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Relationship: 3563

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

SIX1 protein expression, increased leads to Promotion, SIX1 positive progenitor cells in endometrium

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

SIX1 protein expression, increased

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Promotion, SIX1 positive progenitor cells in endometrium

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Early-life estrogen receptor agonism leading to endometrial adenosquamous carcinoma via promotion of sine oculis homeobox 1 progenitor cells	adjacent	High	Not Specified	Travis Karschnik (send email)	Under Development: Contributions and Comments Welcome

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
Homo sapiens	Homo sapiens	High	NCBI
Mus musculus	Mus musculus	High	NCBI
Vertebrates	Vertebrates		NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Female	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
All life stages
Embryo	High
Adult	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Abnormal six1 protein expression during development allows for localization of Six1 transcripts in the endometrium, a location in which it isn't normally found. Establishment and subsequent expansion of these six1+ progenitor populations is a potential contributor to eventual carcinomas.

Suen et al., 2016 and Jefferson et al., 2011 both observed distinct cell-type specific six1 transcript expression differences. “In the vaginal and cervical epithelium, SIX1 localized to the stratified squamous epithelium, with highest expression in the basal and suprabasal layers. In the endocervix, nuclear SIX1 immunolabeling was observed in simple columnar glandular epithelial cells only when there was a layer of progenitor-like basal cells directly subjacent to the luminal cells. SIX1 expression was not observed in endometrial luminal epithelium or morphologically normal glands. SIX1 was present in the uteri of a few control mice but was limited to small focal areas of squamous metaplasia in the uterine body.” (Suen et al., 2016). In contrast, SIX1 was present in the uterus in low numbers after just 5 days of GEN or DES treatment. At 6 months of age, SIX1 localized to basal cell and squamous metaplasia in nonneoplastic endometrial glands of most mice neonatally exposed to GEN or DES.

Suen et al., 2016 also observed SIX1 immunolabeling showed a hot spot distribution in a given metaplastic gland but not in surrounding glands, suggesting a differentiation specific expression pattern potentially as a result of SIX1+ progenitor cells established during the early developmental exposure to ER agonists. In the Jefferson et al., 2011 study they similarly found six1 protein significantly increased in the oviducts of GEN-treated mice compared with controls.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

This Key Event Relationship was part of an Environmental Protection Agency effort to develop AOPs that establish scientifically supported causal linkages between alternative endpoints measured using new approach methodologies (NAMs) and guideline apical endpoints measured in Tier 1 and Tier 2 test guidelines (U.S. EPA, 2024) employed by the Endocrine Disruptor Screening Program (EDSP). A series of key events that represent significant, measurable, milestones connecting molecular initiation to apical endpoints indicative of adversity were identified based on scientific review articles and empirical studies. Additionally, scientific evidence supporting the causal relationships between each pair of key events was assembled and evaluated. The present effort focused primarily on empirical studies with laboratory rodents and other mammals.

McCoy et al., 2009 was used as an originating publication followed by further investigation of the bibliography and google scholar to retrieve full articles.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Six1, like many other transcription factors involved in early development, can regulate its own expression and participate in feed-forward loops. Six1 promotes the expression of genes that maintain progenitor identity (Eya1, Pax3, Pax7, Cyclin D1) and in turn, these progenitors express six1. Six1 expression shifts cellular dynamics towards progenitor cell expansion and maintenance through:

Encouraging proliferation.
Expanding the population of cells that are, or become, progenitors.
Suppressing differentiation.
Stabilizing transcriptional feedback loops (six1/six2) that reinforce progenitor fate and self-renewal.

Empirical evidence for these assertions provided in the section below.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Encouraging proliferation
- Coletta et al., 2004 demonstrated a relationship between six1 overexpression and cyclin A1 mRNA levels and activity in multiple tissues. “Six1 overexpression increases cyclin A1 mRNA levels and activity, cell proliferation, and tumor volume, whereas Six1 down-regulation decreases cyclin A1 mRNA levels and proliferation. Overexpression of Six1 in wild-type mouse embryonic fibroblasts, but not in knockout variants lacking the cyclin A1 gene, induces cell proliferation. Furthermore, inhibition of cyclin A1 in Six1-overexpressing mammary carcinoma cells decreases proliferation. Together these results demonstrate that cyclin A1 is required for the proliferative effect of Six1. We conclude that Six1 overexpression reinstates an embryonic pathway of proliferation in breast cancer by up-regulating cyclin A1.”
Expanding progenitor population
- Six1 expression has been shown to induce epithelia-to-mesenchymal transition (EMT), promoting self-renewal and stem-like behavior. Radisky 2009 is excrpted here. “Isolated primary epithelial cells from Six1-expressing mice showed increased expression of the stem/progenitor cell–associated cell surface markers CD24 and CD29, as well as greatly increased growth as mammospheres, an assay that reflects cell self-renewal. Thus, Six1-induced EMT was associated with increases in the population of stem/progenitor cells and spontaneous tumor growth.”
- McCoy et al., 2009 concluded that “Six1 may play a role in mammary epithelial stem cells, both in the normal gland as well as in tumorigenesis. Mammary epithelial cells from Six1-overexpressing mice are enriched for stem cells and have increased mammosphere-forming capability. Additionally, Six1-driven tumors contain cells of multiple mammary lineages and express Sca-1. Together, these data strongly suggest that Six1 promotes a stem cell phenotype in the normal mammary gland and that Six1-driven tumors arise from a stem/progenitor cell population.”
Suppressing/altering differentiation
- El-Hashash et al., 2011 found “Six1 overexpression in MLE15 lung epithelial cells in vitro inhibited cell differentiation, but increases the expression of progenitor cell markers.”
- Suen et al., 2019 studied female mice exposed to diethylstilbestroL and found “Abnormal endometrial glands express the oncofetal protein sine oculis homeobox 1 (SIX1) and contain cells with basal (cytokeratin [CK]14+/18-) and poorly differentiated features (CK14+/18+), strongly associating SIX1 with aberrant differentiation…”
Stabilizing transcriptional feedback loops (six1/six2) that reinforce progenitor fate and self-renewal.
- O’Brien et al., 2016 highlights an auto/cross regulation relationship between Six1/Six2 genes in human nephron progenitors. However, in the mouse their relationship is independent, with Six1 expression occuring upstream of six2.

