Turkish Journal of Biology Turk J Biol (2014) 38: 772-785 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Review Article doi:10.3906/biy-1407-44

Progesterone receptor modulators in breast cancer

, Ronald D. WIEHLE* ** Repros Therapeutics, The Woodlands, Texas, USA

Received: 11.07.2014 Accepted: 14.10.2014 Published Online: 24.11.2014 Printed: 22.12.2014

Abstract: Breast cancer has been treated successfully with selective estrogen receptor antagonists (SERMs) such as tamoxifen, receptor- depleting agents such as fulvestrant, and aromatase inhibitors such as anastrozole. Selective receptor modulators (SPRMs or PRMs) have not been studied as much and are currently under investigation for inhibition of mammary carcinogenesis in animal models and breast cancer prevention trials in women. They might follow tamoxifen and aromatase inhibitors in the adjuvant treatment of breast cancers with acquired resistance. These uses have not provoked ground-breaking progress or studies and PRMs do not have a high profile. Most in vitro and in vivo studies indicate that PRMs preferentially suppress cell proliferation and also induce apoptosis. In this review we summarized the data on the effects of PRMs and particularly of the antiprogestins RU-486 () and CDB-4124 (Progenta, Proellex or acetate) on breast cancer models. Both agents have been employed in preclinical and clinical studies for prevention and treatment of breast cancer. This author believes that PRMs should be investigated more intensely.

Key words: , PRMs, antiprogestins, mammary carcinogenesis, biomarkers

1. The case for progesterone receptor modulators drivers. Progesterone appears to be one of those drivers (PRMs) for breast cancer but peptide growth factors including EGF and IGF- PRMs have shown benefit in women with uterine 1 need to be considered alongside (Dressing leiomyomas and and those successes and Lange, 2009). We have always known that in clinical trials have led to the realization that the hormone-responsive therapies have not been enough and antiproliferative and pro-apoptotic actions seen may find the current evaluation of new disease usually requires the use in other progesterone-sensitive tissues. As a prime evaluation of Her2/neu status, nodal involvement, tumor developer of a lead PRM, we (Repros Therapeutics) stage and grade, some assessment of proliferation such as soon realized that those young women who used a Ki67, and other markers with many patients opting for an PRM for treatment of fibroids or endometriosis would analysis of tumor markers such as Oncotype DX. Luminal face an extended treatment period of months, if not A is the term we now use to distinguish those tumors with years. Those women needed assurance that the use of the best chance for antihormone treatments, although these hormonal agents was not going to increase their the presence of Her2/neu (Luminal B) adds an additional risk for breast cancer, particularly so in an era when layer of treatment for tumors that appear to be hormone- seemingly innocuous treatments such as hormone responsive. The coming age of individualized medicine for replacement therapy (HRT) with progestins have been breast cancer would seem to require the use of multiple shown to increase the risks of breast cancer (Rossouw markers. et al., 2002; Chlebowski et al., 2003; Rohan, et al., We are accustomed to the linkage of breast cancer with 2008). Any possibility that risks could be mitigated and estrogen and its treatment with agents that oppose estrogen. thus prevention realized would be most welcome, to say This has been a tremendous medical advance for those the least, for women taking a new PRM. Indeed, this women whose breast cancers present as estrogen receptor ‘new world’ of treatments would present a new way of positive (ER+) status at biopsy and surgery. It is known looking at the problem. This paradigm requires giving that progesterone receptor presence (PR+) is estrogen- up a purely estrogen-based or estrocentic view of breast regulated and its function in normal and tumor cells cancer in favor of one that introduces other hormones as requires estrogen and ER signaling (Chabbert-Buffet et al., * Disclaimers: Ronald D. Wiehle is Vice President for Research & Development and a stockholder in Repros Therapeutics Inc. ** Correspondence: [email protected] 772 WIEHLE / Turk J Biol

