AOP ID and Title:

Short Title: AhR activation to metastatic breast cancer

Authors

Xavier Coumoul

Robert Barouki

Meriem Koual

Karine Audouze

Celine Tomkiewicz

Status

Abstract

Breast cancer is the deadliest cancer in women with a poor prognosis in case of metastatic breast cancer. The role of the environments in the formation of metastasis has been suggested. We hypothesized that activation of the AhR (MIE), a xenobiotic receptor, could lead to breast cancer metastasis (AO), through different KEs, constituting a new AOP.

An artificial intelligence tool (AOP-helpfinder), which screens the available literature, was used to collect all existing scientific abstracts to build a novel AOP, using a list of key words. Four hundred and seven abstracts were found containing at least a word from our MIE list and either one word from our AO or KE list. A manual curation retained 113 pertinent articles, which were also screened using PubTator. From these analyses, an AOP was created linking the activation of the AhR to breast cancer related death through decreased apoptosis, inflammation, endothelial cell migration, and increased mortality. These KEs promote an increased tumor growth, angiogenesis and invasion which leads to breast cancer metastasis.

The evidence of the proposed AOP was weighted using the tailored Bradford Hill criteria and the AOP developers’ handbook (https://aopwiki.org/handbooks/). The confidence in our AOP and the biological plausibility was considered strong. Indeed, in vitro and in vivo findings on multiple types of breast cancers (with or without oesrtogen receptors, for instanace) supported our proposed AOP. An in vitro validation must be carried out, but our review proposes a strong relationship between AhR activation and breast cancer metastasis with an innovative use of an artificial intelligence literature search.

 

This work was published in Envionnmental International: https://doi.org/10.1016/j.envint.2022.107323

Background

Breast cancer is a frequent disease, responsible of 2 262 419 new cases and 684 996 deaths in 2020 in the world, making it the deadliest female cancer (Bray et al., 2018). In 70% of cases, the disease is localized, and the prognosis is favorable with a 5-year survival of 99%. However, once the disease spreads (lymph nodes, metastasis), survival is severely altered with a 5-year survival rate of 26% in case of metastasis (Henley et al., 2020). It is therefore of paramount importance to understand the mechanisms of metastasis in breast cancer.

Amongst risk factors clearly established, including obesity, genetic mutations and hormonal exposure, the importance of the role of the environment is currently emerging (Koual et al., 2020 Nov 17). In an epidemiologic study, we found a positive association between the concentrations of 2.3.7.8-TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxine) in the adipose tissue surrounding the tumors, and breast cancer metastasis in overweight and obese patients (Koual et al., 2019). Moreover, we have shown that, using both in vivo and in vitro models, TCDD exposure could promote an aggressive phenotype to breast cancer cells, thus favoring the formation of metastatic cells (Koual et al., 2021). TCDD is a potent ligand of the aryl hydrocarbon receptor (AhR), a transcriptional factor involved notably in the metabolism of xenobiotics (Larigot et al., 2022). Hence, the impact of the environment on breast cancer aggressiveness could be mediated by the activation of the AhR.

Interest is growing on the role of the AhR in breast cancer. First, the AhR is often overexpressed in different breast cancer cell lines (Zudaire et al., 2008, Kim et al., 2000 Nov 16, Li et al., 2014). Interestingly, the level of expression can be correlated to the stage or the molecular sub-type of the disease (Zudaire et al., 2008, Zhao et al., 2013). Second, the AhR pathway has been associated with different pro-metastatic features in breast cancer, such as resistance to apoptosis, invasiveness, modified cell cycle, migration and proliferation (Zudaire et al., 2008, Goode et al., 2013 Dec 15, Kanno et al., 2006). Triple negative cell lines, breast cancer cell lines with the worse prognosis (not over-expressing Her2 receptor or hormonal receptors), over-expressing the AhR seem to develop stem-like characteristics, favoring epithelial-mesenchymal transition (EMT) and thus metastasis (Stanford et al., 2016). Thirdly, the AhR could be involved in the resistance of breast cancer to treatments (Goode et al., 2013 Dec 15, Goode et al., 2014): after AhR knockout, Goode et al. found enhanced sensitivity of paclitaxel (a drug targeting cancer cells) in triple negative breast cancer, a cancer particularly difficult to treat (Goode et al., 2014). Breast cancer patients expressing estrogen receptors (ER-positive) in their cancer cells, can benefit from an efficient endocrine therapy, which greatly improves their survival. Activation of the AhR can lead to the loss of expression of the ER alpha and therefore to the loss of a potential therapeutic target (Safe et al., 2000 Jul).

The mechanisms linking the activation of the AhR to breast cancer aggressiveness are still unclear. Based on the AOP-wiki database (https://aopwiki.org/, last accessed March 2022), the central repository for AOPs, the AhR has already been proposed in several AOPs, but never in one characterized by the AO breast cancer metastasis. Likewise, an AOP linking an MIE to breast cancer aggressiveness has never been proposed. From our expertise and available knowledge, we hypothesize that the activation of the AhR could be a MIE leading to breast cancer metastasis (AO) through different KEs and KERs.

Summary of the AOP

Events

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

Key Event Relationships

Stressors

Overall Assessment of the AOP

The biological plausibility of KERs is defined by the OECD as the « understanding of the fundamental biological processes involved and whether they are consistent with the causal relationship being proposed in the AOP ». The biological plausibility is strong due to the presence of overwhelming evidence present in different studies. A minor setback would be the difficulty to dismiss alternative mechanisms caused by the ligands used for AhR activation. This is detailed in the discussion.

The essentiality of KEs refers to « experimental data for whether or not downstream KEs or the AO are prevented or modified if an upstream event is blocked ». The essentiality of KEs is strong: most works use suppression or inhibition of the AhR (knock out, antagonists and/or silencing) with results coherent with our findings.

Finally, the empirical support of KERs, is often « based on toxicological data derived by one or more reference chemicals where dose–response and temporal concordance for the KE pair can be assessed ». The overall assessment of the empirical support of our KERs is also strong. There is evidence in human cell lines and mice showing a dose–response and temporal concordance for severity of our KE and the presence of metastasis.

 

We propose a simple and robust AOP associating activation of the AhR and breast cancer related death through migration, invasion, inflammation, and neo-angiogenesis.

One of the main limitations of our AOP is the existence of these diverse ligands and pathways, complexifying the definition of ‘AhR activation’ (6,54). Using PubTator, we found that TCDD was by far the most used chemical followed by I3C, alpha-naphthoflavone, polycyclic aromatic hydrocarbons and hexachlorobenzene, all ligands of the AhR. These ligands can activate different pathways after AhR binding and we therefore assumed that these compounds were AhR agonists. It can be difficult to dismiss alternative mechanisms caused by the ligands used for AhR activation. However, the AhR is the only characterized target of TCDD for example, and studies which use several ligands including TCDD, display similar results using the other modulators. Moreover, the concordance of studies using various ligands and the coherence with the AhR inhibition are in favor of the robustness of the proposed AOP. Indeed, to obtain the most accurate AOP possible, the KEs selected had to be present, no matter the ligand used by the study.

Another minor setback of using the AhR, is that the dose response concordance is a non-monotonous curve for several ligands (122,123). Therefore, the tailored Bradford-Hill criteria could sometimes not be fulfilled.

Moreover, the originality of our work lies in the use of artificial intelligence too such as AOP-helpfinder, which enables a thoroughly search of existing knowledge in the PubMed database and PubTator (19–21). Therefore, our literature review was complete and evidence in favor of our proposed AOP was overwhelming. We plan to validate our proposed AOP in a quantitative in vitro work using Integrated Approaches to Testing and Assessment (IATA).

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

The biological applicability domain of the putative AOP concerned mainly females of menstrual of post-menopausal age. Indeed, existing cell lines were derived from women of menstrual of post-menopausal age and in vivo, studies were performed on mice of reproductive age. Only one study used the zebra fish larvae (Narasimhan et al., 2018 May 7). However, it could be extrapolated to men. Indeed, breast cancers in men present similar tumor characteristics and no work has found diverging functions of the AhR between men and women. Moreover, no difference in AhR expression has been characterized between men and women. Furthermore, our AOP concerns ER-positive and triple negative cells lines.

Studies were carried out in humans, mice, and zebrafish (xenotransplant studies, no mammary gland) (i.e. PubTator results) and it can be hypothesized that this AOP is conserved across mammals. Indeed, the AhR is a very conserved and ancient protein (Hahn, 2002 Sep 20). However, since the sensitivity to adverse events are variable among taxa, we can only postulate this AOP in human and mice (Korkalainen et al., 2001 Aug 3, Cohen-Barnhouse et al., 2011 Jan, Doering et al., 2013 Mar).

 

The AhR is a fascinating yet complex receptor since its activation is ligand and cell dependent. To avoid more bias, we decided to limit our AOP to breast cancer. First, this cancer is the most frequent female malignancy, which makes it a major public health concern. Second, this illness is hormonal-dependent and therefore the impact of the environment, through the AhR, can be strongly suggested. However, we have reasons to believe this AOP could be extrapolated to other cancers which share common regulatory pathways (Larigot et al., 2022). The AhR is overexpressed not only in breast cancer but also in lung, liver, stomach, head & neck, cervix, and ovarian cancer (Stanford et al., 2016, DiNatale et al., 2010 Aug 6, Liu et al., 2013 Aug, Stanford et al., 2016 Aug). Moreover, in these cancers, the level of expression is correlated to the stage of the disease (Zudaire et al., 2008, Koliopanos et al., 2002 Sep 5, Chang et al., 2007 Jan 1). Additionally, Moenniks et al. found that mice with constitutively active AhR had more liver tumors than wild type mice (55% versus 6%) (Moennikes et al., 2004 Jul 15). In vitro evidence suggests that the AhR activation could promote a more aggressive phenotype to renal, lung, head and neck, and urothelial cancer through an increase in invasion, migration, and resistance to apoptosis which constitute representative key events of our AOP (Zudaire et al., 2008, Stanford et al., 2016 Aug, Ishida et al., 2015 Jul 15, Ishida et al., 2010 Feb, Diry et al., 2006 Sep 7, John et al., 2014 Oct). Besides, an AOP associating AhR activation and lung cancer initiation is currently under development (AOP, 2021) (https://aopwiki.org/aops/417, accessed May 2022).

Likewise, our AOP covers only breast cancer progression and not initiation. The mechanisms of breast cancer initiation are different from the metastatic pathway, but the AhR could also be involved in breast cancer initiation. In vitro, it was noted that human mammary benign cells with a high level of AhR had an increase in cell proliferation, and migration, and potentially display EMT-like features (Brooks and Eltom, 2011 Jun). In vivo, mice fed with 7,12-dimethylbenz[a]anthracene (DMBA, an AhR activator and a potent mutagen) had an increased risk of mammary tumors, with higher AhR expression (Currier et al., 2005). Strangely in regard of the deadly outcomes associated with aggressive breast tumors, the number of studies focusing on this specific aspect of mammary carcinogenesis is limited and therefore, epidemiological data on the effects of the exposome in breast cancer aggressiveness is scarce. Indeed, occupational exposure is difficult to quantify, and patients are usually exposed to a mixture of pollutants and not a single pollutant in a chronic way. A memory bias cannot be excluded since the half-life of TCDD, for instance, is 7–11 years (Pirkle et al., 1989). Industrial accidents, such as the Seveso incident, studied the increase in breast cancer incidence but did not record breast cancer aggressiveness since it is more complex to quantify. At an early stage, breast cancer has a favorable prognosis whereas the therapeutic challenge lies in the treatment of breast cancer metastases. Therefore, even though epidemiologic and cell evidence suggests that exposure to pollutants and Ahr activation could promote breast cancer initiation, we chose to study breast cancer progression, the most complex situation (Pesatori et al., 2009 Sep, Warner et al., 2002 Jul).

Essentiality of the Key Events

 

KEY EVENT	LEVEL OF ESSENTIALITY	EVIDENCE
KE 1262  : decreased apoptosis	Strong	A decrease in apoptosis is an essentiel element in promoting tumor growth (Hannahan , Fulda). Indeed, in case of a decrease in cell death, the tumor will continue to grow. First, A decrase in apoptosis causes uncontrolled Cell Proliferation. Healthy tissues maintain homeostasis through a balance between cell proliferation and apoptosis. When apoptosis is compromised, cells that should undergo programmed cell death survive and continue dividing. This leads to an unchecked increase in cell number, forming the initial tumor mass. [Hanahan & Weinberg, 2011]. Second, cancer cells sustain proliferative signaling. Many cancers harbor mutations that activate pro-proliferative signaling pathways like Ras or PI3K/Akt. These pathways normally promote cell growth and division. However, mutational dysregulation allows them to continue signaling proliferation even when apoptosis should occur or growth signals are absent. Additionally, reduced apoptosis prevents the activation of pro-apoptotic pathways that normally act as brakes on cell division. [Luo & Heng, 2003] Also, healthy cells respond to cues like density-dependent inhibition and nutrient limitations by activating apoptosis. When apoptosis is compromised, cells can evade these growth-inhibitory signals and continue dividing even when resources are limited or cell density is high. This allows the tumor to expand beyond its boundaries and invade surrounding tissues. [Fulda & Debatin, 2007]. A decrease in apoptosis is therefore essential to maintain tumor growth. However, cell proliferation is also an essentiel element in promoting tumor growth. Yet, due to the presence of diverging evidence on the activation of the AhR and cell proliferation, we chose not to include these in our AOP. Indeed, on one hand, activation of the AhR through ligands such as NK150460, ANI-7, emodine or derivates of revesterol decrease cell proliferation in ER-positive and ER-negative breast cancer cell lines. TCDD has been found to promote cell cycle arrest through phosphorylation of the retinoblastoma protein which binds to E2F. In ER-positive cell lines, beta-naphthoflavone mediated cell cycle arrest through an upregulation of P21. On the other hand, AhR activation could promote cell proliferation. Pearce et al. found that MCDF (6-methyl-1,3,8-trichlorodibenzofuran), an AhR agonist could stimulate cell proliferation with a dose-response concordance. Likewise, I3C, HCB, CPF and licorice could also promote cell proliferation. However, it seems that this cell proliferation is ER-dependent. Indeed, these ligands induced cell proliferation only in ER-positive cells lines with an effect dependent on the level of estrogen present in the medium. Whether this increase in ER-dependent cell proliferation can be independent of the AhR remains unclear. This increase in proliferation could also be mediated by the association of the RelA subunit of NF-kappaB with the AhR resulting in the activation of c-myc gene transcription in breast cancer cells. This would explain why Rodriguez et al. found that proliferation was modulated by the CYP1A1, independently of an exogenous ligand activation of the AhR. These complex effects, highly dependent on the context (cell types, medium content, type of ligand…) were therefore not included in our AOP despite the strong evidence.
KE 1971 :  tumor growth	STRONG	An increase in tumor size is associated with breast cancer metastasis and is essential to the progression of the illness  (Hanahan and Weinberg, 2011 Mar 4). Indeed, clinical evidence suggests that tumor size is directly correlated to the presence of metastasis (Liu Y, He M, Zuo WJ, Hao S, Wang ZH, Shao ZM. Tumor Size Still Impacts Prognosis in Breast Cancer With Extensive Nodal Involvement. Front Oncol and Narod SA. Tumour size predicts long-term survival among women with lymph node-positive breast cancer. Curr Oncol.) Likewise, studies have shown that larger tumor size in colorectal cancer is associated with increased risk of metastasis and poorer overall survival. [Benson et al., 2008]
KE 1241  Increased cell motility	MODERATE	The relation between cell migration and organ invasion is essntial. Organ invasion can be promonted by cell migration, motility and inflammation. Therefore the essentilality of cell motility was classified as moderate since other factors can promote organ invasion. For instance, melanoma cells are known for their high migratory potential, allowing them to invade the surrounding dermis and potentially metastasize to distant organs like the brain and lungs. [Clark et al., 2009] Likewise, breast cancer cells can migrate through the basement membrane and invade surrounding breast tissue, potentially reaching lymph nodes or blood vessels for further dissemination. [Friedl & Weigelin, 2008]
KE 1196: organ invasion	STRONG	Organ invasion is an essential step in promoting breast cancer agressivness and metastasis. Without invasion of the basal membrane, the cancer remains located in an in situ state and does not induce metastasis.  Pancreatic cancer cells are notorious for their invasive nature. They can invade surrounding tissues like the pancreas, blood vessels, and nerves, increasing the risk of metastasis to the liver, lungs, and bones. [Olive et al., 2009] Colorectal cancer cells can invade the bowel wall and potentially reach surrounding blood vessels, allowing them to travel to the liver, lungs, and other distant sites. [Fearon & Vogelstein, 1990]
KE 149  Increased inflammation	MODERATE	Organ invasion can be promonted by cell migration, motility and inflammation. Therefore the essentilality of cell motility was classified as moderate since other factors can promote organ invasion. In angiogenesis, however, increased inflammation is a key factor. Indeed, inflammation, through the secretion of growth factor promotes the creation of blood vessels (VEGF, IL6, COX).
KE 1190 Increased endothelial migration	STRONG	Endothelial cell migration is an essential key event in promoting angiogenesis. Extensive data exists on the essentialitty of this step (Franziska van Zijl, Georg Krupitza, Wolfgang Mikulits, Initial steps of metastasis: Cell invasion and endothelial transmigration, Mutation Research/Reviews in Mutation Research, Volume 728, Issues 1–2, 2011, Pages 23-34, ISSN 1383-5742, https://doi.org/10.1016/j.mrrev.2011.05.002.)
KE 1213: angiogenesis	STRONG	Without the creation of new vessels in order to receive nutrients and energy, the cancer cell cannot survive and create metastatis. It is an essential key event and considered as one of the hallmarks of cancer  (Hanahan and Weinberg, 2011 Mar 4).

