Snail, ZEB and Twist are up-regulated in cancer cells (Diaz et al., 2014). The transcription factors such as Snail, ZEB and Twist bind to E-cadherin (CDH1) promoter and inhibit the CDH1 transcription via the consensus E-boxes (5’-CACCTG-3’ or 5’-CAGGTG-3’), which leads to EMT (Diaz et al., 2014).
KE3 => AO: Epithelial-mesenchymal transition (EMT) leads to treatment-resistant gastric cancer	Biological Plausibility of the KE3 => AO is moderate. Rationale: Some population of the cells exhibiting EMT demonstrates the feature of cancer stem cells (CSCs), which are related to cancer malignancy (Shibue & Weinberg, 2017; Tanabe, 2015a, 2015b; Tanabe et al., 2015). EMT phenomenon is related to cancer metastasis and cancer therapy resistance (Smith & Bhowmick, 2016; Tanabe, 2013). Increase expression of enzymes that degrade the extracellular matrix components and the decrease in adhesion to the basement membrane in EMT induce the cell escape from the basement membrane and metastasis (Smith & Bhowmick, 2016). Morphological changes observed during EMT is associated with therapy resistance (Smith & Bhowmick, 2016).
2. Support for essentiality of KEs
MIE: Increase, ROS	Essentiality of the MIE is high. Rationale for Essentiality of the MIE in the AOP: Sustained ROS contributes to the initiation and development of human gastric cancer (Gu et al., 2018).
KE1: Porcupine-induced Wnt secretion and Wnt signaling activation	Essentiality of the KE1 is moderate. Rationale for Essentiality of the KE1 in the AOP: The Wnt signaling activation is essential for the subsequent b-catenin activation and cancer resistance (Tanabe, 2018).
KE2: beta-catenin activation	Essentiality of the KE2 is moderate. Rationale for Essentiality of the KE2 in the AOP: b-catenin activation is essential for the Wnt-induced cancer resistance (Tanabe, 2018).
KE3: Epithelial-mesenchymal transition (EMT)	Essentiality of the KE3 is moderate. Rationale for Essentiality of the KE3 in the AOP: EMT is essential for the Wnt-induced cancer promotion and acquisition of resistance to anti-cancer drug (Tanabe, 2018, Tanabe et al, 2020a, 2020b, 2023).

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

The AOP298 applies to Homo sapiens (human), all life stages, and both male and female.

 

Essentiality of the Key Events

Sustained ROS contributes to the initiation and development of human gastric cancer (Gu et al., 2018).

Wnt signaling is involved in cancer malignancy (Tanabe, 2018).

Upon stimulation with Wnt ligand to the 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 (Tanabe, 2015c, Tanabe et al, 2020a, 2020b).

Event	Direct Evidence	Indirect Evidence	No experimental evidence
MIE: Increase, ROS	***	*
KE1: Increase, porcupine-induced Wnt secretion and Wnt signaling activation		*	**
KE2: beta-catenin activation		*	**
KE3: Epithelial-Mesenchymal Transition		*	**

Weight of Evidence Summary

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).

MIE => KE1: Increases in cellular ROS leads to porcupine-induced Wnt secretion and Wnt signaling activation	Empirical Support of the MIE => KE1 is moderate. Rationale: Production of ROS and DNA double-strand break cause the tissue damages (Gao et al., 2019). ROS-related signaling induces Wnt/b-catenin pathway activation (Pérez et al., 2017).
KE1 => KE2: Porcupine-induced Wnt secretion and Wnt signaling activation leads to beta-catenin activation	Empirical Support of the KE1 => KE2 is moderate. Rationale: 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; 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 including beta-catenin (Clevers & Nusse, 2012).
KE2 => KE3: beta-catenin activation leads to Epithelial-mesenchymal transition (EMT)	Empirical Support of the KE2 => KE3 is moderate. Rationale: The inhibition of c-MET, which is overexpressed in diffuse-type gastric cancer, induced increase in phosphorylated b-catenin, decrease in b-catenin and Snail (Sohn et al., 2019). The garcinol, that has anti-cancer effect, increases phosphorylated beta-catenin, decreases b-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 b-catenin and Twist expression in human glioblastoma cells (Yang W. et al., 2019). Histone deacetylase inhibitors effect 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 et al., 2007).
KE3 => AO: Epithelial-mesenchymal transition (EMT) leads to Treatment-resistant gastric cancer	Empirical Support of the KE3 => AO is moderate. Rationale: EMT activation induces the expression of multiple members of the ATP-binding cassette (ABC) transporter family, which results in doxorubicin resistance (Saxena et al., 2011; Shibue & Weinberg, 2017). TGFb-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 et al., 2009). Zinc finger E-box-binding homeobox (ZEB1)-induced EMT results in 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).