2005). As a result of endocrine therapy with tamoxifen, ER 299, and ORG 31167, among others. The caution displayed and PR expression may decrease in breast cancer cells, but by major American pharmaceutical companies in the complete receptors loss does not occur, suggesting their 1990s over both the chemistry and the issue of pregnancy potential reactivation and the development of strategies termination seemed to delay further invention on their for their further down-regulation (Bardon et al., 1987). part and we at Repros Therapeutics (then Zonagen, Inc.), The SERMs predominantly affect the ERα isoform, as a small company, were able to move into the space later leading to inhibition of cell cycle progression and eventually with less competition. Another complication was that to cell death (Michna et al., 1989). Given the success of the first compounds were also and SERMS, the analogous and parallel progesterone signal substantial work was done in synthesizing these mixed through PRMs, antiprogesterone therapy would seem to steroids and finding those with activity on one receptor or hold promise even though lagging behind in development. the other, i.e. dissociated binding and biological activity. Importantly, 10 years ago data from the Woman’s Health By 1994, there were many potential PRMs. This entire Initiative (WHI) trial revealed a significant role for story may be best appreciated through a supplement issue progesterone in breast cancer development and growth. to Human Reproduction and a monograph (Beier and Healthy postmenopausal women treated with the Spitz, 1994) published in 1994 based on a symposium held combination of estrogen (E) and progestin (P) were 24% at Mohonk Mountain, New York, in the summer of 1992. more likely to develop invasive breast cancer and had It was recognized early that larger tumors at diagnosis (Chlebowski et al., 2003). Most activity was unwanted in a potential to be used for notably this effect was not seen in hysterectomized women antiprogestational effects. As pointed out early by Horwitz treated with estrogen. Indeed a nonsignificant reduction (Horwitz, 1993), lower antiglucocorticoid activity can in breast cancer incidence was observed with estrogen enhance the usefulness of any drug by allowing higher alone (Rohan et al., 2008), suggesting a specific tumor- dosage. Thus, the early best candidates were judged against promoting effect of combination E+P therapy. Notably the both activities. As an example, it has been shown that RU antiprogesterone RU486 (mifepristone) has been found to 486 was nearly a 10-fold better binder to the reduce proliferation in normal breast tissue (Chabbert- receptor (hGR) than to the hPR and a 3–4-fold better binder Buffet et al., 2005), and preclinical data support the concept to the hGR than (Kloosterboer et al., 1994). of mammary tumor prevention with antiprogesterone In contrast, he found that ZK 98 299 ( or ZK therapy (Bardon et al., 1987; Michna et al., 1989). As a 299) bound the hGR less avidly but with an approximately whole these data support research into antiprogesterone 5-fold selectivity for hGR over hPR. Nevertheless, both therapy for breast cancer therapy and prevention. compounds went into clinical trials. In the case of ZK 299, phase II trials were terminated early due to liver toxicity 2. The introduction of PRMs found by liver function tests (Robertson et al., 1999). We at The relative lack of PRMs may be one reason that clinical Repros Therapeutics have also found liver toxicity at high progress has been relatively slow. The initial antiprogestin doses as well but believe that an optimal dosing regimen in was RU-486 (mifepristone), which was synthesized in the terms of oral dose concentration as well as an appropriate early 1980s by Roussel-Uclaf and described by Philibert schedule is required. in meetings abstracts in 1981 (Philibert et al., 1981) A spin-off of the Schering AG work on PRMs was the and 1982 (Philibert et al., 1982) and in a monograph in compound , which was developed through TAP 1984 (Philibert, 1984). In comparison, the synthesis of (Chen et al., 2006). Although its development appeared tamoxifen was in 1964 and its properties were described to be intended for fibroids and endometriosis, problems by 1966 (Harper and Walpole, 1966). The extensive with uterine bleeding and endometrial changes interfered antiglucocorticoid action of RU 486 was recognized early. with its further development in a 2005 phase III study. Nevertheless, clinical uses were quickly shown by Baulieu’s It is unknown if there is to be further development for group (Herman et al., 1982). An initial indication was oncologic indications. pregnancy termination and that use was constrained by Another PRM compound developed early was CDB- ethical and political opposition that soon found its way 2914 (Xu et al., 2005). A 2012 report in the New England into the mainstream media (Baulieu, 1994). This did Journal of Medicine (Donnez et al., 2012) highlighted much to delay research and development. An early worker a European study that showed efficacy in women with characterized this as ‘political chemistry’ (Hodgen, 1991). fibroids. This drug is approved for emergency contraception The search for new compounds was joined by the although it is not certain if it may be developed for breast labs of C.E. Cook at Research Triangle Park and by the cancer. European firms Schering AG and Onganon. Noteworthy The compound CDB-4124 was synthesized at the early compounds were RTI 3121-012 (CDB-2914), ZK 98 Southwest Foundation for Biomedical Research under a

773 WIEHLE / Turk J Biol contract from the Contraceptive Research Branch, Center 3 mentioned above. Interestingly, Robertson et al. reported for Population Research, NICHD, and studied initially a partial response in 56% of patients and stable disease in under a contract to Bioquals, Inc. Its chemical similarity 11% but symptoms of adrenal insufficiency (Robertson to RU 486 is through its nor-19 steroidal backbone and et al., 1999). This was perhaps not totally unexpected; a 11b substitution. It differs at the C-21 position (Figure 1). rise in serum was seen when RU 486 was used to Another compound from this first set was CDB-4059. These treat patients with meningiomas (Grunberg, 1994) and compounds and others were patented as composition-of- trials of onapristone in women have been closed down matter. In 1999, Repros Therapeutics acquired rights to at due to liver toxicity. Indeed, potential antiglucocorticoid least 44 compounds that were 21-substituted. One of these effects counterindicate the long-term use of RU 486. In a became the lead compound, CDB-4124. We determined study of women treated with RU 486 for endometriosis early that a mono-demethylated metabolite, CDB-4453, (Kettel et al., 1991), patients were anovulatory as was always found along with the parent in animal and expected (Liu et al., 1987) but serum estrogen levels human serum. The metabolite was found to be active remained consistent with midfollicular phase and there relative to the parent. Neither compound demonstrated was evidence for hypercortisolemia and ACTH excess. significant antiglucocorticoid activity but showed high Although RU 486 can serve as the paradigm for this class activity as an antiprogestin. The comparisons of these 2 of antiprogestins, outcomes must be measured against compounds and RU 486 have been shown by Attardi et al. possible antiglucocorticoid effects (Bertagna et al., 1984). In (2002, 2004). a study of 11 postmenopausal women with advanced breast cancer RU-486 induced a short-term clinical response in 1 3. PRMs in breast cancer trials patient and stable disease in 6 others (Bakker et al., 1990). Early progesterone antagonists showed greater antitumor The side effects of RU-486 in this study were mostly related activity than tamoxifen or high-dose progesterone agonists to antiglucocorticoid properties of the drug and increased and synergized with antiestrogens (Klijn et al., 2000). serum estradiol levels. Previous studies suggested that high Clinical data are meager but at least 5 studies have used dose RU-486 can elevate serum estradiol and progesterone antiprogestins in women with breast cancer. One small levels, impacting endometrial cell proliferation (Kettel et al., clinical trial in women with metastatic breast cancer has 1991). shown that RU 486 has some efficacy against the disease CDB-4124 and its metabolites appear to show (Mandelonde, 1987). Two other studies employed RU less antiglucocorticoid activity compared to RU-486, 486 (Romieu et al., 1987; Klijn et al., 1989) as a second- suggesting an advantage in future clinical studies (Attardi line adjuvant therapy. These first 3 trials demonstrated a et al., 2002, 2004). Despite the beneficial effects seen complete or partial response of 12% and stable disease in 46% in women with uterine fibroids (Wiehle et al., 2008) of 33 patients. Two more phase II trials used mifepristone and endometriosis (Spitz, 2009; Spitz et al., 2009), rare, (Perrault et al., 1996) or onapristone (Robertson et al., idiosyncratic liver reactions at high doses (unpublished 1999) as first-line therapy. Perrault et al. (1996) reported data) suggest that lower doses will be required for clinical values for partial response and stable disease similar to the studies.

