Aop: 443

Title

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

Alcohol Induced DNA damage and mutations leading to Metastatic Breast Cancer

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
DNA damage and metastatic breast cancer

Graphical Representation

A graphical representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help
Click to download graphical representation template Explore AOP in a Third Party Tool
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Authors

The names and affiliations of the individual(s)/organisation(s) that created/developed the AOP. More help

Dr Usha S Adiga MD PhD, Professor,Department of Biochemistry,KS Hegde Medical Academy,Nitte Deemed to be University,Mangalore,Karnataka,India

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Usha Adiga   (email point of contact)

Contributors

Users with write access to the AOP page.  Entries in this field are controlled by the Point of Contact. More help
  • Usha Adiga

Status

Provides users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. OECD Status - Tracks the level of review/endorsement the AOP has been subjected to. OECD Project Number - Project number is designated and updated by the OECD. SAAOP Status - Status managed and updated by SAAOP curators. More help
Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite Under Development 1.103 Included in OECD Work Plan
This AOP was last modified on October 21, 2022 02:03

Revision dates for related pages

Page Revision Date/Time
Increased, DNA damage and mutation August 13, 2019 05:41
Inadequate DNA repair October 26, 2021 08:25
Increase, Mutations August 16, 2021 14:41
Increased micro RNA expression March 24, 2022 13:10
Decreased SIRT1 expression March 24, 2022 13:13
Increase activation, Nuclear factor kappa B (NF-kB) March 30, 2021 13:17
Antagonism, Estrogen receptor September 16, 2017 10:14
Induction, Epithelial Mesenchymal Transition January 30, 2019 10:27
metastatic breast cancer March 23, 2022 09:43
Increase Chromosomal Aberrations October 21, 2022 03:03
Increased, DNA damage and mutation leads to Inadequate DNA repair March 24, 2022 23:19
Increased, DNA damage and mutation leads to Increase chromosomal aberrations August 14, 2022 08:50
Increase chromosomal aberrations leads to Increase,miRNA levels August 14, 2022 09:08
Inadequate DNA repair leads to Increase, Mutations June 03, 2020 23:25
Increase, Mutations leads to Increase,miRNA levels March 24, 2022 13:20
Increase,miRNA levels leads to Decrease,SIRT1(sirtuin 1) levels March 24, 2022 13:26
Decrease,SIRT1(sirtuin 1) levels leads to Increase activation, Nuclear factor kappa B (NF-kB) March 24, 2022 13:34
Increase activation, Nuclear factor kappa B (NF-kB) leads to Antagonism, Estrogen receptor March 23, 2022 11:26
Antagonism, Estrogen receptor leads to EMT March 24, 2022 13:42
EMT leads to Metastasis, Breast Cancer October 21, 2022 02:22
Ethyl alcohol March 22, 2022 00:48

Abstract

A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

This adverse outcome pathway details the effect of alcohol as a stressor in metastatic breast cancer. Aim of this AOP is intended to detail the linkage between alcohol and miRNA- SIRT-1 axis induced metastatic breast cancer which represents a knowledge gap as there are not many references available. Consecutive KEs identified are as follows.

Acetaldehyde, which is a metabolite of alcohol is considered a major mutagen which has been determined to induce genotoxic effects on DNA  resulting in increased DNA damage. Inadequate DNA crosslink repair mechanisms leads to accumulation of damaged DNA resulting in impaired DNA synthesis leading to mutations and increased miRNA expression ; leads to disruption of  SIRT-1 signalling . This step is followed by increased acetylation and activity of  NFkB ;  loss of estrogen receptor functions  ; molecular alterations of epithelial cells ; gain of mesenchymal cell features ; eventuating in increased invasion and migration of breast cancer cells resulting in Metastatic breast cancer .

AOP Development Strategy

Context

Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

Alcoholic beverages are classified by the International Agency for Research on Cancer(IARC) as Group 1 carcinogens. Studies have reported alcohol consumption to be a  risk factor for breast cancer in women(Room R et al, 2005). A woman drinking an average of two units of alcohol per day has an 8% higher risk of developing breast cancer than a woman who drinks an average of one unit of alcohol per day[2]. Alcohol is metabolized by alcohol dehydrogenase to acetaldehyde which is a mutagen. Various theories have been proposed which explain the mutagenicity of alcohol. Among them, the most relevant one for carcinoma of the breast has been proposed by Purohita et al, suggesting an alcohol-induced inactivation of the tumor suppressor gene BRCA1 and increased estrogen Responsiveness in breast tissues(Purohit V et al, 2005). Boffetta and Hashibe list plausible mechanisms of breast cancer as a result of the genotoxic effect of acetaldehyde-induced increased estrogen concentration(Boffetta P et al 2006). It has also been found that alcohol stimulates the epithelial-mesenchymal transition (EMT), because of which there is distant metastasis (Forsyth C. B. et al 2010). However, this mechanism needs to be elucidated in detail.

MicroRNAs (miRNAs) are non-coding, single-stranded RNA molecules that regulate target gene expression via post-transcriptional modifications [Mohr A. M& Mott J. L 2015 and Lai E. C. 2002). Several studies indicated the promising role of miRNA in the diagnosis and outcome prediction in several cancers (Mirzaei H et al 2018 and Liu, S. Y et al 2017). miRNA-21 is upregulated and promotes metastasis in several cancers (Kunita, A et al 2018 and Liu Z et al 2015). A Chinese study by Kunita et al proved that plasma levels of miRNA were up-regulated in large B-cell lymphoma patients (Kunita, A et al 2018). A study by Wang et al also proved that plasma levels of miR were upregulated in large B-cell lymphoma patients in China (Chen et al 2014). Although miR-21 was indicated to play a crucial role in the metastasis of lung cancer, ovarian cancer, and head and neck cancer through several signaling pathways, the molecular mechanism of how miR-21 regulates the EMT process in breast cancer is not clear (Liu S. Y et al, Lopez-Santillan et al 2018, Panagal M. et al 2018, Zhou, B.et al 2018, Brabletz T et al and Ye, X. et al 2017).There are a number of miRNAs which regulate SIRT 1 expression. The epithelial-mesenchymal transition (EMT) is a process that which epithelial cells lose their cell polarity and cell adhesion ability, which will lead to cancer metastasis (Vaziri H et al 2001 and Luo, J et al 2001). Epithelial cells exhibit the property of regular cell-cell contacts, adhesion to the surrounding cellular fabric, preventing the detachment of individual cells. Whereas mesenchymal cells do not form intercellular contacts. 

Sirtuins are nicotinamide adenine dinucleotide (NAD+)–dependent deacetylases that function as intracellular regulators of transcriptional activity (Blander G & Guarente L 2004 and Roth M & Chen W 2014). It plays important roles in cell survival, signal transduction, and cell apoptosis by deacetylating key cell signaling molecules and apoptotic related proteins, such as NF-kB, p53, Ku70, and HIFs (Zhao, W et al 2008 and Chen W & Bhatia R 2013). Various studies have inconclusive reports on the role of SIRT1 in cancer, because of its opposite effects as both a tumor activator or suppressor in various human cancers, including breast cancer.  Deng et al found that the expression of SIRT1 was lower in prostate cancer, bladder cancer, ovarian cancer, and glioblastoma when compared with normal tissues (Han, L et al 2013).On the contrary, it was found that, in leukemia and lung cancer, SIRT1 was significantly higher(Riggio M et al 2012 and Lee M S et al 2015).

