Aop: 391

Title

A descriptive title which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Inhibition of Proline/Asparagine Hydroxylation Leads to Breast Cancer

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Inhibition of Hydroxylase leads to Breast Cancer

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Matthew Clinch

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Arthur Author   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Arthur Author

Status

The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite
This AOP was last modified on June 28, 2021 10:45
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Inhibition of Proline and Asparagine Hydroxylation Leads to Stabilization of HIF1-Alpha June 25, 2021 11:44
Stabilization of HIF-1 Alpha June 25, 2021 11:43
Proliferation/ beta-catenin activation September 29, 2020 21:25
Cyclin D1 Overexpression June 14, 2021 15:22
Wnt ligand stimulation June 18, 2021 14:02
N/A, Breast Cancer May 08, 2019 14:14
Increase, Cell Proliferation October 30, 2019 10:55
Inhibition Hydroxylase, HIF-1Alpha Stabilization leads to HIF-1 Alpha Stabilization June 14, 2021 15:19
HIF-1 Alpha Stabilization leads to Wnt ligand stimulation June 25, 2021 11:49
Wnt ligand stimulation leads to Proliferation/ beta-catenin activation June 16, 2021 14:39
Proliferation/ beta-catenin activation leads to Cyclin D1 Overexpression June 14, 2021 15:23
Cyclin D1 Overexpression leads to Increase, Cell Proliferation June 17, 2021 12:02
Increase, Cell Proliferation leads to N/A, Breast Cancer June 17, 2021 12:03

Abstract

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

Hydroxylation often refers to the addition of a hydroxyl group (-OH) into an organic compound. This can be done synthetically and biologically. Most proteins are hydroxylated via 2-oxoglutarate-dependent dioxygenase (Zurlo et al., 2016). Protein hydroxylation can occur on multiple amino acids including lysine, asparagine and aspartate; however, most human protein hydroxylation occurs on proline residues. Some heavy divalent metals have the ability to inhibit this event. Known divalent heavy metals include Cobalt, or Nickle have previously been seen to show these effects (Yuan et al., 2003, Salnikow et al., 2004). Some occupational exposure to divalent metals include smelters, mining activities, hazardous waste sites, and even natural sources (Kaczmarek et al., 2009).

When iron is able to properly function in the hydroxylation of proline and asparagine residues it suppresses HIF transcriptional activity. Hydroxylation of proline residues at positions 405 and 531 on the HIF Alpha Subunit target it for rapid ubiquination by the von Hippel Lindau (VHL) containing E3 ubiquitin ligase (Strowitzki et al., 2019). The HIF alpha subunit is also hydroxylated in an asparagine residue (803) that blocks the binding of the p300 coactivator. These processes result in the degradation of HIF. When divalent metals, such as Cobalt compete for the iron coordination location, HIF alpha is stabilized.

This complex is now able to bind to HRE site and transactivate gene expression. WNTs contain hypoxia response elements within their promoter, which allows for transactivation by Hypoxia Inducible factors (HIFs). This causes WNT ligand induction. The WNT signalling pathway regulates several aspects of cell fate including migration, cell polarity, neural patterning and organogenesis, while organisms undergo embryonic development (Komiya and Habas, 2008). When WNT proteins are induced Beta-catenin is upregulated and translocated in the nucleus (Reya and Clevers, 2005). β-catenin is then able to produce high levels of cyclin D1 messenger RNA and protein (Tetsu and McCormick, 1999). When Cyclin D1 production is not properly regulated, it leads to uncontrolled cell cycle progression (Alt et al., 2000). Accelerated growth or cell proliferation is a hallmark of cancer, including breast cancer (Elmi et al., 2018).

This Adverse Outcome Pathway will display how inhibiton of hydroxylase can lead to breast cancer.

Background (optional)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

Hydroxylation often refers to the addition of a hydroxyl group (-OH) into an organic compound. This can be done synthetically and biologically. Hydroxylation reactions can be facilitated by enzymes called hydroxylases in a biological context. Alcohols can be formed from C-H and an oxygen atom (Huang and Groves, 2017). Other reactions add OH groups to unsaturated substrates (Kolb et al., 1994). The hydroxy groups would be added across the double bond alkene by hydrogen peroxide. In biology, cytochrome P450 is the main hydroxylation agent. However, other known hydroxylating agents include flavins, alpha-ketoglutarate-dependent hydroxylases and diiron hydroxylases (Lehninger et al., 2008).

