Impact of imbalanced PRA/PRB ratio on transcription of genes involved in breast cancer development and metastasis.

<p>Transcriptional changes induced by lack of either PRA or PRB as compared to the pattern obtained in cells co-expressing PR isoforms at equimolar level and potentially enhancing tumor growth and/or metastatic evolution. <i>HBEGF</i> and <i>AREG</i> genes are underlined.</p

Differential Regulation of Breast Cancer-Associated Genes by Progesterone Receptor Isoforms PRA and PRB in a New Bi-Inducible Breast Cancer Cell Line

<div><p>Progesterone receptor isoforms (PRA and PRB) are expressed at equal levels in normal mammary cells. However, alteration in PRA/PRB expression is often observed in aggressive breast cancer suggesting differential contribution of PR isoforms in carcinogenesis. The mechanisms underlying such processes remain to be established mainly due to paucity of appropriate cellular models. To investigate the role of PR isoforms and the impact of imbalanced PRA/PRB ratio in transcriptional regulation, we have generated an original human breast cancer cell line conditionally expressing PRA and/or PRB in dose-dependence of non-steroid inducers. We first focused on PR-dependent transcriptional regulation of the paracrine growth factor gene amphiregulin (<em>AREG)</em> playing important role in cancer. Interestingly, unliganded PRA increases <em>AREG</em> expression, independently of estrogen receptor, yet inhibitable by antiprogestins. We show that functional outcome of epidermal growth factor (EGF) on such regulation is highly dependent on PRA/PRB ratio. Using this valuable model, genome-wide transcriptomic studies allowed us to determine the differential effects of PRA and PRB as a function of hormonal status. We identified a large number of novel PR-regulated genes notably implicated in breast cancer and metastasis and demonstrated that imbalanced PRA/PRB ratio strongly impact their expression predicting poor outcome in breast cancer. In sum, our unique cell-based system strongly suggests that PRA/PRB ratio is a critical determinant of PR target gene selectivity and responses to hormonal/growth factor stimuli. These findings provide molecular support for the aggressive phenotype of breast cancers with impaired expression of PRA or PRB.</p> </div

Inducible expression of PRA and/or PRB in iPRA, iPRB or iPRAB cell lines.

<p>(<b>A</b>) The iPRA or iPRB or iPRAB cells were incubated or not with inducer(s), RheoSwitch Ligand (RSL1) (0.25 µM) and/or doxycycline (Dox, 1 µM) during 24 h and immunoblot analysis of whole cell extracts was performed using anti-PR antibody recognizing both PR isoforms (Novocastra) or anti-tubulin antibody for sample loading control. Inducible PRA and/or PRB electrophoretic bands were compared with endogenously expressed PR isoforms in wild-type T47D cells. (<b>B</b>) Cells were incubated with RSL1 (0.5 µM) or Dox (2 µM) during indicated time intervals and whole cell extracts were immunoblotted using antibodies against PR or loading control tubulin. (<b>C</b>) Cells were incubated with RSL1 (0.5 µM) or Dox (2 µM) during the indicated time intervals (left panels) or after 24 h of inducer exposure (right panels). Cells in the right panels were then rinsed twice with PBS and further incubated until the indicated times with fresh medium without inducer. Both series of cell extracts were immunoblotted as in A. (<b>D</b>) Cells were incubated with increasing concentrations of RSL1 or Dox as indicated for 24 h and immunoblot detection was performed as in A. (<b>E</b>) The iPRAB cells were incubated with RSL1 (1 µM) and Dox (1 µM) during 24 h and PR isoforms expression was compared with T47D cells treated or not with estradiol (E2, 10 nM) during 24 h. (<b>F</b>) The iPRAB cells were incubated with DMSO vehicle or RSL1 (0.5 µM) or Dox (2 µM) during 24 h and immunocytochemical analysis for PR isoforms detection was performed using Novocastra anti-PR antibody as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045993#s4" target="_blank">Materials and Methods</a></i>.</p

Inducibly expressed PR isoforms are functional by regulating reporter and endogenous gene transcription.

