**Progesterone Resistance and Targeting the Progesterone Receptors: A Therapeutic Approach to Endometriosis**

Nick Pullen *Pfizer Global Research & Development, UK* 

### **1. Introduction**

156 Endometriosis - Basic Concepts and Current Research Trends

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Endometriosis is characterised by the benign growth of endometrial glands and stroma on the surface of peritoneal tissues and other organs. It is generally regarded as an aberrant estrogen-dependent growth condition, which presents with symptoms of chronic pelvic pain, bleeding and infertility. Steroidal progestogens are already widely used in the treatment of the condition, dienogest (Visanne) the most recent of which has gained EU approval for clinical use (McCormack, 2010). Progestogens appear to work by both directly inhibiting the functional effects of estrogen on endometrial cell proliferation, and also suppressing ovarian function, to induce anovulatory amenorrhoea. The efficacy of this class of agents in patients with endometriosis, however, is relatively modest and the tolerability (breakthrough bleeding and bloating) as well as concerns on the long term safety (risk of breast cancer and thromboembolism, effect on bone mineral density) has also limited their broader utility. Progesterone receptor antagonists (PRAs) have emerged in recent years as an alternative approach to treating the disease. This class of agents has contrasting effects on reproductive function compared with progestogens. This review will focus on what we know about the PRA mechanism of action from pre-clinical in vitro and in vivo evidence and how clinical data have shaped confidence in this class of agents as a new approach to treating endometriosis symptoms and disease progression1.

#### **2. Progesterone receptor structure & function**

The steroid hormone, progesterone, is a key modulator of normal reproductive function, including ovulation, uterine and mammary gland development and the neurobehavioral expression associated with sexual responsiveness (Clarke & Sutherland, 1990; Lydon et al., 1995). Progesterone is absolutely essential for the maintenance of pregnancy, maintaining uterine quiescence by suppressing expression of genes that mediate increased myometrial

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Progesterone Resistance and Targeting

or PGRMC1 in endometriosis, this is not the case for PR.

association (Painter et al., 2011).

**3. Evidence of progesterone resistance in women with endometriosis** 

orchestrate a more complex pattern of proliferation and differentiation.

The uterus is composed of heterogeneous cell types which undergo synchronous waves of proliferation and differentiation in response to cyclical changes in estrogen (E2) and progesterone levels. The spatiotemporal expression of PRs in epithelial, stromal and myometrial cellular compartments are under the control of estrogen, the primary endometrial mitogen. Progesterone appears to exert proliferative function or induce differentiation depending on the cell type; on epithelia, progesterone, acting via stromal PRs, inhibits estrogen-driven cell proliferation and on stroma progesterone appears to

While the actions of progesterone are critical to the establishment and maintenance of pregnancy, approximately one third of women with endometriosis also present with infertility. For some women, the loss of tubal patency, the modification of the pelvis and the inflammation associated with condition appear to be causally related to the presentation of infertility. However, the characterization of the eutopic endometrium from women with endometriosis has also revealed many defects, including altered patterns of angiogenesis, dyssynchrony with the window of implantation as well as ultra structural abnormalities which may contribute to the infertility. Supporting this, isolated endometrial stromal fibroblasts from women with endometriosis do not appear to undergo a normal decidualisation response (Aghajanova et al., 2010), suggesting an impairment of the progesterone-mediated differentiation programme. Specific alterations in the expression molecular markers of endometrial receptivity have also been widely documented, especially the integrin αvβ3, certain steroid hormone receptors and HOXA10 gene expression, the latter of which has been identified from a genome wide association study of moderate to severe endometriosis as a potential candidate disease locus with proximity to the 7p15.2 SNP

Early studies also pointed to alteration in the normal pathways of estrogen metabolism in ectopic endometrial tissue; specifically expression of 17βHSD-2, the enzyme responsible for

the Progesterone Receptors: A Therapeutic Approach to Endometriosis 159

Beyond the genomic function of progesterone mediated by PR-A and PR-B, progesterone also appears to elicit non-genomic activity. Neurosteroidal function of progesterone and progesterone metabolites, such as allopregnelone (3α-hydroxy-5α-pregnan-20-one), augment GABAergic channel burst durations by increasing the opening frequency through positive allosteric modulation in the hypothalamus (Henderson, 2007). In the female rat, direct administration of allopregnelone in specific regions in the hypothalamus rapidly facilitates lordosis, suggesting a direct non-genomic effect on reproductive function. Outside of the predicted protection from catamenial exacerbation of epileptic seizures and premenstrual dysphoric disorder by neurosteroids (Biagini et al., 2010), the non-genomic effects of progesterone in reproductive function in the female human have been less well characterised. Several other reported progesterone receptors (mPR, PGRMC1 and CatSper, for instance) have also been touted to contribute to the non-genomic effects of progesterone (Dressing et al., 2011; Gellersen et al., 2009; Lishko et al., 2011; Zhu et al., 2003). Based on expression data and some functional characterisation both mPR and PGRMC1 may have a role in reproductive function and are speculated to regulate implantation and myometrial contractility. Whilst there are no evidence reported suggesting a contributing role for mPR