 

Weight of Evidence Summary

KER 2569  Activation of the AhR leads to decreased apoptosis

Several studies have found that the activation of the AhR by stressors such as TCDD, can promote a decrease in apoptosis (KER2569), which is a deleterious event with regards to cancer (Al-Dhfyan et al., 2017 Jan 19, Bekki et al., 2015). Additionally, an increase in cell death was found when blocking the AhR pathway using AhR silencing (RNA interference or knock-out), knockout cell lines or antagonists (CH223191 or alpha-naphthoflavone) (Goode et al., 2013 Dec 15, Al-Dhfyan et al., 2017 Jan 19, Bekki et al., 2015, Regan Anderson et al., 2018). The most frequently used assay to evaluate apoptosis was cytometry with the use of Annexin V: this was performed with ER-positive cells lines (MCF-7, T-47D), triple negative cell lines (MDA-MB-231, HS 578), cells over-expressing the Her2 (SK-BR-3) and cells lines derived from cancer samples from patients (Goode et al., 2013 Dec 15, Al-Dhfyan et al., 2017 Jan 19, Bekki et al., 2015, Regan Anderson et al., 2018, Fujisawa et al., 2011).

The concordance of the evidence was classified as “moderate” since the aim of most studies was to evaluate the capacity to survive in an apoptosis-promoting environment (i.e., chemotherapeutic drugs). Indeed, they assessed the resistance to chemotherapy agents such as doxorubicin and paclitaxel and found that the concomitant inactivation of the AhR pathway could decrease the resistance to these chemotherapy agents through an increase in cell death when compared to cells with a functional (or expressed at sufficient levels) AhR (Goode et al., 2013 Dec 15, Al-Dhfyan et al., 2017 Jan 19, Bekki et al., 2015, Regan Anderson et al., 2018, Fujisawa et al., 2011). Since the environment was modified by the presence of chemotherapy, the hypothesis of an alternative pathway cannot be completely discarded. It must be noticed that the exact biological mechanisms linking the activation of the AhR to the decrease in apoptosis remains unclear. Indeed, Anderson et al. suggested that the AhR interacts with the glucocorticoid receptor (GR) and the hypoxia inducible factor-2α (HIF-2α) (Regan Anderson et al., 2018). The presence of the GR is associated with a poor prognosis, notably in triple negative breast cancer (Pan et al., 2011, Moran et al., 2000 Feb 15). Indeed, this receptor is involved in survival and resistance to chemotherapy through up-regulation of c-myc, Bcl2 and Kruppel-like factor 5 (Pan et al., 2011, Wu et al., 2004, Li et al., 2017). Both GR and HIF 2α could be up regulated by the AhR. They then activate Brk (also known as PTK6), a ligand of EGFR (epidermal growth factor receptor), involved in the inhibition of apoptosis (Regan Anderson et al., 2018, Li et al., 2012). Another possible mechanism suggested by Bekki et al. is that the decrease in apoptosis was caused by the induction of cyclooxygenase 2 (COX-2) and the NF-κB subunit RelB (Bekki et al., 2015). They both prevent apoptosis through induction of Bcl2, an anti-apoptotic factor (Tsujii and DuBois, 1995, Vogel et al., 2007, Thomas et al., 2020, Baud and Jacque, 2008 Dec, Demicco et al., 2005 Nov, Wang et al., 2007 Apr, Liu et al., 2001 May 25).

KER 2577: Decreased apoptosis promotes tumor growth

For KER 2577, in vivo, Goode et al. showed that the knockout of the AhR in mice reduced tumor growth through an increase of cell apoptosis (Goode et al., 2013 Dec 15).

The relationship between decreased apoptosis and increase in tumor growth (KER 2577) is not detailed here due to extensive evidence in the scientific literature (Hanahan and Weinberg, 2011 Mar 4).

KER 2570: Activation of the AhR leads to an increased cell motility

The activation of the AhR can modulate cell motility in different types of breast cancers such as: ER-positive cells lines (MCF-7, T-47D, ZR-75–1), triple negative (MDA-MB-231, MDA-MB-435, HS-578-T, SUM149), and cells overexpressing the Her2 (SK-BR-3) (Goode et al., 2013 Dec 15, Regan Anderson et al., 2018, Parks et al., 2014 Nov, Pontillo et al., 2011 Apr, Qin et al., 2011 Oct 20, Nguyen et al., 2016 Nov 15, Novikov et al., 2016 Nov, Miret et al., 2016 Jul, Shan et al., 2020 Nov, Dwyer et al., 2021 Feb, Narasimhan et al., 2018 May 7, Hsieh et al., 2012 Feb). Activation of the AhR with TCDD, butyl-benzyl phthalate, di-n-butyl phthalate, hexachlorobenzene, and benzo[a]pyrene can promote cell migration in different assays (Parks et al., 2014 Nov, Pontillo et al., 2011 Apr, Qin et al., 2011 Oct 20, Novikov et al., 2016 Nov, Miret et al., 2016 Jul, Shan et al., 2020 Nov, Narasimhan et al., 2018 May 7, Hsieh et al., 2012 Feb). On the other hand, the use of AhR antagonists, AhR silencing or AhR knockout reversed this effect (Goode et al., 2013 Dec 15, Regan Anderson et al., 2018, Parks et al., 2014 Nov, Pontillo et al., 2011 Apr, Qin et al., 2011 Oct 20, Novikov et al., 2016 Nov, Shan et al., 2020 Nov, Narasimhan et al., 2018 May 7, Hsieh et al., 2012 Feb). The most frequently used assays for evaluating cell migration were the scratch wound assay and the transwell chamber assay. Only three works evaluated the dose–response concordance of AhR activation with stressors and cell migration (Pontillo et al., 2011 Apr, Miret et al., 2016 Jul, Shan et al., 2020 Nov). The evidence was therefore classified as “moderate”.

 KER 2572: Activation of the AhR leads to an increased invasion

Due to the extensive robust and concordant literature of the link between activation of the AhR-increased cell motility-increased invasion-breast cancer progression, the confidence in these key events was rated as high. However, due to the use of ligands to activate the AhR, it cannot be completely ruled out that alternative pathways (independent of the AhR) can also contribute to these features. For instance, 2 main pathways seem to explain this increase in migration and invasion: the c-Src/HER1/STAT5b, and ERK1/2 pathways. Yet, these pathways seem only to explain the relation between the AhR activation and cell migration / invasion, when the ligand used is hexachlorobenzene, an organochlorinated pesticide (Pontillo et al., 2011 Apr, Miret et al., 2016 Jul, Pontillo et al., 2013 May 1). Even though alternative mechanisms may present themselves, all studies blocked the AhR pathway and found a decrease in cell migration/invasion. The evidence for alternative mechanisms was therefore classified as “moderate” and the biological plausibility of KER was also classified as “moderate”.

KER 1306: Increased cell motility promotes organ invasion

The relation between cell migration and organ invasion has already been shown (KER-1306, https://aopwiki.org/relationships/1306). Since the 2 are closely linked, most articles studied both cell migration (chemo-tactic) and the capacity to invade the extra-cellular matrix. Cell invasion is indeed defined as the capacity of a cell to migrate and degrade/invade the extracellular matrix. In vitro, this process was evaluated mostly using transwell chamber with Matrigel® and the presence of matrix metalloproteinases (MMP). This effect was found in ER-positive cells, triple negative cell lines and cells overexpressing the Her2.

 KER 2572: Activation of the AhR leads to an increased invasion

The activation of the AhR through the use of different ligands (benzophenone, butyl benzyl phthalate, di-n-butyl phthalate, hexachlorobenzene, chlorpyrifos, TCDD) or the blockage of the AhR (silencing, KO or antagonism) increased or decreased cell invasion, respectively (Parks et al., 2014 Nov, Qin et al., 2011 Oct 20, Nguyen et al., 2016 Nov 15, Miret et al., 2016 Jul, Shan et al., 2020 Nov, Narasimhan et al., 2018 May 7, Hsieh et al., 2012 Feb, Pontillo et al., 2013 May 1, Miller et al., 2005, Belguise et al., 2007 Dec 15, Yamashita et al., 2018 May 1, Miret et al., 2020 May). The dose–response concordance for cell invasion was demonstrated using increasing doses of hexachlorobenzene, benzo[a]pyrene, chlorpyrifos and TCDD (Miret et al., 2016 Jul, Shan et al., 2020 Nov, Pontillo et al., 2013 May 1, Miller et al., 2005, Miret et al., 2020 May). To further explore cell invasion, Nguyen et al. created a model of a lymphatic barrier using a three-dimensional lymph endothelial cell as a monolayer co-cultured with spheroids of MDA-MB231 cells (Nguyen et al., 2016 Nov 15). They found that silencing or antagonizing the AhR (DIM) or activating the AhR (FICZ) respectively decreased or increased invasion of the lymphatic barrier.

On an organ level, in vivo, an increase in metastasis has been found in mice and zebrafish after the activation of the AhR with different ligands (butyl benzyl phthalate, di-n-butyl phthalate, hexachlorobenzene, TCDD) (Goode et al., 2014, Shan et al., 2020 Nov, Narasimhan et al., 2018 May 7, Hsieh et al., 2012 Feb, Pontillo et al., 2013 May 1). In the zebrafish model, Narasimham et al. treated the animals either with triple negative MDA-MB-231 cells only (untreated) or with MDA-MB-231 cells treated with an AhR inhibitor (CB7993113 or CH22319) (Narasimhan et al., 2018 May 7). Untreated fish had significantly more metastasis (OR = 9, IC95%=3–35). Similar results were found using mice models (Goode et al., 2014, Shan et al., 2020 Nov, Narasimhan et al., 2018 May 7, Hsieh et al., 2012 Feb, Pontillo et al., 2013 May 1).

KER 2568: Activation of the AhR leads to an increased inflammation 

In triple negative breast cell lines (MDA-MB436, MDA-MB-231) and ER-positive cell lines, it has been shown that the activation of the AhR can lead to an increase in inflammation. (Bekki et al., 2015, Miller et al., 2005, Yamashita et al., 2018 May 1, Degner et al., 2009 Jan, Vogel et al., 2011 Aug 1, Kolasa et al., 2013 Apr 25, Vacher et al., 2018, Malik et al., 2019 Oct). The stressors mainly used to activate the AhR were TCDD followed by benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhiP). After AhR inhibition (KO or antagonists), a decrease in inflammation biomarkers was found (Miller et al., 2005, Yamashita et al., 2018 May 1, Degner et al., 2009 Jan, Vogel et al., 2011 Aug 1, Kolasa et al., 2013 Apr 25). Assays evaluating cell inflammation were quantitative dosages of IL-6, IL-8 and Cox2 activity/expression. Cox-2 and IL-8 were amongst the top “gene concepts” retrieved by the PubTator Central tool, likewise, “inflammation” was frequently found as a disease concept. The most consensual pathway linking the AhR activation to cell inflammation was the NF-kB pathway (Vogel et al., 2011 Aug 1, Kolasa et al., 2013 Apr 25). Only half of the studies found a dose–response relationship (Miller et al., 2005, Kolasa et al., 2013 Apr 25, Malik et al., 2019 Oct). No studies were carried out in vivo for breast cancer and therefore the concordance and evidence were classified as “moderate”.

AOP 21 also found the association between AhR activation and inflammation via COX 2 (Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2) with a weight of evidence classified as “high”. Indeed, the AhR/ARNT heterodimer links to the dioxin responsive elements which in turn up-regulates COX-2 (66,67].

 KER 2573: Inflammation promotes organ invasion

In the specific setting of AhR activation, only 2 studies showed the continuum between AhR activation – increased inflammation – increased invasion (Miller et al., 2005, Yamashita et al., 2018 May 1). However, in general, there is extensive knowledge on the relationship between cell inflammation and organ invasion. First, COX-2 is expressed at higher levels in triple negative invasive breast cancers than in less aggressive ER-positive cancers (Gilhooly and Rose, 1999 Aug, Liu and Rose, 1996 Nov 15). COX-2 catalyzes the conversion of arachidonic acid into prostaglandin H2, a pro-inflammatory factor, and is therefore considered as a prognosis factor in breast cancer (Ristimäki et al., 2002 Feb 1, Parrett et al., 1997 Mar). Transfection with COX-2 triple negative MDA-MB-435 cells increased cell migration 2-fold compared to control cells in a transwell-Matrigel® assay. Antagonism of COX-2 through an inhibitor (NS-398) reversed this action in a dose-dependent way (Singh et al., 2005 May). Second, in vivo, the use of anti-inflammatory treatments such as celecoxib (COX-2 inhibitor) can reduce tumor growth and spread (Harris et al., 2000 Apr 15). Finally, epidemiologic evidence suggests that inflammatory breast cancers have the worse prognosis. Indeed, the median overall survival of patients with inflammatory breast cancer compared with those with non-inflammatory breast cancer tumors is 4.75 years versus 13.40 years for stage III disease and 2.27 years versus 3.40 years for stage IV disease (Schlichting et al., 2012 Aug, Fouad et al., 2017 Apr).

The mechanism of action of COX-2 are consensual. COX-2 promotes cell invasion through upregulation of MMPs (notably 2 and 9) (Takahashi et al., 1999 Oct 22, Sivula et al., 2005 Feb, Larkins et al., 2006 Jul). Moreover, COX-2 could also activate the urokinase plasminogen activator (uPA) which degrades the basal membrane of epithelia (Singh et al., 2005 May, Takahashi et al., 1999 Oct 22, Larkins et al., 2006 Jul, Guyton et al., 2000 Mar).