This can be explained as follows: SIRT1-mediated deacetylation suppresses the functions of several tumor suppressors including p53, p73, and HIC1, it has been suggested that SIRT1 has a promoting function in tumor development and progression [Pinton G et al 2016, Pillai VB  et al 2014, Wan G et al 2017 and Hwang B et al 2014]. In contrast, SIRT1 may have a suppressive activity in tumor cell growth by suppressing NF-κB, a transcription factor playing a central role in the regulation of the innate and adaptive immune responses and carcinogenesis, the dysregulation of which leads to the onset of tumorigenesis and tumor malignancy(Yuan J et al 2009, Wang R H et al 2008, Chen L F et al 2004 and Greten F R & Karin M 2004). Here, we aim to further explore the role of the SIRT1-NF kB signaling pathway in tumorigenesis of the breast as well as its associated mechanisms.

The nuclear factor-κB (NF- κB)/REL family of transcription factors is comprised of a RELA/p65,c-REL, RELB, p105/NF- κB1 and p100/NF- κB2 (Van Laere S J et al 2007). The p105 and p100 proteins can be processed by proteolytic cleavage into p50 and p52, respectively. Activation of the NF-κB signaling pathway leads to the induction of target genes that can inhibit apoptosis, interaction with cell cycle regulation, cell invasion, contribute to tumorigenesis and metastatic invasion (Shostak K & Chariot 2011). Activation NF-κB in breast cancer is loss of Estrogen Receptor (ER) expression and Human Epidermal Growth Factor Receptor 2 (HER-2) overexpressed via epidermal growth factor receptor (EGFR) and Mitogen-Activated Protein Kinase (MAPK) pathway (Ali S & Coombes R C 2002). Indeed, the binding of epidermal growth factor (EGF) to its receptor (EGFR) also ultimately activates NF-κB and most likely contributes to the enhanced activity of this transcription factor in ER-negative breast cancer cells (Kalkhoven E et al 1996).

Loss of ER function has been associated with constitutive NFkB activity and hyperactive MAPK, because of constitutive secretion of cytokine and growth factors, which ultimately culminates in aggressive, metastatic, hormone-resistant cancers (Merkhofer E C et al 2010). Activation of the progesterone receptor can lead to inhibition of NF-κB driven gene expression (Sethi G et al 2008) reducing its DNA binding and transcriptional activity. HER-2 activates NF-κB through the canonical pathway which surprisingly, involves IKKα (Ito, T et al 2010). Activation of NF-κB promotes the survival of tumor cells. Several gene products that negatively regulate apoptosis in tumor cells are controlled by NF-κB activation (Lee J et al 2010). Estrogen plays an important role in breast cancer initiation and progression. Breast cancer over time acquires different mutations and the proportion of estrogen receptor-negative cells in tumors increases. This transformation confers aggressive biological characteristics to breast cancer such as rapid growth, poor differentiation, and poor response to hormone therapy. NF-κB pathway plays important role in this pathway (Lee J et al 2010).

Expression of SIRT1 is controlled at multiple levels by transcriptional, post-transcriptional, and post-translational mechanisms under physiological and pathological conditions. Emerging evidence indicates that miRs are important regulators of SIRT1 expression (Lovis P et al 2008, Ortega F J et al 2010, Zovoilis A et al 2011, Yamakuchi M et al 2008 and Mullany L E et al 2017). Studies have shown that miR-34a directly binds to the 3′ untranslated region (UTR) of SIRT1 mRNA and reduces its expression (Ortega F J et al 2010).

Study findings support the hypothesis that alcohol consumption is able to influence miRNA expression. Considerable evidence from rodent and human studies demonstrates that disruption of the hepatic SIRT1 signaling by ethanol plays a central role in the development of AFLD (Yin H et al 2014, Li M et al 2014).Ethanol down-regulates SIRT1 in hepatic cells and in the animal livers. The ethanol-mediated disruption of SIRT1 signaling leads to excess fat accumulation and inflammatory responses in the liver of animals and humans. Treatment with resveratrol, a known SIRT1 agonist, can alleviate liver steatosis . Accumulating evidence demonstrates that ethanol-mediated SIRT1 inhibition leads to the development of AFLD largely through disruption of a signaling network mediated by various transcriptional regulators and co-regulators, including nuclear transcription factor-κB (NF-κB)(Yin H et al 2014, Li M et al 2014).

Strategy

Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

Summary of the AOP

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

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 1669 Increased, DNA damage and mutation Increased, DNA damage and mutation
KE 155 Inadequate DNA repair Inadequate DNA repair
KE 185 Increase, Mutations Increase, Mutations
KE 1554 Increase Chromosomal Aberrations Increase chromosomal aberrations
KE 1980 Increased micro RNA expression Increase,miRNA levels
KE 1981 Decreased SIRT1 expression Decrease,SIRT1(sirtuin 1) levels
KE 1172 Increase activation, Nuclear factor kappa B (NF-kB) Increase activation, Nuclear factor kappa B (NF-kB)
KE 112 Antagonism, Estrogen receptor Antagonism, Estrogen receptor
KE 1457 Induction, Epithelial Mesenchymal Transition EMT
AO 1982 metastatic breast cancer Metastasis, Breast Cancer

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Network View

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

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help
Life stage Evidence
Adult, reproductively mature Moderate

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help
Term Scientific Term Evidence Link
human and other cells in culture human and other cells in culture High NCBI
human Homo sapiens Moderate NCBI
mice Mus sp. Moderate NCBI
rat Rattus norvegicus Moderate NCBI
canine heartworm nematode Dirofilaria immitis Moderate NCBI
yeast Saccharomyces cerevisiae Moderate NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Female High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