Hydroxylation is very important in degrading organism compounds in the air, converting lipophilic compounds into hydrophilic compounds and play a critical role in drug activity (Cerniglia, 1992). Hydroxylation can also be categorized as a post-translational modification for proteins. Most proteins are hydroxylated via 2-oxoglutarate-dependent dioxygenase (Zurlo et al., 2016). Protein hydroxylation can occur on multiple amino acids including lysine, asparagine and aspartate; however, most human protein hydroxylation occurs on proline residues. This is because roughly 30% of the proteins in humans are made up of collagen, which contains hydroxyproline at every 3rd residue in the amino acid sequence (Raju, 2019). Hydroxylation of proteins often require iron, molecular oxygen and alpha-ketoglutarate to perform oxidation. Inhibition of hydroxylation of amino acid residues can have many negative effects.

Furthermore, the accumulation of the ROS under normoxic conditions can activate transcription factors known as Hypoxia-Inducible Factors (HIFs), and more specifically, HIF-1a; factors that are normally only found in the cell under hypoxic conditions (Brown et al., 2016). This is done by inactivating HIF-1a’s inhibitor; Prolyl Hydroxylase Domain (PHD) containing hydroxylases (Schieber & Chandel, 2014). Some heavy divalent metals have the ability to inhibit this event. Some known divalent heavy metals such as Cobalt, or Nickle have previously been seen to show these effects (Yuan et al., 2003, Salnikow et al., 2004). PHD hydroxylases and HIFs can mediate transcriptional control of a number of target genes which contain a hypoxia response element (HREs) within their promoters. WNTs contain hypoxia response elements within their promoter, which allows for transactivation by Hypoxia Inducible factors (HIFs). In particular, HIF-1 Alpha has been seen to control the production of WNTs. 

The WNT signalling pathway regulates several aspects of cell fate including migration, cell polarity, neural patterning and organogenesis, while organisms undergo embryonic development (Komiya and Habas, 2008). The WNT proteins activate different intracellular signal transduction pathways (Cadigan et. al., 1997). These pathways can regulate cell proliferation. It has been found that the deregulation of WNT signalling promotes both human degenerative diseases, and several types of cancer, including colorectal, breast, gastric and pancreatic (Logan & Nusse, 2004, Flanagan et al., 2017).

Summary of the AOP

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 1863 Inhibition of Proline and Asparagine Hydroxylation Leads to Stabilization of HIF1-Alpha Inhibition Hydroxylase, HIF-1Alpha Stabilization
2 KE 1864 Stabilization of HIF-1 Alpha HIF-1 Alpha Stabilization
3 KE 1645 Wnt ligand stimulation Wnt ligand stimulation
4 KE 1755 Proliferation/ beta-catenin activation Proliferation/ beta-catenin activation
5 KE 1866 Cyclin D1 Overexpression Cyclin D1 Overexpression
KE 870 Increase, Cell Proliferation Increase, Cell Proliferation
AO 1193 N/A, Breast Cancer N/A, Breast Cancer

Relationships Between Two Key Events (Including MIEs and AOs)

TESTINGThis table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and WoE summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
All life stages Moderate
Adults High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
mammals mammals Moderate NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Mixed Low
Female High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

Risk factors for developing breast cancer include being female (WHO, 2014).  

Breast cancer most commonly develop in cells from the lining of milk ducts (NCI, 2014).

Breast cancer can also be seen in other mammals (Zeng et al., 2020).

Majority of breast cancer in female mammals detected in late adulthood, lower chance of invasive tumor at younger ages (DeSantis et al., 2019).

The domain limiting KE is breast cancer formation in mammalian females during adulthood.