The relationship between inflammation and invasion is well document therefore the evidence was classified as “strong”.

 KER 2574: Inflammation promotes angiogenesis

Likewise, two studies evaluated the specific continuum AhR activation – increased inflammation – increased angiogenesis (Pontillo et al., 2015 Nov 19, Zárate et al., 2020 Aug). As previously mentioned, the AhR activation increases inflammation, notably through an increase in COX 2 (Bekki et al., 2015, Miller et al., 2005, Degner et al., 2009 Jan, Pontillo et al., 2015 Nov 19, Zárate et al., 2020 Aug).

COX-2 can promote angiogenesis through an increase in VEGF (Vascular endothelial growth factor) (Harris et al., 2014 Oct 10, Kirkpatrick et al., 2002). In a pathologic study characterizing 46 breast cancer specimen using immunochemistry, it was found that the density of microvessels was significantly higher in patients with COX-2 expression than in those without expression (p = 0.03) (Costa et al., 2002 Jun). The relationship between COX-2 and angiogenesis has also been shown in gastric and colorectal cancer (Tsujii et al., 1998 May 29, Uefuji et al., 2000 Jan). Indeed, colon carcinoma cells overexpressing COX-2 produce proangiogenic factors (VEGF, bFGF, TBF-β, PDGF, and endothelin-1), and stimulate endothelial migration and the formation of tube vessels. These effects were reversed by an inhibitor (NS-398). In vivo, Diclofenac, a COX-2 inhibitor, decreased angiogenesis in mice presenting a colorectal cancer (Seed et al., 1997 May 1). Likewise, in a murine model of breast cancer, celecoxib (a selective COX-2 inhibitor) reduced metastasis and tumor burden through a decrease of micro vessel density and VEGF (Yoshinaka et al., 2006 Dec, Zhang et al., 2004 Sep). In clinical studies, patients with inflammatory breast cancers have increased levels of genes involved in angiogenesis such as VEGF (Van der Auwera et al., 2004 Dec 1). Patients with an inflammatory breast cancer benefit the most from anti-angiogenic treatment bevacizumab (Pierga et al., 2012 Apr).

The evidence was classified as “moderate” due to the lack of dose response studies.

KER 1266: Activation of the AhR leads to an increased endothelial migration 

The activation of the AhR can lead to an increased endothelial cell migration. This was found when HMEC-1 or EA.hy926 cells were co-cultured with ER-positive MCF-7 cells and triple negative MDA-MB-231 cells (Pontillo et al., 2015 Nov 19, Zárate et al., 2020 Aug). The assay mainly used was the Matrigel® / tube formation assay. Only one study found an increase in endothelial cell proliferation and not migration, therefore it was not kept as a KE (Pontillo et al., 2015 Nov 19). The main pathway explaining this relationship was again related to the activation of COX2 and subsequently to the increase in VEGF. The association between the activation of the AhR and endothelial cell migration was classified as “weak” since only 2 studies explored this feature, and both used hexachlorobenzene as a stressor. However, these works were robust with strong evidence, and both found a reversed association after AhR blockage. No contradicting results were found in the scientific literature.

As opposed to our work, another AOP displayed a link between AhR activation and angiogenesis (AOP 150) and found that activation of the receptor could decrease VEGF production with moderate evidence and quantitative understanding. It must be noted that these AOPs applied only to chicken, zebrafish, and certain rodents whereas our AOP concerns humans. As detailed further, the AhR presents a variability between species which must be considered.

KER 1267:  Increased endothelial migration promotes angiogenesis

Pontillo et al. treated mice with increasing doses of hexachlorobenzene and then calculated the vessel density in mammary fat pads (Pontillo et al., 2015 Nov 19). They found that mice treated with hexachlorobenzene had a higher vessel density with a dose–response concordance. Treatment by AhR antagonists completely reversed this association (Pontillo et al., 2015 Nov 19, Zárate et al., 2020 Aug). The relationship between endothelial migration and angiogenesis was not detailed here since there is existing extensive knowledge (Lamalice et al., 2007 Mar 30, Norton and Popel, 2016 Nov 14, Ausprunk and Folkman, 1977 Jul 1). The KER 12 was considered as “strong”.

 KER 3137, 3138 and 3137: Increased tumor growth, increased invasion, and increased angiogenesis lead to breast cancer metastasis

Due to extensive data in the scientific literature and the empirical evidence in favor of these KERs, these KERs were not detailed here.

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Appendix 1

List of MIEs in this AOP

Event: 18: Activation, AhR

Short Name: Activation, AhR

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The AHR structure has been shown to contribute to differences in species sensitivity to DLCs in several animal models. In 1976, a 10-fold difference was reported between two strains of mice (non-responsive DBA/2 mouse, and responsive C57BL/6 14 mouse) in CYP1A induction, lethality and teratogenicity following TCDD exposure^[3]. This difference in dioxin sensitivity was later attributed to a single nucleotide polymorphism at position 375 (the equivalent position of amino acid residue 380 in chicken) in the AHR LBD^[30][19][31]. Several other studies reported the importance of this amino acid in birds and mammals^{[32][30][22][33][34][35][31][36]}. It has also been shown that the amino acid at position 319 (equivalent to 324 in chicken) plays an important role in ligand-binding affinity to the AHR and transactivation ability of the AHR, due to its involvement in LBD cavity volume and its steric effect^[35]. Mutation at position 319 in the mouse eliminated AHR DNA binding^[35].

The first study that attempted to elucidate the role of avian AHR1 domains and key amino acids within avian AHR1 in avian differential sensitivity was performed by Karchner et al.^[22]. Using chimeric AHR1 constructs combining three AHR1 domains (DBD, LBD and TAD) from the chicken (highly sensitive to DLC toxicity) and common tern (resistant to DLC toxicity), Karchner and colleagues^[22], showed that amino acid differences within the LBD were responsible for differences in TCDD sensitivity between the chicken and common tern. More specifically, the amino acid residues found at positions 324 and 380 in the AHR1 LBD were associated with differences in TCDD binding affinity and transactivation between the chicken (Ile324_Ser380) and common tern (Val324_Ala380) receptors^[22]. Since the Karchner et al. (2006) study was conducted, the predicted AHR1 LBD amino acid sequences were been obtained for over 85 species of birds and 6 amino acid residues differed among species^[14][37] . However, only the amino acids at positions 324 and 380 in the AHR1 LBD were associated with differences in DLC toxicity in ovo and AHR1-mediated gene expression in vitro^[14][37][16]. These results indicate that avian species can be divided into one of three AHR1 types based on the amino acids found at positions 324 and 380 of the AHR1 LBD: type 1 (Ile324_Ser380), type 2 (Ile324_Ala380) and type 3 (Val324_Ala380)^[14][37][16].

• Little is known about differences in binding affinity of AhRs and how this relates to sensitivity in non-avian taxa.
• Low binding affinity for DLCs of AhR1s of African clawed frog (Xenopus laevis) and axolotl (Ambystoma mexicanum) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).
• Among reptiles, only AhRs of American alligator (Alligator mississippiensis) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).
• Among fishes, great differences in sensitivity to DLCs are known both for AhRs and for embryos among species that have been tested (Doering et al 2013; 2014).
• Differences in binding affinity of the AhR2 have been demonstrated to explain differences in sensitivity to DLCs between sensitive and tolerant populations of Atlantic Tomcod (Microgadus tomcod) (Wirgin et al 2011).
  • This was attributed to the rapid evolution of populations in highly contaminated areas of the Hudson River, resulting in a 6-base pair deletion in the AHR sequence (outside the LBD) and reduced ligand binding affinity, due to reduces AHR protein stability.
• Information is not yet available regarding whether differences in binding affinity of AhRs of fishes are predictive of differences in sensitivity of embryos, juveniles, or adults (Doering et al 2013).

The AhR is a very conserved and ancient protein (95) and the AhR is present  in human and mice (96–98). The AhR is present in human physiology and pathology. The AhR is highly expressed at several important physiological barriers such as the placenta, lung, gastrointestinal system, and liver in human (Wakx, Marinelli, Watanabe).  In these tissues, the AhR is involved in both detoxication processes involving xenobiotic metabolizing enzymes such as cytochromes P450, and in immune functions translating chemical signals into immune defence pathways (Marinelli, Stobbe). Moreover, it has a regulatory role in human dendritic cells and myelination (Kado, Shackleford). The lung constitutes another barrier exposed to components of air pollution such as particles and hydrocarbons (air pollution, cigarette smoke). The AhR detects such hydrocarbons and protects the pulmonary cells from their deleterious effects through metabolization. The regulatory effect on blood cells of the AhR, balancing different related cell types, can be extended to the megakaryocytes and their precursors; indeed, StemRegenin 1 (SR1), an antagonist of the AhR increases the human population of CD34+CD41low cells, a fraction of very efficient precursors of proplatelets (Bock). The occurrence of a nystagmus has been subsequently diagnosed in humans bearing a AhR mutation (Borovok).

In human cancer, the AhR has either a pro or con tumor effect depending on the tissue, the ligand, and the duration of the activation (Zudaire, Chang, Litzenburg, Gramatzki, Lin, Wang). In human breast cancer, the AhR is thoughts to be responsible of its progression (Goode, Kanno, Optiz, Novikov, Hall, Subramaniam, Barhoover). In human mammary benign cells, Brooks et al. noted that a high level of AhR was associated with a modified cell cycle (with a 50% increase in population doubling time in cells expressing the AhR by more than 3-fold) and EMT including increased cell migration. Narasimnhan et al. found that suppression of the AhR pathway had a pro-tumorigenic effect in vitro (EMT, tumor migration) in triple negative breast cancer.

Many endogenous and exogenous ligands are present for the AhR in human (Optiz, Adachi, Schroeder, Rothhammer). Indoles, such as indole-3-carbinol or one of its secondary metabolites, 3-3'- Diindolylmethane, are degradation products found in cruciferous vegetables and characterized as AhR ligands (Ema, Kall, Miller) they are also inducers of the human and rat CYP1A1 (Optiz). FICZ is the most potent AhR ligand known to date: it has a stronger affinity than TCDD for the human AhR (TCDD Kd=0.48 nM/FICZ Kd=0.07 nM) (Coumoul).

 

 

Key Event Description

The AHR Receptor

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) superfamily and consists of three domains: the DNA-binding domain (DBD), ligand binding domain (LBD) and transactivation domain (TAD)^[1]. Other members of this superfamily include the AHR nuclear translocator (ARNT), which acts as a dimerization partner of the AHR ^[2][3]; Per, a circadian transcription factor; and Sim, the “single-minded” protein involved in neuronal development ^[4][5]. This group of proteins shares a highly conserved PAS domain and is involved in the detection of and adaptation to environmental change^[4].

Investigations of invertebrates possessing early homologs of the AhR suggest that the AhR evolutionarily functioned in regulation of the cell cycle, cellular proliferation and differentiation, and cell-to-cell communications (Hahn et al 2002). However, critical functions in angiogenesis, regulation of the immune system, neuronal processes, metabolism, development of the heart and other organ systems, and detoxification have emerged sometime in early vertebrate evolution (Duncan et al., 1998; Emmons et al., 1999; Lahvis and Bradfield, 1998).

The molecular Initiating Event

The molecular mechanism for AHR-mediated activation of gene expression is presented in Figure 1. In its unliganded form, the AHR is part of a cytosolic complex containing heat shock protein 90 (HSP90), the HSP90 co-chaperone p23 and AHR-interacting protein (AIP)^[6]. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT^[7]. The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression^[6]. Expression levels of several genes, including phase I (e.g. cytochrome P450 (CYP) 1A, CYP1B, CYP2A) and phase II enzymes (e.g. uridine diphosphate glucuronosyl transferase (UDP-GT), glutathione S-transferases (GSTs)), as well as genes involved in cell proliferation (transforming growth factor-beta, interleukin-1 beta), cell cycle regulation (p27, jun-B) and apoptosis (Bax), are regulated through this mechanism ^[6][8][7][9].

AHR Isoforms

• Over time the AhR has undergone gene duplication and diversification in vertebrates, which has resulted in multiple clades of AhR, namely AhR1, AhR2, and AhR3 (Hahn 2002).
• Fishes and birds express AhR1s and AhR2s, while mammals express a single AhR that is homologous to the AhR1 (Hahn 2002; Hahn et al 2006).
• The AhR3 is poorly understood and known only from some cartilaginous fishes (Hahn 2002).
• Little is known about diversity of AhRs in reptiles and amphibians (Hahn et al 2002).
• In some taxa, subsequent genome duplication events have further led to multiple isoforms of AhRs in some species, with up to four isoforms of the AhR (α, β, δ, γ) having been identified in Atlantic salmon (Salmo salar) (Hansson et al 2004).
• Although homologs of the AhR have been identified in some invertebrates, compared to vertebrates these AhRs have differences in binding of ligands in the species investigated to date (Hahn 2002; Hahn et al 1994).

 

Roles of isoforms in birds:

Two AHR isoforms (AHR1 and AHR2) have been identified in the black-footed albatross (Phoebastria nigripes), great cormorant (Phalacrocorax carbo) and domestic chicken (Gallus gallus domesticus)^[10]. AHR1 mRNA levels were similar in the kidney, heart, lung, spleen, brain, gonad and intestine from the great cormorant but were lower in muscle and pancreas. AHR2 expression was mainly observed in the liver, but was also detected in gonad, brain and intestine. AHR1 levels represented a greater proportion (80%) of total AHR levels than AHR2 in the cormorant liver^[10], and while both AHR isoforms bound to TCDD, AHR2 was less effective at inducing TCDD-dependent transactivation compared to AHR1 in black-footed albatross, great cormorant and domestic chicken^[11][10].

• AhR1 and AhR2 both bind and are activated by TCDD in vitro (Yasui et al 2007).
• AhR1 has greater binding affinity and sensitivity to activation by TCDD relative to AhR2 (Yasui et al 2007).
• AhR1 is believed to mediate toxicities of DLCs, while AhR2 has no known role in toxicities (Farmahin et al 2012; Farmahin et al 2013; Manning et al 2012).

Roles of isoforms in fishes:

• AhR1 and AhR2 both bind and are activated by TCDD in vitro (Bak et al 2013; Doering et al 2014; 2015; Karchner et al 1999; 2005).
• AhR1 has greater sensitivity to activation by TCDD than AhR2 in red seabream (Pagrus major), white sturgeon (Acipenser transmontanus), and lake sturgeon (Acipenser fulvescens) (Bak et al 2013; Doering et al 2014; 2015)
• AhR2 has greater binding affinity or activation by TCDD than AhR1 in zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) (Karchner et al 1999; 2005).
• AhR2 is believed to mediate toxicities in fishes, while AhR1 has no known role in toxicities. Specifically, knockdown of AhR2 protects against toxicities of dioxin-like compounds (DLCs) and polycyclic aromatic hydrocarbons (PAHs) in zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus), while knockdown of AhR1 offers no protection (Clark et al 2010; Prasch et al 2003; Van Tiem & Di Giulio 2011).

Roles of isoforms in amphibians and reptiles:

• Less is known about AhRs of amphibians or reptiles.
• AhR1 is believed to mediate toxicities in amphibians (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015). However, all AhRs of amphibians that have been investigated have very low affinity for TCDD (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015).
• Both AhR1s and AhR2 of American alligator (Alligator mississippiensis) are activated by agonists with comparable sensitivities (Oka et al 2016). AhRs of no other reptiles have been investigated.

