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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

Hormonal regulation of BCL2-L1 protein expression in MDA-iPRA cells.Following 24 h induction of PRA expression using RSL1 (0.5 μM), MDA-iPRA cells were incubated in the presence or absence of P4 (1 nM) and/or UPA(1 μM) in steroid-free medium for 24 h. (a) Western blot analysis of whole cell extracts (35 μg Protein) loaded on a 12% acrylamide gel, membranes were incubated overnight with a 1:1,000 dilution of anti-BCL2-L1 antibody (anti-Bcl-XL antibody, E18, ab32370, Abcam, France), and an anti-PR antibody recognizing both PR isoforms (Novocastra) or anti-GAPDH antibody (G9545—anti-GAPDH antibody produced in rabbit). (b) Band intensities of BCL2-L1 (26 kDa) and GAPDH (36 kDa) used as a sample loading control were quantified and BCL2-L1/GAPDH ratio were determined and expressed in arbitrary units and normalized to vehicle. Relative BCL2L1 protein expression is presented. (c) Band intensities of PRA (94 kDa) and GAPDH (36 kDa) used as a sample loading control were quantified and PRA/GAPDH ratio were determined and expressed in arbitrary units and normalized to vehicle. Relative PRA expression is presented as well. Histograms are means ± SEM of three independent experiments. ** Symbol indicates p<0.01 compared to the vehicle-treated MDA-iPRA cells while xx symbol indicates p<0.01 compared to the P4-treated MDA-iPRA cells (non-parametric Mann Whitney t-tests).
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pone.0140795.g005: Hormonal regulation of BCL2-L1 protein expression in MDA-iPRA cells.Following 24 h induction of PRA expression using RSL1 (0.5 μM), MDA-iPRA cells were incubated in the presence or absence of P4 (1 nM) and/or UPA(1 μM) in steroid-free medium for 24 h. (a) Western blot analysis of whole cell extracts (35 μg Protein) loaded on a 12% acrylamide gel, membranes were incubated overnight with a 1:1,000 dilution of anti-BCL2-L1 antibody (anti-Bcl-XL antibody, E18, ab32370, Abcam, France), and an anti-PR antibody recognizing both PR isoforms (Novocastra) or anti-GAPDH antibody (G9545—anti-GAPDH antibody produced in rabbit). (b) Band intensities of BCL2-L1 (26 kDa) and GAPDH (36 kDa) used as a sample loading control were quantified and BCL2-L1/GAPDH ratio were determined and expressed in arbitrary units and normalized to vehicle. Relative BCL2L1 protein expression is presented. (c) Band intensities of PRA (94 kDa) and GAPDH (36 kDa) used as a sample loading control were quantified and PRA/GAPDH ratio were determined and expressed in arbitrary units and normalized to vehicle. Relative PRA expression is presented as well. Histograms are means ± SEM of three independent experiments. ** Symbol indicates p<0.01 compared to the vehicle-treated MDA-iPRA cells while xx symbol indicates p<0.01 compared to the P4-treated MDA-iPRA cells (non-parametric Mann Whitney t-tests).

Mentions: Since P4 stimulated BCL2-L1 mRNA levels (Fig 3A), we next examined the hormone regulation of the anti-apoptotic factor BCL2-L1, at the protein level. As illustrated in Fig 5A, the 26 kDa band corresponding to BCL2-L1 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 BCL2-L1 expression was antagonized by UPA treatment which, when applied alone, significantly reduced by 2 fold factor the basal BCL2-L1 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 BCL2-L1 expression, both being fully antagonized by UPA.


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)

Hormonal regulation of BCL2-L1 protein expression in MDA-iPRA cells.Following 24 h induction of PRA expression using RSL1 (0.5 μM), MDA-iPRA cells were incubated in the presence or absence of P4 (1 nM) and/or UPA(1 μM) in steroid-free medium for 24 h. (a) Western blot analysis of whole cell extracts (35 μg Protein) loaded on a 12% acrylamide gel, membranes were incubated overnight with a 1:1,000 dilution of anti-BCL2-L1 antibody (anti-Bcl-XL antibody, E18, ab32370, Abcam, France), and an anti-PR antibody recognizing both PR isoforms (Novocastra) or anti-GAPDH antibody (G9545—anti-GAPDH antibody produced in rabbit). (b) Band intensities of BCL2-L1 (26 kDa) and GAPDH (36 kDa) used as a sample loading control were quantified and BCL2-L1/GAPDH ratio were determined and expressed in arbitrary units and normalized to vehicle. Relative BCL2L1 protein expression is presented. (c) Band intensities of PRA (94 kDa) and GAPDH (36 kDa) used as a sample loading control were quantified and PRA/GAPDH ratio were determined and expressed in arbitrary units and normalized to vehicle. Relative PRA expression is presented as well. Histograms are means ± SEM of three independent experiments. ** Symbol indicates p<0.01 compared to the vehicle-treated MDA-iPRA cells while xx symbol indicates p<0.01 compared to the P4-treated MDA-iPRA cells (non-parametric Mann Whitney t-tests).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608808&req=5

pone.0140795.g005: Hormonal regulation of BCL2-L1 protein expression in MDA-iPRA cells.Following 24 h induction of PRA expression using RSL1 (0.5 μM), MDA-iPRA cells were incubated in the presence or absence of P4 (1 nM) and/or UPA(1 μM) in steroid-free medium for 24 h. (a) Western blot analysis of whole cell extracts (35 μg Protein) loaded on a 12% acrylamide gel, membranes were incubated overnight with a 1:1,000 dilution of anti-BCL2-L1 antibody (anti-Bcl-XL antibody, E18, ab32370, Abcam, France), and an anti-PR antibody recognizing both PR isoforms (Novocastra) or anti-GAPDH antibody (G9545—anti-GAPDH antibody produced in rabbit). (b) Band intensities of BCL2-L1 (26 kDa) and GAPDH (36 kDa) used as a sample loading control were quantified and BCL2-L1/GAPDH ratio were determined and expressed in arbitrary units and normalized to vehicle. Relative BCL2L1 protein expression is presented. (c) Band intensities of PRA (94 kDa) and GAPDH (36 kDa) used as a sample loading control were quantified and PRA/GAPDH ratio were determined and expressed in arbitrary units and normalized to vehicle. Relative PRA expression is presented as well. Histograms are means ± SEM of three independent experiments. ** Symbol indicates p<0.01 compared to the vehicle-treated MDA-iPRA cells while xx symbol indicates p<0.01 compared to the P4-treated MDA-iPRA cells (non-parametric Mann Whitney t-tests).
Mentions: Since P4 stimulated BCL2-L1 mRNA levels (Fig 3A), we next examined the hormone regulation of the anti-apoptotic factor BCL2-L1, at the protein level. As illustrated in Fig 5A, the 26 kDa band corresponding to BCL2-L1 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 BCL2-L1 expression was antagonized by UPA treatment which, when applied alone, significantly reduced by 2 fold factor the basal BCL2-L1 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 BCL2-L1 expression, both being fully antagonized by UPA.

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