CH3 H3C H3C O

N N H2C O H3C OH O H3C C CH3 CH3 CH3 O

CH3

O O

Mfeprstone or RU 486 CDB 4124 Figure 1. Structures of RU 486 and CDB-4124.

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4. PRMs in in vivo experimental models argue for the investigation of combined estrogen and Classical experiments have shown DMBA progesterone signaling blockade. Translational clinical (dimethylbenz(a)-anthracene)-induced breast tumors studies to test the central hypothesis that combined ER are inhibited by ovariectomy and maintained by estrogen and PR signaling blockade is safe and clinically effective in and progesterone (Huggins et al., 1962; Welsch, 1985). treating breast cancer have not been performed. A recent Progestins increase the incidence of spontaneous study has shown that the BRCA1 protein inhibits the mammary tumors in dogs (Frank et al., 1979) and mice binding of PR to certain progesterone response elements (Nagasawa et al., 1988). We found that progesterone can without an effect of the binding of ligand to PR (Katiyar promote DMBA-induced mammary carcinogenesis in et al., 2009). rats (Wiehle et al., 2007). Using progestin receptor knock- Our results in the DMBA tumor model (Wiehle et out (PRKO) mice, PR has been shown to be specifically al., 2007) agree with these trends and extend these data: important for DMBA-induced mammary carcinogenesis PRMs CDB-4124 and CDB-4059 as well as RU-486 shrunk (Bakker et al., 1989). When RU-486 was used in DMBA- established tumors and prevented the appearance of new treated rats and in mice that spontaneously developed tumors within the 28 day window of treatment (Table 1). ER+ mammary tumors, a significant reduction in tumor Rats were given DMBA at 50 days of age. Animals were incidence, multiplicity, and size was observed (Michna et weighed and palpated for any sign of lesions or swellings. al., 1989; Chatterton et al., 2002). In a separate study on the Possible tumors were measured in 2 dimensions. If suspect effects of RU-486 on DMBA-induced mammary tumors tumors grew to a size of 10 mm, the individual animal was in rats, a reduction in tumor multiplicity was found randomized into 1 of 15 groups. We treated the animals in 90% of animals versus 75% of animals treated with for 28 days. Rats treated with vehicle alone had the original tamoxifen (Bakker et al., 1989). The combination of both tumor used to activate their treatment regimen and an agents further increased their antitumor potential. The average of 1.67 more tumors, a total of 2.67 tumors per antiprogestins Org 31710 and Org 31806 (Kloosterboer rat. The addition of progesterone increased the average et al., 1994) appear to be more potent than RU 486. In number of tumors per rat to almost 5. CDB-4124 reduced the case of ZK 299, its full agonism may contribute to its tumor number. Since each animal was enrolled into potency. The combination of tamoxifen and RU 486 is a treatment randomly based on finding 1 tumor of a given potent suppresser of tumor growth analogous to chemical size, we conclude that treatment with antiprogestins at castration or ovariectomy (Bakker et al., 1987). The SERM 3 of the 4 top dose levels reduced the number of tumors 164,384 enhances the ability of onapristone to reduce by suppressing the formation of new tumors during the tumors in the MXT mouse mammary tumor model 28-day treatment period. The tumor-inducing effects of (Nishino et al., 1994). progesterone overwhelmed the tumor suppressing effects Interestingly, Dr Eva Lee of the University of California, of CDB-4124 if the dose of progesterone exceeded the dose Irvine, conducted studies demonstrating that progesterone of CDB-4124 by a factor of 5 or more. We measured tumors provides growth signals for breast tumors in mouse weekly during the trial. The data were corrected to exclude models of spontaneously developing tumors carrying the nonmalignant tumor types. Tumors that increased in Brca-1 mutations (Poole et al., 2006). BRCA1, the protein cross-sectional area by at least 33% were considered to be product of the breast cancer susceptibility gene, is known growing. Those that decreased by approximately 33% were to interact directly with ER and PR and modulates both considered to be regressing. Others were considered stable. ligand-dependent and ligand-independent transcriptional Progesterone treatment resulted in aggressively growing activities of ERa and PR. Mice in which both alleles of Brca- tumors. The proportion of tumors regressing in the group 1 and p53 have been knocked out in the mammary tissue given progesterone (8%) is statistically the same as that of exhibited abnormal proliferation of mammary epithelial the controls but the proportion of tumors growing was 10- cells with increased branching and alveolar formation, and fold higher (80%). On the other hand, RU 486 treatment 100% develop breast tumors in a median of 6.6 months. led to no real difference in the proportion of tumors Poole et al. (2006) found the PR levels were markedly growing or regressing. CDB-4124 at 10 mg/kg body weight elevated in the mutant breast cells due to failure of the reduced the proportion of growing tumors but appeared E3 ligase activity of BRCA1 and subsequent proteosomal to exert its prime effects by increasing the proportions of degradation of PR. Treatment of mice with mifepristone regressing tumors. At high doses, CDB-4124 might have for 5 weeks completely blocked tumor formation. These some agonist activity with respect to growth, albeit not groundbreaking animal studies, epidemiologic data, and approaching that of progesterone. observations of the clinical behavior of ER+ positive breast We inferred that all 3 PRMS (RU 486, CDB-4124, and cancers including the observation that aromatase inhibitors CDB-4059) belong to the same class of agents. However, have heterogeneous effects on circulating progestins at equal dose levels, CDB-4124 and CDB-4059 were more

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Table 1. Effect of selective progesterone receptor modulators on tumors.