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Transactivation Reporter Gene Assays (recommended approach)

Transient transfection transactivation

Transient transfection transactivation is the most common method for evaluating nuclear receptor activation^[12]. Full-length AHR cDNAs are cloned into an expression vector along with a reporter gene construct (chimeric luciferase, P-lactamase or CAT reporter vectors containing the appropriate response elements for the gene of interest). There are a number of commercially available cell lines that can serve as recipients for these vectors (CV-1, HuH7, FLC-7, LS174T, LS180 MCF-7, HEC1, LLC-PK1, HEK293, HepG2, and Caco-2 cells)^[12]. The greatest advantage of using transfected cells, rather than primary cell cultures, is the assurance that the nuclear receptor of interest is responsible for the observed induction. This would not be possible in a primary cell culture due to the co-regulation of different receptors for the same target genes. This model makes it easy to compare the responsiveness of the AHR across multiple species under the same conditions simply by switching out the AHR clone. One disadvantage to the transient transfection assay is the inherent variability associated with transfection efficiency, leading to a movement towards the use of stable cell lines containing the nuclear receptor and reporter gene linked to the appropriate response elements^[12].

Luciferase reporter gene (LRG) assay

The described luciferase reporter gene (LRG) assays have been used to investigate activation of AhRs of:

• Humans (Homo sapiens) (Abnet et al 1999) 
• Species of birds, namely chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), and common tern (Sterna hirundo) (Farmahin et al 2012; Manning et al 2013), Mutant AhR1s with ligand binding domains resembling those of at least 86 avian species have also been investigated (Farmahin et al 2013). AhR2s of birds have only been investigated in black-footed albatross (Phoebastria nigripes) and common cormorant (Phalacrocorax carbo) (Yasio et al 2007).
• American alligator (Alligator mississippiensis) is the only reptile for which AhR activation has been investigated (Oka et al 2016), AhR1A, AhR1B, and AhR2 of American alligator were assayed (Oka et al 2016).
• AhR1 of two amphibians have been investigated, namely African clawed frog (Xenopus laevis) and salamander (Ambystoma mexicanum) (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003),
• AhR1s and AhR2s of several species of fish have been investigated, namely Atlantic salmon (Salmo salar), Atlantic tomcod (Microgadus tomcod), white sturgeon (Acipenser transmontanus), rainbow trout (Onchorhynchys mykiss), red seabream (Pagrus major), lake sturgeon (Acipenser fulvescens), and zebrafish (Danio rerio) (Andreasen et al 2002; Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Evans et al 2005; Hansson & Hahn 2008; Karchner et al 1999; Tanguay et al 1999; Wirgin et al 2011).

For demonstrative purposes, a luciferase reporter gene assay used to measure AHR1-mediated transactivation for avian species is described here. However, comparable assays are utilized for investigating AHR1s and AHR2s of all taxa. A monkey kidney cell line (Cos-7) that has low endogenous AHR1 expression was transfected with the appropriate avian AHR1 clone, cormorant ARNT1, a CYP1A5 firefly luciferase reporter construct and a Renilla luciferase vector to control for transfection efficiency. After seeding, the cells were exposed to DLC and luciferase activity was measured using a luminometer. Luminescence, which is proportional to the extent of AHR activation, is expressed as the ratio of firefly luciferase units to Renilla luciferase units ^[13]. This particular assay was modified from its original version to increase throughput efficiency; (a) cells were seeded in 96-well plates rather than Petri dishes or 48- well plates, (b) DLCs were added directly to the wells without changing the cell culture medium, and (c) the same 96-well plates were used to measure luminescence without lysing the cells and transferring to another plate. Similar reporter gene assays have been used to measure AHR1 activation in domestic and wild species of birds, including the chicken, ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), great cormorant, black-footed albatross and peregrine falcon (Falco peregrinus).^{[14][13][15][11][16][17]}

Transactivation in stable cell lines

Stable cell lines have been developed and purified to the extent that each cell contains both the nuclear receptor and appropriate reporter vector, eliminating the variability associated with transfection ^[12]. A stable human cell line containing a luciferase reporter driven by multiple dioxin response elements has been developed that is useful in identifying AhR agonists and antagonists^[18]. An added benefit of this model is the potential to multiplex 3 assays in a single well: receptor activation, cell viability and enzyme activity^[12]. Such assays are used extensively in drug discovery due to their high throughput efficiency, and may serve just as useful for risk assessment purposes.

Ligand-Binding Assays

Ligand binding assays measure the ability of a test compound to compete with a labeled, high-affinity reference ligand for the LBD of a nuclear receptor. It is important to note that ligand binding does not necessitate receptor activation and therefore cannot distinguish between agonists and antagonists; however, binding affinities of AHR ligands are highly correlated with chemical potencies^[19] and can explain differences in species sensitivities to DLCs^[20][21][22]; they are therefore worth mentioning. Binding affinity and efficacy have been used to develop structure-activity relationships for AHR disruption^[20][23] that are potentially useful in risk-assessment. There has been tremendous progress in the development of ligand-binding assays for nuclear receptors that use homogenous assay formats (no wash steps) allowing for the detection of low-affinity ligands, many of which do not require a radiolabel and are amenable to high throughput screening^[24][12]. This author however was unable to find specific examples of such assays in the context of AHR binding and therefore some classic radioligand assays are described instead.

Hydroxyapatite (HAP) binding assay

The HAP binding assay makes use of an in vitro transcription/translation method to synthesize the AHR protein, which is then incubated with radiolabeled TDCPP and a HAP pellet. The occupied protein adsorbs to the HAP and the radioactivity is measured to determine saturation binding. An additional ligand can also be included in the mixture in order to determine its binding affinity relative to TCDD (competitive binding)^[25][22]. This assay is simple, repeatable and reproducible; however, it is insensitive to weak ligand-receptor interactions^[22][21][26].

Whole cell filtration binding assay

Dold and Greenlee^[27] developed a method to detect specific binding of TCDD to whole mammalian cells in culture and was later modified by Farmahin et al.^[21] for avian species. The cultured cells are incubated with radiolabeled TCDD with or without the presence of a competing ligand and filtered. The occupied protein adsorbs onto the filter and the radioactivity is measured to determine saturation binging and/or competitive binding. This assay is able to detect weak ligand-receptor interactions that are below the detection limit of the HAP assay^[21].

Protein-DNA Interaction Assays

The active AHR complexed with ARNT can be measured using protein-DNA interaction assays. Two methods are described in detail by Perez-Romero and Imperiale^[28]. Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions in vivo. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the LRG assay in chicken hepatoma cells dosed with dioxin-like compounds^[29].

In silico Approaches

In silico homology modeling of the ligand binding domain of the AHR in combination with molecular docking simulations can provide valuable insight into the transactivation-potential of a diverse array of AHR ligands.  Such models have been developed for multiple AHR isoforms and ligands (high/low affinity, endogenous and synthetic, agonists and antagonists), and can accurately predict ligand potency based on their structure and physicochemical properties (Bonati et al 2017; Hirano et al 2015; Sovadinova et al 2006).

References

1. ↑ ^{1.0 1.1} Okey, A. B. (2007). An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI. Toxicol.Sci. 98, 5-38.
2. ↑ Hoffman, E. C., Reyes, H., Chu, F. F., Sander, F., Conley, L. H., Brooks, B. A., and Hankinson, O. (1991). Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252, 954-958.
3. ↑ ^{3.0 3.1} Poland, A., Glover, E., and Kende, A. S. (1976). Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. J.Biol.Chem. 251, 4936-4946.
4. ↑ ^{4.0 4.1} Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. Annu.Rev.Pharmacol.Toxicol. 40, 519-561.
5. ↑ Kewley, R. J., Whitelaw, M. L., and Chapman-Smith, A. (2004). The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int.J.Biochem.Cell Biol. 36, 189-204.
6. ↑ ^{6.0 6.1 6.2 6.3} Fujii-Kuriyama, Y., and Kawajiri, K. (2010). Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. Proc.Jpn.Acad.Ser.B Phys.Biol.Sci. 86, 40-53.
7. ↑ ^{7.0 7.1} Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. Biochimica et Biophysica Acta - General Subjects 1619, 263-268.
8. ↑ Giesy, J. P., Kannan, K., Blankenship, A. L., Jones, P. D., and Newsted, J. L. (2006). Toxicology of PCBs and related compounds. In Endocrine Disruption Biological Bases for Health Effects in Wildlife and Humans (D. O. Norris, and J. A. Carr, Eds.), pp. 245-331. Oxford University Press, New York.
9. ↑ ^{9.0 9.1 9.2} Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. Critical Reviews in Toxicology 24, 87-149.
10. ↑ ^{10.0 10.1 10.2} Yasui, T., Kim, E. Y., Iwata, H., Franks, D. G., Karchner, S. I., Hahn, M. E., and Tanabe, S. (2007). Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. Toxicol.Sci. 99, 101-117.
11. ↑ ^{11.0 11.1} Lee, J. S., Kim, E. Y., and Iwata, H. (2009). Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): association with aryl hydrocarbon receptor 1 and 2 isoforms. Toxicol.Appl.Pharmacol. 234, 1-13.
12. ↑ ^{12.0 12.1 12.2 12.3 12.4 12.5} Raucy, J. L., and Lasker, J. M. (2010). Current in vitro high throughput screening approaches to assess nuclear receptor activation. Curr. Drug Metab 11 (9), 806-814.
13. ↑ ^{13.0 13.1 13.2} Farmahin, R., Wu, D., Crump, D., Hervé, J. C., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., and Kennedy, S. W. (2012). Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Environ.Sci.Technol. 46, 2967-2975.
14. ↑ ^{14.0 14.1 14.2 14.3} Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013b). Amino acid sequence of the ligand binding domain of the aryl hydrocarbon receptor 1 (AHR1) predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol.Sci. 131, 139-152.
15. ↑ Fujisawa, N., Ikenaka, Y., Kim, E. Y., Lee, J. S., Iwata, H., and Ishizuka, M. (2012). Molecular evidence predicts aryl hydrocarbon receptor ligand insensitivity in the peregrine falcon (Falco peregrines). European Journal of Wildlife Research 58, 167-175.
16. ↑ ^{16.0 16.1 16.2} Manning, G. E., Farmahin, R., Crump, D., Jones, S. P., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2012). A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the embryolethality of polychlorinated biphenyls in avian species. Toxicol.Appl.Pharmacol. 263, 390-399.
17. ↑ Mol, T. L., Kim, E. Y., Ishibashi, H., and Iwata, H. (2012). In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population. Environ.Sci.Technol. 46, 525-533.
18. ↑ Yueh, M. F., Kawahara, M., and Raucy, J. (2005). Cell-based high-throughput bioassays to assess induction and inhibition of CYP1A enzymes. Toxicol. In Vitro 19 (2), 275-287.
19. ↑ ^{19.0 19.1} Poland, A., and Knutson, J. C. (1982). 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517-554.
20. ↑ ^{20.0 20.1} Hestermann, E. V., Stegeman, J. J., and Hahn, M. E. (2000). Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency. Toxicol. Appl. Pharmacol 168 (2), 160-172.
21. ↑ ^{21.0 21.1 21.2 21.3 21.4} Farmahin, R., Jones, S. P., Crump, D., Hahn, M. E., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2014). Species-specific relative AHR1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency. Comp Biochem. Physiol C. Toxicol. Pharmacol. 161C, 21-25.
22. ↑ ^{22.0 22.1 22.2 22.3 22.4 22.5 22.6} Karchner, S. I., Franks, D. G., Kennedy, S. W., and Hahn, M. E. (2006). The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A 103 (16), 6252-6257.
23. ↑ Lee, S., Shin, W. H., Hong, S., Kang, H., Jung, D., Yim, U. H., Shim, W. J., Khim, J. S., Seok, C., Giesy, J. P., and Choi, K. (2015). Measured and predicted affinities of binding and relative potencies to activate the AhR of PAHs and their alkylated analogues. Chemosphere 139, 23-29.
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26. ↑ Nakai, J. S., and Bunce, N. J. (1995). Characterization of the Ah receptor from human placental tissue. J Biochem. Toxicol. 10 (3), 151-159.
27. ↑ Dold, K. M., and Greenlee, W. F. (1990). Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture. Anal. Biochem. 184 (1), 67-73.
28. ↑ Perez-Romero, P., and Imperiale, M. J. (2007). Assaying protein-DNA interactions in vivo and in vitro using chromatin immunoprecipitation and electrophoretic mobility shift assays. Methods Mol. Med. 131, 123-139.
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30. ↑ ^{30.0 30.1} Ema, M., Ohe, N., Suzuki, M., Mimura, J., Sogawa, K., Ikawa, S., and Fujii-Kuriyama, Y. (1994). Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors. J.Biol.Chem. 269, 27337-27343.
31. ↑ ^{31.0 31.1} Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine aryl hydrocarbon receptor. Mol.Pharmacol. 46, 915-921.
32. ↑ Backlund, M., and Ingelman-Sundberg, M. (2004). Different structural requirements of the ligand binding domain of the aryl hydrocarbon receptor for high- and low-affinity ligand binding and receptor activation. Mol.Pharmacol. 65, 416-425.
33. ↑ Murray, I. A., Reen, R. K., Leathery, N., Ramadoss, P., Bonati, L., Gonzalez, F. J., Peters, J. M., and Perdew, G. H. (2005). Evidence that ligand binding is a key determinant of Ah receptor-mediated transcriptional activity. Arch.Biochem.Biophys. 442, 59-71.
34. ↑ Pandini, A., Denison, M. S., Song, Y., Soshilov, A. A., and Bonati, L. (2007). Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis. Biochemistry 46, 696-708.
35. ↑ ^{35.0 35.1 35.2} Pandini, A., Soshilov, A. A., Song, Y., Zhao, J., Bonati, L., and Denison, M. S. (2009). Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis. Biochemistry 48, 5972-5983.
36. ↑ Ramadoss, P., and Perdew, G. H. (2004). Use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin as a probe to determine the relative ligand affinity of human versus mouse aryl hydrocarbon receptor in cultured cells. Mol.Pharmacol. 66, 129-136.
37. ↑ ^{37.0 37.1 37.2} Head, J. A., Hahn, M. E., and Kennedy, S. W. (2008). Key amino acids in the aryl hydrocarbon receptor predict dioxin sensitivity in avian species. Environ.Sci.Technol. 42, 7535-7541.
38. ↑ ^{38.0 38.1} Denison, M. S., Soshilov, A. A., He, G., DeGroot, D. E., and Zhao, B. (2011). Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. Toxicol.Sci. 124, 1-22.
39. ↑ van den Berg, M., Birnbaum, L. S., Bosveld, A. T., Brunström, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., Kubiak, T. J., Larsen, J. C., Van Leeuwen, F. X. R., Liem, A. K. D., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D. E., Tysklind, M., Younes, M., Wærn, F., and Zacharewski, T. R. (1998). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ.Health Perspect. 106, 775-792.
40. ↑ Cohen-Barnhouse, A. M., Zwiernik, M. J., Link, J. E., Fitzgerald, S. D., Kennedy, S. W., Hervé, J. C., Giesy, J. P., Wiseman, S. B., Yang, Y., Jones, P. D., Wan, Y., Collins, B., Newsted, J. L., Kay, D. P., and Bursian, S. J. (2011b). Sensitivity of Japanese quail (Coturnix japonica), Common pheasant (Phasianus colchicus), and White Leghorn chicken (Gallus gallus domesticus) embryos to in ovo exposure to TCDD, PeCDF, and TCDF. Toxicol.Sci. 119, 93-103.
41. ↑ Farmahin, R., Crump, D., Jones, S. P., Mundy, L. J., and Kennedy, S. W. (2013a). Cytochrome P4501A induction in primary cultures of embryonic European starling hepatocytes exposed to TCDD, PeCDF and TCDF. Ecotoxicology 22(4), 731-739.
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44. ↑ Poland, A., and Glover, E. (1973). Studies on the mechanism of toxicity of the chlorinated dibenzo-p-dioxins. Environ.Health Perspect. 5, 245-251.
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46. ↑ ^{46.0 46.1} Omiecinski, C. J., Vanden Heuvel, J. P., Perdew, G. H., and Peters, J. M. (2011). Xenobiotic metabolism, disposition, and regulation by receptors: from biochemical phenomenon to predictors of major toxicities. Toxicol.Sci. 120 Suppl 1, S49-S75.
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54. ↑ Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., and Fujii-Kuriyama, Y. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645-654.
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Goode GD, Ballard BR, Manning HC, Freeman ML, Kang Y, Eltom SE. 2013. Knockdown of aberrantly upregulated aryl hydrocarbon receptor reduces tumor growth and metastasis of MDA-MB- 231 human breast cancer cell line. Int J Cancer. 133(12):2769–80

