**3.2 Selective ER and PR modulators**

Selective ER modulators (SERMs) function through ERs, acting as agonists or antagonists of E2 depending on the target tissue and modulate the signal transduction pathway to E2 responsive genes. The implementation of SERMs in clinical aspects is wide. They are used to treat or prevent breast cancer and osteoporosis, to cure ovulatory dysfunction in women but also for contraceptive purposes. SERMs have an ability to differently regulate many ERregulated genes (Berrodin et al., 2009; Chang et al., 2010). In general, most SERMs have E2 agonist activity in bone and antagonist activity in the breast, while the activity in the uterus varies among the molecules. The tissue specificity depends on various co-activators (CoA) and co-repressors (CoR) expressed and recruited in different tissues (Riggs et al., 2003). E2 binds to either ERα or ERβ and subsequently binds CoA molecules required to form a transcription complex at EREs located in the promoter region of estrogen-responsive genes. The antiestrogenic action of a SERM results from the inappropriate folding of an ERα or ERβ complex that either cannot recruit CoA molecules or instead recruits CoR molecules. This programmed change in conformation produces antiestrogen action at specific sites like the breast, but estrogen-like effects in the uterus if an excess of CoA molecules is present. SERM–ER complexes may initiate gene transcription to produce an estrogen-like effect, by forming a protein–protein interaction at fos/jun that activates AP-1 sites (Jordan et al., 2001) (Figure 4). Although widely used and with many beneficial effects in treating breast cancer SERMs still battle with several side effects where most common is the stimulation of the endometrium.

The Tissue Specific Role

undiscovered.

Cohen et al., 2000).

(Perez et al., 2006).

bone.

of Estrogen and Progesterone in Human Endometrium and Mammary Gland 45

breast cancer treatment could lead to endometrial cell proliferation later on (Figure 5). Endometrial pathologies associated with TAM use include hyperplasia, polyps, carcinomas and sarcomas (Cohen et al., 2004). The full mechanism of this paradox remains still

Another well-studied antiestrogen, Raloxifen, has an E2-antagonistic effect similarly to TAM but it is reported to have small or no proliferative effect on uterus (Fugere et al., 2000). Although Raloxifen was developed initially for breast cancer treatment, its use was abandoned in the late 1980s because clinical trials showed no activity in TAM-resistant patients (Buzdar et al, 1998). Today, Raloxifen is used specifically to reduce the risk of osteoporosis in postmenopausal women at high risk for osteoporosis (Jordan et al., 2001,

As SERMs have the ability to provide mixed functional ER agonist or antagonist activity, depending on the target tissue, compaunds devoid of agonist activity have been developed. The most known "pure" antiestrogen is Fulverstant (aslo known as ICI 182780) (Bowler et al., 1989; Wakeling et al., 1991). In addition to blocking the ER activity Fulvestrant induces ER degradation by changing its conformation (Dauvois et al., 1993; Gibson et al., 1991; Reese and Katzenellenbogen 1992). This forces the receptor into conformation that it is recognized as being misfolded, which induces its rapid degradation (Wu et al 2005). Fulvestrant is currently licensed for the use in postmenopausal women with ER-positive recurrent disease (Johnston et al., 2010). However, the lack of agonist activity limits its beneficial effects in

SERM might inhibit the ER found in breast cells but activate the ER present in uterine endometrial cells. That would inhibit cell proliferation in breast cells, but stimulate the proliferation of uterine endometrial cells (Figure 5). There are number of decision points that determine the biological response to a SERM, which is linked to its E2-like ability to recruit CoA-s and CoR-s. ER contains a ligand-binding domain, called Activating Function-2 (AF-2), which is essential for the activation of genes that mediate the E2 effect in tissues like breast and uterus. Therefore, the different ligands can induce distinct gene transcription processes. For example, the union of the ligand binding domain with TAM results in partial agonistic effect in the uterus, whereas the same interaction is fully antagonistic in the breast