Quantitative Consideration

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

Considerations for Potential Applications of the AOP (optional)

AOP entitled “Increase in reactive oxygen species (ROS) and chronic ROS leading to human treatment-resistant gastric cancer” 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 the activity of the EMT network would serve as a prediction of the adverse effects of or responsiveness to anti-cancer drugs (Tanabe et al., 2023). The detection methods for increases in ROS in this AOP have future regulatory potentials to assess the human health effects of radiation or ROS-related diseases. The detection methods for human treatment-resistant gastric cancer have future regulatory potentials to diagnose the diseases.

References

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Appendix 1

List of MIEs in this AOP

Event: 1115: Increase, Reactive oxygen species

Short Name: Increase, ROS

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

ROS is a normal constituent found in all organisms, lifestages, and sexes.

Key Event Description

Biological State: increased reactive oxygen species (ROS)

Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.

Reactive oxygen species (ROS) are O₂- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). 
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). 

How it is Measured or Detected

Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.

Yuan, Yan, et al., (2013) described ROS monitoring by using H₂-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H₂-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm.

Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013).

Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS.

On the basis of the pathogenesis of drug-induced phototoxicity, a reactive oxygen species (ROS) assay was proposed to evaluate the phototoxic risk of chemicals. The ROS assay can monitor generation of ROS, such as singlet oxygen and superoxide, from photoirradiated chemicals, and the ROS data can be used to evaluate the photoreactivity of chemicals (Onoue et al. , 2014, Onoue et al. , 2013, Onoue and Tsuda, 2006).  The ROS assay is a recommended approach by guidelines to evaluate the phototoxic risk of chemicals (ICH, 2014, PCPC, 2014).

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List of Key Events in the AOP

Event: 1754: Increase, porcupine-induced Wnt secretion and Wnt signaling activation

Short Name: Increase, porcupine-induced Wnt secretion and Wnt signaling activation

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Oligomerization of FZD and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) activates Wnt/beta-catenin signaling in Homo sapiens (Hua et al., 2018).

Key Event Description

Porcupine, which is a trans-membrane endoplasmic reticulum O-acyl transferase, is important for the secretion of Wnt ligands(Saha et al., 2016a). WNTs are secreted proteins that contain 22-24 conserved cysteine residues (Foulquier et al., 2018). The WNT molecules consist of molecular families including WNT1, WNT2, WNT2B/WNT13, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT10B, WNT11, and WNT16. (Clevers & Nusse, 2012; M. Katoh, 2001; Kusserow et al., 2005)

Wnt proteins consist of 350-400 amino acids (Saito-Diaz et al., 2013).

WNT ligands are known to trigger at least three different downstream signaling cascades including canonical WNT/beta-catenin signaling pathway, non-canonical WNT/Ca²⁺ pathway, and planer cell polarity (PCP) pathway(De, 2011; Lai, Chien, & Moon, 2009; Willert & Nusse, 2012). WNTs bind to Frizzled proteins, which are seven-pass transmembrane receptors with an extracellular N-terminal cysteine-rich domain (Bhanot et al., 1996; Clevers, 2006). Wnt signaling begins with the binding of Wnt ligand towards the Frizzled receptors (Mohammed et al., 2016).

