Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression

The progesterone receptor (PR) with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4) and ulipristal acetate (UPA), a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL 2 -L 1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required.


Introduction
Breast cancer, the most frequent cancer in women, is a hormone-dependent disease, with over 70% of sporadic breast tumors expressing estrogen and/or progesterone receptors (PR) [1]. Systemic anti-hormonal treatments used in clinical practice target the estrogen signaling PRA mediated anti-proliferative effect and could be of interest in the management of human breast cancer patients.

Quantitative Real Time PCR
Total RNA was extracted from cells with TRI Reagent (Applied Biosystem) according to the manufacturer's recommendations. One μg of total RNA was processed for reverse-transcribed-qPCR. Briefly, total RNA was treated with DNase I (Biolabs, Evry, France), then reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). cDNA were analyzed by quantitative RT-PCR using the Power SYBR Green PCR Master Mix (Life Technologies) with the indicated primers (300 nM, final concentration) (S1 Table) and a Ste-pOne Real-Time PCR System (Life Technologies). Relative gene expression was calculated as a ratio of attomoles normalized by ribosomal rRNA 18S expression in femtomoles or by glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) in attomoles.
Control antibodies: anti-α-tubulin antibody (Sigma) and anti-GAPDH (Sigma). Secondary antibodies: Goat anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody (DyLight 800 conjugated, 680 conjugated) and Goat anti-Rabbit IgG (H+L) DyLight 800 conjugated, dilution 1:10,000 (Thermo Fisher Scientific, Rockford, IL, USA). These antibodies were diluted in Phosphate Buffered Saline and 0.1% Tween 20 buffer supplemented with 5% nonfat milk and added to the membranes for 1 h at room temperature or overnight at 4°C followed by incubation with the indicated secondary antibody for 1 h at room temperature. Target proteins were detected using Odyssey1 Fc, Dual-Mode Western Imaging (Li-Cor, Lincoln, NE, USA) by Fluorescence and quantified.

Primer design for genomic amplification assays
Design of primer pairs containing PRbs (Progesterone receptor binding sites) was achieved with the NCBI software Primer BLAST. Human genome was used as database (hg19). Amplicon length was kept between 80 and 150 bp. Primers were from Eurogentec (Angers, France).

Statistical Analysis
All data are mean ± SEM. Non-parametric Mann-Whitney statistical U-tests and ANOVA test (Kruskal-Wallis) were applied to determine all significant differences between experimental conditions, using the software Prism 5 (GraphPad Software, San Diego, CA). Statistical significances are indicated by Ã or x (1-3 symbols corresponding to P<0.05 or <0.01 or <0.001, respectively).

PRA mediated MDA-iPRAB Cell Proliferation
We used the recently established bi-inducible MDA-iPRAB cell model to study the impact of PR isoform on human breast cancer cell proliferation [6]. This model results from genetically modified MDA-MB-231 human breast cancer cell line (PR -), generating various stable cell lines, MDA-iPR -, iPRA and/or iPRB cells. Two non-steroidal inducible systems, Rheoswitch and T-Rex were inserted into the same plasmid pZX-TR [6] (S1A Fig) and transfected into MDA-MB-231 cells generating parental and stable "clone 250" cell line. Subsequently, clone 250 cells were stably transfected with the bi-inducible system, pRheoswitch-PRA and pTRex-PRB, as schematically depicted on Fig 1A and S1B Fig. This bi-inducible system allows a bi-conditional expression of both PR isoforms, PRA at 94 kDa and/or PRB at 114 kDa, upon addition of RSL1 and/or Doxycycline (Dox) for 24 h, respectively (Fig 1B), while in the non-induced DMSO vehicle condition, no PR isoform expression was detected.
