AOP ID and Title:

Short Title: Hypothalamic estrogen receptors inhibition leading to ovarian cancer

Authors

Kalyan Gayen, Department of Chemical Engineering, National Institute of Technology Agartala, India 

Tridib Kumar Bhowmick, Department of Bioengineering, National Institute of Technology Agartala, India 

Status

Abstract

Malfunctioning of sex hormones (e.g., estradiol, estrone and progesterone) may result in ovarian cancer (Fooladi et al. 2020, Meehan and Sadar 2003). Exposure to endocrine-disrupting chemicals (EDCs) in the form of occupational usage of pesticides, fungicides, herbicides, plasticizers, cosmetics, etc. are the causes of ovarian cancer (Samtani, Sharma and Garg 2018). Some stressors molecules (e.g., clomiphene citrate, Tamoxifen, Toremifene) act on neuronal cell in the hypothalamus (molecular initiating event, MIE), where they inhibit hypothalamic Estrogen Receptors selectively and these chemicals increase the risk of ovarian cancer (McLemore et al. 2009). These stressors molecules stimulate the releasing of gonadotropin-releasing hormone (GnRH) from hypothalamic region of brain by the suppression of hypothalamic Estrogen Receptors. Subsequently, secretion of luteinizing hormone (LH) from pituitary becomes high(Cassidenti et al. 1992, Mungenast and Thalhammer 2014a, Tomao et al. 2014). This hormone regulates the synthesis of sex hormones (e.g., estrogens) at cellular level (Shoemaker et al. 2010a, Tomao et al. 2014). These sex hormones are primarily produced in the gonads through a series of enzyme-mediated reactions from cholesterol (precursor) and control through complex signalling pathway along hypothalamus – pituitary –  gonadal (HPG) axis (Shoemaker et al. 2010a, Perkins et al. 2019). High estrogen level increases the risk of ovarian cancer via ovarian epithelial cell hyperplasia (McLemore et al. 2009, Tomao et al. 2014).

Background

Development and progression of certain types of cancer disease (e.g. ovarian cancer, breast cancer, prostate cancer etc.) is related with the hormonal levels in human. Lack of proper diagnosis at early stage of the disease increase the mortality rate of the cancer. Among many types of cancer ovarian cancer has the high mortality rate (~50%) due to the lack of proper diagnosis at early stage of the disease progression. Circulating levels of the steroidal sex hormones in conjunction with the gene expression is related with the progression of this disease. Some important sex hormones which are related with many cancer diseases include oestrogen, progesterone and testosterone. Oestrogen hormone mainly involved in female sex organ development, controlling of menstruation cycle etc. Progesterone also involved in controlling menstrual cycle, maintaining pregnancy and spermatogenesis. Testosterone hormone regulates sexual development, bone mass development, red blood cell production in male.

Summary of the AOP

Events

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

Key Event Relationships

Stressors

Overall Assessment of the AOP

Suppression, Estrogen receptor (ER) activity [Evidence- Strong]: There are number of reports available related to  suppression of Estrogen receptor activity (ER) (Baez-Jurado et al., 2018; Cosman, 2003; Haskell, 2003; Ng et al., 2009; Kang et al., 2001; Roy et al., 1999; Marques P, 2018; Mungenast and Thalhammer, 2014b; Ghasemnejad-Berenji et al., 2020; J. H. Liu, 2020; Oride et al., 2020; Zhang et al., 2020; John F. Kerin et al., 1985b; The Practice Committee of the American Society for Reproductive Medicine, 2013; Moskovic et al., 2012 ; Bryan J. Herzog, 2020). Stressors act on neuronal cell in the hypothalamus, where it inhibits hypothalamic Estrogen Receptors selectively. A number of compounds or molecules (e.g. Clomiphene citrate, Tamoxifen, Toremifene etc.) are detected which show the modulation activity of estrogen receptor in brain leading to high GnRH pulses (Haskell, 2003; Cosman, 2003).

Increased, secretion of GnRH from hypothalamus[Evidence- Strong]: A number of evidencesare found by the researchesthat the increased secretion of gonadotropin-releasing hormone (GnRH)(Shander and Goldman, 1978; Tsourdi et al., 2009). Studies had shown that of inhibition of Estrogen receptor activity (ER) enhances the secretion of GnRH in human (Adashi et al., 1980; Bussenot et al., 1990; JOHN F KERIN et al., 1985a; Tan et al., 1996), rat and mice (Bharti et al., 2013; Kumar and Pakrasi, 1995; Zoeller and Young, 1988). Studies on human patient had shown the application of clomiphene is able to promote response of GnRH secretion (Goerzen et al., 1985; Tan et al., 1996).

Increased, secretion of LH from anterior pituitary [Evidence- Strong]: Good evidence may be acquired from different published articles for the increased secretion of LH increases from anterior pituitary (Plouffe and Siddhanti, 2001; Wright et al., 2012; Shoemaker et al., 2010b). It is also reported that increased secretion of the GnRH in hypothalamus leads to high levelofLH in human (John F Kerin et al., 1985a; Adashi et al., 1980; Bussenot et al., 1990), mice/rat.(Bharti et al., 2013; Kumar and Pakrasi, 1995; Botte et al., 1999) and cow (Fields et al., 2009).

Increased, Steroidogenic acute regulatory protein (StAR) [Evidence- Strong]: Steroidogenic acute regulatory protein (StAR) plays critical role in luteal steroidogenesis by controlling the transport of cholesterol from the outer to inner mitochondrial membrane(Wu et al., 2003; Shoemaker et al., 2010b).It had been reported that increase in LH level leads to increase StAR protein concentration in human(Tsang et al., 1980; Johnson and Bridgham, 2001; Murayama et al., 2012; Rekawiecki et al., 2005), rat(T. Liu et al., 2007; Martinat et al., 2005) and mice(Eacker et al., 2008; Tsuchiya et al., 2003).