Wnt ligands bind to Frizzled (FZD) receptors which are seven transmembrane-domain protein receptors (Nile, Mukund, Stanger, Wang, & Hannoush, 2017). At least 10 FZD receptors are identified in human cells. FZD receptor is activated by Wnt ligand binding (MacDonald, Tamai, & He, 2009). 

How it is Measured or Detected

• Secretion of WNT requires a number of other dedicated factors including the sortin receptor Wntless (WLS), which binds to Wnt and escorts it to the cell surface (Banziger et al., 2006; Ching & Nusse, 2006)
• Wnt signaling is activated by the gene mutations of the signaling components (Ziv et al., 2017).
• Wnt1, Wnt3a, and Wnt5a protein expression are measured by immunoblotting using antibodies for Wnt1, Wnt3a, and Wnt5a, respectively (J. Du et al., 2016; B. Wang et al., 2017).
• WNT2, of which expression is detected by quantitative PCR, immunoblotting, and immunohistochemistry, induces EMT (Zhou et al., 2016).
• Frizzled receptor protein level on the cell surface is measured by flow cytometry with pan-FZD antibody (Jiang et al., 2015; Zeng et al., 2018). DVL protein level is measured by immunoblotting with anti-DVL2 antibodies (Zeng et al., 2018).
• Fzd mRNA level is measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) (Zeng et al., 2018).
• The up-regulation of WNT ligand expression occurs in Homo sapiens (B. Wang et al., 2017).
• The Wnt genes play an important role in the secretion from cells, glycosylation, and tight association with the cell surface and extracellular matrix in Drosophila melanogaster (Willert & Nusse, 2012).

References

Banziger, C., Soldini, D., Schutt, C., Zipperlen, P., Hausmann, G., & Basler, K. (2006). Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell, 125(3), 509-522. doi:10.1016/j.cell.2006.02.049

Bhanot, P., Brink, M., Samos, C. H., Hsieh, J.-C., Wang, Y., Macke, J. P., . . . Nusse, R. (1996). A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature, 382, 225. doi:10.1038/382225a0

Ching, W., & Nusse, R. (2006). A dedicated Wnt secretion factor. Cell, 125(3), 432-433. doi:10.1016/j.cell.2006.04.018

Clevers, H. (2006). Wnt/beta-catenin signaling in development and disease. Cell, 127(3), 469-480. doi:10.1016/j.cell.2006.10.018

Clevers, H., & Nusse, R. (2012). Wnt/beta-catenin signaling and disease. Cell, 149(6), 1192-1205. doi:10.1016/j.cell.2012.05.012

De, A. (2011). Wnt/Ca2+ signaling pathway: a brief overview. Acta Biochim Biophys Sin (Shanghai), 43(10), 745-756. doi:10.1093/abbs/gmr079

Du, J., Zu, Y., Li, J., Du, S., Xu, Y., Zhang, L., . . . Yang, C. (2016). Extracellular matrix stiffness dictates Wnt expression through integrin pathway. Sci Rep, 6, 20395. doi:10.1038/srep20395

Foulquier, S., Daskalopoulos, E. P., Lluri, G., Hermans, K. C. M., Deb, A., & Blankesteijn, W. M. (2018). WNT Signaling in Cardiac and Vascular Disease. Pharmacol Rev, 70(1), 68-141. doi:10.1124/pr.117.013896

Hua, Y., Yang, Y., Li, Q., He, X., Zhu, W., Wang, J., & Gan, X. (2018). Oligomerization of Frizzled and LRP5/6 protein initiates intracellular signaling for the canonical WNT/beta-catenin pathway. J Biol Chem, 293(51), 19710-19724. doi:10.1074/jbc.RA118.004434

Jiang, X., Charlat, O., Zamponi, R., Yang, Y., & Cong, F. (2015). Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases. Mol Cell, 58(3), 522-533. doi:10.1016/j.molcel.2015.03.015

Katoh, M. (2001). Molecular cloning and characterization of human WNT3. International journal of oncology, 19(5), 977-982. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/11604997

Kusserow, A., Pang, K., Sturm, C., Hrouda, M., Lentfer, J., Schmidt, H. A., . . . Holstein, T. W. (2005). Unexpected complexity of the Wnt gene family in a sea anemone. Nature, 433(7022), 156-160. doi:10.1038/nature03158