Increased DNA damage and mutations [Evidence: high]DNA damage refers to any modification in the physical and/or chemical structure of DNA resulting in an altered DNA molecule that is different from the original DNA molecule with regard to its physical, chemical, and/or structural properties".External factors to the cell such as environmental or potentially aggressive factors produced by the normal cell metabolism can damage the DNA. The effects caused by the action of endogenous factors may be more serious and/or more extensive than the effect of most of the exogenous DNA damaging factors. Evidence suggested that prolonged alcohol intake is positively associated with an increased risk of cancer.  It can cause changes in the sequence of genomic DNA, which may act as a tumor promoter as well. Alcohol consumption can result in the generation of DNA-damaging molecules such as reactive oxygen species (ROS), lipid peroxidation products, and acetaldehyde. Strand breaks and oxidative base damage in DNA can be produced by hydroxyl radicals which are both mutagenic and cytotoxic. Alcohol is a known inducer of microsomal oxidizing system, which includes a specific ethanol-inducible form of cytochrome P450, referred to as CYP2EL (Lieber C 1992). This effect on the enzyme system has been associated with liver pathology induced by alcohol (Morimoto M et al 1993, French S et al 1993, Nanji A et al 1994, Albano E et al 1996). Again the damaging effects of high levels of CYP2E1 may be mediated by the generation of ROS (Cederbaum 1989, Reinke L et al 1990, Ishii H et al 1989). ROS that is highly reactive, include the oxygen radicals superoxide anion and hydroxyl radicals and can react with lipids, proteins, and DNA and thereby damage them (Knecht K et al 1990).  It has been confirmed in vivo experiments that hydroxyethyl radical formation takes place after ethanol exposure (Albano E et al 1996, Moore D et al 1995, Clot, P et al 1996, Thurman R 1973). Chronic exposure to ethanol also results in increased production of H202, (Kukieka E et al 1992, Kukielka, E., & Cederbaum, A. I. 1994) which can react with metal ions (such as iron in the Fenton reaction); thus resulting in the production of the highly reactive hydroxyl radicals. DNA is very sensitive to the attack by the hydroxyl radical. A sensitive assay for hydroxyl radical formation from CYP2E1 uses DNA damage (strand breakage) as an endpoint (Breen AP, Murphy JA 1995). Apart from this, more than twenty different types of DNA base damage with diverse biological properties are produced by hydroxyl radical (Moriya M 1993).8-hydroxy-2'-deoxyguanosine, is one such DNA lesion brought about by oxidative stress.  This is mutagenic, due to the tendency of DNA polymerases to misincorporate deoxyadenosine residues opposite this oxidized base (Song B 1996).

Inadequate DNA cross-link repair mechanisms [Evidence:high]:

As a result of DNA damage, DNA repair activities change. A variety of genotoxic agents, such as N-nitrosodimethylamine, aflatoxin B1, and 2-acetylaminofluorene induce the protein, O6-Alkylguanine-DNA alkyltransferase (ATase), are responsible for the repair of DNA alkylation damage in rats (O’Connor, 1989; Chinnasamy et al.,1996). Grombacher and Kaina (1996) reported an increased human ATase mRNA expression by alkylating agents like N-methyl-N′-nitro-N-nitrosoguanidine and methyl methanesulphonate and by ionizing radiation via the induction of the ATase promoter. ATase mRNA expression was increased in response to treatment with 2-acetylaminofluorene in rat liver (Potter et al., 1991; Chinnasamy et al., 1996). In another study, it was demonstrated that ATase gene induction is p53 gene-dependent: ATase activity was induced in mouse tissues following γ-irradiation in p53 wild-type mice, but not in p53 null animals (Rafferty et al., 1996). Alkylating agents and X-rays also induce DNA glycosylase, alkylpurine-DNA-N-glycosylase (APNG)  (Lefebvre et al., 1993; Mitra and Kaina, 1993).

Increased mutations [Evidence: moderate]Inadequate repair causes damaged DNA to be retained and used as a template during DNA replication. Incorrect nucleotides may be inserted during the replication of damaged DNA, and these nucleotides become 'fixed' in the cell after replication. The mutation propagates to more cells as a result of further replication. Non-homologous end joining (NHEJ) is one of the repair methods employed in human somatic cells to repair DNA double-strand breaks (DSBs). (Petrini et al., 1997; Mao et al., 2008). However, this mechanism is prone to errors and may result in mutations during the DNA repair process. (Little, 2000). As it does not use a homologous template to repair the DSB, NHEJ is considered error-prone. Many proteins work together in the NHEJ pathway to bridge the DSB gap by overlapping single-strand termini that are typically less than 10 nucleotides long. (Anderson, 1993; Getts & Stamato, 1994; Rathmell & Chu, 1994). Errors are introduced during this process, which can result in mutations like insertions, deletions, inversions, or translocations.

Increased micro RNA expression [Evidence: moderate]DNA damage-responsive transcription factors, such as NF-kB, E2F, and Myc, are also involved in miRNA transcription regulation. The p53 protein also functions as a transcriptional repressor by binding to miRNA promoters and preventing the recruitment of transcriptional activators. For example, p53 prevents the TATA-binding protein from binding to the TAATA site in the promoter of the miR-17-92 cluster gene, suppressing transcription. Under hypoxic conditions, the miR-17-92 cluster is suppressed by a p53-dependent mechanism, making cells more susceptible to hypoxia-induced death (Yan et al.,2009).

Decreased SIRT1 expression [Evidence: moderate]There are several signaling pathways that establish the role of increased miRNA expression in downregulating the SIRT 1 gene few of which are listed as follows; Butyrate has been demonstrated to cause apoptosis and reduce carcinogenesis in a variety of cancers (Tailor et al.,2014; Rahmani et al.,2002). Although butyrate has been shown to suppress SIRT1 gene expression in various cancers, this has yet to be proven in hepatocellular carcinoma (HCC) (Iglesias et al., 2007). In HCC, miR-22 was found to be downregulated, and its low levels aided carcinogenesis (Zhang et al.,2010). The Huh7 cells' in vitro proliferation was decreased by miR-22 expression, which activated apoptosis. In Huh7 cells, on the other hand, SIRT1 expression was high, which enhanced the expression of antioxidants such as superoxide dismutase (SOD), allowing cell growth to continue (Chen et al.,2012). Butyrate upregulated miR-22 in Huh7 cells, which binds directly to the 3′UTR region of SIRT1 and suppresses its expression.Notch3–SIRT1–LSD1–SOX2 Signaling Pathway in metastasis (Wang et al.,2016; Wu et al .,2017).MiR-486 inhibits HCC invasion and tumorigenicity by directly targeting and suppressing SIRT1 expression. This reduced the tumorigenic and chemo-resistant features of LCSCs and inhibited HCC invasion and tumorigenicity (Yan et al.,2019).

Increased activity of NF kB [Evidence: moderate]SIRT1 deacetylates  NFkB. In the context of NFkB, all of the evidence so far points to its signaling being inhibited after SIRT1 deacetylation (Morris, 2012). According to Yeung et al, SIRT1 can directly interact with and deacetylate the RelA/p65 component of the NF-B complex (Yeung et al.,2004). NF-B can be activated by cytokines (TNF-, IL-1), growth factors (EGF), bacterial and viral products (lipopolysaccharide (LPS), dsRNA), UV and ionizing radiation, reactive oxygen species (ROS), DNA damage, and oncogenic stress from inside the cells. Almost all stimuli eventually activate a large cytoplasmic protein complex called the inhibitor of B (IB) kinase (IKK) complex via a so-called "canonical pathway." The exact composition of this complex is unknown, however, it has three fundamental components: IKK1/IKK, IKK2/IKK, and NEMO/IKK. IB is phosphorylated by the activated IKK complex, which marks it for destruction by the -transducin repeat-containing protein (-TrCP)-dependent E3 ubiquitin ligase-mediated proteasomal degradation pathway (Liu et al., 2012;Li et al., 2002). As a result, unbound NF-B dimers can go from the cytoplasm to the nucleus, bind to DNA, and control gene transcription.