Increased, estrogens [Evidence- Strong]: Aromatase is a key enzyme for estrogen formation in human tissues. In female, one of the important sites of estrogen enzyme synthesis is ovarian granulose cells(Holesh et al., 2017; Shoemaker et al., 2010b). Although ovarian aromatase enzyme expression in postmenopausal female is very low, high estrogen level is maintained in the blood through aromatase expression in other tissues. A number of researches had shown increased synthesis of StAR Protein increases the estrogen in ovarian granulosa cellsin human (Kiriakidou et al., 1996; Fang et al., 2016; Men et al., 2017), rat (Ronen-Fuhrmann et al., 1998; Nimrod, 1981) and fish (Kusakabe et al., 2002).

Increased, circulating estrogen levels [Evidence- Strong]: Researches had shown increased synthesis of estrogen in ovarian granulosa cells leads to maintain the high circulating estrogen levels in blood (Holesh et al., 2017; Shoemaker et al., 2010b).

Hyperplasia, ovarian epithelium [Evidence- High]: Ovarian surface is covered by the epithelium cells often called as ovarian mesothelium tissue. High evidence is available which supports that hyperplasia of the stromal cells might lead towards the hyperplasia of the ovarian epithelium tissue(Nyboe Andersen et al., 2008; Kang et al., 2001).

Promotion, ovarian adenomas[Evidence- Moderate]: Ovarian adenoma or cystadenoma is classified as benign tumor in the epithelial tissue. Evidence on the promotion of ovarian adenoma due to the hyperplasia in the ovarian epithelial tissue is available.

Promotion, ovarian cancer [Evidence- Strong]: Promotion of ovarian adenomas  leads to the phenotype outcome of ovarian cancer at individual level (Johansson et al., 2022, Christine Stewart et al., 2019).

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Sex: This particular AOP is mainly applicable for the females. Sex hormone regulation in female is more complex compare to the male. Development and growth of the ovaries depend on the hormonal balance in the body. This hormonal balance in female changes often observed during the menstrual cycle and pregnancy. Imbalance in the hormonal levels leads to the abnormal function of the ovaries.Predominant form of estrogen (estradiol) hormone also found in male and plays critical role in sexual behavior and spermatogenesis. However, males more likely experiences imbalance in testosterone hormonelevels.

Life stage: This AOP is closer to the adult female. In particular the females (at the age of 45-55) going through the menopause are having greater chance of developing ovarian cancer compared to the young adult female. Young female undergoing through the hormonal therapy (usually estrogen) also having high risk of developing ovarian cancer. Risk factor of ovarian cancer is high in case of adult females who are taking ovulation stimulating drugs to increase fertility.

Taxonomic: For this AOP taxonomic domain is applicable to the different species like mice, rat, guinea pig and human.

Essentiality of the Key Events

In this AOP the essentiality of the proposed events are supported by a number of scientific works.

Kettel et al., had shown the treatment of seventeen females with clomiphene citrate with 150mg/day dose for 5 days enhance the estrogen levels. Analysis of the other hormones (follicle-stimulating hormone, luteinizing hormone, gonadotropin-releasing hormone) levels suggest the clomiphene citrate involved in the modulation in hormonal secretion at the hypothalamic site (Kettel et al., 1993). 

Koch et al, had shown female rat injected with the clomiphene citrate (1-100 ng/kg) for 20 days increase the gonadotropin-releasing hormone (GnRH) release in the hypothalamus region (Koch et al., 1971).

Research by Kurosawa et al., on 293T cells (transfectable derivative of human embryonic kidney 293 cells, revealed that effect of clomiphene citrate depend on the concentration of the molecule. Clomiphene citrate at higher concentration (10^-10 - 10^-12 M) showed the estrogenic activity. However at higher concentration (10^-6 - 10^-12 M) no estrogenic activity was observed. Results of the study also suggest that clomiphene citrate either act as agonist or as an antagonist depends on the presence of 17β-estradiol (E2) receptor(Kurosawa et al., 2010).

Weight of Evidence Summary

Overall assessment of the biological plausibility, empirical support and quantitative understanding of the KEs and KERs associated with this AOP shows that molecular mechanism or signaling pathway of tumor development in the female ovaries due to the suppression of estrogen receptors activities in the hypothalamus is still unclear.

Empirical evidence is available which shows the release of gonadotropin-releasing hormone (GnRH) depends on the concentration of the Selective Estrogen Receptors Modulator (SERM) compound (e.g. clomiphene citrate). However, molecular mechanism for the enhancement of GnRH by suppression of Estrogen receptor activity is poorly known.A number of researches had shown secretion of luteinizing hormone (LH) from anterior pituitary depends on the GnRH concentration or dose. Scientific reports have shown the both stimulatory and inhibitory effects on the GnRH secretion exhibited by the estradiol depending on the concentration of stressor (clomiphene) molecules and presence of types of receptors. The requirement of the GnRH dose for the secretary release of the LH in the different species varies widely.