Given that the impact of P4 on cell proliferation was reported to be highly dependent on the cellular context [5,29], we examined the influence of P4 on MDA-iPRAB cell proliferation. Unexpectedly, and unlike what we had published before with P4 at 10 nM, here we showed through repeated cell proliferation tests that P4 (1 nM) significantly increased MDA-iPRA cell proliferation at Day 5 ( Fig 1C, S2A Fig). Of note, no effect of RSL1 and P4 was observed on cell proliferation of the parental clone 250, excluding a potential non-specific effect of these compounds ( Fig 1C, left histograms). Moreover, P4 did not induce cell proliferation when none of the PR isoform was induced. In contrast, MDA-iPRB cell proliferation was significantly  Anti-proliferative effect of UPA on MDA-iPRA cells We next examined the effect of the SPRM, UPA, on MDA-iPRA cell proliferation (Fig 2) and found that UPA at 1 μM alone or in association with P4 significantly inhibited cell proliferation as early as Day 3 (see also S2A Fig). This was consistent with our previous report showing that RU486 (Mifepristone) inhibits MDA-iPRA cell proliferation [6]. Of note, in the absence of PR, UPA has no effect, while UPA inhibited PRB-expressing cells and PRAB-expressing cells proliferation (S2B and S2C Fig). UPA has synergistic effects with P4 in MDA-iPRB cells.
Taken together, we reported the first cellular model in which P4 triggers PRA mediated human breast cancer cell proliferation in vitro. In this model, we unambiguously demonstrated that PRA isoform mediated P4-induced cell proliferation that was antagonized by UPA, highlighting the anti-proliferative role of UPA on human breast cancer cells.
Key PRA target genes involved in human breast cancer cell proliferation: Effects of P4 and UPA We have previously identified by microarray analysis on the same MDA-iPRAB cell model, a subset of potential target gene candidates regulated in a P4-dependent and/or PR isoform specific manner [6]. Given the importance of PRA isoform, its impact on P4 signaling in mammary tumorigenesis processes such as cell growth, death and migration, and taking into account its role in mediating cell proliferation, we chose to focus our attention on some PRAdependent upregulated (Table 1) or downregulated genes (Table 2) in the presence of P4. It would be thus possible to correlate P4-stimulated MDA-iPRA cell proliferation to the corresponding gene expression and to further evaluate the activity of the antiprogestin UPA in these processes. Among PRA-targeted genes transcriptionally stimulated (Fig 3) or inhibited by P4 (Fig 4), we examined the expression of few genes (BCL 2 -L 1 , DUSP6, WNT5A, LGR4, TGFβ2, EREG, F3, F2RL1), owing to their role in apoptosis, cell proliferation, metastasis, invasiveness, angiogenesis (Tables 1 and 2). We first validated the regulated expression of these genes by RT-qPCR analysis, after a 6 hr treatment by P4 following the same experimental duration as previously described [6]. We found that P4 significantly enhanced expression of BCL 2 -L 1 by 25% (Fig 3A), that of DUSP6 by 50% (Fig 3B), that of WNT5A by 30% ( Fig 3C) while LGR4 expression was increased by 60% ( Fig 3D). Interestingly, we demonstrated that the expression of these P4-upregulated genes was repressed by UPA alone or in combination with P4, confirming the antagonistic activity of UPA (Fig 3).
In contrast, P4 treatment significantly reduced the expression of TGFβ2 by 60% (Fig 4A), and that of EREG, F3 and F2RL1 by 70% (Fig 4B, 4C and 4D) in MDA-iPRA cells, as revealed RT-qPCR analysis. Unexpectedly, UPA alone also inhibited the expression of TGFβ2, F3 and F 2 RL 1 (Fig 4A, 4C and 4D) but not that of EREG ( Fig 4B). However, the P4-dependent downregulation of EREG expression was completely prevented by UPA (Fig 4B), consistent with a full PR antagonist activity of UPA. Of note, a strict correlation was observed between gene expression in transcriptomic and RT-qPCR analysis (Tables 1 and 2). The expression of PRA-regulated genes was also analyzed in MDA-iPRB expressing cells. As shown in S3 and S4 Figs, expression of these genes did not vary in the presence of P4 and/or UPA treatment. Thus, we confirmed that BCL 2 -L 1 , DUSP6, WNT5A, LGR4, TGFβ2, EREG, F3, F2RL1 are indeed PRA specific genes and that UPA exerts an inhibitory effect by down regulating their expression only when PRA is expressed (MDA-iPRA).