Similarly to anti-estrogens, anti-progestins or Selective Progesterone Receptor Modulators (SPRMs) are developed in order to antagonize the processes activated by P4. Mifepristone (RU-486) acts as a P4 antagonist by competing with endogenous P4 for receptor binding and has three primary pharmacological effects: endometrial, gonadotropic, and adrenocortical (Goldberg et al., 1998). It has 2 to 10 time higher affinity compared to P4 to bind PRs (Brogden et al., 1993). Because PRs are found primarily in reproductive organs, Mifepristone exerts its principal effect on the uterus. More precisely, Mifepristone blocks the effects of natural P4 on the endometrium and decidua. While P4 is supposed to support the pregnancy, anti-P4 leads to degeneration and shedding of the endometrial lining, thereby preventing or disrupting implantation of the conceptus. Mifepristone also increases both uterine production of prostaglandins and uterine sensitivity to the contractile effects of prostaglandins, stimulating uterine contractions. It is postulated that Mifepristone acts directly on the uterine muscle through an entirely separate mechanism, perhaps by increasing gap junctions in the myometrium (Weiss et al., 1993). Tissue culture studies have shown that Mifepristone continues to display procontractile effects on the uterus even when

Fig. 4. The signal transduction pathways available to E2 or a SERM to initiate gene transcription. E2 receptors – ERα, ERβ, selective ER modulator-SERM, coactivator –CoA, corepressor CoR, E2 response element-ERE, activating protein -1-AP-1 (Figure adapted from Jordan et al., 2001).

Tamoxifen (TAM), the first SERM available for clinical use, is regarded as a highly effective agent for the prevention and treatment of breast cancer in premenopausal and postmenopausal women. TAM has been used in women to treat breast cancer for over 40 years (Fisher et al., 1998; Fisher et al., 2005). This compound binds with high affinity to ER, thereby blocking the action of native E2. Subsequently it inhibits or modifies the interaction of ER with DNA, which impedes the transcriptional activation of target genes (Berry et al., 2005). TAM strongly counteracts E2 effects, including secretion of several growth factors and growth controlling enzymes, so that a woman's own E2 cannot stimulate growth of the tumor cells. TAM has been a successful drug especially in treating hormone-responsive breast cancer, being one of the main reasons why ER-positive breast cancer patients have a better prognosis compared to those with an ERα-negative breast tumours. Another positive effect was noticed when postmenopausal women's bone density increased after breast cancer treatment (Love et al., 1992). One of the most significant side effects of the treatment with the TAM appears to be its proliferative effect on the endometrium (estrogen-agonistic effect; Buzdar et al., 1998; Bergman et al., 2000). The use of TAM results significant 3.3 fold increase of endometrial cancer (Fisher 2005). The repression of the cell proliferation during

Fig. 4. The signal transduction pathways available to E2 or a SERM to initiate gene transcription. E2 receptors – ERα, ERβ, selective ER modulator-SERM, coactivator –CoA, corepressor CoR, E2 response element-ERE, activating protein -1-AP-1 (Figure adapted from

Tamoxifen (TAM), the first SERM available for clinical use, is regarded as a highly effective agent for the prevention and treatment of breast cancer in premenopausal and postmenopausal women. TAM has been used in women to treat breast cancer for over 40 years (Fisher et al., 1998; Fisher et al., 2005). This compound binds with high affinity to ER, thereby blocking the action of native E2. Subsequently it inhibits or modifies the interaction of ER with DNA, which impedes the transcriptional activation of target genes (Berry et al., 2005). TAM strongly counteracts E2 effects, including secretion of several growth factors and growth controlling enzymes, so that a woman's own E2 cannot stimulate growth of the tumor cells. TAM has been a successful drug especially in treating hormone-responsive breast cancer, being one of the main reasons why ER-positive breast cancer patients have a better prognosis compared to those with an ERα-negative breast tumours. Another positive effect was noticed when postmenopausal women's bone density increased after breast cancer treatment (Love et al., 1992). One of the most significant side effects of the treatment with the TAM appears to be its proliferative effect on the endometrium (estrogen-agonistic effect; Buzdar et al., 1998; Bergman et al., 2000). The use of TAM results significant 3.3 fold increase of endometrial cancer (Fisher 2005). The repression of the cell proliferation during

Jordan et al., 2001).

breast cancer treatment could lead to endometrial cell proliferation later on (Figure 5). Endometrial pathologies associated with TAM use include hyperplasia, polyps, carcinomas and sarcomas (Cohen et al., 2004). The full mechanism of this paradox remains still undiscovered.