A number of articles had shown that release of LH from the anterior pituitary regulates the steroidogenic function of cells by controlling the cholesterol transportation to the mitochondria. Biological plausibility of this event is very high as a number of studies have shown the similar results using different biological models (e.g.  granulosa cells of adult female, bovine luteal cells, leydig cells of mice and rat etc.) in their study. Estradiol synthesis during menstrual cycle is governed via expression of StAR protein synthesis. Quantitative estimation of the event has been performed through indirect measurement (e.g. Northern blot analysis of mRNA collected from ovarian follicle granulosa cells). Therefore in many studies finding results are inconsistent. Circulating estrogen levels increases due to the increased estradiol synthesis and concentration controlled by the negative feedback loop of the other steroidal hormone synthesis.Biological evidence of tumor formation in the ovarian granulose cells due to the high circulating estrogen levels in the plasma is pretty high. High circulating estrogen drives the endometrial hyperplasia towards the progression of endometrial cancer.

Quantitative Consideration

Quantitative understanding in many KEs and KERs are available. However, exploitation of different biological models and use of different assay techniques provide incoherent results. Inconsistent results also have been mentioned in many KEs and KERs. A few assay techniques such as radioimmunoassay, radioreceptor assay, estrogen receptor binding assay etc. are sensitive enough to measure the concentration of a molecule at pictogram level. Some other techniques such as quantitative real time PCR (qRT-PCR), northern blot analysis of RNA also have been used for quantitative estimation of molecules at low concentration. Some indirect methods such as immunohistochemistry also have been employed for identification and quantitative estimation of biological molecule.

Considerations for Potential Applications of the AOP (optional)

This AOP provides the valuable informations regarding chemical messengers and different glands of endocrine system that are related for the risk and promotion of ovarian cancer. Linkage of qualitative and quantitative informations of different chemical messengers for the promotion of ovarian cancer would be beneficial for the cancer therapy and cancer drug development. Further, this AOP would be helpful to evaluate the hazardous long-term effects of the endocrine-disrupting chemicals and drugs which may lead towards the development of the ovarian cancer. This AOP would also help to regulate the uses of these stressor molecules which have inhibitory effects on the hypothalamic Estrogen Receptors.   Understanding of the molecular events related with this AOP would help to screen these molecules and provide guideline to access the risk associated with these stressors.

References

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Appendix 1

List of MIEs in this AOP

Event: 1046: Suppression, Estrogen receptor (ER) activity

Short Name: Suppression, Estrogen receptor (ER) activity

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

Clomiphene citrate (a stressor) at 10^-10 - 10^-12 M concentrations exhibits approximately 30% of the estrogenic activity which is same from 17β-estradiol (at 10^-10 M) in ERα-expressing cells where as no activity in ERβ cells.

Clomiphene citrate at the concentration of  10^-10 M reveals weak estrogen agonist activity in the presence of 17 β -estradiol (E2) at the concentration of 10^-14 M in ERα-expressing cells, and no activity was found in ERβ cells.

Clomiphene citrate at lower doses (10^-10 - 10^-12 M), but not higher doses (10^-6 - 10^-8 M) showed estrogenic activity via ERα. However, clomiphene citrate at concentrations between 10^-6 M and 10^-12 M did not reveal any estrogenic activity via ERβ. In the presence of E2, clomiphene citrate worked as either as an agonist or an antagonist through ERα depending on the concentrations of E2.  Clomiphene citrate worked as antagonistic when it is combined with the higher E2 concentrations and worked as agonistic with the lower E2 concentrations. On the other hand, via ER β, clomiphene citrate acted as an estrogen antagonist irrespective of the concentration of E2. (Kurosawa et al., 2010).

 

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Neuronal cell in Hypothalamus

Key Event Description

Estrogen receptors are produced in all vertebrates and located in either the cell cytoplasm or nucleus(Bondesson et al., 2015; Eick and Thornton, 2011). Estrogen receptors are localized either in cytoplasm, or on the cell surface.

 

Site of action: Stressors (e.g., clomiphene) act on neuronal cell in the hypothalamus, where it inhibits hypothalamic Estrogen Receptors selectively.

Responses at the macromolecular level: Stressors activate the Estrogen Receptor α in the presence of lower level of estrogen and partially blocks the same for higher level of estrogen and works as antagonist for the Estrogen Receptor β(Trost and Khera, 2014). Stressors appear to act in the brain's pituitary gland to secrete an increased amount of gonadotropins hormone (GnRH) in hypothalamus leading towards increased GnRH level in blood.

Estrogen Receptor α: ERα (Estrogen Receptor α or NR3A1 or ESR1) - A nuclear receptor and it is activated by the estrogen (sex hormone). Estrogen located at chromosome number 6 ( 6q25.1)

Estrogen Receptor β:  ERβ (Estrogen Receptor β or NR3A2 or  ESR2) – This is also nuclear receptor and  activated by the sex hormone estrogen which is located at chromosome number 14 (14q23.2). I ERβ has  both N-terminal has DNA binding domain and C-terminal has ligand binding domain. This  is localized to the nucleus, cytoplasm, and mitochondria. Selective estrogen receptor modulators (SERM) inhibits the ERβ. Drugs used as SERM are clomiphene, tamoxifen, raloxifene etc.

Biological compartments:  Estrogen receptors (ER) are present in the plasma membrane. Both ERα and ERβ have diverse functions depending on cells and organs. ERs have also been loacated in cytoplasmic organelles including mitochondria and the endoplasmic reticulum(Levin, 2009).  