Collectively, we demonstrated that the selected genes were specifically regulated by PRA isoform in the presence of its natural ligand P4. In addition, we also showed that UPA generally displayed PRA antagonist properties while, in some cases, UPA exerted a partial progesterone agonist activity consistent with the SPRM feature of his compound [11,30].
Regulation of BCL 2 -L 1 protein expression by P4 and UPA in MDA-iPRA cells Since P4 stimulated BCL 2 -L 1 mRNA levels (Fig 3A), we next examined the hormone regulation of the anti-apoptotic factor BCL 2 -L 1 , at the protein level. As illustrated in Fig 5A, the 26 kDa band corresponding to BCL 2 -L 1 protein was significantly increased by 23% in MDA-iPRA cells upon 24 h exposure to P4 as revealed to quantification of band intensities relative to GAPDH (36 kDa) (Fig 5B). We also found that P4-stimulated BCL 2 -L 1 expression was antagonized by UPA treatment which, when applied alone, significantly reduced by 2 fold factor the basal BCL 2 -L 1 expression (Fig 5B). PRA expression was also quantified relative to loading control GAPDH ( Fig 5C). As anticipated, PRA was significantly downregulated upon P4 exposure while UPA alone was ineffective in modulating PRA expression in MDA-iPRA cells. Taken together, we provide direct evidence of P4-stimulated MDA-iPRA cell proliferation, associated with BCL 2 -L 1 expression, both being fully antagonized by UPA.

PRA recruitment on BCL 2 -L 1 gene
To better elucidate the molecular mechanisms by which P4-activated PRA regulated BCL 2-L 1 gene expression in MDA-iPRA cells and based on previous studies performed in T47D cells [27], ChIP assays were performed to examine PRA recruitment on one genomic target motif, already established on BCL 2 -L 1 gene [31]. We chose the intragenic PR-binding sequence located at +58 kb downstream Transcription Start Site (TSS) of BCL 2 -L 1 gene as schematically depicted in the genomic structure of the human BCL 2 -L 1 gene (Fig 6A). P4 treatment induced a significant 40-fold increase in PRA recruitment on the well-documented +58 kb response element of the BCL 2 -L 1 gene, in comparison to the vehicle condition. Furthermore, UPA in combination with P4 markedly reduced P4-stimulated PRA enrichment on this PR-binding site (P< 0.001). Interesting enough, UPA alone was able to stimulate PRA recruitment by a 10-fold factor (Fig 6) as well as a substantial recruitment of the well-known SRC1 coactivator however to a lesser extent than that induced by P4 (2 vs 8 Fold) (S5A Fig). However, under these experimental conditions, as opposed to P4, UPA was unable to promote RNA Polymerase type II

Discussion
We have exploited an original human breast cancer cell-based system in which cell proliferation is stimulated by progesterone through PR, using the recently established bi-inducible, ER negative, MDA-iPRAB cell line [6]. Several breast cancer cell models have been generated to study steroid effects, mostly focusing on the impact of estrogens and anti-estrogens [32][33][34]. The effects of progesterone and progestins on breast cancer cell proliferation in vitro drew contradictory results [35], although proliferative effects of progesterone have been reported in the ER + , PR + T47D and MCF-7 breast cancer cells, without distinction between PR isoforms  (review in [36]). Cell proliferation was evaluated in ER negative MDA-MB 231 cells transfected with each PR isoforms and cell proliferation inhibition by progesterone was observed in both cases [11]. In this report, progesterone was used at 0.1 μM and cells cultured in red phenol free DMEM supplemented with 5% charcoal treated fetal calf serum, while we used 2.5% FCS and 1 nM progesterone [11]. Progestin-stimulated cell proliferation has been reported to depend on progesterone concentration, mode of administration (continuous vs discontinuous) and may involve cross-talks with growth factors [36]. This may explain the discrepancy between our results and this study. We previously published that unliganded MDA-iPRA cells proliferate to a lesser extent than MDA-iPRB cells [6], in this study, we confirmed these findings (data not shown). We