Another well-studied antiestrogen, Raloxifen, has an E2-antagonistic effect similarly to TAM but it is reported to have small or no proliferative effect on uterus (Fugere et al., 2000). Although Raloxifen was developed initially for breast cancer treatment, its use was abandoned in the late 1980s because clinical trials showed no activity in TAM-resistant patients (Buzdar et al, 1998). Today, Raloxifen is used specifically to reduce the risk of osteoporosis in postmenopausal women at high risk for osteoporosis (Jordan et al., 2001, Cohen et al., 2000).

As SERMs have the ability to provide mixed functional ER agonist or antagonist activity, depending on the target tissue, compaunds devoid of agonist activity have been developed. The most known "pure" antiestrogen is Fulverstant (aslo known as ICI 182780) (Bowler et al., 1989; Wakeling et al., 1991). In addition to blocking the ER activity Fulvestrant induces ER degradation by changing its conformation (Dauvois et al., 1993; Gibson et al., 1991; Reese and Katzenellenbogen 1992). This forces the receptor into conformation that it is recognized as being misfolded, which induces its rapid degradation (Wu et al 2005). Fulvestrant is currently licensed for the use in postmenopausal women with ER-positive recurrent disease (Johnston et al., 2010). However, the lack of agonist activity limits its beneficial effects in bone.

SERM might inhibit the ER found in breast cells but activate the ER present in uterine endometrial cells. That would inhibit cell proliferation in breast cells, but stimulate the proliferation of uterine endometrial cells (Figure 5). There are number of decision points that determine the biological response to a SERM, which is linked to its E2-like ability to recruit CoA-s and CoR-s. ER contains a ligand-binding domain, called Activating Function-2 (AF-2), which is essential for the activation of genes that mediate the E2 effect in tissues like breast and uterus. Therefore, the different ligands can induce distinct gene transcription processes. For example, the union of the ligand binding domain with TAM results in partial agonistic effect in the uterus, whereas the same interaction is fully antagonistic in the breast (Perez et al., 2006).

Similarly to anti-estrogens, anti-progestins or Selective Progesterone Receptor Modulators (SPRMs) are developed in order to antagonize the processes activated by P4. Mifepristone (RU-486) acts as a P4 antagonist by competing with endogenous P4 for receptor binding and has three primary pharmacological effects: endometrial, gonadotropic, and adrenocortical (Goldberg et al., 1998). It has 2 to 10 time higher affinity compared to P4 to bind PRs (Brogden et al., 1993). Because PRs are found primarily in reproductive organs, Mifepristone exerts its principal effect on the uterus. More precisely, Mifepristone blocks the effects of natural P4 on the endometrium and decidua. While P4 is supposed to support the pregnancy, anti-P4 leads to degeneration and shedding of the endometrial lining, thereby preventing or disrupting implantation of the conceptus. Mifepristone also increases both uterine production of prostaglandins and uterine sensitivity to the contractile effects of prostaglandins, stimulating uterine contractions. It is postulated that Mifepristone acts directly on the uterine muscle through an entirely separate mechanism, perhaps by increasing gap junctions in the myometrium (Weiss et al., 1993). Tissue culture studies have shown that Mifepristone continues to display procontractile effects on the uterus even when

The Tissue Specific Role

family history of BRCA mutations.