Lai, S. L., Chien, A. J., & Moon, R. T. (2009). Wnt/Fz signaling and the cytoskeleton: potential roles in tumorigenesis. Cell Res, 19(5), 532-545. doi:10.1038/cr.2009.41

MacDonald, B. T., Tamai, K., & He, X. (2009). Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell, 17(1), 9-26. doi:10.1016/j.devcel.2009.06.016

Mohammed, M. K., Shao, C., Wang, J., Wei, Q., Wang, X., Collier, Z., . . . Lee, M. J. (2016). Wnt/beta-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance. Genes Dis, 3(1), 11-40. doi:10.1016/j.gendis.2015.12.004

Nile, A. H., Mukund, S., Stanger, K., Wang, W., & Hannoush, R. N. (2017). Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding. Proc Natl Acad Sci U S A, 114(16), 4147-4152. doi:10.1073/pnas.1618293114

Saha, S., Aranda, E., Hayakawa, Y., Bhanja, P., Atay, S., Brodin, N. P., . . . Pollard, J. W. (2016). Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury. Nature Communications, 7(1), 13096. doi:10.1038/ncomms13096

Saito-Diaz, K., Chen, T. W., Wang, X., Thorne, C. A., Wallace, H. A., Page-McCaw, A., & Lee, E. (2013). The way Wnt works: components and mechanism. Growth Factors, 31(1), 1-31. doi:10.3109/08977194.2012.752737

Wang, B., Tang, Z., Gong, H., Zhu, L., & Liu, X. (2017). Wnt5a promotes epithelial-to-mesenchymal transition and metastasis in non-small-cell lung cancer. Biosci Rep, 37(6). doi:10.1042/BSR20171092

Willert, K., & Nusse, R. (2012). Wnt proteins. Cold Spring Harb Perspect Biol, 4(9), a007864. doi:10.1101/cshperspect.a007864

Zeng, H., Lu, B., Zamponi, R., Yang, Z., Wetzel, K., Loureiro, J., . . . Cong, F. (2018). mTORC1 signaling suppresses Wnt/beta-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level. Proc Natl Acad Sci U S A, 115(44), E10362-E10369. doi:10.1073/pnas.1808575115

Zhou, Y., Huang, Y., Cao, X., Xu, J., Zhang, L., Wang, J., . . . Zheng, M. (2016). WNT2 Promotes Cervical Carcinoma Metastasis and Induction of Epithelial-Mesenchymal Transition. PLoS One, 11(8), e0160414. doi:10.1371/journal.pone.0160414

Ziv, E., Yarmohammadi, H., Boas, F. E., Petre, E. N., Brown, K. T., Solomon, S. B., . . . Erinjeri, J. P. (2017). Gene Signature Associated with Upregulation of the Wnt/beta-Catenin Signaling Pathway Predicts Tumor Response to Transarterial Embolization. J Vasc Interv Radiol, 28(3), 349-355 e341. doi:10.1016/j.jvir.2016.11.004

Event: 1755: beta-catenin activation

Short Name: beta-catenin activation

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Beta-catenin is stabilized and translocated into nucleus in Homo sapiens (Huang et al., 2019).

Beta-catenin is activated in Homo sapiens (Huang et al., 2019) (Naujok et al., 2014).

Key Event Description

Upon the Wnt signaling activation, beta-catenin is stabilized and activated via inhibition of the phosphorylation by GSK3beta (Huang et al., 2019). Once the beta-catenin is stabilized, it translocates into the nucleus and enhances the expression of target genes of Wnt/beta-catenin signaling pathway (Huang et al., 2019). Beta-catenin activation is related to cancer (Tanabe, 2014).