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Schlosser et al., 2008 found “The effects of overexpression of Eya1 or Six1 are dose dependent. At higher levels, Eya1 and Six1 expand the expression of SoxB1 genes (Sox2, Sox3), maintain cells in a proliferative state and block expression of neuronal determination and differentiation genes. At lower levels, Eya1 and Six1 promote neuronal differentiation, acting downstream of and/or parallel to Ngnr1.”

Six1 can promote specific types of differentiation. For example:

Mesenchymal stem cells into brown adipocytes (Brunmeir et al., 2016).
Human embryonic stem cells can be induced to differentiate into lacrimal gland epithelial-like cells by simultaneous overexpression of three transcription factors, SIX1, PAX6, and FOXC1 (Hirayama et al., 2017).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

The aforementioned Schlosser et al., 2008 study, noted a dose dependent effect of overexpression of Eya1 or Six1, where low doses encouraged differentiation and high doses suppressed it, used doses of 250 to 2000 pg of mRNA injected into Xenopus laevis embryos.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

In both neonatally GEN and DES-exposed groups, nuclear six1 was present in low numbers of scattered luminal and basal-type epithelial cells on the 5th day of treatment (Suen et al., 2016). Nuclear six1 was not observed at the same time on the control organisms.

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Taxonomic Applicability

The evidence presented here are sourced from human and mouse tissues.

Lifestage Applicability

Empirical observations cited here were made during development and adulthood.

Sex Applicability

The downstream event here is organ specific (endometrium) so the Sex applicability is limited to females.

Generally speaking, six1 protein expression leading to the promotion of six1 positive progenitors cells in the endometrium could be expected to occur in any species where six1 expression occurs in animals that have a uterus.

References

List of the literature that was cited for this KER description. More help

Brunmeir R, Wu J, Peng X, Kim SY, Julien SG, Zhang Q, et al. Comparative Transcriptomic and Epigenomic Analyses Reveal New Regulators of Murine Brown Adipogenesis. PLoS genetics 2016;12:e1006474

Coletta, R. D., Christensen, K., Reichenberger, K. J., Lamb, J., Micomonaco, D., Huang, L., ... & Ford, H. L. (2004). The Six1 homeoprotein stimulates tumorigenesis by reactivation of cyclin A1. Proceedings of the National Academy of Sciences, 101(17), 6478-6483.

El-Hashash, A. H., Al Alam, D., Turcatel, G., Rogers, O., Li, X., Bellusci, S., & Warburton, D. (2011). Six1 transcription factor is critical for coordination of epithelial, mesenchymal and vascular morphogenesis in the mammalian lung. Developmental biology, 353(2), 242-258.

Hirayama M, Ko SBH, Kawakita T, Akiyama T, Goparaju SK, Soma A, et al. Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells. NPJ aging and mechanisms of disease 2017;3:1

McCoy, E. L., Iwanaga, R., Jedlicka, P., Abbey, N. S., Chodosh, L. A., Heichman, K. A., ... & Ford, H. L. (2009). Six1 expands the mouse mammary epithelial stem/progenitor cell pool and induces mammary tumors that undergo epithelial-mesenchymal transition. The Journal of clinical investigation, 119(9), 2663-2677.

O'Brien, L. L., Guo, Q., Lee, Y., Tran, T., Benazet, J. D., Whitney, P. H., ... & McMahon, A. P. (2016). Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators. Development, 143(4), 595-608.

Radisky, D. C. (2009). Defining a role for the homeoprotein Six1 in EMT and mammary tumorigenesis. The Journal of clinical investigation, 119(9), 2528-2531.

Schlosser, G., Awtry, T., Brugmann, S. A., Jensen, E. D., Neilson, K., Ruan, G., ... & Moody, S. A. (2008). Eya1 and Six1 promote neurogenesis in the cranial placodes in a SoxB1-dependent fashion. Developmental biology, 320(1), 199-214.

Suen, A. A., Jefferson, W. N., Wood, C. E., & Williams, C. J. (2019). SIX1 regulates aberrant endometrial epithelial cell differentiation and cancer latency following developmental estrogenic chemical exposure. Molecular Cancer Research, 17(12), 2369-2382.

Suen, A. A., Jefferson, W. N., Wood, C. E., Padilla-Banks, E., Bae-Jump, V. L., & Williams, C. J. (2016). SIX1 oncoprotein as a biomarker in a model of hormonal carcinogenesis and in human endometrial cancer. Molecular Cancer Research, 14(9), 849-858.