Antagonism of estrogen receptor [Evidence: moderate]Activation NF-κB in breast cancer leads to loss of Estrogen Receptor (ER) expression and Human Epidermal Growth Factor Receptor 2 (HER-2) overexpressed via epidermal growth factor receptor (EGFR) and Mitogen-Activated Protein Kinase (MAPK) pathway (Laere et al.,2007). Indeed, the binding of epidermal growth factor (EGF) to its receptor (EGFR) activates NF-B, which most likely contributes to this transcription factor's increased activity in ER-negative breast cancer cells (Shostak et al.,2011). Because of the constitutive production of cytokines and growth factors, loss of ER function has been linked to constitutive NF-kB activity and hyperactive MAPK, resulting in aggressive, metastatic, hormone-resistant malignancies (Ali et al., 2002). Activation of the progesterone receptor can reduce DNA binding and transcriptional activity by inhibiting NF-B-driven gene expression (Kalkhoven et al., 1996). HER-2 stimulates NF-B via the conventional route, which includes IKK (Merkhofer et al., 2010).

Epithelial-mesenchymal transition cell [Evidence: high]Estrogen/ERa signaling maintains an epithelial phenotype and suppresses EMT.ERa signaling promotes proliferation and epithelial differentiation and opposes EMT. Various studies support this finding (Eeckhoute et al.,2007, Kouros-Mehr et al.,2008, Nakshatri et al., 2009, Taylor et al.,2010). ER-a negative was related to activation of genes implicated in Wnt, Sonic Hedgehog, and TGF-b signaling, indicating epithelial-mesenchymal transition (EMT)(Wik et al.,2013).

Metastatic breast cancer [Evidence: high]The “epithelial-mesenchymal transition” (EMT), a key developmental regulatory program, has been reported to play critical and intricate roles in promoting tumor invasion and metastasis in epithelium-derived carcinomas in recent years. Some of the cells undergoing EMT have the characteristics of cancer stem cells (CSCs), which are linked to cancer malignancy (Shibue & Weinberg, 2017; Shihori Tanabe, 2015a, 2015b; Tanabe, Aoyagi, Yokozaki, & Sasaki, 2015). Cancer metastasis and cancer therapeutic resistance are linked to the EMT phenomenon (Smith & Bhowmick, 2016; Tanabe, 2013). EMT causes the cell to escape from the basement membrane and metastasize by increasing the production of enzymes that break down extracellular matrix components and decreasing adherence to the basement membrane (Smith & Bhowmick, 2016). 

Overall Assessment:

overall assessment of the AOP was based on the biological domain of the applicability, the essentiality of all KEs, Biological plausibility of each KER, Empirical support for each KER, and Quantitative weight of evidence considerations optional.

MIE 1669

KE 155

KE 185

KE1980

KE1981

KE 1172

KE 112

KE1457

AO1982

Sex/Life stage /Taxa

Female/Reproductive/Human,human cell line,mice,rat

Female/Reproductive/Rat/rat cel lines/mouse

Female/Reproductive/Mice,yeast,hman cel line

Female/Reproductive/canine,mouse,human cell line

Female/Reproductive/human,human cell ine

Female/Reproductive/human,human cell ine,mice

Female/Reproductive/human,human cell ine,mice

Female/Reproductive/hman,hman cell line

Female/Reproductive/hman,hman cell line,mice

Essentiality of KEs

Direct Evidence

Direct Evidence

Direct Evidence

Direct Evidence

Direct Evidence

Direct Evidence

Direct Evidence

Direct Evidence

Direct Evidence

Empirical Support of KER

High for MIE1669 to KE155

High for KE 155 to KE 185

Moderate for KE 185 to KE1980

Moderate for KE1980 to KE1981

Moderate for KE1981 to KE 1172

Moderate for KE 1172 to KE 112

High for KE 112 to KE1457

High for KE 1457 to AO 1982

-

Biological plausibiity of KER

High for MIE1669 to KE155

High for KE 155 to KE 185

Moderate for KE 185 to KE1980

Moderate for KE1980 to KE1981

Moderate for KE1981 to KE 1172

Moderate for KE 1172 to KE 112

Moderate for KE 112 to KE1457

High for KE 1457 to AO 1982

-

Quantitative assessment

PCR-RFLP

OHdG – ELISA &RT- PCR, Western Blot

MAPK assay, Immunoprecipitation,

Western immunoblotting

  1.  

Quantification of ATase activity – BSA method

APNG assay,

OXOG glycosylase activity assay,

Western immunoblotting,

Immunohistochemical detection of ATase.

Acetaldehyde assay,

Extract preparation and Western blotting,

N2- Ethyl dGuo quantitation

Western blotting,clonal survival assay,FACs

qRT-PCR,Western blotting,Luciferase reporter assay

Micro-array

qRT-PCR,immunohistochemistry

qRT-PCR, immunohistochemistry ()

IHC,micro array,qPCR, SNP array

qRT-PCR,,Luciferase reporter assay ,immunoblotting,immunoprecipitation,cell invasion assay,cell migration assay,

References

Chen CH et al 2011,

Panida Navasumrit et al, 2001),Kotova N et al, 2013,Garaycoechea JI et al, 2012

Abraham J et al 2011,Garaycoechea JI et al, 2018,Voordeckers K et al, 2020

van Jaarsveld MT et al 2014,

Abdelfattah, N et al, 2018,Liu Z et al, 2017,Zhang X et al,2011 Wan G et al, 2013,Bulkowska M et al, 2017

Shen ZL et al 2016,Guo S et al 2020,Bae HJ et al 2014,Zhou J et al 2017,Fu H et al 2018,,Lian B et al 2018,Guan Y et al 2017,Yang X et al 2014,Jiang G et al 2016,Luo J et al 2017,Tian Z et al 2016,Yan X et al 2019,Zhang S et al 2016

McGlynn LM et al 2014,Paul T. Pfluger et al 2008,Yeung F et al 2004

Sampepajung E et al 2021, Van Laere SJ et al 2007,

Singh S  et al 2007,Holloway JN  et al 2004,Biswas DK  et al 2000, Song RX et al 2005,Scherbakov AM et al 2009,Allred DC and Mohsin SK 2000

Biswas DK et al 2001

Wik E et al 2013,,Bouris P et al 2015, Liu Y et al 2015,Al Saleh S et al 2011,Zeng Q et al 2014,Ye Y et al 2010,

Lin, HY et al 2018

Liang et al., 2013;Liu et al., 2016;Zhang et al.,2015; Chen et al., 2015;Yue et al.,2019;Wang et al.,  2018;Yu et al.,2017

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

Sex:The AOP is appicable to women.However study suggests that the relative risk of breast cancer in men is comparable to that in women for alcohol intakes below 60 g per day. It continues to increase at high consumption levels not usually studied in women (Guénel P et al 2004). 

Life stage:There are no research articles which highlight the role alcohol in a particular life stage.In addition, age-related differences in response to alcohol exposure are neither uniform nor linear. The data available is insufficient which direct the construction of a catalog of “appropriate” tests or to define all the factors which influence nonlinear effects (Squeglia LM  et al 2014).

Taxonomic:The evidences for the key events of this AOP are available in various species ike rat,mice and humans.