Dishevelled (DVL), a positive regulator of Wnt signaling, forms the complex with FZD and leads to trigger the Wnt signaling together with Wnt coreceptor low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (Clevers & Nusse, 2012; Jiang, et al., 2015). DVL, however, has a controversial role to promote Wnt receptor degradation (Jiang et al., 2015). Meanwhile, DVL-dependent regulation of FZD level is involved in mTORC1 signaling suppression via Wnt/beta-catenin signaling (Zeng et al., 2018). The recruitment of Axin to the DVL-FZD complex induces the beta-catenin stabilization and activation. The stabilized beta-catenin translocates into the nucleus, which forms the complex with TCF to induce the up-regulated expression of proliferation-related genes.

How it is Measured or Detected

The beta-catenin level in nucleus is measured by immunoblotting with anti-beta-catenin antibody (Huang et al., 2019).

The beta-catenin nuclear translocation is measured by immunofluorescence assay (Huang et al., 2019).

Activity of beta-catenin is measured by Wnt/beta-catenin activity assay, in which the vector containing the firefly luciferase gene controlled by TCF/LEF binding sites is transfected in the cells (Naujok et al., 2014).

References

Clevers, H., & Nusse, R. (2012). Wnt/beta-catenin signaling and disease. Cell, 149(6), 1192-1205. doi:10.1016/j.cell.2012.05.012

Huang, J. Q., Wei, F. K., Xu, X. L., Ye, S. X., Song, J. W., Ding, P. K., . . . Gong, L. Y. (2019). SOX9 drives the epithelial-mesenchymal transition in non-small-cell lung cancer through the Wnt/beta-catenin pathway. J Transl Med, 17(1), 143. doi:10.1186/s12967-019-1895-2

Jiang, X., Charlat, O., Zamponi, R., Yang, Y., & Cong, F. (2015). Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases. Mol Cell, 58(3), 522-533. doi:10.1016/j.molcel.2015.03.015

Naujok, O., Lentes, J., Diekmann, U., Davenport, C., & Lenzen, S. (2014). Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. BMC Res Notes, 7, 273. doi:10.1186/1756-0500-7-273

Tanabe, S. (2014). Role of mesenchymal stem cells in cell life and their signaling. World journal of stem cells, 6(1), 24-32. doi:10.4252/wjsc.v6.i1.24

Zeng, H., Lu, B., Zamponi, R., Yang, Z., Wetzel, K., Loureiro, J., . . . Cong, F. (2018). mTORC1 signaling suppresses Wnt/beta-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level. Proc Natl Acad Sci U S A, 115(44), E10362-E10369. doi:10.1073/pnas.1808575115

Event: 1457: Epithelial Mesenchymal Transition

Short Name: EMT

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The key event is applicaple in Homo sapiens:

• Wnt5a expression leads to epithelial-mesenchymal transition (EMT) and metastasis in non-small-cell lung cancer in Homo sapiens (Wang et al., 2017).
• WNT2 expression lead to EMT induction in Homo sapiens (Zhou et al., 2016).
• EMT is induced in cancer and involved in cancer metastasis in Homo sapiens (Suarez-Carmona, Lesage, Cataldo, & Gilles, 2017) (Du & Shim, 2016).

Regulation of miRNA expression by DNA replication,damage and repair responses,transcription and translation has been proved in animals like mice,canine and cell line experiments.

Key Event Description

Epithelial-mesenchymal transition (EMT) is a phenomenon in which the cells transit from epithelial-like into mesenchymal-like phenotypes (Huan et al., 2022; Tanabe, 2017; Tanabe et al., 2015). In cancer, cells exhibiting EMT features contribute to metastasis and drug resistance.

It is known that D-2-hydroxyglurate induces EMT (Guerra et al., 2017; Jia et al., 2018; Mishra et al., 2018; Sciacovelli & Frezza, 2017). D-2-hydroxyglurate, an inhibitor of Jumonji-family histone demethylase, increased the trimethylation of histone H3 lysine 4 (H3K4) in the promoter region of the zinc finger E-box-binding homeobox 1 (ZEB1), followed by the induction of EMT (Colvin et al., 2016).

Wnt5a induces EMT and metastasis in non-small-cell lung cancer (Wang et al., 2017).

EMT is related to Wnt/beta-catenin signaling and is important for treatment-resistant cancer (Tanabe et al., 2016).