repeatedly and reproducibly observed that progesterone stimulated MDA-iPRA cell proliferation in 6 independent experiments (n = 6 per experiment). This is in accordance with PRA cells expressing increased responsiveness to stimulatory effects of progestins, as compared to cells expressing PRB reported by others [36][37]. , were fixed and lysed and chromatin extracts subjected to ChIP assays using PR antibody (Anti-PR, sc-7208, Santa Cruz Biotechnology, CA) or unrelated rabbit IgG antibodies used as negative control. Immunoprecipitated and eluted DNA fragments were analyzed by real-time qPCR using primer pair encompassing genomic sequence of the +58 kb site of the BCL 2 -L 1 gene. Histograms represent the fold induction of PRA enrichment compared to vehicle condition arbitrary set at 1 and are means ± SEM of four independent experiments performed in triplicates. Statistical difference is indicated as compared to vehicle condition for PRA-induced cells treated by V (***, p<0.001), or by P4 (xxx, p<0.001) (Non-parametric Mann Whitney t-tests). doi:10.1371/journal.pone.0140795.g006 One major advantage of our cell-based system is that one might selectively evaluate the effects of PR, its isoforms and ligands independently of estrogenic effects, since proliferation of this ERcell line is estrogen-independent. Moreover, the bi-inducible PR expression by nonsteroid inducers is genomically stable, the non-induced cell line constituting a reliable negative control [6]. We cannot exclude that the absence of ERα (and estrogens) might modify the expression of co-regulators required for PR signaling and thus influencing transcriptional responses to PR isoforms. However data from previous reports did not show any influence of ERα transfection on PR ligands induced morphological changes in PR transfected MDA-MB 231 cells [11]. Taking into account these limitations, our studies provide new insights into the understanding of the functional consequences of imbalanced PRA/PRB ratio in the context of human breast cancer cells.
Interestingly, as opposed to the already known P4-biphasic regulation of cell proliferation observed in estrogen dependent T47D-YB cell line constitutively expressing PR-B [38], we report that P4 stimulates cell proliferation of bi-inducible MDA-iPRAB cells only when PRA isoform is expressed, while P4 exerts an anti-proliferative effect in PRB-expressing cells, without any biphasic effect. This underlines intrinsic differences in cell lines, in particular the MDA MB cell line is derived from a basal breast cancer [39] while the T47D cell line is derived from a luminal breast cancer [40].
In this model as well, PRA and PRB exhibit distinct transcriptional activities and regulate specific gene transcription. We have previously shown that PRA coexpression potentiated PRB-mediated cancer cell migration and that PR isoforms differentially regulated expression of major players of cell migration [41] such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1 (PAI-1), uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. PRA, as well as PRB, interacted with FAK complexes, whose degradation was coupled to a progestin-dependent turnover of PRA and PRB.
Our previous transcriptomic data allowed us to select PRA specific target genes [6]. Among PRA-modulated genes involved in breast tumorigenesis (Tables 1 and 2), we more specifically evaluated by quantitative RT-PCR and confirmed that BCL 2 -L 1 , DUSP6, WNT5A, LGR4 expression were upregulated in the presence of progesterone, and that TGFβ2, EREG, F3, F2RL1 were down regulated in the presence of progesterone. BCL 2 -L 1 is one of the most important regulators of programmed cell death. Deregulation of BCL 2 -L 1 expression contributes to oncogenic transformation of normal cells [42][43]. In our MDA-iPRA model, P4 stimulates BCL 2 -L 1 transcript and protein expression, while BCL 2 -L 1 expression was not regulated by P4 in the MDA-iPRB cell line. Induction of anti-apoptotic factors of the Bcl family by progestins has been reported in endogenously PR-expressing T47D cells [35,44], however the specific role of PR isoforms has not been directly evaluated.