General role in biology: Estrogen receptors (both estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) binds the estrogens to promote the the biological functions of estrogens. Depending upon a balance between ERα and ERβ activities in target organs, estrogen signaling is selectively stimulated or inhibited (Welboren et al., 2009). ERβ has a high degree of sequence homology with the classical estrogen receptor. Interestingly, ERβ is detected in many tissues, including those previously assumed to be estrogen insensitive. In tissues where both ERs are expressed, such as the hypothalamus, uterus, mammary glands, and immune system, ERα promotes proliferation whereas ERβ has pro-apoptotic and pro-differentiating functions(Morani et al., 2008). ERα is present mainly in ovary (thecal cells) where as ERβ is found mainly in  ovary (granulosa cells)(Paterni et al., 2014). ERα and ERβ is identical approximately 97% in the DNA-binding domain and approximately 56% in the ligand-binding domain(Dahlman-Wright et al., 2006).

How it is Measured or Detected

Radioreceptor assay/The estrogen receptor binding assay (using Rat Uterine Cytosol): This assay identifies chemicals that have the potential to interact with the estrogen receptor (ER) in vitro.  Principle of this particular assay is based on the competitive protein-binding methods. A radiolabelled ligand and an unlabelled ligand are presented together to a specific receptor. The radioactivity measurement provides the quantitative estimation of the bound and unbound fraction of the ligand with the receptor. All cytosolic estrogen receptor subtypes that are expressed in the specific tissue, including ERα and ERβ are used for the determination of estrogen receptor binding. This assay is simple and rapid to perform when optimal conditions for binding are determined. Assay determines if a ligand/chemical can interact and displace the endogenous hormone 17β-estradiol (Freyberger et al., 2010).

References

Adashi, E. Y., Hsueh, A. J., & Yen, S. S. (1980). Alterations induced by clomiphene in the concentrations of oestrogen receptors in the uterus, pituitary gland and hypothalamus of female rats. J Endocrinol. , 87(3), 383-92.

Bharti, S., Misro, M., & Rai, U. (2013). Clomiphene citrate potentiates the adverse effects of estrogen on rat testis and down-regulates the expression of steroidogenic enzyme genes. Fertility and sterility, 99(1), 140-148. e5.

Bondesson, M., Hao, R., Lin, C.-Y., Williams, C., & Gustafsson, J.-Å. (2015). Estrogen receptor signaling during vertebrate development. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1849(2), 142-151.

Bussenot, I., Parinaud, J., Clamagirand, C., Vieitez, G., & Pontonnier, G. (1990). Effect of clomiphene cirate on oestrogen secretion by human granulosa cells in culture. Human Reproduction, 5(5), 533-536.

Dahlman-Wright, K., Cavailles, V., Fuqua, S. A., Jordan, V. C., Katzenellenbogen, J. A., Korach, K. S., et al. (2006). International union of pharmacology. LXIV. Estrogen receptors. Pharmacological reviews, 58(4), 773-781.

Dominguez, R., & Micevych, P. (2010). Estradiol rapidly regulates membrane estrogen receptor alpha levels in hypothalamic neurons. J Neurosci, 30(38), 12589-96. doi:30/38/12589 [pii]10.1523/JNEUROSCI.1038-10.2010.

Eick, G. N., & Thornton, J. W. (2011). Evolution of steroid receptors from an estrogen-sensitive ancestral receptor. Molecular and cellular endocrinology, 334(1-2), 31-38.

Freyberger, A., Wilson, V., Weimer, M., Tan, S., Tran, H. S., & Ahr, H. J. (2010). Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation. Reprod Toxicol, 30(1), 50-9. doi:S0890-6238(10)00003-1 [pii].

Kerin, J. F., Liu, J. H., Phillipou, G., & Yen, S. S. (1985). Evidence for a hypothalamic site of action of clomiphene citrate in women. J Clin Endocrinol Metab. , 61(2), 65-68.

Kettel, L. M., Roseff, S. J., Berga, S. L., Mortola, J. F., & Yen, S. S. (1993). Hypothalamic-pituitary-ovarian response to clomiphene citrate in women with polycystic ovary syndrome. Fertil Steril. , 59(3), 532-38.

Koch, Y., Dikstein, S., Superstine, E., & Sulman, F. G. (1971). THE EFFECT OF PROMETHAZINE AND CLOMIPHENE ON GONADOTROPHIN SECRETION IN THE RAT. Journal of Endocrinology, 49(1), 13-17. doi:10.1677/joe.0.0490013.

Kurosawa, T., Hiroi, H., Momoeda, M., Inoue, S., & Taketani, Y. (2010). Clomiphene citrate elicits estrogen agonistic/antagonistic effects differentially via estrogen receptors αand β. Endocrine journal, 57(6), 517-521.

Levin, E. R. (2009). Plasma membrane estrogen receptors. Trends in Endocrinology & Metabolism, 20(10), 477-482.

Morani, A., Warner, M., & Gustafsson, J. Å. (2008). Biological functions and clinical implications of oestrogen receptors alfa and beta in epithelial tissues. Journal of internal medicine, 264(2), 128-142.

Oride, A., Kanasaki, H., Tumurbaatar, T., Zolzaya, T., Okada, H., Hara, T., et al. (2020). Effects of the Fertility Drugs Clomiphene Citrate and Letrozole on Kiss-1 Expression in Hypothalamic Kiss-1-Expressing Cell Models. Reproductive sciences (Thousand Oaks, Calif.), 27. doi:10.1007/s43032-020-00154-1.

Paterni, I., Granchi, C., Katzenellenbogen, J. A., & Minutolo, F. (2014). Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids, 90, 13-29.

Sutaria, U., Crooke, A., Bertrand, P., & Hodgson, C. (1980). Clomiphene citrate and human chorionic gonadotropin in the treatment of anovulatory infertility. International Journal of Gynecology & Obstetrics, 18(6), 435-437.