TGFbeta induces EMT (Wendt et al., 2010).

ZEB is one of the critical transcription factors for EMT regulation (Zhang et al., 2015).

SNAI1 (Snail) is an important transcription factor for cell differentiation and survival. The phosphorylation and nuclear localization of Snail1 induced by Wnt signaling pathways are critical for the regulation of EMT (Kaufhold & Bonavida, 2014).

Transcription factors SNAI1 and TWIST1 induce EMT (Hodge et al., 2018) (Mani et al., 2008).

It is suggested that Sp1, a transcription factor involved in cell growth and metastasis, is induced by cytochrome P450 1B1 (CYP1B1), and promotes EMT, which leads to cell proliferation and metastasis (Kwon et al., 2016).

Biological state   An epithelial-mesenchymal transition (EMT) is a biologic process in which epithelial cells are polarized, interact through their basal surface with basement membrane, and undergo biochemical changes to assume a mesenchymal cell phenotype. This phenotypic transformation has various characters such as enhanced migratory capacity, high invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components (Kalluri,  R.,  and  Neilson,  E.G.  2003). The completion of an EMT is signalled by the degradation of the underlying basement membrane and the formation of a mesenchymal cell that can migrate away from the epithelial layer in which it originated.    EMT has a number of distinct molecular processes like activation of transcription factors, expression of specific cell surface proteins, reorganization and expression of cytoskeletal proteins, production of ECM-degrading enzymes, and changes in the expression of specific microRNAs. These factors are used as biomarkers to demonstrate the passage of a  cell through an EMT.   Biological compartment Cellular Role in General Biology: Excessive proliferation of epithelial cells and angiogenesis mark the initiation and early growth of primary epithelial cancers. (Hanahan, D., and Weinberg, R.A. 2000). The subsequent acquisition of invasiveness, initially manifest by invasion through the basement membrane, is thought  to herald the onset of the last stages of the multi-step process that  leads eventually to metastatic dissemination, with life-threatening  consequences.  There has been an intense research going on in the genetic controls and biochemical mechanisms underlying the acquisition of the invasive phenotype and the subsequent systemic spread of the cancer cell.  Activation of an EMT program has been proposed as the critical mechanism for the acquisition of malignant phenotypes by epithelial cancer cells (Thiery, J.P. 2002).  Pre-clinical experiments such as mice models and cell culture experiments  has demonstrated  that carcinoma cells can acquire a mesenchymal phenotype and express mesenchymal markers such as α-SMA, FSP1, vimentin,  and desmin (Yang,  J.,  and  Weinberg,  R.A.  2008). These cells  are seen at the invasive front  of primary tumors and are considered to be the cells that eventually  enter into subsequent steps of the invasion-metastasis cascade, i.e.,  intravasation, transport through the circulation, extravasation, formation of micro metastases, and ultimately colonization (the growth  of small colonies into macroscopic metastases) (Thiery, J.P. 2002, Fidler, I.J., and Poste, G. 2008, Brabletz, T., et al. 2001). An  apparent  paradox  comes  from  the  observation  that  the  EMT-derived migratory cancer cells typically establish secondary colonies at distant sites that resemble, at the histopathological  level, the primary tumor from which they arose; accordingly,  they no longer exhibit the mesenchymal phenotypes ascribed to  metastasizing  carcinoma  cells.  