Essentiality of the Key Events

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

Direct evidence is available for all the suggested key events. However the strength of weight of evidence varies from moderate to high. however, some inconsistencies are also available. majority of the experimental evidence is available in rats, mice, canine and human cell lines. only a few studies are available on human subjects.

  • Human normal hepatocytes (HL-7702) were subjected to escalating doses of N,N-dimethylformamide for 24 hours (C. Wang et al., 2016). At all concentrations, a concentration-dependent increase in ROS was detected; the rise was statistically significant when compared to control (6.4, 16, 40, 100 mM). Until the highest two concentrations (40 and 100 mM), no significant rise in 8-oxodG was seen, indicating inadequate repair at these dosages. Excision repair genes (XRCC2 and XRCC3) were considerably up-regulated at 6.4 and 16 mM, well below the doses that significantly produced 8-oxodG, indicating that adequate DNA repair was possible at these low concentrations. These findings show that repair is competent at low concentrations (removing 8-oxodG quickly), but that repair is swamped (i.e., insufficient) at larger doses, where 8-oxodG greatly increases. AS52 Chinese hamster ovary cells (wild type and OGG1-overexpressing) were exposed to varying doses of ultraviolet A (UVA) radiation (Dahle et al., 2008). Formamidopyrimidine glycosylase (Fpg)-sensitive sites were quantified using alkaline elution after increasing repair times (0, 1, 2, 3, 4 h) following 100 kJ/m2 UVA irradiation. OGG1-overexpressing AS52 cells (OGG1+): Fpg-sensitive sites reduced to 71% within half an hour and down to background levels at 4h.Wild type AS52 cells: at 4h, 70% of the Fpg-sensitive sites remained, indicating accumulation of oxidative lesions. Mutations in the Gpt gene was quantified in both wild type and OGG1+ cells by sequencing after 13-15 days following 400 kJ/mUVA irradiation.G:C→T:A mutations in UVA-irradiated OGG1+ cells were completely eliminated (thus, repair was sufficient when repair overexpressed).G:C→T:A mutation frequency in wild type cells increased from 1.8 mutants/million cells to 3.8 mutants/million cells following irradiation – indicating incorrect repair or lack of repair of accumulated 8-oxo-dG.
  • There is evidence from knock-out/knock-down studies indicating there is a strong link between DNA repair adequacy and the frequency of mutations. Defects in proteins involved in DNA repair resulted in altered mutation frequencies in all of the instances studied when compared to wild-type cases. In cell lines deficient in LIG4 (Smith et al., 2003) and Ku80 (Feldmann et al., 2000), there were significant decline in the frequency and accuracy of DNA repair; rescue experiments performed with these two cell lines further confirmed that inadequate DNA repair was the cause of the observed decreases in repair frequency and accuracy (Feldmann et al., 2000; Smith et al., 2003). There was more spontaneous DNA damage in Nibrin-deficient mouse cells than in wild-type controls, implying insufficient DNA repair. In vivo mutation frequencies were also observed to be higher in Nibrin-deficient mice than in wild-type mice using the corresponding Nibrin-deficient and wild-type mice (Wessendorf et al., 2014). Furthermore, depending on the XPC status of cancer patients, mutation densities in certain genomic areas were influenced differentially. In XPC-wild-type patients, mutation frequencies were higher at DHS promoters and 100 bp upstream of TSS than in cancer patients missing functional XPC (Perera et al., 2016). Finally,it was found that radiation exposure caused four times more mutations in WKT1 cells with lower repair capacity than in TK6 cells with normal repair capacity in a research (Amundson and Chen, 1996). 
  • There are findings that strongly link the different elements of DNA damage and repair events to the expression of miRNA. Zhang and coworkers examined genome-wide mature miRNA expression in Atm+/+ and Atm-/- littermate mouse embryonic fibroblasts to see how miRNAs are regulated in the DNA damage response (MEFs)(Zhang et al.,2011). MEFs were given neocarzinostatin (NCS), a radiomimetic medication that causes DSBs (Ziv et al., 2006). Mouse miRNA microarray analysis was used to determine miRNA expression profile in each sample, which was done at several time points (0–24 hr). As many as 71 distinct miRNAs were found to be considerably (2-fold) upregulated in the NCS-treated Atm+/+ MEFs, but not in the corresponding Atm-/- MEFs, implying that DNA damage stress causes broad-spectrum changes in miRNA expression. According to Wan et al., regulatory RNA-binding proteins in the Drosha and Dicer complexes, such as DDX5 and KSRP, drive posttranscriptional processing of primary and precursor miRNAs after DNA damage. The findings show that nuclear export of pre-miRNAs is increased in an ATM-dependent manner after DNA damage. The ATM-activated AKT kinase phosphorylates Nup153, a main component of the nucleopore, resulting in enhanced interaction between Nup153 and Exportin-5 (XPO5) and increased nuclear export of pre-miRNAs. These findings demonstrate that DNA damage signalling is important for miRNA transport and maturation. In agreement with previous reports showing that ATM-activated p53 and KSRP promote miRNA expression (Suzuki et al., 2009; Zhang et al., 2011), the study found 61 p53-dependent miRNAs and 29 KSRP-dependent miRNAs among the ATM-induced miRNAs.
  • In Jiang et al's study, the cellular function and molecular mechanism of miR2045p in hepatocellular cancer were investigated (HCC)(Jiang et al.,2016). Shen et al showed that downregulation of miR-199b is associated with distant metastasis in colorectal cancer via activation of SIRT1 and inhibition of CREB/KISS1 signalling(Shen et al., 2016). A study by Tian et al found that MicroRNA-133b targets Sirt1 and suppresses hepatocellular carcinoma cell progression(Tian et al., 2016). In liver cancer, Yan et al discovered that MicroRNA 486 5p acts as a tumour suppressor of proliferation and cancer stem-like cell characteristics by targeting Sirt1(Yan et al.,2019). Zhang et al reported that MicroRNA-22 functions as a tumor suppressor by targeting SIRT1 in renal cell carcinoma (Zhang et al., 2016). 
  • According to Lu et al, SIRT1 inhibited the growth of gastric cancer through inhibiting the activation of STAT3 and NF-B (Lu et al.,2014). The goal was to look at SIRT1's regulatory effects on gastric cancer (GC) cells (AGS and MKN-45) as well as the links between SIRT1 and STAT3 and NF-B activation in GC cells. The SIRT1 activator (resveratrol RSV) was discovered to contribute to the repression of viability and increase of senescence, which was reversed by SIRT1 inhibitor (nicotinamide NA) and SIRT1 depletion using the CCK-8 and SA-β-gal assays, respectively. SIRT1 activation (RSV supplement) reduced not only STAT3 activation, including STAT3 mRNA level, c-myc mRNA level, phosphorylated STAT3 (pSTAT3) proteins, and acetylizad STAT3 (acSTAT3) proteins, but also pNF-B p65 and acNF-B p65 suppression. The effects of RSV were reversed by NA. Furthermore, when STAT3 or NF-B were knocked down, neither RSV nor NA could affect cellular survival or senescence in MKN-45 cells. Overall, the outcomes of the study revealed that SIRT1 activation could cause GC in vitro to lose viability and senescence. Furthermore, our findings demonstrated that SIRT1 inhibited proliferation in GC cells and was related to deacetylation-mediated suppression of STAT3 and NF-B protein activation. The levels of SIRT1 protein expression in non-small-cell lung cancer (NSCLC) cell lines were examined in a study by Yeung et al.,2004. In comparison to immortalised epithelial human lung NL-20 cells, NSCLC cells exhibit significant quantities of SIRT1 protein, as reported by other researchers (Luo et al, 2001; Vaziri et al, 2001). Pharmacological modulators of Sirtuin activity were employed to see if NF-kB transcription was regulated by Sirtuins (Landry et al, 2000; Bedalov et al, 2001; Howitz et al, 2003). Transient luciferase reporter experiments revealed that cells pretreated with resveratrol had very minimal NF-kB transcription following the presence of TNFa. TNFa-induced NF-kB activity was boosted when cells were pretreated with the Sirtuin inhibitors nicotinamide or splitomicin. NF-kB transcription was also potentiated in cells treated with trichostatin A (TSA), an HDAC class I and class II inhibitor, as expected.
  • In specific subclasses of human breast cancer cells and tumour tissue specimens, an enhanced level of activated NF-kB is found, primarily in erbB2-overexpressing ER-negative breast cancer (Biswas et al 2000;2003). Singh et al explored a variety of methods to inhibit NF-kB activation in ER-negative breast cancer cells and looked at the effects on cell proliferation, apoptosis, and tumour growth in xenografts(Singh et al.,2007). In a prospective cohort study, Sampepajung et al used immunohistochemistry (IHC) to examine NF-B expression and intrinsic subtypes of breast cancer tissue and found a significant correlation between negative ER and overexpression of NF-B (p 0.05), with overexpression of NF-B being higher in negative ER (77.3 percent) compared to positive ER (47.4 percent )( Sampepajung et al., 2021). Laere et al suggested that activation of NF-kB in inflammatory breast cancer (IBC) is associated with loss of estrogen receptor (ER) expression, indicating potential crosstalk between NF-kB and ER(Laere et al.,2007). Differential Sensitivity of ER α and ERβ Cells to the NF-kB Inhibitor Go6976 was tested. A differential sensitivity to Go6976 by ER α and ERβ breast cancer cells was observed (Holloway et al.,2004). The ER α cells were more sensitive and less viable after treatment with this NF-kB inhibitor. The IC50 (50% killing) by Go6976 was 1 mM for Era of MDA-MB435 and MDA-MB231 breast cancer cells, whereas it was greater than 10 mM for ERa of MCF-7 and T47D or the normal mammary epithelial H16N  cells. At 10 mM Go6976, about 80% of the ERa cells were killed, whereas only 15–30% of ERa and normal H16N cells were sensitive to this compound. The relative resistance of the H16N normal human mammary cells indicates a possible high therapeutic index of Go6976 against ERa cancer cells.
  • Endogenous ER silencing causes EMT in ER-positive breast cancer cells. ER-positive MCF-7 cells were infected with ER shRNA lentiviral particles and stable clones were selected with puromycin (optimal dose of 0.8 g/mL) to knock down ER gene expression (Zheng et al.,2014). When the number of cell passages was increased following infection, the expression of ER was gradually knocked down. Saleh et al. hypothesise that loss of oestrogen receptor function, which causes endocrine resistance in breast cancer, also causes trans-differentiation from an epithelial to a mesenchymal phenotype, which causes enhanced aggressiveness and metastatic tendency(Saleh et al., 2011). 
  •  EMT is the most crucial step in initiating metastasis, including metastasis to lymph nodes, because tumour cell movement is a pre-requisite for the metastatic process (Da et al., 2017). Multiple signalling pathways cause cancer cells to lose their cell-to-cell connections and cellular polarity during EMT, increasing their motility and invasive ness (Huang et al., 2017). MMPs cause E-cadherin to be cleaved, which increases tumour cell motility and invasion (Pradella et al., 2017). Chen et al investigated the potential function of MDM2 in ovarian cancer SKOV3 cells' EMT and metastasis(Chen et al.,2015). TGFbeta and Twist induce EMT by upregulating the expression of EMT markers such Snail, Vimentin, N-cadherin, and ABC transporters like ABCA3, ABCC1, ABCC3, and ABCC10 (Saxena et al., 2011).In the treatment with about 0.3, 3, 30 mM of doxorubicin, human mammary epithelial cells (HMLE) stably expressing Twist, FOXC2 or Snail demonstrate increased cell viability compared to control HMLE, dose-dependently (Saxena et al., 2011). 