contractility, such as the oxytocin receptor. The physiological effects of progesterone (P4) are principally mediated by interaction with two specific intracellular receptors termed PR-A and PR-B. PR-A and PR-B are members of the nuclear receptor superfamily of transcription factors (Mangelsdorf et al., 1995; McKenna et al., 2009; O'Malley & Conneely, 1992; Tsai & O'Malley, 1994). Nuclear hormone receptors regulate gene transcription by discriminative binding to specific DNA sequences, so-called progesterone response elements (PREs) Specific interactions with co-activator and/or co-repressor proteins, induced by ligand binding, trigger interactions with the RNA polymerase complex (McKenna & O'Malley, 2001). PR-A and PR-B are expressed from a single gene as a result of transcription from two alternative promoters and translation initiation at two alternative initiation codons (Kraus et al., 1993). The human PR-A and PR-B are identical except for an additional N-terminal 164 amino acid sequence present in PR-B. Both isoforms have two activation function domains, AF-1 proximal to the DNA-binding domain, and a ligand-dependent AF-2 domain in the C terminus (Tetel et al., 1999). By virtue of the longer N-terminus, PR-B also has a unique AF-3 domain that may contribute to its differential trans-activation properties compared with PR-A (Tung et al., 2006).

Ligand binding (progesterone as well as other synthetic PR ligands such as progestogens (e.g. dienogest, tanaproget, medroxyprogesterone acetate), the progesterone receptor antagonists (PRAs, such as RU-486) and modulators (e.g. J-867)) triggers a conformational change that causes the dissociation of bound heat shock proteins, receptor phosphorylation, receptor dimerisation, nuclear translocation and DNA binding. Binding occurs at specific PRE sequences in promoters of progesterone response genes, and alters transcriptional activity, negatively or positively, depending on PRE sequence, the conformation of the ligand-bound complex and cell-specific context. In the presence of steroidal PRAs, such as RU-486, the complex becomes transcriptionally inactive due to recruitment of co-repressors such as NCoR1 (Wagner et al., 1998), whereas selective PR modulators, such as J-867, elicit a mixed agonist/antagonist response (Elger et al., 2000; Madauss et al., 2007).

Studies in mice with selective ablation of PR isoforms have revealed that PR-A is necessary for ovulation and modulates the anti-proliferative effects of progesterone in the uterus. In contrast PR-B knockout mice are fertile and sustain a normal pregnancy, but PR-B appears to be required for normal mammary gland development and function (Mulac-Jericevic & Conneely, 2005). In an attempt to understand the function of PR-B in the endometrium, one group has used PRAs and siRNA to knockdown gene expression in an immortalised human endometrial stromal cell line (Wu & Guo, 2006; Wu et al., 2008). Ablation of PR-B promoted cellular proliferation, by approximately 20% compared with control, supporting the notion that PR-B acts as a break on progesterone function. It is noteworthy that breast and endometrial malignancy is often accompanied by disruption of PR-A and PR-B expression or altered functional PR responses (Arnett-Mansfield et al., 2004; De Vivo et al., 2002; Kobayashi et al., 2010; McGowan et al., 2004), an observation which has triggered interest in the potential utility of PRAs for oncology (Fuhrmann et al., 2000; Poole et al., 2006; Tieszen et al., 2011; Wiehle et al., 2011). Recent evidence has also confirmed the existence of a functional third isoform, PR-C which lacks AF-2 and AF-3 domains and appears to act as a sink for progesterone and have a function in regulating the onset of parturition (Condon et al., 2006).