Taheripanah, R., Kabir-Salmani, M., Favayedi, M., Zamaniyan, M., Malih, N., & Taheripanah, A. (2020). Effects of clomiphene citrate plus estradiol or progesterone on endometrial ultrastructure: An RCT. International Journal of Reproductive BioMedicine, 18(3), 201.

Trost, L. W., & Khera, M. (2014). Alternative treatment modalities for the hypogonadal patient. Current urology reports, 15(7), 1-12.

Wahab, O. A., Princely, A. C., Oluwadamilare, A. A., Ore-Oluwapo, D. O., Blessing, A. O., & Alfred, E. F. (2019). Clomiphene citrate ameliorated lead acetate-induced reproductive toxicity in male Wistar rats. JBRA assisted reproduction, 23(4), 336-343. doi:10.5935/1518-0557.20190038.

Welboren, W.-J., Sweep, F. C., Span, P. N., & Stunnenberg, H. G. (2009). Genomic actions of estrogen receptor?: what are the targets and how are they regulated? Endocrine-related cancer, 16(4), 1073.

 

 

List of Key Events in the AOP

Event: 1047: Increased, secretion of GnRH from hypothalamus

Short Name: Increased, secretion of GnRH from hypothalamus

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Event: 1050: Increased, secretion of LH from anterior pituitary

Short Name: Increased, secretion of LH from anterior pituitary

Key Event Component

AOPs Including This Key Event

Biological Context

Event: 1972: Increased, Steroidogenic acute regulatory protein (StAR)

Short Name: Increased, Steroidogenic acute regulatory protein (StAR)

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

In Granulosa cells

Key Event Description

Biological state: Steroidogenic acute regulatory protein (StAR) plays important role in luteal steroidogenesis(Christenson and Devoto, 2003). Steroidogenic acute regulatory protein (StAR) controls the transport of cholesterol from the outer to inner mitochondrial membrane(Stocco, 2000). There are several pathways involved for the transport of cholesterol from different subcellular pools into the inner mitochondria(Martin et al., 2016).

Biological compartments: Cholesterol is one type of lipid which is crystalline solid with yellow colour.  It is biosynthesized by animal cells and is an essential structural component of animal cell membranes (Hanukoglu, 1992). It is the precursor molecule for the synthesis all steroid hormones(Payne and Hales, 2004).  Cytochrome P450 enzymes are present in most tissues of the body, and play important roles in hormone synthesis in mitochondria using cholesterol as precursor(Poderoso et al., 2013).

General role in biology:  It is been reported that high  in cholesterol levels in mitochondrial resulted several diseases like cancer, neurodegenerative diseases, steatohepatitis ischemia, and influence disease (Martin et al., 2016). The alteration in mitochondrial cholesterol import may change the cholesterol concentrations that may lead to proper mitochondrial function along with biophysical properties of mitochondrial membranes. In absence of StAR protein, cholesterol transport into the mitochondria did not occurs leading to  no conversion of progesterone from cholesterol precursors doesn’t occur(Kiriakidou et al., 1996; Pescador et al., 1996). All Steroidogenic acute regulatory protein (StAR) promoters contain steroidogenic factor 1 binding sites which is responsible for sex hormones regulation(Manna et al., 2002).

One of the important function of the steroid hormones is maintaining reproductive capacity. For this purpose, steroidogenic cells must move large amounts of cholesterol from the outer mitochondrial membrane to the inner membrane. In the granulosa cells, this cholesterol is ultimately converted to progesterone. The initial transport of cholesterol across the mitochondrial membrane requires Steroidogenic Acute Regulatory (StAR) protein. Expression of StAR protein in preovulatory cells of the developing follicle is low. The dramatic upregulation of StAR protein expression within the dominant follicle is found after the luteinizing hormone (LH) surge. This upregulation allows the corpus luteum to produce substantial amounts of progesterone to maintain the reproductive capacity in human/animal (Men et al., 2017; Stocco, 2000).

How it is Measured or Detected

StAR protein is measure by quantitative real time PCR (qRT-PCR):

For qRT-PCR analyses, cDNA is synthesized using reagent kit in a 20-μl reaction containing 0.5 μg of total RNA collected from human ovarian granulosa tumor cell line ( KGN cells ), mouse Leydig cells. qPCR is performed in a 25-μl reaction containing 0.5 to 1.5 μl of cDNA using fluorescein in real-time PCR detection systems. PCR was performed by initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C. The threshold cycle values of each sample are used to calculate mRNA levels. The PCR primers for the indicated human and mouse genes are as follows (Men et al., 2017).

Human H19 forward: 5′-GCACCTTGGACATCTGGAGT

Human H19 reverse: 5′-TTCTTTCCAGCCCTAGCTCA

Human StAR forward: 5′-GGCATCCTTAGCAACCAAGA

Human StAR reverse: 5′-TCTCCTTGACATTGGGGTTC

Mouse StAR forward: 5′-TTGGGCATACTCAACAACCA

Mouse StAR reverse: 5′-GAAACACCTTGCCCACATCT

Indirect immunohistochemistry for the detection of Steroidogenic Acute Regulatory Protein (StAR):

Ovarian or peritoneal tissues from the human patients are collected. Ovarian or peritoneal tissues from the patient are fixed using 10% paraformaldehyde. Tissues are embedded in paraffin. Serial sections of 5 µm are made using microtome. Tissue sections are prepared by microwave heating in 10× citrate buffer, pH 6.0, for 10 min. Tissues are rinsed three times in 20 mM phosphate buffered saline (PBS), pH 7.2, for 10 min each, before incubation with 1:200 dilutions of polyclonal anti-human StAR antibodies at 37°C for 60 min. Tissue sections were washed three times in 20 mM PBS, pH 7.2, for 2 min each, before incubation with a 1:1000 dilution of secondary mouse– anti-rabbit antibody at 37°C for 30 min. Indirect immunohistochemistry kits were used according to the manufacturer’s instructions to visualize StAR protein stained tissue under microscope and image collected. A pathological image analysis system is used to measure mean optical density (MOD) analysis under high-magnification (×400) microscopy. The MOD, which reflected the positive staining intensity, and the positive staining ratio (area %) of every positively stained area, are measured. The area % is calculated as ([the area of positive staining]/[total nuclear area in the field of view]) × 100. The MOD and area % are used to calculate the expression index, EI (%) = MOD × area %(Tian et al., 2009).