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

Essentiality of KE 1: Stabilization of HIF-1 Alpha

Evidence to assess the essentiality of HIF-1 Alpha stabilization can be done by both experimentally blocking and exacerbating the event. Direct evidence includes endothelial cell specific inactivation of HIF-1 Alpha. Studies have done crossbreeding of Tie2-Cre transgenic mice with HIF-1 Alpha +f/+f mice homozygous for HIF-1 Alpha allele with exon 2 flanked by loxp (Ryan et al., 1998). This produced HIF-1 Alpha Endothelial cell knockout mice (Li et al., 2017). Conformation of this knockout was confirmed using PCR. This can be shown in the figure below.

Figure 1: PCR analysis of homozygous (Homo), heterogygous (Het) and wild-type (WT) pups illustrating the presence of HIF-1 α in WT and its absence of the HIF-1 α EC KO (Li et al., 2017).

Quantitative western blot analysis of wild type (WT) and knockout (KO) endothelial cell lysates showing the presence (WT) and absence (KO) of HIF-1 α protein expression under both normoxic (Nx) and low oxygen (Hx) conditions was shown. A figure is provided below.

Figure 2: Western blot analysis of wild type (WT) and knockout (KO) endothelial cell lysates showing the presence (WT) and absence (KO) of HIF-1 α protein expression under both normoxic (Nx) and low oxygen (Hx) (Li et al., 2017).

Each of these studies display that blocking the stabilization of HIF-1 Alpha causes the reduced expression at the protein and genetic level. This would prove essential blocking of the event. Further indirect evidence can support that when cells are exposed to known hypoxic mimetics such as Dimethyloxaloylglycine (DMOG), HIF-1 Alpha can be stabilized (Sen and Sen, 2016). This can be seen in TBI mice in the following study.

Figure 3: Western Blot analysis of expression of HIF-1 Alpha and HIF-1 Beta with increasing DMOG concentrations in TBI Mice (Sen and Sen, 2016).

HIF-1 Alpha can also be overexpressed under low oxygen or hypoxic conditions. In the following study hypoxia displays increased HIF-1 alpha mRNA and protein expression in C57BL/6J mice (BelAiba et al., 2007).

Figure 4: Hypoxia (1%) increases HIF-1 Alpha mRNA and protein levels (BelAiba et al., 2007).

Based on the finding from Figure 3 and Figure 4 it can be seen that HIF-1 Alpha can be overexpressed by hypoxic conditions and other chemicals that induce a hypoxic response. This would display the essentiality of exacerbating HIF-1 Alpha stabilization.

Weight of Evidence for KE1: Stabilization of HIF-1 Alpha

The defining question guiding essentiality here is whether downstream KEs or the AO is prevented if a KE upstream is blocked.

Table 1: Weight of Evidence for KE1 (Stabilization of HIF-1 Alpha)

Event

Direct

Evidence

Indirect

Evidence

No Exp.

Evidence

Contradictory

Exp.

Evidence

KE1

HIGH

  • Knockout Studies
  • Protein (Western)
  • Gene (PCR)

HIGH

  • Hypoxic Mimetics
  • Hypoxia
  • mRNA expression
  • Protein expression
N/A N/A

Based on the table above, high weight of evidence is shown in direct and indirect studies for stabilization of HIF-1 Alpha. 

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help
KER Summart of Bio. Plausibility Evidence WOE Call
KER1

Inhibition hydroxylase leads to HIF-1 alpha stabilization, potentially due to divalent metals (Davidson et al., 2005).

Studies have proposed potential mechanism, however they are theoretical (Yuan et al., 2003).

Moderate
KER2

HIF-1 Alpha Stabilization leads to WNT ligand stimulation is fairly well defined in literature.

Hypoxic Response Elements Within the WNT10 gene promoter (Park et al., 2013).

HIF-1 Alpha expression increased by hypoxia and other hypoxic mimetics lead to increase in WNT11 mRNA and protein (Mori et al., 2016).

Similar studies have shown that HIF-1 Alpha can directly promote WNT7A gene and protein expression, done with increase luciferase activity (Cirillo et al., 2020).

High
KER3

WNT ligand stimulation leads to Proliferation/beta catenin.