<p>(<b>A</b>) Following 24 h induction of PRA and/or PRB using RSL1 (0.5 µM) and/or Dox (2 µM) in steroid-free medium, iPRAB cells were transiently co-transfected with vector encoding PRE₂-luciferase reporter gene and internal standard βGal expression vector as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045993#s4" target="_blank">Materials and Methods</a></i>. Following 24 h of transfection, cells were treated with vehicle (ethanol) or 10 nM progesterone (P4) during 6 h in steroid free medium. Cells were then rinsed twice with PBS, luciferase activity was determined and results (mean ± SEM) from six independent cell cultures are normalized to protein content (graphs). Statistical tests on difference either with PR− cells treated by vehicle (*) or between the indicated groups (x) are presented as described in <i>Material and Methods</i>. Western blot analysis of whole cell extract showing PR isoforms expression under similar induction and treatment conditions is presented (lower panel). (<b>B</b>) The iPRAB cells were induced as in A and then treated with vehicle (ethanol) or P4 (10 nM) during 6 h in steroid-free medium and quantitative real-time RT-PCR analysis was performed for determining <i>FKBP5</i> or <i>SGK1</i> transcripts levels as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045993#s4" target="_blank">Materials and Methods</a></i>. Data (mean ± SEM) from three independent cell cultures measured in duplicate are presented as fold increase in transcript levels as compared to vehicle-treated uninduced (PR−) cells. Statistical differences are presented as in A.</p

Antiproliferative efficacy of antiprogestin RU486 depends on PRA or PRB isoform expression.

<p>(<b>A</b>) Approximately 5,000 iPRAB cells per well were cultured in 96-well plates in steroid-free medium during 24 h. On Day 0, 2 and 4, fresh steroid-free medium was replaced containing or not RSL1 (0.5 µM) and/or Dox (2 µM) to respectively induce expression of either PRA or PRB or both (PRAB) or none of them (PR−). Cell proliferation assays were performed using CellTiter 96® AqueousOne Solution as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045993#s4" target="_blank">Materials and Methods</a></i> and absorbance at 490 nm, representative of total number of living cells was determined at the indicated time (days). Data (mean ± SEM) from six independent cell cultures (n = 6) are presented. Stars (*) represent statistical difference between uninduced (PR−) and PR isoforms(s) expressing cells in the absence of ligand. (<b>B</b>) PR isoforms(s) expression was induced as above then cells were treated by vehicle or P4 (10⁻⁸ M) or RU486 (10⁻⁶ M). Cell proliferation was determined on day 1, 3 and 5 as in A. Data (mean ± SEM) from six independent cell cultures (n = 6) are presented, the low error bars being masked by point symbols (maximal SEM for the four graphs was 0.028 for PRA under P4 at day 5). Statistical differences at p<0.01 with either vehicle (**) or P4 alone (xx) -treated cells are indicated and were calculated by Mann-Whitney test. For clarity, only one statistical test is presented for the closest or overlapped points giving similar p-values.</p

PR isoforms distinctly regulate transcription of <i>HBEGF</i> and <i>AREG</i> genes.

<p>(<b>A</b>) Following 24 h induction of PRA and/or PRB expression using RSL1 (0.5 µM) and/or Dox (2 µM) in steroid-free medium, cells were treated with vehicle or P4 (10 nM) during 6 h and qRT-PCR analysis was performed for determining <i>HBEGF</i> and <i>AREG</i> mRNA levels as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045993#s4" target="_blank">Materials and Methods</a></i>. Data (mean ± SEM) from three independent cell cultures measured in duplicate are presented as fold change in transcript levels as compared to that in vehicle-treated uninduced (PR−) cells. Statistical difference is indicated as compared to vehicle condition for either uninduced (PR−) cells (*) or the corresponding PR-induced cells (x). (<b>B</b>) Cells were incubated with RSL1 (0.25 µM) and/or Dox (1 µM) during 24 h in steroid-free medium and then treated with vehicle or P4 (10 nM) or EGF (30 ng/ml) or both during 6 h. qRT-PCR analysis was performed as above and data (mean ± SEM) are presented as fold change with respect to the corresponding induced vehicle-treated cells. Statistical difference is indicated either as star (*) for the indicated treatment groups or as cross (x) for P4+EGF <i>vs</i> P4-treated cells.</p

Functional impact of unliganded and liganded PR isoform(s) on cellular functions and cancer processes.

<p>Genes regulated by selective PR isoform(s) expression and hormonal conditions were analyzed using the Ingenuity Pathway Analysis system (IPA) as defined in <i>Material and Methods</i>. The transcriptional effects on biofunctions (cut-off FC ±1.3) for each conditional expression of PRA A), PRB (B) or PRA plus PRB (AB) in the absence (−) or presence (+) of P4 were evaluated by an impact factor (IF) corresponding to –log(IPA p-value). (<b>A</b>) The impact of isoforms on main cellular functions are represented by a polar chart where each curve corresponds to variation of IF for a given condition. (<b>B</b>) The impact on cancer, breast cancer, metastasis (left panel) and molecular mechanisms of cancer (right panel) is represented on a polar chart where each axis symbolizes the indicated condition, and each curve corresponds to variation of IF for a given disease. The vertical axis corresponds to equimolar expression of PRA and PRB (AB), while the oblique axes represent the unbalanced ratio conditions for PRA (A) or PRB (B). The hormonal status is symbolized at the opposite poles of each axis (− : no ligand.; +: P4). The corresponding number of genes is indicated.</p

Gene expression profiles following PR isoform conditional expression and hormonal treatment.