**4. Genome-wide E2 and P4 signalling** 

extraction.

of Estrogen and Progesterone in Human Endometrium and Mammary Gland 47

studies have found elevated risk for ovarian cancer too but probably the risk was already higher prior to the first IVF (Källen et al., 2005; Kristiansson et al., 2007). However, there are publications, which have not found a relation between infertility treatments and any cancer development (Potashnik et al., 1999, Doyle et al., 2002; Dor et al., 2002; Lerner-Geva et al., 2010; Brinton et al., 2004). For example, a case-control study (1380 pairs) showed no risk for IVF treatment even among women who carry mutations in breast cancer susceptibility gene 1 (BRCA1) or BRCA2 gene (Kotsopoulos et al., 2008). The common opinion today is that the use of fertility medications does not increase the risk of breast cancer among those with

A more recent study, published by Källen and colleagues using Swedish cancer register, showed that there was no or significantly low cancer risk among women udergoing IVF treatment compared to general population. The study included 24 058 women who had been treated with IVF where 1279 women later appeared in the cancer register. For comparison, total of 1 394 061 women in the general population were studied as a control

The phrase "healthy patient effect" has emerged saying that women who choose IVF treatment might be more aware of risks or more health conscious at the time of conception compared to non-IVF women (Venn et al., 2001). In addition, there are numerous confounding factors which could influence the outcome of the study like the age at the time of the first IVF cycle or the first delivery, the number of the unsuccessful cycles and the follow up time after last IVF treatment. It is obvious that the question needs to be studied in more detail involving large number of women and with attention to precise subgroups.

There are hundreds of studies presenting how expression of a single gene could change upon E2 or P4 treatment in different cell culture. Knock out studies with transgenic animals have confirmed the importance of ERs and PRs in reproductive system and cancer development. To understand the broad role of steroid hormones in humans it is mandatory to study their action in genome-wide level. Recently, the development of large-scale genomic methods to analyse gene expression and factor binding to DNA enable us to study steroid hormone dependent gene expression changes and transcription regulation in the entire genome. As ERs and PRs are acting as TFs they have an ability to regulate the expression of proximal and distal genes by binding hormone responsive elements. Chromatin immunoprecipitation (ChIP) analysis has been broadly used for identification TF binding regions on DNA. ChIP assay can be followed by polymerase chain reaction (PCR), hybridization the probes on a microarray (ChIP-on-chip) or high throughput (HTP) sequencing (ChIP-Seq) to establish the genomic regions occupied by a specific TF. To understand whether TF binding has a positive or negative impact on gene expression microarrays, sequencing (RNA-Seq) and RT-qPCR are commonly used followed to mRNA

ChIP is a technique for assaying protein-DNA interactions *in vivo* (Weinmann et al., 2002). This analysis allows identifying regions of the genome bound directly to ERs or PRs as well as regions bound indirectly via other TFs or co-regulators. During the procedure proteins are cross-linked to DNA and the chromatin is thereafter sonicated to small fragments

group where 95 775 women had registered cancer (Källen et al., 2011).

**4.1 Genome-wide identification of TF binding regions, ChIP-Seq** 

the effects of prostaglandins are neutralized (Brogden et al., 1993). Most research and clinical experience with Mifepristone involves its use as an aborted material. Several studies reported its effectiveness in softening and dilation of the cervix prior to surgical abortion, decrease of pain in women with diagnosed endometriosis and in labour inducement (Goldberg et al., 1998). In the absence of P4, however, Mifepristone can act as a partial agonist (Spitz et al., 1993) and upregulate P4-responsive genes, such as p53, and through this possesses a slight anticarcinogenic effect.

Fig. 5. **The opposite effect of SERM on breast and uterine cell proliferation.** SERM might inhibit the ER found in breast cancer cells but activate the ER present in uterine endometrial cells. A SERM of this type would inhibit cell proliferation in breast cells, but stimulate the proliferation of uterine endometrial cells. (Figure adapted from internet: http://www.cancer.gov/cancertopics/understandingcancer/estrogenreceptors/page14)