Animals Tumor weight Effects on weight

Group TX with tumors in group % Tumors TB/animal mean median* MWW test*

N g G Compared to controls 1 No TX 12 / 13 92 32 4.7 1.91 0.80 vehicle 2 No DMBA 0 / 10 0 0 0 0 0 vehicle 3 RU486 13 / 14 93 27 2.47 1.19 0.16 P = 0.012 10 4 P4 10 / 10 100 49 7.34 1.5 0.5 P = 0.42 10 5 4124 13 / 13 100 20 4.71 3.4 0.125 P = 0.066 20 6 4124 10 / 11 91 18 0.48 0.26 0.075 P = 0.0003 10 7 4124 11 / 12 92 17 4.61 2.98 0.09 P = 0.012 2 8 4124 9 / 11 82 16 0.94 0.53 0.17 P = 0.0009 1 9 4124 12 / 12 100 29 5.1 2.26 0.77 P = 0.99 0.1 10 4124/P4 8 / 10 90 18 0.92 0.46 0.18 P = 0.005 20/10 11 4124/P4 10 / 12 83 14 3.82 2.73 0.185 P = 0.013 10/10 12 4124/P4 10 / 11 91 34 5.8 1.96 0.45 P = 0.19 2/10 13 4124/P4 14 / 15 93 45 4.28 1.19 0.49 P = 0.27 1/10 14 4124/P4 11 / 11 100 41 6.16 1.65 0.49 P = 0.66 0.1/10 15 4059 9 / 11 82 12 1.24 0.93 0.17 P = 0.10 10 effective than RU 486 in provoking tumor regression. 4059 may be as effective. These effects are mimicked by The order of tumor-suppressing activity by these criteria effects on proliferation and apoptosis (see section below). was CDB-4059 ~ CDB-4124 > RU 486. When CDB-4124 Christov et al. used a rat MNU model to show is given alone at moderate concentrations or in excess predicative value in breast cancer prevention/therapy for of progesterone, its effects predominate on growth and compounds that are both antiproliferative and proapoptotic regression. When progesterone is given alone or in excess (e.g. SERMs and AIs) and as compared to castration and over CDB-4124 its effects are strongly growth-enhancing. an aromatase inhibitor (Christov et al., 2007). Tamoxifen The effects of CDB-4124 are consistent with decreasing the had proven itself useful in this model (Christov et al., size of DMBA-induced mammary tumors in the rat. CDB- 2003). We treated nascent mammary tumors with CDB-

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4124 in this breast tumor model of prevention (Wiehle et and 3) suppressing lobular proliferation. These results in al., 2011). In our case, female Sprague Dawley rats at 50 the mammary gland are reminiscent of those seen in the days of age were intravenously injected with N-methyl- mammary gland of PRKO mice (Bakker et al., 1989). CDB- N-nitrosourea (MNU). Randomly selected animals were 4124 also decreased proliferative activity of mammary placed into 2 arms of the study (20 rats per arm) and epithelial cells (Table 3), but did not significantly affect received CDB-4124 at a high (30 mg) or low (3 mg) dose apoptosis. in the form of a subcutaneous pellet beginning 5 days after MNU. A third arm received a placebo pellet. The treatment 5. Evidence for the cellular mode of action: uterine was for 90 days. The occurrence of mammary tumors was proliferation and apoptosis monitored by palpation, starting 4 weeks after carcinogen Under investigation is whether progesterone antagonist administration. Paraffin tissue sections of tumors were may induce apoptosis by affecting pro- or antiapoptotic stained for identification of tumor morphology as well as proteins. This topic has been covered with respect to CDB- with antibodies for assessment of ER, PR, and proliferation- 4124 in a recent review (Christov and Wiehle, 2012) and and apoptosis-related biomarkers. Blood samples were will be mentioned below for breast tissue. At the same taken for the determination of estrogen and progesterone. time, we believe much may be gained by looking at the As shown in Table 2 and Figure 2, treatment with CDB- effects of PRMs on other progesterone-sensitive tissues 4124 resulted in dose-dependent reduction in mammary such as those in the female reproductive tract. gland tumor size and multiplicity. CDB-4124 prevented the Asoprisnil, a PRM with mixed progesterone agonist/ appearance and reduced the size of tumors in the 90-day antagonist activities, was able to reduce uterine leiomyoma period following carcinogen insult. The higher dose level volume in a dose-dependent manner in the presence of of CDB-4124 was more effective. CDB-4124 treatment follicular phase estrogen (Chen et al., 2006). Studies in vitro had no effect on the serum estrogen level in the animals on cultured human uterine leiomyoma cells and matched compared to controls but the highest dose of CDB-4124 normal myometria showed that asoprisnil decreased significantly reduced circulating progesterone by 45%. the PCNA-positive rate and PCNA protein expression Thus, the effects were not due to estrogen suppression. in cultured leiomyoma cells. Asoprisnil increased the In the course of drug development, Repros TUNEL-positive rate, cleaved caspase-3, and cleaved Therapeutics conducted a 2-year carcinogenesis study poly (adenosine 5’-diphosphate-ribose) polymerase (MPI1155-006). The mammary gland of animals treated expression and decreased Bcl-2 protein expression in for 2 years (the mean lifetime) with CDB-4124 showed cultured leiomyoma cells. These effects were dose- and fewer tumors and abnormalities than controls. However, time-dependent. However, in cultured myometrial cells, placebo-treated animals developed spontaneously asoprisnil did not affect cell proliferation and apoptosis. hyperplastic and premalignant mammary lesions, as CDB-2914, another SPRM, was also shown to increase well as cystic formations (Wiehle et al., 2011). This was apoptosis and suppress cell proliferation in leiomyomas essentially the opposite of the aim of this carcinogenicity (Xu et al., 2005). In that study cultured human uterine study, which was to disclose overt tumor development leiomyoma cells were subcultured for 120 h and then across the animal organs and lifetime. We reviewed stepped down to serum-free conditions for 12, 24, 48, and slides for mammary gland histology, proliferation (Ki-67 96 h in the absence or presence of graded concentrations staining), and apoptosis markers from these tissues. Based of CDB-2914 between 10–6 and 10–9 M. Compared with on these data, CDB-4124, when given for 2 years to female untreated control cultures, CDB-2914 decreased the Sprague Dawley rats, remodeled mammary morphology number of viable cultured leiomyoma cells and the by: 1) decreasing lobular differentiation of mammary PCNA-positive rate in those cells and increased the epithelial cells, as shown by a prevalence of single lobules; TUNEL-positive (apoptotic) cells in a dose-dependent 2) stimulating cystic formations in the mammary gland; manner. Western blot analysis revealed that treatment

Table 2. CDB-4124 inhibits the incidence, multiplicity, and weight of mammary tumors.