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

Increased, DNA damage and mutation leads to Inadequate DNA repair

DNA base excision repair (BER) and, to a lesser extent, nucleotide excision repair (NER)  are used to repair oxidative DNA damage. Previous research has found thresholded dose-response curves in oxidative DNA damage and attributed these findings to a lack of repair capability at the curve's inflection point (Gagne et al., 2012; Seager et al., 2012). Following chemical exposures, in vivo, a rise and buildup of oxidative DNA lesions was seen despite the activation of BER, suggesting poor repair of oxidative DNA lesions beyond a certain level(Ma et al., 2008).

Empirical Evidence (include consideration of temporal concordance ) has been documented in several studies as follows;

Compound class

Species

Study type

Dose

KER findings

Reference

N,N-dimethylformamide

Homosapiens hepatocyte cell line

In vitro

Experimental

mM

Increased DNA damage leads to decreased DNA cross link repair mechanisms

Wang et al.,2016

UV radiation

Cricetulus griseus(Chinese hamster)

-do-

kJ/m2

-do-

Dahle et al.,2008

X-rays

Human leukemia cell line

-do-

Gy/min

-do-

Li et al.,2013

X rays

Mice

In vivo

Experimental

Gy/min

-do-

Li et al.,2013

Aniline

Rat

-do-

Kg/day

-do-

Ma et al.,2008

Inadequate DNA repair leads to Increase, Mutations

There will be no increase in mutation frequency if DNA repair is capable of appropriately and efficiently repairing DNA lesions caused by a genotoxic stressor.

For alkylated DNA, for example, efficient AGT removal will result in no increases in mutation frequency. However, once AGT reaches a certain dose, it becomes saturated and can no longer effectively remove alkyl adducts. Mutation occurs when O-alkyl adducts are replicated. The evidence that unrepaired O-alkylated DNA replication induces mutations in somatic cells is vast and has been evaluated. (Basu and Essigmann 1990; Shrivastav et al. 2010).