References

Baker, B. Y., Epand, R. F., Epand, R. M., & Miller, W. L. (2007). Cholesterol binding does not predict activity of the steroidogenic acute regulatory protein, StAR. J Biol Chem, 282(14), 10223-32. doi:S0021-9258(19)57693-1 [pii]

Chaffin, C., Dissen, G., & Stouffer, R. (2000). Hormonal regulation of steroidogenic enzyme expression in granulosa cells during the peri-ovulatory interval in monkeys. Molecular human reproduction, 6(1), 11-18.

Christenson, L. K., & Devoto, L. (2003). Cholesterol transport and steroidogenesis by the corpus luteum. Reproductive Biology and Endocrinology, 1(1), 1-9.

Hanukoglu, I. (1992). Steroidogenic enzymes: structure, function, and role in regulation of steroid hormone biosynthesis. The Journal of steroid biochemistry and molecular biology, 43(8), 779-804.

Hasegawa, T., Zhao, L., Caron, K. M., Majdic, G., Suzuki, T., Shizawa, S., et al. (2000). Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice. Mol Endocrinol, 14(9), 1462-71. doi:10.1210/mend.14.9.0515.

Kiriakidou, M., Mcallister, J. M., Sugawara, T., & Strauss 3rd, J. (1996). Expression of steroidogenic acute regulatory protein (StAR) in the human ovary. The Journal of Clinical Endocrinology & Metabolism, 81(11), 4122-4128.

Manna, P. R., Dyson, M. T., Eubank, D. W., Clark, B. J., Lalli, E., Sassone-Corsi, P., et al. (2002). Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family. Molecular Endocrinology, 16(1), 184-199.

Martin, L. A., Kennedy, B. E., & Karten, B. (2016). Mitochondrial cholesterol: mechanisms of import and effects on mitochondrial function. Journal of bioenergetics and biomembranes, 48(2), 137-151.

Men, Y., Fan, Y., Shen, Y., Lu, L., & Kallen, A. N. (2017). The Steroidogenic Acute Regulatory Protein (StAR) Is Regulated by the H19/let-7 Axis. Endocrinology, 158(2), 402-409. doi:10.1210/en.2016-1340.

Payne, A. H., & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine reviews, 25(6), 947-970.

Pescador, N., Soumano, K., Stocco, D. M., Price, C. A., & Murphy, B. D. (1996). Steroidogenic acute regulatory protein in bovine corpora lutea. Biology of reproduction, 55(2), 485-491.

Poderoso, C., Duarte, A., Cooke, M., Orlando, U., Gottifredi, V., Solano, A. R., et al. (2013). The spatial and temporal regulation of the hormonal signal. Role of mitochondria in the formation of a protein complex required for the activation of cholesterol transport and steroids synthesis. Molecular and cellular endocrinology, 371(1-2), 26-33.

Sreerangaraja Urs, D. B., Wu, W.-H., Komrskova, K., Postlerova, P., Lin, Y.-F., Tzeng, C.-R., et al. (2020). Mitochondrial function in modulating human granulosa cell steroidogenesis and female fertility. International journal of molecular sciences, 21(10), 3592.

Stocco, D. (2000). The role of the StAR protein in steroidogenesis: challenges for the future. Journal of Endocrinology, 164(3), 247-253.

Tian, Y., Kong, B., Zhu, W., Su, S., & Kan, Y. (2009). Expression of steroidogenic factor 1 (SF-1) and steroidogenic acute regulatory protein (StAR) in endometriosis is associated with endometriosis severity. J Int Med Res, 37(5), 1389-95. doi:10.1177/147323000903700513.

Event: 1973: Increased, estrogens

Short Name: Increased, estrogens

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

It is applicable in reproduction system, cell growth and cell function

Key Event Description

Biological state: The most predominant form of estrogens is 17β-estradiol (E2) which is sex hormone. In women having premenopausal it is mainly produced in the ovaries. For postmenopausal women, it E2 primarily is sythesized from testosterone by aromatase enzyme in extragonadal tissues(Simpson, 2003). Estradiol stimulates both cell growth and cholesterogenesis in the MCF-7 line (breast cancer cell line) (Cypriani et al., 1988). Cholesterol increases neuronal estradiol release into the medium through synapse formation(Fester et al., 2009).

Biological compartments: Estrogen is considered as the risk of developing cholesterol gallstones by enhancing the hepatic secretion of biliary cholesterol leading to an increase in cholesterol(Wang et al., 2009).

General role in biology: When estrogen levels decline, levels of low-density lipoprotein, the harmful kind of cholesterol increases, and levels of high-density lipoprotein, the positive kind of cholesterol decrease, due to which fat build up in the body and cholesterol in the arteries that causes heart attack and stroke(Fåhraeus, 1988; Wahl et al., 1983). Granulosa cells are the primary cell which provides the support and microenvironment required for the developing oocyte in the ovary(Sen and Hammes, 2010; Sterneck et al., 1997).