Groups Dose-mg Animals-no. Incidence-% Multiplicity Burden-g 1 0 20 85 3.0 2.60 2 3.0 20 60 2.2 0.62* 3 30.0 20 35* 1.1* 0.26*

*, significant (P < 0.05).

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2.50 Table 3. Effect of various agents on proliferation in cynomolgous monkeys. 2.00 Uterus Breast

1.50 Epithelium Stroma 1.00 TXT BrdU-% BrdU-% BrdU-% Tumors per rat 0.50 Control 10.0 ± 2.5 2.6 ± 0.6 2.4 ± 1.1 Lupron 3.1 ± 0.8* 2.2 ± 1.0 0.3 ± 0.1* 0.00 35 42 49 55 59 62 66 71 76 80 84 Autopsy RU 486 12.6 ± 1.8 3.1 ± 1.0 0.9 ± 0.3 Days post-MNU CDB-4124 2.1 ± 2.2* 1.1 ± 0.25* 1.9 ± 0.7 Figure 2. CDB-4124 inhibits the multiplicity of mammary * P < 0.05, compared to controls (t-test). tumors. The filled squares are animals receiving control pellets. The filled diamonds are animals receiving pellets containing 3 mg of CDB-4124; the filled triangles are animals receiving 30 mg the progesterone activation required growth factor action of CDB-4124. rather than classical action through the PR. The lack of a coactivator specific for growth in these tumors due to culturing remains possible although an unsatisfying with CDB-2914 significantly decreased PCNA and Bcl- answer and one difficult to answer. Subtle changes at the 2 and increased cleaved caspase-3 and cleaved PARP cellular level can have major effects. The phosphorylation expression in a dose-dependent manner, suggesting the and sumolyation of PR by intracellular mechanisms can involvement of apoptosis in leiomyoma growth inhibition impact PR action (Daniel and Lange, 2009). Fibroids are and disintegration. understood to be clonal in nature and thus may vary from The effects of CDB-4124 on cell proliferation and one cell line to another. In any case, this lack of effect may apoptosis were assessed in cultured human uterine emerge for some patients under treatment for fibroids with leiomyoma smooth muscle (LSM) cells and control a PRM. We know not all fibroids respond the same way. myometrial smooth muscle (MSM) cells in matched This may justify thoroughly characterizing any subject uterine cells (Luo et al., 2010). CDB4124 (10−8–10−6 M) or on a PRM with a heterogeneous response, i.e. if a subject vehicle were added to the culture medium for 24, 48, or with multiple fibroids shows a different response for her 72 h. CDB-4124 significantly decreased PCNA antigen, individual fibroids. the number of viable LSM cells, and the antiapoptotic PRMs may affect not only the growth and progression protein Bcl-2. CDB-4124 also increased apoptosis related of leiomyomas but also suppress clinical symptoms biomarkers cleaved polyadenosine 5′-diphosphate-ribose of endometriosis. However, little is known on the polymerase and the tumor suppressor Krüppel-like morphological base of endometrial changes and the transcription factor 11 in a dose- and time-dependent biomarkers mostly affected. Recent studies by a panel manner in LSM cells. In matched MSM cells, however, of pathologists have found that women treated with CDB-4124 did not affect cell proliferation or apoptosis, various PRMs given at different doses for different time indicating that this PRM selectively targets leiomyoma but intervals develop cystic glandular formations previously not normal uterine smooth muscle cells. not known. They also reported apoptotic cells in cystic Not all cell systems and conditions show these effects formations, suggesting antiprogestins may induce (Roeder et al., 2011). In order to investigate the role of apoptosis in endothelial cells and thus suppress their apoptosis in mediating the effects of PRMs on uterine proliferation (Mutter et al., 2008). In a recent study 58 leiomyomas, PR+ fibroid cells, known to be capable premenopausal women with endometriosis or uterine of apoptosis, were grown to 80% confluence in serum leiomyomas were treated for 3 or 6 months with different containing and treated for 48 h with 0, 10, 100, or 1000 doses of CDB-4124. Endometrial biopsies were taken and nmol/L CDB-4124. However, no evidence for increased were independently examined by 3 pathologists (Ioffe apoptosis or elevated caspase-3, determined by Western et al., 2009). They confirmed previous observations for blotting, was found. The reasons for this lack of effect in cystic formations among endometrial glands and reported this system were not identified. It was also unexpected lack of endometrial hyperplasia or carcinomas in treated in the face of clinical and in vitro data to the contrary. patients. The condition of months-long PRM treatment It is possible that the cells employed were resistant to did not provoke a condition similar to unopposed estrogen PRMs, although they were PR+. It is also possible that but rather to what they characterized as PAEC, PRM-

