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

Esber N, Le Billan F, Resche-Rigon M, Loosfelt H, Lombès M, Chabbert-Buffet N - PLoS ONE (2015)

Bottom Line: P4 significantly stimulated MDA-iPRA expressing cells proliferation.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.Further confirmation in the clinical setting is required.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-Scientifique 1185, Faculté de Médecine Paris Sud, Le Kremlin-Bicêtre, France; Université Paris-Sud, Faculté de Médecine Paris Sud, Unité Mixte de Recherche-Scientifique 1185, Le Kremlin-Bicêtre, France; HRA-Pharma, Paris, France.

ABSTRACT
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 BCL2-L1 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.

No MeSH data available.


Related in: MedlinePlus

Progesterone enhances PRA recruitment on the intragenic region of BCL2-L1 gene.(a) Schematic representation of BCL2-L1 gene structure. It contains 3 exons. The genomic PR target motif previously identified [31] is located downstream the 3’end at position +58 kb of the transcription start site of BCL2-L1 gene. The proposed PR response element (PRE) is illustrated on which the pre-initiation complex containing PR isoform, steroid receptor coactivator 1 (SRC1) as well as the RNA polymerase type 2 (Pol II) binds. (b) MDA-iPRA cells treated for 1 h with P4 (10 nM) and/or UPA (1 μM), 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 BCL2-L1 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).
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pone.0140795.g006: Progesterone enhances PRA recruitment on the intragenic region of BCL2-L1 gene.(a) Schematic representation of BCL2-L1 gene structure. It contains 3 exons. The genomic PR target motif previously identified [31] is located downstream the 3’end at position +58 kb of the transcription start site of BCL2-L1 gene. The proposed PR response element (PRE) is illustrated on which the pre-initiation complex containing PR isoform, steroid receptor coactivator 1 (SRC1) as well as the RNA polymerase type 2 (Pol II) binds. (b) MDA-iPRA cells treated for 1 h with P4 (10 nM) and/or UPA (1 μM), 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 BCL2-L1 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).

Mentions: To better elucidate the molecular mechanisms by which P4-activated PRA regulated BCL2-L1 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 BCL2-L1 gene [31]. We chose the intragenic PR-binding sequence located at +58 kb downstream Transcription Start Site (TSS) of BCL2-L1 gene as schematically depicted in the genomic structure of the human BCL2-L1 gene (Fig 6A). P4 treatment induced a significant 40-fold increase in PRA recruitment on the well-documented +58 kb response element of the BCL2-L1 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 recruitment into the pre-initiation complex assembly (S5B Fig), required for transcriptional regulation. This finding is consistent with the UPA-dependent repression of BCL2-L1 expression (see Figs 3 and 5).


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

Esber N, Le Billan F, Resche-Rigon M, Loosfelt H, Lombès M, Chabbert-Buffet N - PLoS ONE (2015)

Progesterone enhances PRA recruitment on the intragenic region of BCL2-L1 gene.(a) Schematic representation of BCL2-L1 gene structure. It contains 3 exons. The genomic PR target motif previously identified [31] is located downstream the 3’end at position +58 kb of the transcription start site of BCL2-L1 gene. The proposed PR response element (PRE) is illustrated on which the pre-initiation complex containing PR isoform, steroid receptor coactivator 1 (SRC1) as well as the RNA polymerase type 2 (Pol II) binds. (b) MDA-iPRA cells treated for 1 h with P4 (10 nM) and/or UPA (1 μM), 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 BCL2-L1 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).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608808&req=5

pone.0140795.g006: Progesterone enhances PRA recruitment on the intragenic region of BCL2-L1 gene.(a) Schematic representation of BCL2-L1 gene structure. It contains 3 exons. The genomic PR target motif previously identified [31] is located downstream the 3’end at position +58 kb of the transcription start site of BCL2-L1 gene. The proposed PR response element (PRE) is illustrated on which the pre-initiation complex containing PR isoform, steroid receptor coactivator 1 (SRC1) as well as the RNA polymerase type 2 (Pol II) binds. (b) MDA-iPRA cells treated for 1 h with P4 (10 nM) and/or UPA (1 μM), 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 BCL2-L1 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).
Mentions: To better elucidate the molecular mechanisms by which P4-activated PRA regulated BCL2-L1 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 BCL2-L1 gene [31]. We chose the intragenic PR-binding sequence located at +58 kb downstream Transcription Start Site (TSS) of BCL2-L1 gene as schematically depicted in the genomic structure of the human BCL2-L1 gene (Fig 6A). P4 treatment induced a significant 40-fold increase in PRA recruitment on the well-documented +58 kb response element of the BCL2-L1 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 recruitment into the pre-initiation complex assembly (S5B Fig), required for transcriptional regulation. This finding is consistent with the UPA-dependent repression of BCL2-L1 expression (see Figs 3 and 5).

Bottom Line: P4 significantly stimulated MDA-iPRA expressing cells proliferation.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.Further confirmation in the clinical setting is required.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-Scientifique 1185, Faculté de Médecine Paris Sud, Le Kremlin-Bicêtre, France; Université Paris-Sud, Faculté de Médecine Paris Sud, Unité Mixte de Recherche-Scientifique 1185, Le Kremlin-Bicêtre, France; HRA-Pharma, Paris, France.

ABSTRACT
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 BCL2-L1 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.

No MeSH data available.


Related in: MedlinePlus