 

Chang JT, Chang H, Chen P-H, Lin S-L, Lin P. 2007. Requirement of aryl hydrocarbon receptor overexpression for CYP1B1 up-regulation and cell growth in human lung adenocarcinomas. Clin Cancer

Res. 13(1):38–45

 

Kanno Y, Takane Y, Izawa T, Nakahama T, Inouye Y. 2006. The inhibitory effect of aryl hydrocarbon receptor repressor (AhRR) on the growth of human breast cancer MCF-7 cells. Biol Pharm Bull. 29(6):1254–57

 

Goode G, Pratap S, Eltom SE. 2014. Depletion of the aryl hydrocarbon receptor in MDA-MB- 231 human breast cancer cells altered the expression of genes in key regulatory pathways of cancer. PLoS One. 9(6):e100103

 

Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, et al. 2011. An endogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Nature. 478(7368):197–203

 

Novikov O, Wang Z, Stanford EA, Parks AJ, Ramirez-Cardenas A, et al. 2016. An Aryl Hydrocarbon Receptor-Mediated Amplification Loop That Enforces Cell Migration in ER-/PR-/Her2- Human Breast Cancer Cells. Mol Pharmacol. 90(5):674–88

 

Litzenburger UM, Opitz CA, Sahm F, Rauschenbach KJ, Trump S, et al. 2014. Constitutive IDO expression in human cancer is sustained by an autocrine signaling loop involving IL-6, STAT3 and the AHR. Oncotarget. 5(4):1038–51

 

Hall JM, Barhoover MA, Kazmin D, McDonnell DP, Greenlee WF, Thomas RS. 2010. Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation. Mol Endocrinol. 24(2):359–69

 

Gramatzki D, Pantazis G, Schittenhelm J, Tabatabai G, K.hle C, et al. 2009. Aryl hydrocarbon receptor inhibition downregulates the TGF-beta/Smad pathway in human glioblastoma cells. Oncogene. 28(28):2593–2605

 

Subramaniam V, Ace O, Prud’homme GJ, Jothy S. 2011. Tranilast treatment decreases cell growth, migration and inhibits colony formation of human breast cancer cells. Exp Mol Pathol. 90(1):116–22

 

Rothhammer V, Borucki DM, Kenison JE, Hewson P, Wang Z, et al. 2018. Detection of aryl hydrocarbon receptor agonists in human samples. Sci Rep. 8(1):4970

 

Lin P, Chang H, Tsai W-T, Wu M-H, Liao Y-S, et al. 2003. Overexpression of aryl hydrocarbon receptor in human lung carcinomas. Toxicol Pathol. 31(1):22–30

 

Barhoover MA, Hall JM, Greenlee WF, Thomas RS. 2010. Aryl hydrocarbon receptor regulates cell cycle progression in human breast cancer cells via a functional interaction with cyclin-dependent kinase 4. Mol Pharmacol. 77(2):195–201

 

Wang K, Li Y, Jiang Y-Z, Dai C-F, Patankar MS, et al. 2013. An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells. Cancer Lett. 340(1):63–71

 

Borovok N, Weiss C, Sharkia R, Reichenstein M, Wissinger B, et al. 2020. Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation. Front Genet. 11:582796

List of Key Events in the AOP

Event: 149: Increase, Inflammation

Short Name: Increase, Inflammation

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic: appears to be present broadly, with representative studies focused on mammals (humans, lab mice, lab rats).

 

Extensive data exists on the presence of inflammation in human (Coussens, Aggarwal, Hannhan, Mantovani..) In human, many examples of chronic inflammation leading to cancer or cancer progression exist. For instance, Helicobacter pylori infection leads to gut cancer (Wang).

 

Key Event Description

Inflammation is complex to define. 

Villeneuve et al. (2018) analyzed the varied biological responses, provided guidance to simplify the  process representing inflammation in adverse outcome pathways, and recommended 3 key steps: 1. Tissue resident cell activation 2. Increased Pro-inflammatory mediators 3. Leukocyte recruitment/activation.  Tissue resident cell activation generally occurs when healthy tissue is exposed to a stressor, or when damage occurs, initiating a signal response of pro-inflammatory mediators (ex. cytokines).  Pro-inflammatory mediators result in the production of lipids and proteins, signaling, and initiate leukocyte recruitment/activation.  Leukocyte recruitment/activation initiate inflammation and other morphological changes. 

In cancer, inflammation is a cascade of events created by the host in response to the spread of the cancer (Coussens and Werb, 2002). In response to an injury or the presence of cancer, the host heals itself through inflammation. Indeed, the activation and the migration of  leukocytes (neutrophils, monocytes and eosinophils) to the wound induces the healing process. These inflammatory cells provide an extracellular matrix that forms upon which fibroblast and endothelial cells proliferate and migrate in order to recreate a normal environment. Damage to the epithelial layer initiate inflammatory reactions (Palmer et al. 2011).  In cancer, this inflammatory state induces cell proliferation, increases the production of reactive oxygen species leading to oxidative DNA damage, and reduces DNA repair (Coussens and Werb, 2002).  For review of inflammation caused by microplastics in mammals, see Wright and Kelly (2017).

 

 

Inflammation can be defined as the response of the organism to a tissue injury (Coussens). Indeed, in order to heal this injury, a multitude of chemical signals initiate and maintain a host response. Leukocytes (neutrophils, monocytes and eosinophils) are recruited to the site of the damage through the attraction by chemokines (TNF-α (tumour necrosis factor-α), interleukines…). A provisional extracellular matrix (ECM) is created, and fibroblast and endothelial cells proliferate and migrate to it. Wound healing is an example of physiological inflammation and is self-limiting (Coussens). In case of a dysregulation, inflammation can lead to pathologies. Inflammation can be caused by physical injury, ischemic injury, infection, exposure to toxins, or other types of trauma (Singh).

Inflammation was described as one of the hallmarks of cancer by Hannahan et al. as a response to tumor invasion through mainly two mechanisms: promoting genetic instatbility and supply pro-tumorogenic factors.

First, inflammation in cancer promotes genetic instability (Mantovani, colotta). Macrophages, in contact with the inflammatory site can be responsible of a reactive stress oxygen reaction (ROS) (Maeda, Pollard, Grivennikov). Indeed, they generate high levels of reactive oxygen and nitrogen species which produce mutagenic agents (peroxynitrite), which in turn causes DNA mutations.

Second, in inflammation, the tumor micro environment plays a critical role (Coussens). Indeed, in can supply growth factors, survival factors, proangiogenic factors, extracellular matrix-modifying enzymes that facilitate angiogenesis, invasion, and metastasis, and inductive signals that lead to activation of EMT and other hallmark-facilitating programs (Hannahan). For example, macrophages can become tumor associated macrophage which promote cell proliferation, angiogenesis, and invasion (Singh, Lin, Qian).

Moreover, chronic inflammation can also lead to tumorigenesis (Karin, Singh). Indeed, since 1863, Virchow has hypothesized that chronic inflammation causes cell proliferation (Balkwill). According to Aggarwall, several pro-inflammatory markers such as TNF and members of its superfamily, IL-1alpha, IL-1beta, IL-6, IL-8, IL-18, chemokines, MMP-9, VEGF, COX-2, and 5-LOX mediate suppression of apoptosis, proliferation, angiogenesis, invasion, and metastasis (Aggarwal).

How it is Measured or Detected

Inflammation is generally detected in histopathological examination of organs (ex. liver, intestines) or in changes in gene expression (ex. interleukins).  Activation of the innate immune response and the release of various inflammatory cytokines can also be assessed (Flake and Morgan, 2017).

 

Several assays can be used to measure inflammation:

• Histopathology on samples. Several scoring tools exist (Goeboes)
• Measuring chemokines in the blood (ELISA, multiplex bead assays : interleukines (IL1, IL6), TNF, interferon… ) (Brenner) and histopathology samples
• Measuring Prostaglandin levels, COX-2 (ELISA
  Liquid chromatography/tandem mass spectrometry, IHC)
• Transcription factors : STAT3 Activation, NF-κB Activation (ELISA
  RtPCR to measure mRNA
• Biomarkers (white cell count, CRP) ratios, and predictive score using
• Measuring ROS(DCFDA, horseradish peroxidase (HRP)-oxidizing substrates, SOD-inhibitable reduction of cytochrome c) (Murphy).

 

Methods are extensively reviewed in Marchand et al and Murphy et al.

References

Flake, G.P., and Morgan, D.L. 2017. Pathology of diacetyl and 2,3-pentanedione airway lesions in a rat model of obliterative bronchiolitis. Toxicology, 388, 40–47. https://doi.org/10.1016/j.tox.2016.10.013

Palmer, S.M., Flake, G.P., Kelly, F.L., Zhang, H.L., Nugent, J.L., Kirby, P.J., Zhang, H.L., Nugent, J.L., Kirby, P.J., Foley, J.F., Gwinn, W.M., and Morgan, D.L. 2011. Severe airway epithelial injury, aberrant repair and Bronchiolitis obliterans develops after diacetyl instillation in rats. PLoS ONE, 6(3). https://doi.org/10.1371/journal.pone.0017644

 

Wang F, Meng W, Wang B, Qiao L. Helicobacter pylori-induced gastric inflammation and gastric cancer. Cancer Lett. 2014 Apr 10;345(2):196-202. doi: 10.1016/j.canlet.2013.08.016. Epub 2013 Aug 24. PMID: 23981572.

Naylor MS, Stamp GW, Foulkes WD, Eccles D, Balkwill FR. Tumor necrosis factor and its receptors in human ovarian cancer. Potential role in disease progression. J Clin Invest. 1993;91:2194–206.

Coussens L.M. and Werb Z. Inflammation and cancer. Nature. 2002 Dec 19-26;420(6917):860-7. doi: 10.1038/nature01322. PMID: 12490959; PMCID: PMC2803035.

Wright, S.L. and Kelly, F.J.  2017.  Plastic and human health: a micro issue?  Enviromental Science and Technology 51: 6634-6647.

Villeneuve, D.L., Landesmann, B., Allavena, P., Ashley, N., Bal-Price, A., Corsini, E., Halappanavar, S., Hussell, T., Laskin, D., Lawrence, T., Nikolic-Paterson, D., Pallary, M., Paini, A., Pietrs, R., Roth, R., and Tschudi-Monnet, F.  2018.  Toxicological Sciences 163(2): 346-352.

 

 

Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell. 2010 Apr 2;141(1):39-51. doi: 10.1016/j.cell.2010.03.014. PMID: 20371344; PMCID: PMC4994190.

Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230.

Karin M. Nuclear factor-kappaB in cancer development and progression. Nature. 2006;441:431–6.

Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G. Inflammation and cancer: How hot is the link? Biochem Pharmacol. 2006;72:1605–21

Singh N, Baby D, Rajguru JP, Patil PB, Thakkannavar SS, Pujari VB. Inflammation and cancer. Ann Afr Med. 2019 Jul-Sep;18(3):121-126. doi: 10.4103/aam.aam_56_18. PMID: 31417011; PMCID: PMC6704802.

Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420:860–7

Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet. 2001;357:539–545

Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature. 2008;454:436–44

Cancer-related inflammation, the seventh hallmark of cancer: Links to genetic instability. 

Maeda H, Akaike T. Nitric oxide and oxygen radicals in infection, inflammation, and cancer. Biochemistry (Mosc) 1998;63:854–65.

Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer. 2004;4:71–8

Lin, Y., Xu, J. & Lan, H. Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications. J Hematol Oncol 12, 76 (2019). https://doi.org/10.1186/s13045-019-0760-3

Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010 Mar 19;140(6):883-99. doi: 10.1016/j.cell.2010.01.025. PMID: 20303878; PMCID: PMC2866629.

 

Murphy, M.P., Bayir, H., Belousov, V. et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab 4, 651–662 (2022). https://doi.org/10.1038/s42255-022-00591-z

Geboes K, Riddell R, Öst A, et al

A reproducible grading scale for histological assessment of inflammation in ulcerative colitis

Gut 2000;47:404-409.

Brenner DR, Scherer D, Muir K, Schildkraut J, Boffetta P, Spitz MR, Le Marchand L, Chan AT, Goode EL, Ulrich CM, Hung RJ. A review of the application of inflammatory biomarkers in epidemiologic cancer research. Cancer Epidemiol Biomarkers Prev. 2014 Sep;23(9):1729-51. doi: 10.1158/1055-9965.EPI-14-0064. Epub 2014 Jun 24. PMID: 24962838; PMCID: PMC4155060.

Event: 1262: Apoptosis

Short Name: Apoptosis

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

・Apoptosis is induced in human prostate cancer cell lines (Homo sapiens) [Parajuli et al., 2014].

・Apoptosis occurs in B6C3F1 mouse (Mus musculus) [Elmore, 2007].

・Apoptosis occurs in Sprague-Dawley rat (Rattus norvegicus) [Elmore, 2007].

・Apoptosis occurs in the nematode (Caenorhabditis elegans) [Elmore, 2007].

• Apoptosis occurs in breast cancer cells, human and mouse (Parton)

 

 

Key Event Description

Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called “physiological cell death”, is involved in cell turnover, physiological involution, and atrophy of various tissues and organs [Kerr et al., 1972]. The formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances [Kerr et al., 1972]. Apoptosis is characterized by DNA ladder and chromatin condensation. Several stimuli such as hypoxia, nucleotides deprivation, chemotherapeutical drugs, DNA damage, and mitotic spindle damage induce p53 activation, leading to p21 activation and cell cycle arrest [Pucci et al., 2000]. The SAHA or TSA treatment on neonatal human dermal fibroblasts (NHDFs) for 24 or 72 hrs inhibited proliferation of the NHDF cells [Glaser et al., 2003]. Considering that the acetylation of histone H4 was increased by the treatment of SAHA for 4 hrs, histone deacetylase inhibition may be involved in the inhibition of the cell proliferation [Glaser et al., 2003]. The impaired proliferation was observed in HDAC1^-/- ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz et al., 2010]. An AOP focuses existes on p21 pathway leading to apoptosis, however, alternative pathways such as NF-kappaB signaling pathways may be involved in the apoptosis of spermatocytes [Wang et al., 2017].

Apoptosis is defined as a programmed cell death.  A decrease in apoptosis or a resistance to cell death is noted is described as a hallmark of cancer by Hanahan et al. It is widely admitted as an essential step in tumor proliferation (Adams, Lowe).  Apoptosis occurs after activation of a number of intrinsic and extrinsic signals which activate the protease caspase system which in turn activates the destruction of the cell.