WNT induction leads to cytoplasmic upregulation of Beta-catenin (Pai et al., 2017)

High
KER4

Proliferation/Beta-catenin activation leads to Cyclin D1 overexpression.

The canonical WNT pathway increasing beta-catenin levels signals the increase in expression of CYCLIN D1 and cMYC. CYCLIN D1 induces the G1 phase whereas cMYC induces the S phase. (Lecarpentier et al., 2019).

High
KER5

Cyclin D1 overexpression leads to Cell proliferation.

The high expression of Cyclin D1 drives unchecked cellular proliferation promoting tumor growth, thus, the Cyclin D1 carries out a central role in the pathogenesis of cancer (Montalto and De Amicis, 2020). 

Nuclear cyclin D1 accumulation leads to uncontrolled cell cycle progression (Alt et al., 2000).

Cyclin D1 is frequently deregulated in various cancers, including malignant hemopathies, and tumor cells display uncontrolled cell proliferation (Bustany et al., 2015).

High
KER6

Increase Cell Proliferation leads to Breast cancer. This is a charateristic of breast cancer but is not the only reason for breast cancer tumor growth.

Accelerated growth is a hallmark of cancer, including breast cancer (Elmi et al., 2018).

Moderate
KER Summary of Empirical Evidence WOE Call
KER1

Inhibition hydroxylase leads to HIF-1 alpha stabilization, potentially due to divalent metals.

Temporal Concordance: Low

Dose Concordance: Low

Incidence Concordance: Low

Difficult to directly quantify inhibition of hydroxylases. Most cases stabilization of HIF-1 alpha is onset by some other factor that leads to the inhibiton of hydroxylation. This includes known hypoxic mimetics, low oxygen and some divalent metals (Davidson et al., 2005).

Low
KER2

HIF-1 Alpha Stabilization leads to WNT ligand stimulation.

Temporal Concordance: High

Studies have shown that HIF-1 Alpha stabiliaztion occurs before WNT11 and WNT10 ligand induction by mRNA expression and protein expression after treatment with consistent DMOG or Hypoxia; known HIF-1 alpha stabilizaers (Mori et al., 2016, Park et al., 2013).

Dose Concordance: Low

With increasing Dose HIF-1 Alpha stabilizes, however data was not found for WNTs. It cannot be said with concrete data that WNT induction occurs at a higher dose compared to HIF-1 Alpha (Peng et al., 2014).

Incidence Concordance: Moderate 

Relative expression of HIF-1 alpha and WNT3a were compared in bar graphs. This data displayed that their is a higher incidence in HIF-1 alpha at specific time points compared to the same timepoints for WNT3a (Li et al., 2020).

Protein concentrations of HIF-1 alpha have been shown in pg/ug and molecules/cell (Park et al., 2014). Further information for WNT protein concentration would need to be done to determine concrete incidence concordance.

Moderate
KER3

WNT ligand stimulation leads to Proliferation/beta catenin.

Temporal Concordance: Low

Dose Concordance: Low

Incidence Concordance: Low

WNT ligand stimulation is tighlty regulated to Beta-catenin production. Many studies monitor WNT upregulation via Beta-catenin expression, therefore it is difficult to display temporal, dose and incidence concordance. However, the pathway of WNT leading to beta-catenin accumulation is well known in literature.

A characteristic feature of the canonical Wnt pathway is tight regulation of the level of β-catenin (Gao et al., 2014)

Low
KER4

Proliferation/Beta-catenin activation leads to Cyclin D1 overexpression.

Temporal Concordance: Low

Similar to KER3, beta-catenin and cyclin D1 expression is tighly regulated and would be difficult to monitor. In many studies WNT acticity can be shown and compared to CDK (cyclin dependent Kinases). 

Dose Concordance: Moderate

Indirect evidence could possibly show that when WNT ligand is uregulated beta-catenin is also increased. 

Incidence Concordance: Low 

Difficult to measure incidence concordance since beta-catenin is tightly regulated to WNT. Multiple cycline dependent kinases can be measured.

β-catenin produce high levels of cyclin D1 messenger RNA and protein (Tetsu and McCormick, 1999).