How it is Measured or Detected

Radioimmunoassay (RIA) and analytical method based on mass spectroscopic are used for estrogen measurement present in serum (Smy and Straseski, 2018; Giese, 2003).   

References

Adashi, E., & Hsueh, A. (1982). Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells. Journal of Biological Chemistry, 257(11), 6077-6083.

Cypriani, B., Tabacik, C., & Descomps, B. (1988). Effect of estradiol and antiestrogens on cholesterol biosynthesis in hormone-dependent and-independent breast cancer cell lines. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 972(2), 167-178.

Darabi, M., Rabbani, M., Ani, M., Zarean, E., Panjehpour, M., & Movahedian, A. (2011). Increased leukocyte ABCA1 gene expression in post-menopausal women on hormone replacement therapy. Gynecological Endocrinology, 27(9), 701-705.

Fåhraeus, L. (1988). The effects of estradiol on blood lipids and lipoproteins in postmenopausal women. Obstetrics and gynecology, 72(5 Suppl), 18S-22S.

Fester, L., Zhou, L., Bütow, A., Huber, C., Von Lossow, R., Prange‐Kiel, J., et al. (2009). Cholesterol‐promoted synaptogenesis requires the conversion of cholesterol to estradiol in the hippocampus. Hippocampus, 19(8), 692-705.

Giese, R. W. (2003). Measurement of endogenous estrogens: analytical challenges and recent advances. Journal of Chromatography A, 1000(1), 401-412. doi:https://doi.org/10.1016/S0021-9673(03)00306-6.

Mao, Z., Li, J., & Zhang, W. (2018). Hormonal regulation of cholesterol homeostasis. Cholesterol-Good, Bad and the Heart.

Park, Y., Maizels, E. T., Feiger, Z. J., Alam, H., Peters, C. A., Woodruff, T. K., et al. (2005). Induction of cyclin D2 in rat granulosa cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from Smad. Journal of Biological Chemistry, 280(10), 9135-9148.

Sen, A., & Hammes, S. R. (2010). Granulosa cell-specific androgen receptors are critical regulators of ovarian development and function. Molecular endocrinology, 24(7), 1393-1403.

Simpson, E. R. (2003). Sources of estrogen and their importance. The Journal of steroid biochemistry and molecular biology, 86(3-5), 225-230.

Smy, L., & Straseski, J. A. (2018). Measuring estrogens in women, men, and children: Recent advances 2012-2017. Clin Biochem, 62, 11-23.

Sterneck, E., Tessarollo, L., & Johnson, P. F. (1997). An essential role for C/EBPβ in female reproduction. Genes & development, 11(17), 2153-2162.

Wahl, P., Walden, C., Knopp, R., Hoover, J., Wallace, R., Heiss, G., et al. (1983). Effect of estrogen/progestin potency on lipid/lipoprotein cholesterol. New England Journal of Medicine, 308(15), 862-867.

Wang, H. H., Liu, M., Clegg, D. J., Portincasa, P., & Wang, D. Q.-H. (2009). New insights into the molecular mechanisms underlying effects of estrogen on cholesterol gallstone formation. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1791(11), 1037-1047.

Event: 1076: Increased, circulating estrogen levels

Short Name: Increased, circulating estrogen levels

Key Event Component

AOPs Including This Key Event

Biological Context

Event: 1052: Hyperplasia, ovarian epithelium

Short Name: Hyperplasia, ovarian epithelium

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

List of Adverse Outcomes in this AOP

Event: 1053: Promotion, ovarian adenomas

Short Name: Promotion, ovarian adenomas

Key Event Component

AOPs Including This Key Event

Biological Context

Event: 2092: Promotion, Ovarian Cancer

Short Name: Promotion, Ovarian Cancer

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

It is applicable in ovary for reproductive matured female.

Key Event Description

Biological state: Ovarian cancer is fatal gynecological malignancy and ranked as fifth most commonly diagnosed cancer among women.  Generally, mortality rate is highest (~ 50 %) from this cancer as there is lack of proper diagnosis at early stage(Siegel et al. 2019). Ovarian cancers are broadly categorised into three types based on origin of cells namely epithelial, stromal and germ cell cancers (Gilks and Prat 2009). Recent research efforts revealed that numbers of molecular level (genome, transcriptome and proteome level) perturbations are responsible for the development and progression of ovarian cancer (Cheng and Zhan 2017).  There is need to develop a molecular level biomarker for early detection, treatment and development of personalized medicine. Understanding of molecular level interactions in large and complex biological networks using systems biology approach will be key factors to identify the major regulatory motifs (Zhang et al. 2018). This approach not only reduces the animal experiments substantially, but will able to quick detect of key perturbations

Biological compartments: Recent studies have suggested that FSH stimulates the proliferation and invasion of ovarian cancer cells, inhibits apoptosis and facilitates neovascularisation (Tao et al. 2013). Earlier studies also have established that the estrogen (ER) and progesterone (PR) receptors are important prognostic indicators of breast and endometrial cancers, and epithelial ovarian cancer. Despite acceptance regarding the influence of reproductive hormones on ovarian cancer risk and considerable advances in the understanding of epithelial ovarian carcinogenesis on a molecular level, complete understanding of the biologic processes underlying malignant transformation of ovarian surface epithelium is still lacking  (Gharwan et al. 2015).