Reconciling this behaviour with the proposed role of EMT as a facilitator of metastatic dissemination requires the additional notion that metastasizing cancer cells must shed their mesenchymal phenotype via a MET during  the course of secondary tumor formation (Zeisberg, M et al 2005). The tendency of  disseminated cancer cells to undergo EMT likely reflects the local  microenvironments that they encounter after extravasation into  the parenchyma of a distant organ, quite possibly the absence of  the heterotypic signals they experienced in the primary tumor that  were responsible for inducing the EMT in the first place (Thiery, J.P. 2002, Jechlinger, M et al 2002, Bissell, M.J et al 2002). These evidences indicate that induction of an EMT is likely to be a centrally important mechanism for the progression of carcinomas to a metastatic stage and implicates MET during the subsequent colonization process. However, many steps of this mechanistic model still require direct experimental validation. It remains unclear at present whether these phenomena and molecular mechanisms relate to and explain the metastatic dissemination of non-epithelial cancer cells. The entire spectrum of signaling agents that contribute to EMTs of carcinoma cells remains unclear. One  theory suggests that  the genetic and epigenetic alterations undergone by cancer cells during the course of primary tumor formation render them especially responsive to EMT-inducing heterotypic signals originating in the tumor-associated stroma. Oncogenes induce senescence, and recent studies suggest that cancer cell EMTs may also play a role in preventing senescence induced by oncogenes, thereby facilitating subsequent aggressive dissemination (Smit, M.A., and Peeper, D.S. 2008, Ansieau, S., et al. 2008, Weinberg, R.A. 2008).  In  the case of many carcinomas, EMT-inducing signals emanating  from the tumor-associated stroma, notably HGF, EGF, PDGF,  and TGF-β, appear to be responsible for the induction or functional  activation  in  cancer  cells  of  a  series  of  EMT-inducing  transcription factors, notably Snail, Slug, zinc finger E-box binding homeobox 1 (ZEB1), Twist, Goosecoid, and FOXC2 (Thiery, J.P. 2002, Jechlinger, M  et al 2002, Shi, Y., and Massague, J. 2003, Niessen, K., et al. 2008, Medici, D et al 2008, Kokudo,  T.,  et  al.  2008). Once expressed and activated, each of these transcription factors can act pleiotropically to choreograph the complex EMT program, more often than not with the help of other members of this cohort of transcription factors. The actual implementation by these cells of their EMT program depends on a series of  intracellular signaling networks involving, among other signal- transducing  proteins,  ERK,  MAPK,  PI3K,  Akt,  Smads,  RhoB,  β-catenin, lymphoid enhancer binding factor (LEF), Ras, and c-Fos  as well as cell surface proteins such as β4 integrins, α5β1 integrin, and αVβ6 integrin (Tse,  J.C.,  and  Kalluri,  R.  2007). Activation of EMT programs is also facilitated by the disruption of cell-cell adherens junctions and the cell-ECM adhesions mediated by integrins (Yang,  J.,  and  Weinberg,  R.A.  2008, Weinberg, R.A. 2008, Gupta, P.B  et al 2005, Yang,  J et al 2006, Mani, S.A., et al. 2007, Mani, S.A., et al. 2008, Hartwell, K.A., et al. 2006, Taki, M et al 2006)..