Low
KER5

Cyclin D1 overexpression leads to Cell proliferation.

Temporal Concordance: Moderate

Dose Concordance: Moderate 

Incidence Concordance: Low

Cyclin D1 is not alone in acting as a transcriptional regulator to control cell proliferation. Cyclin D1 and its binding partner Cdk4 act as transcriptional regulators to control cell proliferation and migration (Fusté et al., 2016). Abnormal Cyclin D1/Cdk4 expression promotes tumour growth and metastasis. Therefore, could be difficult to measure only cyclin D1 leading to proliferation. However, overexpressing Cyclin d1 in cell culture and monitoring cell proliferation could be done at different concentrations/overtime quite easily. 

Moderate
KER6

Increase Cell Proliferation leads to Breast cancer.

Temporal Concordance: Moderate

Dose Concordance: Low

Incidence Concordance: Low

Cell proliferation is most definetly a characteristic of breast cancer and tumor progression as a whole (Elmi et al., 2018). It could be assumed that Temproral and possibly Incidence concordance could be determined based on monitoring breast cancer tumors over time and determine if increased cell proliferation lead to the tumor. Epidemiology studies could determine if increasing cell proliferation in epethilial cells is higher compared to the breast cancer production (Mester and Redeuilh, 2008).

Moderate
KER

Summary of Quantitative Understanding

WOE Call
KER1

Inhibition hydroxylase leads to HIF-1 alpha stabilization, potentially due to divalent metals.

How much Change KEup: Hydroxylation would need to be monitored after introduction of known hydroxylation inhibitors (divalent metals, hypoxia or hypoxia mimetics). Amount of hydroxylation inhibiton could then display lowest observed HIF-1 alpha stabilization.

How long: The same experiment would need to be done at different times to determine how much time KEup needs to form the change in KEdown. 

Low
KER2

HIF-1 Alpha Stabilization leads to WNT ligand stimulation.

How much Change KEup: At 3% oxygen HIF-1 Alpha is shown to stabilize more compard to WNT10 ligand induction (Gultice et al., 2009, Park et al., 2013).

How long: HIF-1 Alpha is shown to stabilize approximately 6 days before WNT10 ligand induction (Park et al., 2013).

High
KER3

WNT ligand stimulation leads to Proliferation/beta catenin.

How much Change KEup: Since WNT and B-catenin is very tighlty regulated it would be difficult to show how much change in KEup needed for change in KEdown.

How long: Since WNT and B-catenin is very tighlty regulated it would be difficult to show how much change in KEup needed for change in KEdown.

A characteristic feature of the canonical Wnt pathway is tight regulation of the level of β-catenin (Gao et al., 2014)

Low
KER4

Proliferation/Beta-catenin activation leads to Cyclin D1 overexpression.

How much Change KEup: Similar to KER3, beta-catenin and cyclin D1 expression is tighly regulated and would be difficult to monitor. In many studies WNT acticity can be shown and compared to CDK (cyclin dependent Kinases). Although, overexpressing Beta-catenin at increasing amounts and monitoring Cyclin D1 expression could be done.

How long: Similar experiments could be done at one LOAEL of Beta-catenin that produces Cyclin D1 expression over time.

Moderate
KER5

Cyclin D1 overexpression leads to Cell proliferation.

How much Change KEup: Overexpressing Cyclin d1 in cell culture and monitoring cell proliferation could be done at different concentrations easily.

How long: Overexpressing Cyclin d1 in cell culture and monitoring cell proliferation could be done at different overtime quite easily. Cyclin D1 could be introduced to cells at the LOAEL and monitored for cell proliferation over time.

Moderate
KER6

Increase Cell Proliferation leads to Breast cancer.

How much Change KEup: Uncontrolled cell proliferation is a well known charateristic of Breast cancer (Elmi et al., 2018). Determining the LOAEL of cell prolifertation that causes breast cancer would prove how much proliferation causes the tumor.

How long: Exposing cells to a factor that induces cell proliferation at the LOAEL over time could also display how long cell proliferation/metastasis of the tumor, which would lead to breast cancer formation. Theoretical.

Low

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

References

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

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