Empirical Evidence (include consideration of temporal concordance ) has been documented in several studies as follows;

Compound class

Species

Study type

Dose

KER findings

Reference

UV radiation

Chinese hamster

In vitro

kJ/m2

inadequate DNA repair leads to increased mutations

Dahle et al.,2008

Mice

In vivo

-do-

Klungland et al., 1999

X ray

human

In vitro

Gy

-do-(dose-incidence)

Mcmohan et al., 2016

Increase, Mutations leads to Increase,miRNA levels

Evidences suggest that transcription pathway for miRNAs is regulated in the DNA damage response (DDR).Inadequate repair and mutations increase miRNA expression.DNA damage-responsive transcription factors, such as NF-kB, E2F, and Myc, are also involved in miRNA  transcription regulation.The p53 protein also functions as a transcriptional repressor by binding to miRNA promoters and preventing the recruitment of transcriptional activators.The empirical and dose response evidence for increased mutations inducing miRNA expression has been documented as follows;

Compound class

Species

Study type

Dose

KER findings

Reference

Neocarzinostatin

Mouse

Fibroblast

In vitro

Ng/ml

Increased mutation leads to increased microRNA expression

  

Ziv et al.,2006

Neocarzinostatin

Mouse

Fibroblast

In vitro

Ng/ml

-do-

Zhang et al.,2011

Cisplatin and IR

Human mammary epithelial cells

In vitro

mM and Gy

-do-

Jaarsveld et al., 2014

Increase,miRNA levels leads to Decrease,SIRT1(sirtuin 1) leves

There are several pathways which suggest suppression of SIRT1  expression when miRNA is elevated.SIRT1 was downregulated at the mRNA and protein levels when miR-138 expression was increased. MiR-138 binds to the SIRT1 gene's 3′UTR unique complimentary site and inhibits SIRT1 expression directly, preventing HCC proliferation, migration, and invasion (Luo et al.,2017).When compared to the normal hepatic cell line L02, SIRT1 is overexpressed, while miR-138 levels are lowered in HepG2, SMMC7721, Bel7404, and HCCM3 .

The evidence for this fact has been listed as follows;

Compound class

Species

Study type

KER findings

Reference

Human HCC Cell lines

In vitro

Increased miRNA leads to Reduced SIRT1

Jiang et al.,2016; Luo et al.,2017; Tian et al., 2016; Yan et al.,2019; Bae et al.,2014;Zhou et al.,2017

Human CRC cell lines

In vitro

-do-

Shen et al., 2016;Lian et al.,2018

Human RCC Cell lines

In vitro

-do-

Zhang et al., 2016;Fu et al.,2018

Astragalus Polysachcharide

Prostate cancer cell lines

In vitro

-do-

Guo et al.,2020;Yang et al.,2014

Lung cancer cell lines

In vitro

-do-

Guan et al.,2017

Decrease,SIRT1(sirtuin 1) leves leads to Increase activation, Nuclear factor kappa B (NF-kB)

SIRT1 suppresses NF-B signalling either directly by deacetylating the RelA/p65 subunit or indirectly by triggering repressive transcriptional complexes, which frequently involve heterochromatin formation at NF-B promoter regions. SIRT1 expression and signalling are both inhibited by NF-B.

Zhang et al.  found that overexpressing RelA/p65 protein increased SIRT1 expression at both the transcriptional and protein levels (36 h treatment), whereas knocking down RelA/p65 expression decreased TNF-induced SIRT1 expression (8 h treatment)(Zhang et al.,2010). They also discovered that the RelA/p65 protein may bind to the SIRT1 promoter's NF-B motifs. These findings suggest that NF-B may promote SIRT1 expression. Given that SIRT1 induction appeared to occur much later than NF-B activation, it appears that this action could represent a feedback response limiting inflammation and eventually generating endotoxin tolerance.

Evidences supporting this key event is as follows;

Compound class

Species

Study type

KER findings

Reference

nicotinamide

Human gastric cancer cell lines

In vitro

Decreased, SIRT1 leads to increased NF kB activity

Lu et al.,2014

nicotinamide or splitomicin

non-small-cell lung cancer (NSCLC) cell lines

In vitro

Decreased, SIRT1 leads to increased NF kB activity

Yeung et al.,2004; Luo et al, 2001; Vaziri et al, 2001

Increase activation, Nuclear factor kappa B (NF-kB) leads to Antagonism, Estrogen receptor

NF-kB activation in breast cancer has been extensively documented in oestrogen receptor negative (ER) breast tumours and ER breast cancer cell lines, implying a significant inhibitory interaction between both signalling pathways (Biswas et al, 2000, 2001, 2004; Zhou et al, 2005). A rise in both NF-kB DNA-binding activity (Nakshatri et al, 1997) and expression of NF-kB target genes such IL8 coincides with a transition from oestrogen dependence to oestrogen independence in breast cancer, indicating inhibitory cross-talk. The fact that some breast tumours that are resistant to the tumoricidal action of anti-estrogens become sensitised to apoptosis and show a drop in NF-kB activity after treatment with oestrogen supports the inverse relationship between ER and NF-kB activity.

-This shows that oestrogen's proapoptotic actions in these tumours are mediated via NF-kB suppression.

Both in vivo and in vitro studies support the finding;

Compound class

Species

Study type

Dose

KER findings

Reference

Bortezomib

Breast cancer cell lines

In vitro

Increased activity of NF kB,  leads to Reduced Estrogen receptor expression

Singh et al.,2017; Holloway et l.,2004

Human Breast tissue

In vivo

-do-

Biswas et al 2000;2003

Human Breast tissue

In vivo

-do-

Sampepajung et al., 2021; Laere et al.,2007; Indra et al.,2021;

Antagonism, Estrogen receptor leads to EMT

E2/ERa signalling, in part through transcriptional activation of luminal/epithelial-related transcription factors, promotes the development of mammary epithelia along a luminal/epithelial lineage. GATA3 and ERa both promote each other (Eeckhoute et al.,2007). In normal breast epithelia, GATA3 is needed for luminal differentiation(Kouros-Mehr et al.,2008) and GATA3 and ERa control many of the same genes (Wilson et al.,2008).  In mice, forcing GATA3 expression in mesenchymal breast cancer cells produces mesenchymal–epithelial transition (MET), a reversible mechanism analogous to EMT, and prevents tumour metastasis (Yan et al.,2010). Another ERa-interacting transcription factor, FOXA1, is essential for luminal lineage in mammary epithelia and stimulates ductal development in mice (Bernardo et al.,2010). FOXA1 enhances ERa gene expression by increasing the accessibility of estrogen-response regions for ERa binding (Nakshatri et al., 2009). In breast cancer cells, on the other hand, E2 appears to increase FOXA1 expression. Importantly, ERa, FOXA1, and GATA3 are all positive breast cancer prognostic factors(Nakshatri et al.,2009).

Ye et al.  investigated the impact of ERa overexpression in ERa-negative breast cancer cell lines (MDA-MB-468, MDA-MB-231) or ERa knockdown in ERa-positive cell lines (MCF-7, T47D) on Slug and Snail expression and phenotypes in ERa-positive cell lines (MCF-7, T47D)(Ye et al., 2010). Slug is repressed, E-cadherin is increased, and cells develop as adherent colonies with less invasiveness when ERa is forced to get expressed. ERa knockdown, on the other hand, causes an increase in Slug expression, a decrease in E-cadherin, and spindle-shaped invasive cells.