<p>The iPRAB cells were treated for 24 h by RSL1 (0.25 µM) and/or Dox (1 µM) to induce expression of either PRA (A) or PRB (B) or PRA plus PRB (AB) expression and then incubated with vehicle (−) or 10⁻⁸ M progesterone (+) for 6 h. Control cells were not treated by inducers (O). PR isoforms expression was analyzed by western-blot (upper left inset). PRA/PRB ratio value corresponding to each condition was estimated according to ligand binding assays and western blot quantification as described in <i>Material and Methods</i> (A: 45; B: 0.04; AB: 1). The corresponding total RNAs (O−, O+, A−, A+, B−, B+, AB−, AB+) were extracted and gene expression profiling was done using Agilent 44K-oligonucleotide microarrays as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045993#s4" target="_blank">Materials and Methods</a></i>. Venn diagrams showing the total number of genes regulated for each subset and clustering analyses (heat maps) of differentially expressed genes in each experimental condition were performed as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045993#s4" target="_blank">Materials and Methods</a></i>. Each conditional expression as compared to the reference RNA is indicated on the top of heat maps. The gene clusters corresponding to Venn diagram areas are indicated on the left side of heat maps (−: ligand-independent; + ligand-dependent; ±: mixed regulation; &: intersection). As indicated in the lower right panel, color intensities reflect the log₂ (ratio) values obtained for down-regulated (green) and up-regulated (red) genes, and were saturated at log₂ (ratio) of ±2 corresponding to FC ±4.</p

Unliganded PRA induces continuously elevated <i>AREG</i> mRNA levels.

<p>(<b>A</b>) Cells were incubated with DMSO vehicle or RSL1 (0.5 µM) in steroid-free medium for indicated time periods and qRT-PCR analysis was performed for <i>AREG</i> or cyclin D1 transcript levels as indicated. Data (mean ± SEM) from three independent cell cultures measured in duplicate are presented as fold change with respect to transcript levels at 1 h time period in uninduced cells. Star (*) represents statistical difference with uninduced (PR−) cells for the corresponding time point. (<b>B</b>) Following 24 h exposure to DMSO vehicle or RSL1 (0.5 µM), cells were incubated with actinomycin D (4 µM) during indicated time periods and qRT-PCR analysis of AREG mRNA expression was performed. Star (*) represents statistical difference with uninduced (PR−) cells for the corresponding time point. (<b>C</b>) Cells were induced or not for 24 h as in B and then treated with vehicle or antiprogestin RU486 (10 nM) during 6 h. AREG mRNA was quantified by qRT-PCR and data (mean ± SEM) from three independent cell cultures in duplicate are presented as fold change with respect to vehicle-treated uninduced cells (PR−). Statistical differences with either uninduced PR− cells (*) or vehicle-treated PRA-induced cells (x).</p

Down-regulated PRA–target genes in the presence of P4.

<p>MDA-iPRAB cells were cultured during 24 h in steroid-free medium containing RSL1 (0.5 μM) and/or Dox (2 μM) to induce expression of PRA and/or PRB. Cells were treated by vehicle or P4 (1 nM) for 6 hours and a genome wide transcriptomic studies was performed as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140795#pone.0140795.ref006" target="_blank">6</a>]. A total of 999 genes were affected by PR expression. These genes were separated into three groups: A and B and AB (co-expressed at similar levels relative to physiological conditions) regrouping genes that are regulated by either by PRA, PRB or PRAB. Tables above represent class <i>A+</i> genes regulated by PRA in the presence of the ligand; Class <i>A+&AB+</i>, genes responsive to liganded PRA and their expression may or may not be influenced by PRB co-expression; Class <i>A±&AB±</i> genes responsive to both liganded and unliganded PRA, and their expression may be influenced by PRB co-expression; Class <i>A+&B+& AB+</i>, genes that are commonly regulated by liganded PRA, PRB and PRAB. FC calculated either by microarray studies or by RT-qPCR analysis and their function in the cancer. FC: Transcriptional Fold Change calculated by microarray studies (FC*) and by RT-qPCR (FI, Fold Induction).</p><p>Down-regulated PRA–target genes in the presence of P4.</p