WNT5A and LGR4 regulate cell polarity, proliferation, survival and aberrant regulation often leads to pathological conditions including cancer [45]. In T47D cells, it has been demonstrated that PRB-induced transcriptional up-regulation of WNT1 leading to activation of epidermal growth factor receptor (EGFR) and c-Src and Erk1/2 [46]. Here, we report that PRA mediates the P4-induction and UPA-inhibition of the expression of both WNT5A and LGR4, suggesting an essential function of PRA-mediated, P4 triggered-breast cancer cell proliferation.
DUSP6 (the dual specificity phosphatase 6, MKP3) is a negative regulator of the MAPK pathway [47]. Previous studies demonstrated a direct interaction between PRB and DUSP6 [47], our study confirms that DUSP6 is a PRA-target gene up-regulated by P4 in MDA cells.
TGFβ2 and EREG (Epiregulin) were shown to be down regulated by progesterone in the presence of PRA in our model [48][49]. This is in accordance with previous data suggesting that PR isoforms expression correlated negatively with other members of the same gene family associated with tumor aggressiveness such as EREG and TGF alpha [50].
Our results thus suggest a specific role of PRA in breast carcinogenesis, including modulation of regulatory factors of cell proliferation, invasion, apoptosis, and angiogenesis. This may constitute a trail for further molecular characterization of breast tumors, where the evaluation of a panel of specific PRA target genes could be used to further identify poor prognosis tumors. Indeed, breast cancer is now considered as a plural disease whose molecular signature analysis is routinely used to define personalized therapeutic schemes [51].
In addition, our data bring new insights in the isoform specific molecular mechanisms of action of PR and its ligands. It has been shown that over 60% of PR binding sites are located more than 10 kb from the transcription start site [52]. Here we showed that PRA is recruited to an intragenic PR-binding site (PRbs) located at +58 Kb downstream transcription start site of BCL 2 -L 1 and close to the 3'-end of the gene. This PRb was previously reported to serve as a highly active hormone-dependent binding site [31]. The canonical coactivator SRC1 and the Pol II were also recruited to this +58 Kb site, in a P4 enhanced way. To our knowledge, this is the first report of a direct evaluation of PRA interaction with molecular partners in PR + basal breast cancer cells, while almost all previous PR microarrays studies were performed on luminal breast cancer cells [53]. This issue has been only addressed once [54], functional analyses were conducted in estrogen-dependent T47D cells, however ChIP experiments were conducted in non mammary HeLa cells and did not specifically evaluate PRA recruitment.
To evaluate the potential activity of PR antagonists on PRA-mediated cell proliferation, we used the recently marketed selective progesterone receptor modulator (SPRM), ulipristal acetate (UPA), a specific, highly selective PR antagonist with no agonist activity towards other steroid receptors [55], with mixed PR agonist/antagonist properties depending on the tissue [56]. Of interest, we showed that UPA inhibited MDA-iPRA cell proliferation. The anti-proliferative effect of UPA was restricted to PRA-expressing cells, independently of the presence of P4 while it exerts partial agonist activity on MDA-iPRB cells. ChIP experiments demonstrated that UPA partially inhibited PRA, SRC1 and Pol II recruitment on BCL 2 -L 1 PR-binding site in the presence of P4, while UPA alone weakly enhanced PRA and SRC1 recruitment only. This absence of Pol II recruitment in the presence of UPA correlated with the lack of UPA-induced PRAmediated BCL 2 -L 1 expression, and is consistent with UPA inhibitory effect on both P4-induced BCL 2 -L 1 transcript and protein. This may be correlated as well to the inhibition of cell proliferation in the presence of UPA, as already observed for other tumor types in vivo [24]. PRB interaction with SRC1 in the presence of a non-commercially available SPRM (asoprisnil) has been reported using transactivation assays in T47D cells [57]. To our knowledge this is the first report on the molecular mechanism of action of UPA.