778 WIEHLE / Turk J Biol associated endometrial changes, characterized by rare Table 4. Effect of various agents on apoptosis in cynomolgous mitoses, atrophic/cystic structures, and apoptotic bodies. monkeys. We have compared the effect of RU486, CDB412, and Lupron (a GnRH agonist often used to treat human Uterus Breast endometriosis) on the endometrium of cynomolgous monkeys (Macaca fascicularis). This was done to model Epithelium Stroma endometriotic lesions of women and disclose effects of TXT Apo % Apo % Apo % PRMs. The peritoneal cavity of each monkey received Control 0.2 ± 0.1 0.7 ± 0.2 0.5 ± 0.3 minced pieces of the monkey’s own endometrium. Certain Lupron 0.2 ± 0.1 0.2 ± 0.1 1.4 ± 0.7 of these fragments attached and established themselves in the cavity as lesions resembling endometriosis in size and RU 486 0.5 ± 0.1* 0.5 ± 0.1 1.2 ± 0.6 appearance. Groups of 7 monkeys were treated orally for CDB-4124 0.5 ± 0.2* 0.5 ± 0.1 2.6 ± 0.9* 36 weeks with CDB-4124 at 3 different doses of RU-486 or with placebo. Another group received Lupron IM. Animals * P < 0.05, compared to controls (t-test). were inspected visually after 18 and 36 weeks of treatment and sacrificed at the end of the study. Monkeys receiving suggested a set of pathways for regulation that included Lupron, RU486, and CDB-4124 clearly stopped cycling MAPK-cyclin D1. The authors thought the fact that these and had lowered levels of progesterone. Lupron decreased cells were ER– may have been a major contributor to the serum estrogen compared to control and pretreatment effects seen, an effect that implied cross-talk between ER follicular phase levels but serum estrogen levels were not and PR in a cellular system with converging signals. different in the monkeys receiving CDB-4124 and RU The use of xenographs of breast cancer is a way of 486. Although showing little change in size, lesions in looking at human cancer cells in an animal model. Liang monkeys treated with CDB-4124 differed in appearance, et al. (2007) used BT-474 and T47D cells without Matrigel occurring often as clear cysts. Three monkeys in each as xenographs in nude mice. These cells will regress group were injected with BrdU within 24 h of sacrifice to soon after inoculation but that regression is held up if assess tissue proliferation. Full thickness uterine sections progesterone or medroxy-progesterone acetate is given. were stained and examined microscopically for evidence The effects are sensitive to RU 486. Most interesting is the of proliferation in terms of the % cells incorporating BrdU apparent dependency on VEGF. (Table 3). Lupron and CDB-4124 decreased proliferation When given by subcutaneous injection over a wide in the epithelial cells of the endometrium and CDB- range of concentrations, CDB-4124 suppressed tumor 4124 also decreased stromal proliferation. RU-486 did growth of DMBA-induced mammary tumors in rats, dose- not decrease proliferation in either cell type. RU-486 and dependently as already described (Wiehle et al., 2007). CDB-4124 increased apoptosis in the epithelial cells of Progesterone, on the other hand, stimulated DMBA- the endometrium and CDB-4124 also increased apoptosis induced mammary carcinogenesis and tumor growth and in the breast (Table 4). Lupron did not alter apoptosis in this was associated with increased cell proliferation and either cell type. No treatment appeared to alter the stroma. decreased apoptosis, suggesting that PRMs may act as This dichotomy between RU 486 and CDB-4124 in terms inhibitors of apoptosis. CDB-4124 significantly decreased of proliferation may indicate a functional difference cell proliferation in DMBA-induced mammary tumors, as between these 2 antiprogestins. evaluated by Ki-67 antibody reactivity (Table 5). In order better to understand cellular mechanisms 6. Cellular mode of action: mammary proliferation and of CDB-4124-induced inhibition of mammary apoptosis carcinogenesis, proliferation activity and apoptosis were By employing MDA-MB-231 cells that are ER– and PR–, examined in mammary tumors from the Sprague Dawley researchers transfected cells with PR-complementary rats treated with N-methyl-N-nitrosourea (MNU) (Wiehle DNA (Lin et al., 1999). They found that PR, in the et al., 2011). As mentioned above, treatment increased absence of ER, affected cell morphology and cell latency, and, as shown in Table 2, decreased incidence, differentiation biomarkers. Progesterone induced multiplicity (Figure 2), and tumor burden. Decreased E-cadherin, cytokeratins, vimentin, and STAT 1, 3, 5a, size of mammary tumors was a consequence of decreased and 5b expression, all cell differentiation biomarkers. The cell proliferation and induced apoptosis. Apoptosis was more interesting effect was the negative effect on growth. evaluated by TUNEL assay and cell proliferation by Ki- Follow on studies (Lin et al., 2003) indicated inhibition 67 antibody that recognized the cells in all phases of the of the p42/44 MAPK pathway, an effect unlike previous cell cycle. As shown in Table 6, CDB-4124 significantly work they cited where progestin activated MAPK. This suppressed Ki-67 positive cells in mammary tumors, from

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Table 5. Effects of CDB-4124 on Ki-67 on rat DMBA tumors.

Compared to Group Treatment % cells positive n controls (t-test) 1 Control tumors 13.5 ± 7.8 12 3 RU 486 (10 mg/kg) 12.9 ± 7 P = 0.85 10 4 P4 (Progesterone) (10 mg/kg) 25.7 ± 5.8 P = 0.0007 9 6 4124 (10 mg/kg) 5.1 ± 4.2 P = 0.00 7 11 4124 + P4 (10 + 10) 15.5 ± 12.2 P = 0.66 8

ANOVA = 0.0001 [P4 > Control, RU486, 4124 + P4 > 4124].

Table 6. Effects of CDB-4124 on cell proliferation and apoptosis in mammary tumors.

Treatment Dose mg/kg Animals no. Ki-67 LI% P Apoptosis LI-% P__ Control 0 19 30.5 ± 7.1 0.7 ± 0.4 Proellex 3.0 20 25.4 ± 14.4 1.4+0.8 0.05 Proellex 30.0 20 10.3 ± 4.5 0.001 1.6 ± 0.8 0.01

The differences in the values are significant (P < 0.05) as compared to those of the control animals (Student-Fisher t-test).

