Progesterone receptor isoforms implication in breast cancer metastasis : in vitro studies in MDA-MB 231 cells

Le récepteur de la progestérone (PR) est un acteur majeur du développement de la glande mammaire. Dans les cellules épithéliales normales, PR est exprimé sous deux isoformes PRA (94 kDa) et PRB (116 kDa) de façon équimolaire. Il est établi que celles-ci ont un impact important sur le développement des cancers du sein, mais leurs rôles dans l’évolution métastatique reste très mal connus. Le ratio d’expression PRA/PRB étant souvent déséquilibré dans les tumeurs mammaires, nous avons analysé le turnover des deux isoformes. Nous avons démontré que PRA et PRB sont les cibles de modifications post-traductionnelles dirigées par les MAPK qui tendent à stabiliser l’une ou l’autre isoforme de manière sélective. Ainsi, Erk1/2 (p42/44) inhibe la dégradation de PRB tandis que la p38 stabilise PRA. Il en résulte que le ratio PRA/PRB varie de façon importante en fonction des signalisations extracellulaires impliquant les facteurs de croissance et les cytokines inflammatoires souvent exacerbés dans les cancers. Pour mieux étudier les effets différentiels de PRA et PRB, nous avons établi un modèle cellulaire original exprimant de manière bi-inductible l’une ou l’autre isoforme de PR, à partir de la lignée cellulaire MDA-MB 231 provenant d’une métastase de cancer du sein. En étudiant les variations induites par PRA ou PRB sur le transcriptome de ces cellules, nous avons identifié les gènes cibles spécifiques de ces isoformes. Parmi-eux se trouvent de nombreux gènes impliqués dans les cancers, notamment agissant sur la prolifération, la survie et la motilité cellulaires comme uPA et PAI-1. De plus, en analysant la migration cellulaire, nous avons mis en évidence un effet pro-migratoire de PRB particulièrement important en absence d’hormone. En recherchant la cause de ces effets, nous avons découvert que PRB était colocalisé et interagissait avec la kinase d’adhésion focale (FAK) qu’il active au niveau des points d’adhésion focaux. Ces travaux soulignent l’incidence du ratio PRA/PRB et du statut du ligand sur les métastases du cancer du sein, aussi bien au niveau de la sélectivité transcriptionnelle que celui des régulations non génomiques impactant la migration cellulaire. Nous suggérons la possibilité de cibler les tumeurs mammaires par des antagonistes sélectifs de PR et des inhibiteurs des voies de signalisation des MAPK ou de FAK.Progesterone receptor (PR) is a major actor of mammary gland development. PR is equally expressed as two main isoforms PRA (94 kDa) and PRB (116kDa) in the mammary gland epithelium. However, breast cancer progression has been associated with abnormalities of their expression ratio through undefined mechanisms. In this study, using a stably transfected cell line, we showed that PRA and PRB stabilizations are differentially regulated by Erk1/2 (p42/44) and p38 MAPKs respectively, leading to strongly influence PRA/PRB ratio. These results highlight the impact of growth factors and inflammatory cytokines on PRA/PRB imbalance in cancer cells. To study the differential effect of PR isoforms, we established an original bi-inducible cell line expressing either PRA and/or PRB. By analyzing variations induced by PRA and PRB on such cell transcriptomes, we identified the isoform-specific targets genes by DNA microarrays. Most of them are implicated in cancers, notably acting on cell proliferation, survival and motility. Furthermore, focusing our studies on cell migration, we showed that PRB acts as a pro-migratory factor particularly powerful in the absence of ligand. We discovered that PRB colocalized and interacted with the focal adhesion kinase (FAK) that was activated in focal adhesion points. Our results highlight the impacts of both PRA/PRB ratio and ligand status on metastatic evolution, in the contexts of transcriptional regulation as well as non-genomic events. We suggest the possibility to target mammary tumors by PR-selective antagonists and/or inhibitors of MAPK and FAK signalings

Impact des isoformes du récepteur de la progestérone sur la progression métastatique du cancer du sein (étude in vitro de la motilité de la lignée MDA-MB-231)