Wik et al used integrated molecular profiling to examine Endometrial cancer samples from a primary investigation cohort and three independent validation cohorts (Wik et al.,2013). Patient survival was closely linked to ER-a immunohistochemical staining and receptor gene (ESR1) mRNA expression. In the study cohort, ER-a negative was related with activation of genes implicated in Wnt, Sonic Hedgehog, and TGF-b signalling, indicating epithelial–mesenchymal transition (EMT)

EMT leads to Metastasis, Breast Cancer

The “epithelial–mesenchymal transition” (EMT), a key developmental regulatory program, has been reported to play critical and intricate roles in promoting tumor invasion and metastasis in epithelium-derived carcinomas.

EMT is marked by a decrease in E-cadherin and β- -catenin expression and an increase in vimentin, fibronectin, and N-cadherin expression (Irani et al., 2018). EMT is a master mechanism in cancer cells that allows them to lose their epithelial characteristics and gain mesenchymal-like qualities. EMT is the most crucial step in initiating metastasis, including metastasis to lymph nodes, because tumour cell movement is a pre-requisite for the metastatic process (Da et al., 2017). Multiple signalling pathways cause cancer cells to lose their cell-to-cell connections and cellular polarity during EMT, increasing their motility and invasive ness (Huang et al., 2017). MMPs cause E-cadherin to be cleaved, which increases tumour cell motility and invasion (Pradella et al., 2017).

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help
Modulating Factor (MF) Influence or Outcome KER(s) involved
     

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

The techniques used for quantifying KE's were reliable with repeatability and reproducibility. Assays were fit for the purpose.

MIE 1669

KE 155

KE 185

KE1980

KE1981

KE 1172

KE 112

KE1457

AO1982

Human

PCR-RFLP

8-OHdG – ELISA & MDA

(Chen CH et al 2011)

-

Acetaldehyde assay,

Extract preparation and Western blotting,

N2- Ethyl dGuo quantitation

Abraham J et al 2011

-

-

qRT-PCR,immunohistochemistry (McGlynn LM et al 2014)

qRT-PCR, immunohistochemistry (Sampepajung E et al 2021, Van Laere SJ et al 2007,)

IHC,micro array,qPCR, SNP array(Wik E et al 2013)

Liang et al., 2013;Liu et al., 2016;Zhang et al.,2015; Chen et al., 2015;Yue et al.,2019;Wang et al.,  2018;Yu et al.,2017

Human Tissues

-

-

-

-

qRT-PCR,Western blotting,Luciferase reporter assay H2,H4,H7,H8,H9

Micro-array (Shen ZL et al 2016)

-

-

-

-

Human Cell lines

RT- PCR, Western Blot

MAPK assay, Immunoprecipitation,

Western immunoblotting

Thymidine uptake

ECL-SDS PAGE,

RIA

Adduct removal measurements,

DNA isolation,

TLC,

LCMS

Acetaldehyde estimation,

DNA adducts – LC-ESI-MS/ MS-SRM, Western blotting

Western blotting, enzymatic assay,

LC-ESI-MS/ MS-SRM

DNA oxidative damage by ELISA, Immunofluorescence, cell culture,

8-OHdG – ELISA & Ph2Aλ–fociformation assays,

P53 luciferase assays,

qPCR,

Western Blotting

(Elise A. Triano et al 2003,

Etique.Nicolas etiqu et al 2004,

Izevbigie EB et al 2002,

Przylipiak A et al 1996,

Singletary KW et al 2001,

Singletary KW et al 2004,

Abraham J et al 2011,

Zhao M et al 2017,

Jessy Abraham J et al 2011)

-

-

Western blotting,clonal survival assay,FACs(van Jaarsveld MT et al 2014)

Micro-array, qRT-PCR,Western blotting,Luciferase reporter assay

(Guo S et al 2020,

Bae HJ et al 2014,

Zhou J et al 2017,

Fu H et al 2018,

Lian B et al 2018

Guan Y et al 2017

Yang X et al 2014)

qRT-PCR,,Luciferase reporter assay

Cell based HDAC assay(Yeung F et al 2004)

qPCR, western blotting, immunoprecipitation, immunofluorescent microscopy, Luciferase reporter assay

EMSA,

IHC,Cell viability assay (Singh S et al 2007, Holloway JN  et al 2004,

Biswas DK et al 2000,

Song RX et al 2005,

Scherbakov AM et al 2005,

Scherbakov AM et al 2009)

qRT-PCR,cell viability assay,

Western blotting,EdU incorporation assay(Bouris P et al 2015,

Liu Y et al 2015,

Al Saleh S et al 2011,

Zeng Q et al 2014,

Ye Y et al 2010,

Lin, HY et al 2018)

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,

Gujral et al.,2014;Cui et al.,2013;Shiota et al.,2012;Gao et al.,2018;Chen et al.,2017;Liu et al.,2020;Casas et al.,2011;Jackstadt et al.,2013;Kong et al.,2016;Zhang et al.,2014;Huang et al.,2014

Rat

Free radical assay

GC-MS-SIM

 (Hackney JF et al 1992,

McDermott EW et al 1992)

Quantification of ATase activity – BSA method

APNG assay,

OXOG glycosylase activity assay,

Western immunoblotting,

Immunohistochemical detection of ATase. (Kotova N et al, 2013)

-

Free radicCyQuant cell Proliferation assay (Abdelfattah, N. et al 2018)

-

-

-

-

-

Rat Cell lines

-

Flow cytometric micronucleus assay,

Cell cycle analysis,

Replication fork elongation assay,

Cytotoxicity assay,

Recombination assay, (Panida Navasumrit et al, 2001)

-

-

-

-

-

-

-

Mice

Comet assay,

ROS generation assay.

(Lei  Guo et al 2008)

FISH karyotyping,

Invivo point mutation assay,

Whole genome sequencing of HSC clones. (Garaycoechea JI et al, 2012)

In vivo point mutation assay

Garaycoechea JI et al, 2018

Free radicCyQuant cell Proliferation assay (Abdelfattah, N. et al 2018)

RNA sequence analysis,Immuno staining,immunoblotting,Flowcytometry,COMET assay,qRT PCR(Liu Z et al 2017)

Microarray (Zhang X et al 2011)

qRT PCR,RIP assay,Immunogold EM(Wan G et al 2013)

qRT-PCR,Western blotting,Luciferase reporter assay,ELISA,cell culture

Bai XZ et al 2018

qRT-PCR,Southern and northern blotting, reporter gene  assay(Paul T et al 2008)

EMSA,Autoradiography,Immunofluorescent microscopy, Westernblotting (Biswas DK et al 2001)

-

Chen et al.,2017; Gumireddy et al.,2009; Yu et al., 2016; Sarkar et al.,2015

Canine

-

-

-

micro array(Bulkowska M et al 2017)

-

-

-

-

-

Yeast

-

-

Fluctuation assay

Voordeckers K et al, 2020

-

-

-

-

-

-

Considerations for Potential Applications of the AOP (optional)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

Intended uses of this AOP:

  • Helpful for risk assessors, in assessing the risk of alcohol on metastatic breast cancer
  • If the causal relationship is established between key events, it may be useful drug targets
  • An alternative model to animal model based test methods

References

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

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