30.5 ± 5.4% in the placebo group to 25.4 ± 14.4% in the treatment (Engman et al., 2008). A significant reduction low-dose and further to 10.3 ± 4.5% in the high-dose in proliferating breast epithelial cells (Ki-67 positive) was group of treated animals. Both doses of CDB-4124 induced observed in RU-486 treated vs. placebo treated patients, apoptosis in mammary tumors, where 0.7 ± 0.4% apoptotic suggesting that antiprogestin treatment can prevent the cells were detected in the control group vs. 1.4 ± 0.8% in development and progression of ER+ and PR+ breast the low-dose and 1.6 ± 0.8%, in the high-dose group. These cancer by inhibiting mammary epithelial cell proliferation. data correlated with decreased proportion of PR+ tumor Apoptosis was not examined in these patients. cells and with decreased serum progesterone. CDB-4124 did not affect serum estradiol. We examined PR expression 7. Humoral mode of action: serum steroids in mammary tumors by immunohistochemistry. CDB- One of the observations we made purposefully was the 4124 decreased the proportion of PR+ cells from 48 ± 11% effect of CDB-4124 on serum steroid levels. A frequent in placebo treated to 32 ± 12% in CDB-4124 (30 mg/kg) finding was the presence of estrogen but a loss of treated animals. Although trending lower, CDB-4124 did progesterone along with cycling in animals and menstrual not significantly affect the proportion of ER+ cells in the cycling in women. In an unpublished safety study (ZP- samples. 204), we determined that normal young women who were Other work has shown the link between PRMs and being assessed for evidence of liver toxicity provided little apoptosis. CDB-4124 suppressed the growth of ER- and evidence for LH surges or serum progesterone over 10 PR-expressing T47D cells and induced apoptosis (Wiehle weeks of treatment dependent on dose. There was some et al., 2011). Cells were treated with 0, 0.1, 1.0, and 10.0 µM evidence of cycling at the lowest oral daily dose, 1 mg/ CDB-4124 for 3 days or 6 days, and cell number in triplicate day, but none at daily at doses of 3 mg, 6 mg, 9 mg, or 12 was determined by cell counter. In addition, caspase 3 as a mg. Serum progesterone in women at visit 12, i.e. in the biomarker of apoptotic cell death was determined in cells last week of active dosing, as compared to the follow up grown on cover slips. CDB-4124 at 1.0 µM and 10.0 µM (FU) month when they were no longer taking the drug, suppressed cell growth in a dose-dependent manner and demonstrated a major difference (Figure 3). Even though the high dose induced cell death in both 3- and 6-days these women could no longer be considered cycling by treated cells. vaginal bleeding or LH surges, it could be argued that we Women with leiomyoma treated with 50 mg of RU- “missed” the luteal phases at visit 12. This seems unlikely to 486 every second day for 3 months underwent fine needle be true for all groups and all individuals in the face of the breast biopsies before initiation and after termination of FU subjects who demonstrated serum progesterone levels

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E ect of Proellex on Serum signal-modifying agents could break out of the current Progesterone dilemma where estrogen therapies fail because tumors 8 have circumvented antiestrogen or estrogen depletion 7 treatments. First line therapy for women with ER–/PR+ tumors should be considered as well, even though the 6 number of women with such tumors is small. Window- 5 of-opportunity trials such as currently underway at 4 Northwestern under Seema Khan (NU 12B09, Pre-surgical

gesterone ng/mL Phase IIB Trial of Oral CDB-4124 vs. Placebo in Women ro 3 V12 with Stage I-III Primary Breast Cancer) can highlight 2 rum p FU possible efficacies while mitigating risk. Neoadjuvant use Se 1 may find a place if strong antiproliferative effects can be documented. 0 0 3 6 912 We would propose a chronic prevention paradigm. mg of daly Proellex for 10 weeks The lesson taught here is from both the DMBA and the MNU studies in rats where potential tumors seem to be Figure 3. Serum progesterone in women in the last week of active dosing, as compared to the follow up (FU) month when less likely to be detected by difference between groups. The they were no longer taking the drug. effects seen on the mammary gland itself, on hormones, and proliferation/apoptosis are provocative and may be predictive. Taken together, these data indicate that typical of the luteal phase and with a mean progesterone CDB-4124, among others, can suppress the development level in excess of 3 ng/mL. Other studies showed similar of precancerous lesions and ER+ mammary tumors effects at higher doses. in rats, and may have implications for prevention and While we were not surprised at the effects at the higher treatment of human breast cancer. Women treated with doses, the efficacy at the lower doses was unexpected. This PRMs for benign gynecologic indications (fibroids and suggested a sensitivity of CDB-4124 towards provoking endometriosis) for relatively long periods are a set of meaningful changes in the gonadotropin hormones that individuals who may be monitored. are responsible for cycling and for hormones, especially We would propose the inhibitory effect of CDB-4124 progesterone from the corpus lutea. This had been seen in on mammary carcinogenesis to be a consequence, in part, early studies of RU 486 (Ortmann et al., 1994). The effects of decreased progesterone action in the mammary gland. on cycling suggest a central effect on the hypothalamus/ Perhaps this was shown already by the changes in ductal- pituitary with major effects that result in acyclicity. The lateral branching or stem cells, which is promoted by hypothalamus receives multiple inputs and the KNDy physiological levels of serum progestins. During pregnancy neurons of that tissue possess both ER and PR receptors, the high blood levels of progesterone stimulate ductal- allowing them to respond to both up- and down-regulatory lateral branching, leading to lobular development and milk signals and control LH release by the pituitary by way of production. This has been supported by recent data on kisspeptins and/or GnRH (Skorupskaite et al., 2014). BRCA1/p53-transgenic mice that have shown that RU-486 We believe that the effects on serum progesterone suppressed mammary carcinogenesis by inhibiting ductal- constitute an additional source of effects on sensitive lateral branching and lobular differentiation of mammary tissues. This drug-induced hormonal suppression broadly epithelial cells (Poole et al., 2006). These data also suggest affects progesterone action. PRMs should be more effective that patients with mutations in BRCA1 may benefit from when the level of ovarian progesterone is low. Thus, a PRM antiprogestin therapy in breast cancer prevention and like CBD-4124 attenuates its own effects by reducing the treatment studies. In a different study, PRKO mice have competition from endogenous progesterone. also shown distinctive mammary gland architecture with the presence of ducts, but lack of alveoli and lobules 8. Conclusions (Bakker et al., 1989). With the cessation of breast feeding We would propose a second line adjuvant therapy for and drop in the progesterone circulation level, apoptosis patients with hormone-dependent breast cancer for plays a critical role in disintegration of these structures. women who fail estrogen-based therapies. That could be Castration of mice and rats similarly induces apoptosis composed of the use of a progesterone antagonist such as and reduction of mammary lobular structures. CDB-4124. This is supported by the DBMA results in rats and by the studies demonstrating effects on proliferation 9. New Directions and apoptosis in many systems. CDB-54124 when used Future studies should also address the role of CDB-4124 alone or in combination with other growth factor or cell- alone, or in combination with tamoxifen or other SERMs,