How it is Measured or Detected

Loss of E-cadherin and cell polarity is considered to be a fundamental event in epithelial-mesenchymal transition. The simultaneous expression of epithelial (e.g. E-cadherin) and mesenchymal markers (e.g. N-cadherin and vimentin) within the airway epithelium are indicative for ongoing transition (Borthwick et al. 2009, 2010).

	Method/ measurement referenc	Reliability	Strength of evidence	Assay fit for purpose	Repeatability/ reproducibility	Direct measure
Human cell line	qRT-PCR,cell viability assay, Western blotting,EdU incorporation assay	+	Strong	Yes	Yes	Yes
Human	IHC,micro array,qPCR, SNP array	+	Moderate	Yes	Yes	Yes

• EMT can be detected by immunostaining with pro-surfactant protein-C (pro-SPC) and N-cadherin in idiopathic pulmonary fibrosis (IPF) lung in vivo (Kim et al., 2006).
• EMT can be detected by immunostaining with vimentin in lung alveola in vivo (Kim et al., 2006).
• EMT can be detected as the increased level of the transcription factors, zinc finger E-box-binding homeobox (ZEB), Twist and Snail (Huang et al., 2022).

 

References

Borthwick, L. A., Parker, S. M., Brougham, K. A., Johnson, G. E., Gorowiec, M. R., Ward, C., … Fisher, A. J. (2009). Epithelial to mesenchymal transition (EMT) and airway remodelling after human lung transplantation. Thorax, 64(9), 770–777. https://doi.org/10.1136/thx.2008.104133

Borthwick, L. A., McIlroy, E. I., Gorowiec, M. R., Brodlie, M., Johnson, G. E., Ward, C., … Fisher, A. J. (2010). Inflammation and epithelial to mesenchymal transition in lung transplant recipients: Role in dysregulated epithelial wound repair. American Journal of Transplantation, 10(3), 498–509. https://doi.org/10.1111/j.1600-6143.2009.02953.x

Al Saleh, S., Al Mulla, F., & Luqmani, Y. A. (2011). Estrogen receptor silencing induces epithelial to mesenchymal transition in human breast cancer cells. PloS one, 6(6), e20610.

Bissell, M. J., Radisky, D. C., Rizki, A., Weaver, V. M., & Petersen, O. W. (2002). The organizing principle: microenvironmental influences in the normal and malignant breast. Differentiation, 70(9-10), 537-546.

Bouris, P., Skandalis, S. S., Piperigkou, Z., Afratis, N., Karamanou, K., Aletras, A. J., ... & Karamanos, N. K. (2015). Estrogen receptor alpha mediates epithelial to mesenchymal transition, expression of specific matrix effectors and functional properties of breast cancer cells. Matrix Biology, 43, 42-60.

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List of Adverse Outcomes in this AOP

Event: 1651: Treatment-resistant gastric cancer

Short Name: Resistant gastric cancer

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Drug resistance occurs in Homo sapiens (Du & Shim, 2016).

Key Event Description

It is known that diffuse-type gastric cancer, which has a poor prognosis, is treatment-resistant and more malignant compared to intestinal-type gastric cancer (Tanabe et al., 2014). Drug resistance is involved in EMT, which is an important phenomenon exhibiting features similar to cancer stem cells (CSCs) (Du & Shim, 2016).

EMT is involved in metastasis and cancer therapy resistance (Smith & Bhowmick, 2016).

How it is Measured or Detected

Treatment-resistant gastric cancer and EMT can be detected with biomarkers (Zeisberg & Neilson, 2009).

Treatment-resistant gastric cancer which exhibits EMT phenotype can be detected as the increased level of the transcription factors, zinc finger E-box-binding homeobox 1/2 (ZEB1/2), SNAI1/2, and TWIST2 which are associated with the activation of EMT-related genes (Tanabe et al., 2022a and 2022b).

Regulatory Significance of the AO

Drug resistance is very important in cancer treatment since cancer metastasis and recurrence are some of the main obstacles to treating cancer. Cancer stem cells that share the phenotype of EMT may be targeted in anti-cancer drug development. 

References

Du, B., & Shim, J. S. (2016). Targeting Epithelial-Mesenchymal Transition (EMT) to Overcome Drug Resistance in Cancer. Molecules, 21(7). doi:10.3390/molecules21070965

Smith, B. N., & Bhowmick, N. A. (2016). Role of EMT in Metastasis and Therapy Resistance. J Clin Med, 5(2). doi:10.3390/jcm5020017

Tanabe, S., Aoyagi, K., Yokozaki, H., Sasaki, H. (2014). Gene expression signatures for identifying diffuse-type gastric cancer associated with epithelial-mesenchymal transition. International journal of oncology, 44(6), 1955-1970. doi:10.3892/ijo.2014.2387

Tanabe, S., Quader, S., Cabral, H., Ono, R. (2020a). Interplay of EMT and CSC in Cancer and the Potential Therapeutic Strategies. Front Pharmacol, 11, 904. doi:10.3389/fphar.2020.00904

Tanabe S, Quader S, Ono R, Cabral H, Aoyagi K, Hirose A, Yokozaki H., Sasaki, H. (2020b). Molecular Network Profiling in Intestinal- and Diffuse-Type Gastric Cancer. Cancers (Basel), 12(12), 3833. doi:10.3390/cancers12123833

Zeisberg, M., & Neilson, E. G. (2009). Biomarkers for epithelial-mesenchymal transitions. J Clin Invest, 119(6), 1429-1437. doi:10.1172/JCI36183

Appendix 2

List of Key Event Relationships in the AOP