Our data suggest that PRA expression is associated with increased human breast cancer cell proliferation, further stimulated by P4. We also show that UPA is able to decrease cell proliferation in this PRA expressing model. This effect of UPA includes basal as well as progesterone stimulated cell proliferation. Thus, our findings suggest that UPA might be of interest in the adjuvant treatment of breast cancer, more specifically in ER-PR+ tumors.
In sum, we propose that future characterization of breast cancer should include PR isoforms evaluation to better specify tumor phenotype, and/or its molecular signature independently of estrogen signaling. Most notably, this should evaluate PRA target genes to identify poor prognosis tumors, and potential candidates for SPRM adjuvant therapy. This obviously requires prior confirmation in the clinical setting.
Supporting Information S1 Fig. Schematic representation of stably transfected plasmids inserted into the parental clone 250 and the bi-inducible system applied to generate MDA-iPRAB cells for the conditional PR isoform expression in MDA-MB-231 cells [6]. (a) The two regulatory proteins systems RheoSwitch and T-Rex were inserted in the same plasmid pZX-TR besides the zeocin resistance gene. (b) PRA and/or PRB expression was induced under the control of two promoters used to establish the secondary stable MDA-iPRAB cell line. Addition of RSL1 and/or Dox selectively induces PRA or PRB isoform, respectively. In their absence, no PR expression is induced. (EPS) Cells were treated for 6 h with vehicle, P4 (1 nM) and/or UPA (1 μM) in steroid-free medium, following 24 h induction of PRB expression using Dox (2μM). RT-qPCR analyses were performed as described in Materials and Methods. Data are expressed as fold induction compared to vehicle condition arbitrarily set at 1, and are means ± SEM from three independent cell cultures measured in duplicate. No statistical difference was detected. (EPS) S5 Fig. SRC1 and Pol II recruitment to the BCL 2 -L 1 gene. MDA-iPRA cells treated for 1 h with P4 (10 nM) or UPA (1 μM), were fixed and lysed and chromatin extracts subjected to ChIP assays using SRC1 antibody (Rabbit anti-SRC1 antibody, M-341, sc-8995, Santa Cruz Biotechnology) or Pol II antibody (Rabbit anti-Polymerase II antibody, H-224, sc-9001, Santa Cruz Biotechnology). Immunoprecipitated and eluted DNA fragments were analyzed by realtime qPCR using primer pair encompassing genomic sequence of the +58 kb site of the BCL 2 L 1 gene. Histograms represent the fold induction of SRC1 (a) or Pol II (b) enrichment compared to vehicle condition arbitrary set at 1 and are means ± SEM of three independent determinations. (EPS) S6 Fig. Regulation of FKBP5, a PRB-dependent gene. mRNA expression levels were determined in MDA-iPRB cells. Cells were treated for 6 h with vehicle, P4 (1 nM) and/or UPA (1 μM) in steroid-free medium, following 24 h induction of PRB expression using Dox (2μM). RT-qPCR analyses were performed as described in Materials and Methods. Data are expressed as fold change compared to vehicle condition arbitrarily set at 1, and are means ± SEM from three independent cell cultures measured in duplicate. Ã , ÃÃ , ÃÃÃ symbols indicate p<0.05, 0.01 and 0.001 respectively compared to the vehicle-treated MDA-iPRBcells, while x, xx, xxx symbols indicate p< 0.05, 0.01 and 0.001 respectively compared to the V or P4-treated MDA-iPRB cells (non-parametric ANOVA test (Kruskal-Wallis). (EPS) S1