contractility, such as the oxytocin receptor. The physiological effects of progesterone (P4) are principally mediated by interaction with two specific intracellular receptors termed PR-A and PR-B. PR-A and PR-B are members of the nuclear receptor superfamily of transcription factors (Mangelsdorf et al., 1995; McKenna et al., 2009; O'Malley & Conneely, 1992; Tsai & O'Malley, 1994). Nuclear hormone receptors regulate gene transcription by discriminative binding to specific DNA sequences, so-called progesterone response elements (PREs) Specific interactions with co-activator and/or co-repressor proteins, induced by ligand binding, trigger interactions with the RNA polymerase complex (McKenna & O'Malley, 2001). PR-A and PR-B are expressed from a single gene as a result of transcription from two alternative promoters and translation initiation at two alternative initiation codons (Kraus et al., 1993). The human PR-A and PR-B are identical except for an additional N-terminal 164 amino acid sequence present in PR-B. Both isoforms have two activation function domains, AF-1 proximal to the DNA-binding domain, and a ligand-dependent AF-2 domain in the C terminus (Tetel et al., 1999). By virtue of the longer N-terminus, PR-B also has a unique AF-3 domain that may contribute to its differential trans-activation properties compared with PR-

Ligand binding (progesterone as well as other synthetic PR ligands such as progestogens (e.g. dienogest, tanaproget, medroxyprogesterone acetate), the progesterone receptor antagonists (PRAs, such as RU-486) and modulators (e.g. J-867)) triggers a conformational change that causes the dissociation of bound heat shock proteins, receptor phosphorylation, receptor dimerisation, nuclear translocation and DNA binding. Binding occurs at specific PRE sequences in promoters of progesterone response genes, and alters transcriptional activity, negatively or positively, depending on PRE sequence, the conformation of the ligand-bound complex and cell-specific context. In the presence of steroidal PRAs, such as RU-486, the complex becomes transcriptionally inactive due to recruitment of co-repressors such as NCoR1 (Wagner et al., 1998), whereas selective PR modulators, such as J-867, elicit a mixed agonist/antagonist response (Elger et al., 2000;

Studies in mice with selective ablation of PR isoforms have revealed that PR-A is necessary for ovulation and modulates the anti-proliferative effects of progesterone in the uterus. In contrast PR-B knockout mice are fertile and sustain a normal pregnancy, but PR-B appears to be required for normal mammary gland development and function (Mulac-Jericevic & Conneely, 2005). In an attempt to understand the function of PR-B in the endometrium, one group has used PRAs and siRNA to knockdown gene expression in an immortalised human endometrial stromal cell line (Wu & Guo, 2006; Wu et al., 2008). Ablation of PR-B promoted cellular proliferation, by approximately 20% compared with control, supporting the notion that PR-B acts as a break on progesterone function. It is noteworthy that breast and endometrial malignancy is often accompanied by disruption of PR-A and PR-B expression or altered functional PR responses (Arnett-Mansfield et al., 2004; De Vivo et al., 2002; Kobayashi et al., 2010; McGowan et al., 2004), an observation which has triggered interest in the potential utility of PRAs for oncology (Fuhrmann et al., 2000; Poole et al., 2006; Tieszen et al., 2011; Wiehle et al., 2011). Recent evidence has also confirmed the existence of a functional third isoform, PR-C which lacks AF-2 and AF-3 domains and appears to act as a sink for progesterone and have a function in regulating the onset of parturition (Condon et

A (Tung et al., 2006).

Madauss et al., 2007).

al., 2006).

Beyond the genomic function of progesterone mediated by PR-A and PR-B, progesterone also appears to elicit non-genomic activity. Neurosteroidal function of progesterone and progesterone metabolites, such as allopregnelone (3α-hydroxy-5α-pregnan-20-one), augment GABAergic channel burst durations by increasing the opening frequency through positive allosteric modulation in the hypothalamus (Henderson, 2007). In the female rat, direct administration of allopregnelone in specific regions in the hypothalamus rapidly facilitates lordosis, suggesting a direct non-genomic effect on reproductive function. Outside of the predicted protection from catamenial exacerbation of epileptic seizures and premenstrual dysphoric disorder by neurosteroids (Biagini et al., 2010), the non-genomic effects of progesterone in reproductive function in the female human have been less well characterised. Several other reported progesterone receptors (mPR, PGRMC1 and CatSper, for instance) have also been touted to contribute to the non-genomic effects of progesterone (Dressing et al., 2011; Gellersen et al., 2009; Lishko et al., 2011; Zhu et al., 2003). Based on expression data and some functional characterisation both mPR and PGRMC1 may have a role in reproductive function and are speculated to regulate implantation and myometrial contractility. Whilst there are no evidence reported suggesting a contributing role for mPR or PGRMC1 in endometriosis, this is not the case for PR.