The Bcl-2 is a protein family suppressing apoptosis by binding and inhibiting two proapoptotic proteins (Bax and Bak) and transferring them to the mitochondrial outer membrane. In the absence of inhibition by Bcl2, Bax and Bak destroy the mitochondrial membrane and releases proapoptotic signaling proteins, such as cytochrome c which activated the caspase system. An increased expression of these antiapoptotic proteins (Bcl-2, Bcl-x_L) occurs in cancer (Hanahan, Adams, Lowe). Several others pathways such as the loss of TP53 tumor suppressor function, or the increase of survival signals (Igf1/2), or decrease of proapoptotic factors (Bax, Bim, Puma) can also increase tumor growth (Hanahan, Juntilla).

In breast cancer a decrease in apoptosis and a resistance to cell death has been described thoroughly, especially using a dysregulation of the Bcl2 system or TP53 (Parton, Williams, Shahbandi).

How it is Measured or Detected

Apoptosis is characterized by many morphological and biochemical changes such as homogenous condensation of chromatin to one side or the periphery of the nuclei, membrane blebbing and formation of apoptotic bodies with fragmented nuclei, DNA fragmentation, enzymatic activation of pro-caspases, or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteomic and genomic methods, and spectroscopic techniques [Archana et al., 2013; Martinez et al., 2010; Taatjes et al., 2008; Yasuhara et al., 2003].

・DNA fragmentation can be quantified with comet assay using electrophoresis, where the tail length, head size, tail intensity, and head intensity of the comet are measured [Yasuhara et al., 2003].

・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli et al., 2014].

・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli et al., 2014].

・Apoptotic nucleosomes are detected using Cell Death Detection ELISA kit, which was calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli et al., 2014].

・Cleavage of PARP is detected with Western blotting [Parajuli et al., 2014].

・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu et al., 2016].

・Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry [Wu et al., 2016].

・Apoptosis is detected with the Terminal dUTP Nick End-Labeling (TUNEL) method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks [Kressel and Groscurth, 1994].

・For the detection of apoptosis, the testes are fixed in neutral buffered formalin and embedded in paraffin. Germ cell death is visualized in testis sections by Terminal dUTP Nick End-Labeling (TUNEL) staining method [Wade et al., 2008]. The incidence of TUNEL-positive cells is expressed as the number of positive cells per tubule examined for one entire testis section per animal [Wade et al., 2008]

References

Archana, M. et al. (2013), "Various methods available for detection of apoptotic cells", Indian J Cancer 50:274-283

Elmore, S. (2007), "Apoptosis: a review of programmed cell death", Toxicol Pathol 35:495-516

Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163

Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257

Kressel, M. and Groscurth, P. (1994), "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA", Cell Tissue Res 278:549-556

Martinez, M.M. et al. (2010), "Detection of apoptosis: A review of conventioinal and novel techniques", Anal Methods 2:996-1004

Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313

Pucci, B. et al. (2000), "Cell cycle and apoptosis", Neoplasia 2:291-299

Taatjes, D.J. et al. (2008), "Morphological and cytochemical determination of cell death by apoptosis", Histochem Cell Biol 129:33-43

Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831

Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143

Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One 11:e0167052

Yasuhara, S. et al. (2003), "Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis", J Histochem Cytochem 51:873-885

Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181

 

Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230

Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981.

Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004). https://doi.org/10.1038/nature03098

Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.

Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747.

Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133.

Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.

Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230

Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981.

Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004). https://doi.org/10.1038/nature03098

Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.

Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747.

Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133.

Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.

 

Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.

Event: 1241: Increased, Motility

Short Name: Increased, Motility

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Cell motility has been largely described in human breast cancer cell lines, mice and fish (Stuelten)

Key Event Description

Cell motility is the capacity of cells to translocate onto a solid substratum.

 

In order to move several actions such as : cell–substrate adhesion, cell–cell adhesion, cell cortex rigidity (membrane and cytoskeleton), actin polymerization-mediated protrusion and actomyosin contractility (Stuelten, Lauffenburger, Montell).

Several key factors contribute to cell motility in cancer (Friedl, Lamouille, Sahail):

• Actin Cytoskeleton Dynamics: The actin cytoskeleton plays a crucial role in cell motility. Remodeling of the actin cytoskeleton is essential for cell shape changes, protrusion formation, and cell migration. This process is tightly regulated by proteins such as actin polymerization factors, focal adhesion proteins, and myosin motors.
• Cell Adhesion and Extracellular Matrix (ECM) Interactions: Integrins and other cell adhesion molecules mediate the interaction between cancer cells and the ECM. These interactions activate signaling pathways that influence cell motility. Changes in adhesion molecules can enhance or inhibit the migratory potential of breast cancer cells.
• Epithelial-Mesenchymal Transition (EMT): EMT is a biological process in which epithelial cells acquire mesenchymal characteristics, including increased motility. EMT is associated with the invasive behavior of cancer cells, allowing them to detach from the primary tumor and migrate to distant sites.
• Chemotaxis and Gradients: Cancer cells can respond to chemical gradients, a process known as chemotaxis. Growth factors and cytokines in the tumor microenvironment can attract or repel cancer cells, influencing their direction of movement.
• Proteolytic Enzymes and Matrix Metalloproteinases (MMPs): Proteolytic enzymes, especially MMPs, are involved in degrading the ECM, facilitating cancer cell invasion. The degradation of the surrounding matrix creates space for cell movement and allows cancer cells to penetrate adjacent tissues.

In breast cancer, cell motility can favor metastasis through different steps: loss of epithelial polarity, breakdown of tissue architecture, breach of the basement membrane, intravasation, extravasation, migration into new tissues, and expansion of metastatic colonies (Stuelten). For instance, an increase in invasion of the surrounding tissues and blood vessels. Once cancer cells have invaded the local tissue, they may enter the bloodstream through a process called intravasation. Subsequently, they must migrate through the vasculature to reach distant organs, a process known as extravasation (Chambers). Once in the circulation, cells utilize chemotaxis, responding to chemokines and other signals in the microenvironment, to navigate through the bloodstream and reach specific distant organs. The ability of cancer cells to home in on specific organs depends on their motility and the interactions with the target tissue (Psaila, Labelle). Once cancer cells reach a distant organ, they need to extravasate and establish micrometastases. Motility enables cancer cells to navigate through the tissue, invade the local environment, and form secondary tumor foci (Nguyen).

How it is Measured or Detected

Several assays can be used to measure cell motility, and the choice depends on the specific requirements and characteristics of the cells being studied. Here are some commonly used assays for measuring cell motility (Justus)

• Wound Healing Assay (Scratch Assay):

Principle: Create a controlled "wound" or scratch in a cell monolayer and monitor the closure of the gap over time.

Measurement: Quantify the rate of cell migration by measuring the reduction in the wound area.

• Transwell Migration Assay:

Principle: Cells migrate through a porous membrane from one side to the other in response to a chemoattractant.

Measurement: Count the number of cells that have migrated through the membrane or quantify fluorescence if cells are labeled.

• Boyden Chamber Assay:

Principle: Similar to the Transwell assay, cells migrate through a membrane towards a chemoattractant.

Measurement: Assess the migrated cells on the lower surface of the membrane.

• Time-Lapse Microscopy:

Principle: Track the movement of individual cells over time using live-cell imaging.

Measurement: Analyze cell trajectories, speed, and directionality.

• Collagen Invasion Assay:

Principle: Assess cell invasion through a three-dimensional collagen matrix.

Measurement: Quantify the extent of cell invasion into the matrix

• Fluorescence Recovery After Photobleaching (FRAP):

Principle: Measure the mobility of fluorescently labeled molecules or proteins within cells.

Measurement: Assess the recovery of fluorescence in a photobleached region over time.

• Single-Cell Tracking:

Principle: Monitor individual cell movements using time-lapse microscopy.

Measurement: Analyze parameters such as speed, persistence, and directionality for each tracked cell.

• Electric Cell-Substrate Impedance Sensing (ECIS):

Principle: Measure changes in electrical impedance as cells migrate and interact with a substrate.

Measurement: Quantify impedance-based parameters to assess cell motility.

• Bead-Based Motility Assay:

Principle: Attach beads to cells and track their movement using microscopy.

Measurement: Analyze the displacement of beads to determine cell motility.

Selecting the most appropriate assay depends on factors such as the nature of the cells, the desired readout, and the specific aspects of cell motility being investigated. Researchers often use a combination of these assays to gain a comprehensive understanding of cell motility in different contexts

References

Stuelten, C., Parent, C. & Montell, D. Cell motility in cancer invasion and metastasis: insights from simple model organisms. Nat Rev Cancer 18, 296–312 (2018). https://doi.org/10.1038/nrc.2018.15

Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV. In vitro cell migration and invasion assays. J Vis Exp. 2014 Jun 1;(88):51046. doi: 10.3791/51046. PMID: 24962652; PMCID: PMC4186330.

Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell. 1996 Feb 9;84(3):359-69. doi: 10.1016/s0092-8674(00)81280-5. PMID: 8608589.

Chambers, A. F., Groom, A. C., & MacDonald, I. C. (2002). Dissemination and growth of cancer cells in metastatic sites. Nature Reviews Cancer, 2(8), 563–572. doi:10.1038/nrc865

Montell DJ. Morphogenetic cell movements: diversity from modular mechanical properties. Science. 2008 Dec 5;322(5907):1502-5. doi: 10.1126/science.1164073. PMID: 19056976.

Friedl, P., & Wolf, K. (2003). Tumour-cell invasion and migration: diversity and escape mechanisms. Nature Reviews Cancer, 3(5), 362–374. doi:10.1038/nrc1075

Lamouille, S., Xu, J., & Derynck, R. (2014). Molecular mechanisms of epithelial-mesenchymal transition. Nature Reviews Molecular Cell Biology, 15(3), 178–196. doi:10.1038/nrm3758

Sahai, E. (2005). Mechanisms of cancer cell invasion. Current Opinion in Genetics & Development, 15(1), 87–96. doi:10.1016/j.gde.2004.12.002

Psaila, B., & Lyden, D. (2009). The metastatic niche: adapting the foreign soil. Nature Reviews Cancer, 9(4), 285–293. doi:10.1038/nrc2621

Labelle, M., & Hynes, R. O. (2012). The initial hours of metastasis: the importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discovery, 2(12), 1091–1099. doi:10.1158/2159-8290.CD-12-0329

Nguyen, D. X., & Bos, P. D. (2009). Massagué, J. (2009). Metastasis: from dissemination to organ-specific colonization. Nature Reviews Cancer, 9(4), 274–284. doi:10.1038/nrc2622

Quail, D. F., & Joyce, J. A. (2013). Microenvironmental regulation of tumor progression and metastasis. Nature Medicine, 19(11), 1423–1437. doi:10.1038/nm.3394

Event: 1190: Increased, Migration (Endothelial Cells)

Short Name: Increased, Migration (Endothelial Cells)

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human, breast cancer cell lines

Mice

Key Event Description

Endothelial cell migration refers to the movement of endothelial cells, which are the cells lining the inner surface of blood vessels, across tissues. This dynamic process is essential for various physiological functions, including vascular development, tissue repair, and angiogenesis (Michaelis, Fonseca)..

 

During migration, endothelial cells undergo a series of coordinated steps, including sensing chemotactic signals, altering their cytoskeleton to form protrusions (Extension of finger-like projections (filopodia) at the leading edge of the cell to sense the environment), adhering to the extracellular matrix through molecules such as integrins,contraction (pulling the cell forward using actin) and finally detachment for movement (Michaelis, Fonseca). These movements are crucial for the remodeling and maintenance of blood vessels. This is regulated by chemical signs (VEGG, integrins) and physical cues (Michaelis, Fonseca, Norton).

 

The role of endothelial cell migration is in (Michaelis, Fonseca).:

• Angiogenesis: One of the primary roles of endothelial cell migration is in angiogenesis, the formation of new blood vessels. In response to signals from growth factors like vascular endothelial growth factor (VEGF), endothelial cells migrate towards the site of angiogenesis, contributing to the expansion of the vascular network (Michaelis, Lamalis).
• Tissue Repair: Endothelial cell migration is crucial for repairing damaged blood vessels. In response to injury or inflammation, endothelial cells migrate to the site of damage, facilitating the restoration of vascular integrity (Michaelis).
• Vascular Development: During embryonic development, endothelial cell migration is essential for the formation and remodeling of blood vessels (Scarpa). This process helps establish the intricate vascular network required for organ development.
• Immune Response: Endothelial cells play a role in immune responses by facilitating the migration of immune cells across blood vessel walls to sites of infection or injury (Sturtzel).
• Lymphangiogenesis: Endothelial cell migration is involved in lymphangiogenesis, the formation of new lymphatic vessels. This process is crucial for fluid drainage, immune surveillance, and can also play a role in cancer metastasis (Pengchung).
• Wound Healing:Endothelial cells contribute to wound healing by migrating to the site of injury and participating in the formation of new blood vessels, a process known as neovascularization (Lamalis, Amersfoort).
• Cancer Metastasis: In cancer, endothelial cell migration is hijacked by tumors to support their growth and metastasis. Tumor cells release angiogenic factors, inducing the migration of endothelial cells to form new blood vessels that supply nutrients to the growing tumor (Lamalis).

How it is Measured or Detected

Assays used to study endothelial cell migration (Guo):  

• Boyden chamber: evaluates the ability of cells to migrate through a porous membrane towards a chemoattractant (substance that attracts cells) placed in the lower chamber.
• Scratch wound assay: collective movement of endothelial cells to close a "wound" created by scratching a confluent monolayer of cells.
• Microfluidic assay: microfluidic channels to create controlled environments that mimic the physiological flow conditions experienced by endothelial cells in vivo (Shih)
• Tube formation: assays evaluate the ability of endothelial cells to form tube-like structures, mimicking the process of blood vessel formation (angiogenesis) (Guo)
• Collagen Invasion Assay: Assess the invasive capacity of endothelial cells through a three-dimensional collagen matrix
• Time-lapse microscopy: using live-cell imaging
• 3D spheroid migration
• In vivo: vessel density in fat pads

References

Mierke CT. Role of the endothelium during tumor cell metastasis: is the endothelium a barrier or a promoter for cell invasion and metastasis? J Biophys. 2008;2008:183516. doi: 10.1155/2008/183516. Epub 2009 Mar 5. PMID: 20107573; PMCID: PMC2809021.

Michaelis UR. Mechanisms of endothelial cell migration. Cell Mol Life Sci. 2014 Nov;71(21):4131-48. doi: 10.1007/s00018-014-1678-0. Epub 2014 Jul 20. PMID: 25038776.

 

Guo S, Lok J, Liu Y, Hayakawa K, Leung W, Xing C, Ji X, Lo EH. Assays to examine endothelial cell migration, tube formation, and gene expression profiles. Methods Mol Biol. 2014;1135:393-402. doi: 10.1007/978-1-4939-0320-7_32. PMID: 24510881; PMCID: PMC4035906.

 

Shih, HC., Lee, TA., Wu, HM. et al. Microfluidic Collective Cell Migration Assay for Study of Endothelial Cell Proliferation and Migration under Combinations of Oxygen Gradients, Tensions, and Drug Treatments. Sci Rep 9, 8234 (2019). https://doi.org/10.1038/s41598-019-44594-5

 

Fonseca CG, Barbacena P, Franco CA. Endothelial cells on the move: dynamics in vascular morphogenesis and disease. Vasc Biol. 2020 Jul 2;2(1):H29-H43. doi: 10.1530/VB-20-0007. PMID: 32935077; PMCID: PMC7487603.