781 WIEHLE / Turk J Biol with agents like fulvastrant, and aromatase inhibitors One issue that is specific to the use of PRMs and on the modulation of ER and PR signaling. The potential presents a new situation is the realization that women taking involvement of specific coactivators and corepressors will a drug like CDB-4124 achieve a long-term state of low need to be pursued (Katzenellenbogen et al., 1996) as well as progesterone, a progestopenia. Given that that a PRM usually the challenging proposals that PR can work at the same sites has antagonist activity, perhaps the term aprogestogenic state as ER or cross-talk with the ER or other nuclear receptors is more appropriate. This is going to be the case for young (Daniel et al., 2014; Lin et al., 2003; Vicent et al., 2006). The women regardless of whether they are under treatment for involvement of peptide growth factors such as EGF and benign gynecology or cancer. As pointed out, this lowering IGF-1 needs to be addressed (Dressing and Lange, 2009). of progesterone contributes to drug efficiency but may have Interaction with the membrane PRs (Charles et al., 2010) that downsides. Pre- and postmenopausal females have neither provoke early responses is entirely open to investigation. The high estrogen nor progesterone levels. The absolute level development of resistance in ER+ breast carcinomas after that characterizes partial from total lack of progesterone will be established fully as we utilize the more sensitive LC- treatment with tamoxifen may offer additional possibilities mass spectroscopy for hormone analysis. As this becomes for clinical applications of progesterone receptor antagonists, widespread, we will be able to delineate levels that are alone or cooperatively with other SERMs. We know that meaningfully low and sort out the low levels from the very CDB-4124 is compatible with the use of aromatase inhibitors low levels with reference to physiologic effects. At the same from in vitro studies (Gupta et al., 2013). Any potential that time, women on PRMs should retain tonic levels of estrogen progestins play a role in BRCA-1/-2 cancer is a high priority in the absence of progesterone. While this might suggest the given the few choices faced by those women and the results establishment of a state of unopposed estrogen, the direct that suggest the involvement of progesterone in that process. effect of PRMs on the endometrium producing PAEC (Ioffe The main challenges for clinical applications of PR et al., 2009) avoids unopposed estrogenic stimulation. modulators are their potential toxicity and their potential The downsides to low estrogen for young women are effects on the modulation of . The topical considerable in terms of well-being and include vaginal use of PRMs for breast cancer, an approach much like the dryness, hot flashes, and bone loss. The downsides to low vaginal suppository use for gynecologic disorders currently progesterone are somewhat harder to identify. These women underway in clinical trials at Repros Therapeutics, is one are amenorrheic but the condition is not unprecedented. way to avoid the first pass through the liver and metabolism Certain kinds of popular oral contraceptives allow ovulation and liver toxicity. and menstruation 3 to 4 times a year, a situation not unlike Development of novel PR modulators with high receptor the use of CDB-4124 with off-drug holidays. Anovulation is binding affinity and low incidences of toxic effects is highly the downside; these women are likely to be contracepted but desirable for further study in the prevention and treatment without the estrogen that is in the usual oral formulation. This of breast cancer. For completely new chemical entities to may not a problem for women who smoke and are in danger be brought out, agents should be evaluated with tools that of clotting disorders: these individuals would welcome a new will allow us to recognize their activity in breast tissues and contraceptive that does not contain an estrogen. In a world breast cancer. The determination of their binding in cell- where women seek oral contraception, another form is not free extracts of PR+ tissues has always been a first step, as a drawback unless lowering progesterone itself and blocking has the use of reporter assays that show potential activation residual progesterone activities have negative effects. This of a convenient gene through a proximal promoter that is should be an area of investigation to be followed. In the last 10 years, it has become recognized that responsive to progestins. The classical way to differentiate play a role in mammary proliferation and new PRMs has been to test their antiprogestational activity in breast cancer presents a new challenge. Taken on the rabbit uterus. Given the nature of tissue-specificity inherent broadest possible terms, monthly cyclic progesterone and, in in PRMs, a method that demonstrates direct effects on breast the modern world, exogenous progestins seem to pose a risk tissue would be preferred. The xenographic model of Liang et for breast cancer. Is breast cancer in older women the price al. would be favorable (Liang et al., 2007). The strong effects women pay for being fertile when they were young? If that seen in virgin mammary glands of mice and rats suggest that were the trade-off, widespread fertility trumps the marginal the PRM could be disclosed in that living system as well. risk of dread disease after the child-bearing years, taking the Clearly, the tissue specificity of PRMs and the discovery of long view of history with a nod to evolution. Certainly, given specific progesterone-proliferative modulatory genes and that Homo sapiens spent most of its history in small isolated their suppression by PRMs would be a considerable advance. groups where child-bearing and nurturing were existential, This adds not only to the rationalization of progesterone’s the deadly bargain makes a kind of perverse sense. However, effects in the breast and breast tumors but also the increases it is not a good bargain for women in our times and perhaps the possibilities that markers of progestin and PRM action there is an opportunity to cheat that fate, if that is indeed can be discerned. what we face.

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