General role in biology: Malfunctioning of sex hormones (e.g., estradiol, estrone and progesterone) may result ovarian cancer (Fooladi et al. 2020, Meehan and Sadar 2003). Exposure to endocrine-disrupting chemicals (EDCs) in the form of occupational usage of pesticides, fungicides, herbicides, plasticizers, cosmetics, etc. are the cause of ovarian cancer (Samtani et al. 2018). Clomiphene which is used as drug to treat infertility and it is reported that this chemical increases the risk of ovarian cancer (McLemore et al. 2009). Clomiphene (molecular initiating event, MIE) stimulates the releasing of gonadotropin-releasing hormone (GnRH) from hypothalamic. Also, it stimulates the secretion of the Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from pituitary (Cassidenti et al. 1992, Mungenast and Thalhammer 2014, Tomao et al. 2014). These hormones regulate the synthesis of sex hormons (e.g., estrogen) level (Shoemaker et al. 2010, Tomao et al. 2014). These sex hormones are primarily produced in the gonads through a series of enzyme-mediated reactions from cholesterol (precursor) and control through complex signalling pathway along hypothalamus – pituitary - gonadal (HPG) axis (Perkins et al. 2019, Shoemaker et al. 2010).  The series of complex signalling pathways in ovary include G-protein cycle, G-protein activation, adenylate cyclase (AC) activation, cyclic AMP (cAMP) activation, protein kinase A (PKA) activation, steroidogenic factor 1 (SF1) and StAR transcription. Ultimately, this signalling pathway activates the StAR protein which regulates the intake of cholesterol into the inner mitochondria where synthesis of sex hormones takes place. It may be noted that cholesterol is the precursor of the sex hormones synthesis.  Again, releasing of LH is regulated by estradiol and testosterone level resulting complex signalling pathway that includes genes, transcritome, proteome and metabolites (Perkins et al. 2019, Shoemaker et al. 2010). Under clomiphene exposure, synthesis of estrogen level becomes high resulting risk of ovarian cancer (McLemore et al. 2009, Tomao et al. 2014). Therefore, perturbations of GnRH, FSH and LH can result adverse phenotype as ovarian cancer.

How it is Measured or Detected

Gossmann et al., had shown the effects of angiogenesis inhibition on tumor microvascular permeability was monitored with the help of magnetic resonance imaging (MRI) technique in a rat model of human ovarian cancer (Gossmann et al. 2000).

Gitsch et al., had developed gamma-ray detection probe for overcoming the conventional radio-immunoscintigraphy problems for the detection of ovarian cancer in female patients (Gitsch and Pateisky 1989).

Kim et al., had used the detection of magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) for the detection of ovarian tumor in human patient. Sensitivity and accuracy of the PET/CT technique for detecting the ovarian tumor was reported 73% and 91%. Whereas, the sensitivity and accuracy of the MRI technique was reported 81% and 89% (Kim et al. 2007).

Harrington et al., had used immunotechniques (Anti-CDCP1 immuno-conjugates) for detection of the ovarian cancer. Expression and binding properties of the cell surface protein was detected in ovarian cancer cell (in vitro) using flow cytometry and western blot technique (Harrington et al.).

Regulatory Significance of the AO

Informations related with ovarian cancer will be helpful for the regulatory authorities to develop monographs, frame the rules of assesments and monitoring of the process.

References

Cassidenti, D.L., Paulson, R.J., Lobo, R.A. and Sauer, M.V. (1992) The synergistic effects of clomiphene citrate and human menopausal gonadotrophin in the folliculogenesis of stimulated cycles as assessed by the gonadotrophin-releasing hormone antagonist Nal-Glu. Hum Reprod 7(3), 344-348.

Cheng, T. and Zhan, X. (2017) Pattern recognition for predictive, preventive, and personalized medicine in cancer. EPMA J 8(1), 51-60.

Fooladi, S., Akbari, H., Abolhassani, M., Sadeghi, E. and Fallah, H. (2020) Estradiol, des-acylated, and total ghrelin levels might be associated with epithelial ovarian cancer in postmenopausal women. medRxiv.

Gharwan, H., Bunch, K.P. and Annunziata, C.M. (2015) The role of reproductive hormones in epithelial ovarian carcinogenesis. Endocr Relat Cancer 22(6), R339-363.

Gilks, C.B. and Prat, J. (2009) Ovarian carcinoma pathology and genetics: recent advances. Hum Pathol 40(9), 1213-1223.

Gitsch, E. and Pateisky, N. (1989) Radio-immunoscintigraphy and intraoperative tumour detection by means of anti-tumour antibodies in patients with ovarian cancer. Baillieres Clin Obstet Gynaecol 3(1), 31-36.

Gossmann, A., Helbich, T.H., Mesiano, S., Shames, D.M., Wendland, M.F., Roberts, T.P., Ferrara, N., Jaffe, R.B. and Brasch, R.C. (2000) Magnetic resonance imaging in an experimental model of human ovarian cancer demonstrating altered microvascular permeability after inhibition of vascular endothelial growth factor. Am J Obstet Gynecol 183(4), 956-963.

Harrington, B.S., He, Y., Khan, T., Puttick, S., Conroy, P.J., Kryza, T., Cuda, T., Sokolowski, K.A., Tse, B.W., Robbins, K.K., Arachchige, B.J., Stehbens, S.J., Pollock, P.M., Reed, S., Weroha, S.J., Haluska, P., Salomon, C., Lourie, R., Perrin, L.C., Law, R.H.P., Whisstock, J.C. and Hooper, J.D. Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer. Theranostics 10(5), 2095-2114.

Hollis, R.L.J.C.L. (2023) Molecular characteristics and clinical behaviour of epithelial ovarian cancers. 216057.

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Appendix 2

List of Key Event Relationships in the AOP