 

Yu P, Wu G, Lee HW, Simons M. Endothelial Metabolic Control of Lymphangiogenesis. Bioessays. 2018 Jun;40(6):e1700245. doi: 10.1002/bies.201700245. Epub 2018 May 11. PMID: 29750374; PMCID: PMC6237195.

 

Amersfoort, J., Eelen, G. & Carmeliet, P. Immunomodulation by endothelial cells — partnering up with the immune system?. Nat Rev Immunol 22, 576–588 (2022). https://doi.org/10.1038/s41577-022-00694-4

 

Sturtzel C. Endothelial Cells. Adv Exp Med Biol. 2017;1003:71-91. doi: 10.1007/978-3-319-57613-8_4. PMID: 28667554.

 

Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res. 2007 Mar 30;100(6):782-94. doi: 10.1161/01.RES.0000259593.07661.1e. PMID: 17395884.

 

Scarpa E, Mayor R. Collective cell migration in development. J Cell Biol. 2016 Jan 18;212(2):143-55. doi: 10.1083/jcb.201508047. PMID: 26783298; PMCID: PMC4738384.

 

Michaelis UR. Mechanisms of endothelial cell migration. Cell Mol Life Sci. 2014 Nov;71(21):4131-48. doi: 10.1007/s00018-014-1678-0. Epub 2014 Jul 20. PMID: 25038776.

 

Norton KA, Popel AS. Effects of endothelial cell proliferation and migration rates in a computational model of sprouting angiogenesis. Sci Rep. 2016 Nov 14;6:36992. doi: 10.1038/srep36992. PMID: 27841344; PMCID: PMC5107954.

Event: 1196: Increased, Invasion

Short Name: Increased, Invasion

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human 

Mice

Key Event Description

Cell invasion refers to the active movement of cells into and through tissues, barriers, or extracellular matrices (ECM) (Friedl). It involves a series of coordinated processes by which cells penetrate physical barriers, navigate through the extracellular environment, and potentially reach distant locations (Hynes). It is regulated by growth factors (VEGF), signaling pathways and cell-cell interactions.

 

Key Steps in Cell Invasion:

• Detachment: Detachment of cells to the extracellular matrix (ECM) or neighboring cells through interactions with adhesion molecules, including integrins and cadherins.
• Proteolysis: Degradation of ECM components by proteolytic enzymes, such as matrix metalloproteinases (MMPs), secreted by invasive cells. This process creates pathways for cell movement.
• Motility: Dynamic changes in the cell's cytoskeleton, involving the formation of actin-rich structures like lamellipodia and filopodia, which facilitate cell movement.
• Intravasation: Invasion of cells into blood vessels or lymphatic vessels, allowing them to enter the circulatory system and potentially spread to distant sites.
• Extravasation: Exit of invasive cells from the bloodstream or lymphatic vessels at a secondary site, facilitating colonization and the formation of secondary tumors.
• Adhesion: Cells form new attachments to the ECM at the leading edge, allowing for continued movement.

 

There are many roles for cell invasion:

• Development and Tissue Repair: Cell invasion is crucial during embryonic development for processes such as tissue patterning and organ formation. In adults, invasion is essential for tissue repair and regeneration.
• Embryonic development: During development, cells migrate to form different organs and tissues, shaping the intricate structure of the organism (Heisenberg).
• Immune Response: Immune cells use invasion to migrate to sites of infection or injury, where they participate in immune responses.
• Angiogenesis: Endothelial cells migrate to form new blood vessels, delivering oxygen and nutrients to growing tissues or healing wounds (Carmeliet, Lamalice).
• Wound Healing: Invasive migration of cells is essential for wound healing, allowing cells to move into the wounded area and contribute to tissue repair (Grinnell).
• Cancer Metastasis: In cancer, invasion is a hallmark of malignancy and a critical step in metastasis. Cancer cells acquire the ability to invade surrounding tissues, enter blood or lymphatic vessels, and establish secondary tumors at distant sites (Krakhmal).

How it is Measured or Detected

Several assays can be used to study cell invasion (Justus):

• Transwell Invasion Assay: Cells migrate through a porous membrane coated with ECM proteins toward a chemoattractant (Hulkower).
• Boyden Chamber Assay: cell migration and invasion through a porous membrane in response to a gradient of chemoattractants.
• 3D Spheroid Invasion Assay: spheroids embedded in a 3D matrix, and invasion is assessed as cells migrate out from the spheroid into the surrounding matrix (Pijuan).
• Collagen Invasion Assay: Cells invade through a collagen matrix, simulating the extracellular environment.
• Matrigel Invasion Assay: Cells invade through Matrigel, a basement membrane matrix rich in ECM proteins.
• Zymography: Assess the activity of matrix metalloproteinases (MMPs), enzymes involved in ECM degradation and cell invasion.
• Electric Cell-Substrate Impedance Sensing (ECIS): Measure changes in electrical impedance as cells invade and interact with a substrate.
• Microfluidic Invasion Assays: Use microfluidic devices to create controlled environments for studying cell invasion (Fonseca).
• In Vivo Invasion Assays: Intravital imaging or xenograft models to study cell invasion in vivo.

References

Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV. In vitro cell migration and invasion assays. J Vis Exp. 2014 Jun 1;(88):51046. doi: 10.3791/51046. PMID: 24962652; PMCID: PMC4186330.

 

Fonseca CG, Barbacena P, Franco CA. Endothelial cells on the move: dynamics in vascular morphogenesis and disease. Vasc Biol. 2020 Jul 2;2(1):H29-H43. doi: 10.1530/VB-20-0007. PMID: 32935077; PMCID: PMC7487603.

 

Pijuan J, Barceló C, Moreno DF, Maiques O, Sisó P, Marti RM, Macià A, Panosa A. In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis. Front Cell Dev Biol. 2019 Jun 14;7:107. doi: 10.3389/fcell.2019.00107. PMID: 31259172; PMCID: PMC6587234.

 

Hulkower KI, Herber RL. Cell migration and invasion assays as tools for drug discovery. Pharmaceutics. 2011 Mar 11;3(1):107-24. doi: 10.3390/pharmaceutics3010107. PMID: 24310428; PMCID: PMC3857040.

 

Friedl, P., & Weigelin, B. (2008). Interstitial cell migration and invasion in tumorous environments: Past, present and future. Cell adhesion & migration, 2(1), 115-125. https://pathsocjournals.onlinelibrary.wiley.com/doi/full/10.1002/path.3031

 

Hynes, R. O. (2009). The extracellular matrix in action. Cell, 137(5), 910-921. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4185430/

 

Krakhmal NV, Zavyalova MV, Denisov EV, Vtorushin SV, Perelmuter VM. Cancer Invasion: Patterns and Mechanisms. Acta Naturae. 2015 Apr-Jun;7(2):17-28. PMID: 26085941; PMCID: PMC4463409.

 

Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res. 2007 Mar 30;100(6):782-94. doi: 10.1161/01.RES.0000259593.07661.1e. PMID: 17395884.

 

Heisenberg, C. P., & Bellairs, R. (2013). Cell migration in development and disease. Nature reviews. Molecular cell biology, 14(7), 481-494. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457291/

 

Grinnell, F. (2003). Fibroblast biology: From contraction to proliferation. Journal of cell physiology, 197(1), 301-303. https://pubmed.ncbi.nlm.nih.gov/8106541/

 

Carmeliet, P., & Jain, R. K. (2011). Angiogenesis in disease and the angiogenic switch. Nature medicine, 17(7), 755-763

 

 

 

Event: 1376: Increase, angiogenesis

Short Name: Increase, angiogenesis

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human

Mice

Key Event Description

Angiogenesis is the physiological process through which new blood vessels form from existing vessels. This complex and tightly regulated process involves the proliferation and migration of endothelial cells, the remodeling of the extracellular matrix, and the recruitment of pericytes and smooth muscle cells.

 

Key Steps in Angiogenesis:

• Stimulus for Angiogenesis: Angiogenesis is triggered by specific signals, such as growth factors, released in response to tissue hypoxia, injury, or other physiological needs.
• Activation of Endothelial Cells: Endothelial cells in existing blood vessels become activated in response to angiogenic signals, leading to changes in gene expression and cell behavior.
• Proliferation and Migration: Activated endothelial cells proliferate and migrate toward the angiogenic stimulus, guided by chemotactic signals.
• Tube Formation: Endothelial cells organize into tube-like structures, forming capillaries. This process involves the creation of lumens within the tubes.
• Vessel Maturation: The newly formed vessels undergo maturation processes, including the recruitment of pericytes and smooth muscle cells. This maturation is crucial for the stability and functionality of the vasculature.
• Integration with Circulatory System: The newly formed blood vessels integrate into the existing circulatory system, providing increased blood flow to the target tissues.

 

Angiogenesis is regulated by both pro and anti-angiogenic factors. The most common pro angiogenic factors are VEGF and FGF (Folkman).

 

Angiogenesis is a fundamental mechanism in development, tissue repair, and various pathological conditions, including cancer :

• Development: During embryonic development, angiogenesis is critical for establishing the vascular network necessary for organ and tissue formation (Ribatti).
• Tissue Repair and Regeneration: Angiogenesis plays a key role in tissue repair and regeneration after injury or damage. The formation of new blood vessels helps supply nutrients and oxygen to the healing tissue (Ribatti).
• Menstrual Cycle and Pregnancy: In the female reproductive system, angiogenesis is a normal part of the menstrual cycle and is essential for the development of the placenta during pregnancy (Hoier).
• Inflammatory Response: Angiogenesis is involved in the inflammatory response, facilitating the influx of immune cells to sites of infection or injury.
• Cancer Growth and Metastasis : In cancer, angiogenesis is hijacked by tumors to support their growth and metastasis. Tumors release pro-angiogenic factors, promoting the formation of new blood vessels that supply nutrients and oxygen to the growing cancer cells (Nishida).
• Ischemic Diseases: Angiogenesis is a therapeutic target in diseases involving inadequate blood supply, such as ischemic heart disease and peripheral artery disease (Ferrara)

How it is Measured or Detected

Several assays are commonly employed (Staton, Stryker, Irvin):

• Endothelial Cell Proliferation Assays: Assays like BrdU (bromodeoxyuridine) incorporation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, or EdU (5-ethynyl-2'-deoxyuridine) incorporation can be used
• Endothelial Cell Migration Assays:  Transwell migration assays, scratch/wound healing assays, or microfluidic devices to study directed migration.
• Tube Formation Assay: Assess the ability of endothelial cells to form capillary-like structures (Stryker)
• Chorioallantoic Membrane (CAM) Assay: In vivo assay utilizing the chick embryo CAM to observe angiogenesis (Staton).
• Matrigel Plug Assay: In vivo assay involving the subcutaneous injection of Matrigel containing angiogenic inducers or cells (Tahergorabi).
• Aortic Ring Assay
• Corneal Neovascularization Assay
• Angiogenesis Imaging: as confocal microscopy or intravital microscopy to visualize blood vessel formation.
• Quantitative PCR (qPCR) for Angiogenic Markers
• ELISA for Angiogenic Factors

References

Staton CA, Stribbling SM, Tazzyman S, Hughes R, Brown NJ, Lewis CE. Current methods for assaying angiogenesis in vitro and in vivo. Int J Exp Pathol. 2004 Oct;85(5):233-48. doi: 10.1111/j.0959-9673.2004.00396.x. PMID: 15379956; PMCID: PMC2517524.

 

Irvin MW, Zijlstra A, Wikswo JP, Pozzi A. Techniques and assays for the study of angiogenesis. Exp Biol Med (Maywood). 2014 Nov;239(11):1476-88. doi: 10.1177/1535370214529386. Epub 2014 May 28. PMID: 24872440; PMCID: PMC4216737.

 

Tahergorabi Z, Khazaei M. A review on angiogenesis and its assays. Iran J Basic Med Sci. 2012 Nov;15(6):1110-26. PMID: 23653839; PMCID: PMC3646220.

 

Stryker ZI, Rajabi M, Davis PJ, Mousa SA. Evaluation of Angiogenesis Assays. Biomedicines. 2019 May 16;7(2):37. doi: 10.3390/biomedicines7020037. PMID: 31100863; PMCID: PMC6631830.

 

Nishida N, Yano H, Nishida T, Kamura T, Kojiro M. Angiogenesis in cancer. Vasc Health Risk Manag. 2006;2(3):213-9. doi: 10.2147/vhrm.2006.2.3.213. PMID: 17326328; PMCID: PMC1993983.

 

Ribatti, C. (2016). The crucial role of angiogenesis in embryogenesis. Life Sciences, 157, 17-22

 

Ribatti, C. (2014). The role of angiogenesis in wound healing. Journal of Vascular Research, 51(1), 2-11.

 

Reynolds, L. P., & Grazul-Balsas, J. L. (2010). Angiogenesis in the corpus luteum. Endocrine Reviews, 31(2), 226-240. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820779/

 

Hoier, J. D., & Hellström, M. (2014). Regulation of skeletal muscle angiogenesis by exercise training. Journal of Physiology, 592(11), 2523-2533. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104895/

 

Ferrara, N. (2005). VEGF as a therapeutic target in cancer. Cancer Cell, 8(6), 399-407.

 

Folkman, J. (2002). Angiogenesis: an essential step in tumor progression. Seminars in Oncology, 29(6), 315-322. [https://pubmed

Staton CA, Stribbling SM, Tazzyman S, Hughes R, Brown NJ, Lewis CE. Current methods for assaying angiogenesis in vitro and in vivo. Int J Exp Pathol. 2004 Oct;85(5):233-48. doi: 10.1111/j.0959-9673.2004.00396.x. PMID: 15379956; PMCID: PMC2517524.

 

Irvin MW, Zijlstra A, Wikswo JP, Pozzi A. Techniques and assays for the study of angiogenesis. Exp Biol Med (Maywood). 2014 Nov;239(11):1476-88. doi: 10.1177/1535370214529386. Epub 2014 May 28. PMID: 24872440; PMCID: PMC4216737.

 

Tahergorabi Z, Khazaei M. A review on angiogenesis and its assays. Iran J Basic Med Sci. 2012 Nov;15(6):1110-26. PMID: 23653839; PMCID: PMC3646220.

 

Stryker ZI, Rajabi M, Davis PJ, Mousa SA. Evaluation of Angiogenesis Assays. Biomedicines. 2019 May 16;7(2):37. doi: 10.3390/biomedicines7020037. PMID: 31100863; PMCID: PMC6631830.

 

Nishida N, Yano H, Nishida T, Kamura T, Kojiro M. Angiogenesis in cancer. Vasc Health Risk Manag. 2006;2(3):213-9. doi: 10.2147/vhrm.2006.2.3.213. PMID: 17326328; PMCID: PMC1993983.

 

Ribatti, C. (2016). The crucial role of angiogenesis in embryogenesis. Life Sciences, 157, 17-22

 

Ribatti, C. (2014). The role of angiogenesis in wound healing. Journal of Vascular Research, 51(1), 2-11.

 

Reynolds, L. P., & Grazul-Balsas, J. L. (2010). Angiogenesis in the corpus luteum. Endocrine Reviews, 31(2), 226-240. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820779/

 

Hoier, J. D., & Hellström, M. (2014). Regulation of skeletal muscle angiogenesis by exercise training. Journal of Physiology, 592(11), 2523-2533. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104895/

 

Ferrara, N. (2005). VEGF as a therapeutic target in cancer. Cancer Cell, 8(6), 399-407.

 

Folkman, J. (2002). Angiogenesis: an essential step in tumor progression. Seminars in Oncology, 29(6), 315-322. [https://pubmed

Event: 1971: Increased, tumor growth

Short Name: tumor growth

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human, mice

Key Event Description

Tumor growth refers to the increase in size of a cancer due to the uncontrolled proliferation of cells. The mechanisms have been detailed in Hanahan et al. hallmarks of cancer:

• Initiation: Tumor growth often begins with the initiation of genetic alterations in normal cells. This can result from mutations caused by various factors such as exposure to carcinogens, genetic predisposition, or viral infections.
• Uncontrolled Cell Proliferation: One of the hallmark features of tumor growth is uncontrolled cell division. Initiating mutations in key regulatory genes, such as oncogenes and tumor suppressor genes, disrupt normal cell cycle control, leading to continuous and unregulated cell proliferation. The PI3K/AKT/mTOR pathway regulates cell growth, proliferation, and survival. Mutations in genes like PTEN, a negative regulator of this pathway, can lead to its hyperactivation, promoting tumor growth (Janaku, Paplomatta). The MAPK is involved in cell proliferation, differentiation, and survival. Mutations in genes like BRAF and KRAS can activate this pathway, contributing to uncontrolled cell growth and tumor development (Steelman, Guo).
• Angiogenesis: Tumors require a blood supply for sustained growth. Angiogenesis, the formation of new blood vessels, is induced by the tumor to ensure a nutrient and oxygen supply. Tumor cells release pro-angiogenic factors, promoting the development of a network of blood vessels within and around the tumor (Nishida).
• Metabolic Adaptations: Tumor cells often exhibit altered metabolism, characterized by increased glycolysis even in the presence of oxygen (Warburg effect). This metabolic shift supports the high energy demands of rapidly dividing cells (Pham).
• Tumor Microenvironment: Tumor growth involves interactions with the surrounding microenvironment, including stromal cells, immune cells, and the extracellular matrix. Tumor cells can influence their microenvironment to promote their survival and expansion. Fibroblasts transform into cancer associated fibroblasts to support tumor growth by producing growth factors and promoting angiogenesis (Asif).
• Immune Evasion: Malignant tumors can develop mechanisms to evade the immune system. This may involve downregulation of antigens, inhibitory signals to immune cells, or the recruitment of immunosuppressive cells, allowing the tumor to escape immune detection and attack (Hiam).
• Invasion and Metastasis: Malignant tumors can invade nearby tissues and, in advanced stages, metastasize to distant organs. Invasion involves the penetration of tumor cells into surrounding tissues, while metastasis is the spread of cancer cells to other parts of the body via the bloodstream or lymphatic system.
• Tumor Dormancy: In some cases, tumor growth may enter a state of dormancy, where the proliferation of cancer cells is temporarily halted. Dormant tumors can later resume growth, posing challenges in terms of early detection and treatment (Endo).

 

 

Detailed here are key molecular mechanisms associated with breast tumor growth (Hanahan):

• Genetic Mutations: Genetic alterations in key oncogenes (e.g., HER2, MYC, PIK3CA) promote cell proliferation whereas mutations in tumor suppressor genes (e.g., TP53, BRCA1, BRCA2) remove inhibitory controls on cell growth. (Knudson)
• Hormone Receptor Signaling: ER-positive breast cancers (70% of cancers) respond to estrogen stimulation, promoting cell proliferation. Endocrine therapies targeting ER signaling are effective in treating these cancers (Elikatkin).
• HER2/Neu overexpression : Amplification or overexpression of the human epidermal growth factor receptor 2 (HER2) promotes cell growth and survival (Slamon, Elikatkin).
• PI3K/AKT/mTOR Pathway Activation: Mutations in the PIK3CA gene or activation of PI3K signaling pathway promotes cell survival and proliferation. Phosphoinositide 3-kinase (PI3K) activation leads to downstream signaling through AKT and mTOR, promoting cell growth and protein synthesis (Janku, Paplomata)
• MAPK pathway: This pathway is involved in cell proliferation, differentiation, and survival. Mutations in this pathway can also contribute to breast cancer development (Steelman).
• Cell Cycle Regulation: Dysregulation of cyclin-dependent kinase (CDK) and cyclin complexes controls the cell cycle progression. Inactivation of the p16 tumor suppressor and retinoblastoma protein (pRB) pathway contributes to uncontrolled cell cycle progression (Witkiewicz).
• Apoptosis Evasion: Overexpression of anti-apoptotic proteins (e.g., Bcl-2, Bcl-xL) inhibits programmed cell death. Mutations or inactivation of pro-apoptotic proteins (e.g., p53) hinders apoptotic responses.
• Angiogenesis Stimulation: Vascular endothelial growth factor (VEGF) and its receptors stimulate angiogenesis, ensuring a blood supply for tumor growth. Hypoxia-inducible factor 1-alpha (HIF-1α) activates angiogenic responses in low-oxygen conditions.
• Epithelial-Mesenchymal Transition (EMT): Downregulation of adhesion molecules (e.g., E-cadherin) leads to increased cell mobility. Acquisition of mesenchymal characteristics enhances the ability of tumor cells to invade surrounding tissues (Drasin).
• Extracellular Matrix (ECM) Remodeling: Overexpression of MMPs facilitates ECM degradation, enabling tumor invasion.
• Metastasis Formation: Tumor cells invade surrounding tissues and enter blood or lymphatic vessels. Ability of tumor cells to survive in the bloodstream. Tumor cells exit circulation, invade distant tissues, and establish secondary tumors.

How it is Measured or Detected

Many different assays can be used to measure tumor growth directly:

• Clinical measurement and palpation
• Histopathology with fluorescence imaging, dyes or weight
• Serum Biomarkers
• Imagery using caliper measurement on Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Positron Emission Tomography (PET), or ultrasound can provide detailed images for volume calculation.
• Positron Emission Tomography (PET) Imaging : measurement of metabolic activity using radioactive tracers.
• In vivo models: xenograft tumor models, orthotopic models, genetically engineered mouse models

 

 

Indirect assays can also be used:

• Bioluminescence Imaging (BLI): Measurement of light emitted by luciferase-expressing tumor cells.
• Flow Cytometry: Quantification of tumor cells based on DNA content.
• Cell Proliferation Assays (MTT/MTS, BrdU)
• Colony formation

References

Asif PJ, Longobardi C, Hahne M, Medema JP. The Role of Cancer-Associated Fibroblasts in Cancer Invasion and Metastasis. Cancers (Basel). 2021 Sep 21;13(18):4720. doi: 10.3390/cancers13184720. PMID: 34572947; PMCID: PMC8472587.

 

Witkiewicz AK, Knudsen ES. Retinoblastoma tumor suppressor pathway in breast cancer: prognosis, precision medicine, and therapeutic interventions. Breast Cancer Res. 2014 May 7;16(3):207. doi: 10.1186/bcr3652. PMID: 25223380; PMCID: PMC4076637.

 

Eliyatkın N, Yalçın E, Zengel B, Aktaş S, Vardar E. Molecular Classification of Breast Carcinoma: From Traditional, Old-Fashioned Way to A New Age, and A New Way. J Breast Health. 2015 Apr 1;11(2):59-66. doi: 10.5152/tjbh.2015.1669. PMID: 28331693; PMCID: PMC5351488.

 

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List of Adverse Outcomes in this AOP

Event: 1982: metastatic breast cancer

Short Name: Metastasis, Breast Cancer

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Increased metastasis of cancerous cells  is known to be highly conserved throughout evolution and is present from humans to invertebrates.

Key Event Description

Processs: metastasis of cancer cells                             Object:metastasis                    Process:Increased

Biological state: 

Dissemination of the cancer cells from one organ to another which is not directly connected to the primary site is called metastasis. It has a crucial role in the prognosis of cancer patients. In the initial stage of metastasis, cancer cells detach from the primary tumor and disseminate in the tissue. Subsequently cancer cells enter the vascular or lymphatic channels (23-25). The establishment of micro-metastasis mainly depends on the survival of the circulating tumor cells (CTCs) inside the lymphatic or blood channels. Extravasation of cancer cells through the vessel wall takes place resulting in the proliferation of cancer cells in the secondary site.  Various signalling pathways are involved in each of the above mentioned   process. Few theories have been proposed to explain the mechanism of metastasis.  The “organ selection concept” theory suggests that the growth factors establish a successful metastasis in the metastatic site (26,27) whereas the “adhesion theory” proposes the tissue specific adhesion molecules are expressed on endothelial cells of recipient organs  which will anchor the migrating cancer cells,  providing the a pre-metastatic niche. The role of chemokine receptor has been explained in “chemo-attraction theory” while Paget   reported the theory of “seed” for metastatic tumor cells and of “soil” for the secondary site. As per this concept the organ distribution is determined by the site and histopathological type of the primary tumor.  The recent understanding suggested, pre-metastatic niche has been indicated to explain metastasis.  It is interesting to note that prior to co-localization, the primary tumor induces the micro environment of secondary site by CTCs.

 Subsequently, a metastatic niche is generated to support disseminated tumor cells (DTCs) and localize them to develop a metastasis.  The most recent theory describes a bidirectional relationship between the primary and secondary sites. According to this theory, the surviving cancer cells in the metastatic tumor can return to the primary site to promote the primary tumor progression (28,29). Efficient and  direct  blood  flow  can  explain  the  probability  of metastasis  to  the  specific  organs  like  hepatic  metastasis in patients  with colon cancer which  receive direct blood  flow  from  the  primary  site .Vascular  permeability is also the other factor  which  significantly  promotes extravasation  at  the metastatic  site. However at present, understanding of molecular mechanisms of metastasis remains incomplete.

Biological compartment

Organs,Cellular

Role in general biology

Epithelial-  mesenchymal  transition  (EMT) and its reverse  mesenchymal-epithelial  transition (MET)  are characteristics  of cellular  plasticity  during embryogenesis  and  tumor metastasis  (30).  There has been decreased expression of  E-cadherin  and  β-catenin  and  elevated  expression  levels of  vimentin,  fibronectin  and  N-cadherin in EMT   (31).  In cancers, EMT  is a major  process  by which  cancer cells  lose their epithelial  characteristics  to acquire mesenchymal-like properties.  Tumor cell  migration  is a pre-requisite for the metastatic process in which, EMT is  the most critical step to  initiate  metastasis including metastasis to  lymph nodes  (32).  During   EMT, cancer  cells lose their  cell-to-cell junctions and cellular  polarity via multiple  signaling pathways which  increase  the motilities and invasive phenotype of them (33). Cleavage of  E-cadherin mediated by the MMPs  increases  the tumor cell  motility and invasion . Apart from this ,EMT has a  key role  in  drug resistance.  This is supported by the finding that  high levels of vimentin was found  in adriamycin and  vinblastine  resistant  breast  cancer cell  lines  (34). EMT  promotes CSCs  motility, cancer cell invasion, tumor  metastasis and recurrence and drug resistance.   Expression  of  stem cell like  markers  and formation of tumor spheres by CSCs are enhanced by EMT  process.  CSCs acquire mesenchymal  features by undergoing EMT phenomenon. By acquiring mesenchymal features,  CSCs become resistant  to anti-cancer therapies; hence, they can  survive and cause cancer recurrence.  In addition to this ,CSCs invade to the adjacent  stromal  tissues, enter the  vascular channels,  and  finally  reach  the  distant  organs.  In  the target organs, CSCs  cause MET phenomenon  which results in the acquisition of  epithelial  characteristics.  MET  phenomenon also   increases the  cell-to-cell attachment, cancer cells proliferation and differentiation to form  metastatic lesions  (35).  Altogether , EMT induces  CSC properties   and metastatic  activities. On the other hand, EMT  and CSCs collaborate in invasion capacity   hence targeting  the EMT/CSC  phenotype can be a therapeutic  approach for the treatment of metastasis and tumor recurrence (36).

 

EMT programs are regulated by a network of signal- ling pathways that involve components such as growth factors (transforming growth factor-β [TGF-β], epider- mal growth factor [EGF]) and their associated signalling proteins (Wnt, Notch, Hedgehog, nuclear-factor kappa B [NF-κB], extracellular signal-regulated kinase [ERK], and phosphatidylinositol 3-kinase [PI3K]/Akt) in response to stresses involved in tumorigenesis, including hypoxia, oncogenic or metabolic stress, inflammation, and physical constraints [37-41].

These signals activate EMT-inducing transcription factors, including Snail/Slug, ZEB1/δEF1, ZEB2/SIP1, Twist1/ 2, and E12/E47 [42-44]. EMT-inducing transcription factors regulate the expression of proteins involved in cell polarity, cell-cell contact, cytoskeletal structural maintenance, and extracellular matrix (ECM) degradation, and they sup- press key epithelial genes. Loss of E-cadherin is considered a hallmark of EMT; these EMT-inducing transcription factors bind to E-box elements in the E-cadherin gene promoter to repress its transcription. Of particular note, Snail is an early marker of EMT that is involved in the initial cell-migratory phenotype, and it occasionally induces other factors .

 

During EMT, epithelial cells reorganize cytoskeleton and resolve cell–cell junctions, which are accompanied with switching off the expression of epithelial markers and turning on mesenchymal genes. Although changes in epithelial and mesenchymal markers during EMT can vary significantly in different biologic contexts, a network of transcription factors, including TWIST1/2, SNAIL1/2, ZEB1/2, and FOXC2, are consistently required to orchestrate the EMT program (45). The expression of these transcription factors is associated with poor prognosis and distant metastasis in various human cancers has been documented in various studies. (46). Besides its role in promoting tumor cell invasion, EMT is shown to confer tumor cells with resistance to apoptosis  and anoikis (47), thus allowing cell survival in the blood stream after intravasation. EMT could also facilitate tumor cells' escape from the senescence program, especially through TWIST1 and ZEB1 (48,49). Furthermore, EMT has been shown to  cancer cells with cancer stem cell (CSC)–like features, which further aid tumor dormancy and chemo resistance (50,51).Tumor samples or experimental tumor xenograft models have provided convincing evidence for the activation of EMT in various primary epithelial tumors in various studies. . Interestingly, more recent studies reveal a dynamic requirement of EMT in tumor metastasis: activation of EMT promotes local tumor invasion, intravasation, and extravasation of the systemic circulation, whereas reversion of EMT is essential to establish macrometatasis in distant organs (52,53).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

How it is Measured or Detected

	Method/ measurement reference	Reliability	Strength of evidence	Assay fit for purpose	Repeatability/ reproducibility	Direct measure
Cell line,humans,Human cell line studies	qRT-PCR,,Luciferase reporter assay ,immunoblotting,immunoprecipitation,cell invasion assay,cell migration assay, bioluminesence imaging,wound healing assay,Wound scratch & Transwell assay, Microarray,Immunofluorescence, Immunohistochemistry,	+	Strong	Yes	Yes	Yes

Regulatory Significance of the AO

The Adverse Outcome Pathway (AOP) holds substantial regulatory significance as a structured framework for understanding and predicting the biological sequence of events leading from DNA damage to a metastatic breast cancer. By elucidating the causal relationships between key events along the pathway, AOP offer a comprehensive understanding of toxicological mechanisms and provide a basis for informed decision-making in risk assessment and regulatory decision-making. AOPs facilitate the integration of diverse scientific data, enabling regulators to evaluate the potential impact of chemical exposures on human health and the environment. These pathways empower the development of targeted testing strategies, alternative methods, and safer chemical design, ultimately enhancing the efficiency and accuracy of risk assessment and regulatory policies.

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Appendix 2

List of Key Event Relationships in the AOP