Le récepteur de la progestérone (PR) est un acteur majeur du développement de la glande mammaire. Dans les cellules épithéliales normales, PR est exprimé sous deux isoformes PRA (94 kDa) et PRB (116 kDa) de façon équimolaire. Il est établi que celles-ci ont un impact important sur le développement des cancers du sein, mais leurs rôles dans l évolution métastatique reste très mal connus. Le ratio d expression PRA/PRB étant souvent déséquilibré dans les tumeurs mammaires, nous avons analysé le turnover des deux isoformes. Nous avons démontré que PRA et PRB sont les cibles de modifications post-traductionnelles dirigées par les MAPK qui tendent à stabiliser l une ou l autre isoforme de manière sélective. Ainsi, Erk1/2 (p42/44) inhibe la dégradation de PRB tandis que la p38 stabilise PRA. Il en résulte que le ratio PRA/PRB varie de façon importante en fonction des signalisations extracellulaires impliquant les facteurs de croissance et les cytokines inflammatoires souvent exacerbés dans les cancers. Pour mieux étudier les effets différentiels de PRA et PRB, nous avons établi un modèle cellulaire original exprimant de manière bi-inductible l une ou l autre isoforme de PR, à partir de la lignée cellulaire MDA-MB 231 provenant d une métastase de cancer du sein. En étudiant les variations induites par PRA ou PRB sur le transcriptome de ces cellules, nous avons identifié les gènes cibles spécifiques de ces isoformes. Parmi-eux se trouvent de nombreux gènes impliqués dans les cancers, notamment agissant sur la prolifération, la survie et la motilité cellulaires comme uPA et PAI-1. De plus, en analysant la migration cellulaire, nous avons mis en évidence un effet pro-migratoire de PRB particulièrement important en absence d hormone. En recherchant la cause de ces effets, nous avons découvert que PRB était colocalisé et interagissait avec la kinase d adhésion focale (FAK) qu il active au niveau des points d adhésion focaux. Ces travaux soulignent l incidence du ratio PRA/PRB et du statut du ligand sur les métastases du cancer du sein, aussi bien au niveau de la sélectivité transcriptionnelle que celui des régulations non génomiques impactant la migration cellulaire. Nous suggérons la possibilité de cibler les tumeurs mammaires par des antagonistes sélectifs de PR et des inhibiteurs des voies de signalisation des MAPK ou de FAK.Progesterone receptor (PR) is a major actor of mammary gland development. PR is equally expressed as two main isoforms PRA (94 kDa) and PRB (116kDa) in the mammary gland epithelium. However, breast cancer progression has been associated with abnormalities of their expression ratio through undefined mechanisms. In this study, using a stably transfected cell line, we showed that PRA and PRB stabilizations are differentially regulated by Erk1/2 (p42/44) and p38 MAPKs respectively, leading to strongly influence PRA/PRB ratio. These results highlight the impact of growth factors and inflammatory cytokines on PRA/PRB imbalance in cancer cells. To study the differential effect of PR isoforms, we established an original bi-inducible cell line expressing either PRA and/or PRB. By analyzing variations induced by PRA and PRB on such cell transcriptomes, we identified the isoform-specific targets genes by DNA microarrays. Most of them are implicated in cancers, notably acting on cell proliferation, survival and motility. Furthermore, focusing our studies on cell migration, we showed that PRB acts as a pro-migratory factor particularly powerful in the absence of ligand. We discovered that PRB colocalized and interacted with the focal adhesion kinase (FAK) that was activated in focal adhesion points. Our results highlight the impacts of both PRA/PRB ratio and ligand status on metastatic evolution, in the contexts of transcriptional regulation as well as non-genomic events. We suggest the possibility to target mammary tumors by PR-selective antagonists and/or inhibitors of MAPK and FAK signalings.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

: PR-dependent breast cancer cell motility

International audienceProgesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes

p38 and p42/44 MAPKs Differentially Regulate Progesterone Receptor A and B Isoform Stabilization.: Distinct MAPKs regulate PRA and PRB stability

International audienceProgesterone receptor (PR) isoforms (PRA and PRB) are implicated in the progression of breast cancers frequently associated with imbalanced PRA/PRB expression ratio. Antiprogestins represent potential antitumorigenic agents for such hormone-dependent cancers. To investigate the mechanism(s) controlling PR isoforms degradation/stability in the context of agonist and antagonist ligands, we used endometrial and mammary cancer cells stably expressing PRA and/or PRB. We found that the antiprogestin RU486 inhibited the agonist-induced turnover of PR isoforms through active mechanism(s) involving distinct MAPK-dependent phosphorylations. p42/44 MAPK activity inhibited proteasome-mediated degradation of RU486-bound PRB but not PRA in both cell lines. Ligand-induced PRB turnover required neosynthesis of a mandatory down-regulating partner whose interaction/function is negatively controlled by p42/44 MAPK. Such regulation strongly influenced expression of various endogenous PRB target genes in a selective manner, supporting functional relevance of the mechanism. Interestingly, in contrast to PRB, PRA stability was specifically increased by MAPK kinase kinase 1-induced p38 MAPK activation. Selective inhibition of p42/p44 or p38 activity resulted in opposite variations of the PRA/PRB expression ratio. Moreover, MAPK-dependent PR isoforms stability was independent of PR serine-294 phosphorylation previously proposed as a major sensor of PR down-regulation. In sum, we demonstrate that MAPK-mediated cell signaling differentially controls PRA/PRB expression ratio at posttranslational level through ligand-sensitive processes. Imbalance in PRA/PRB ratio frequently associated with carcinogenesis might be a direct consequence of disorders in MAPK signaling that might switch cellular responses to hormonal stimuli and contribute towards pathogenesis

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

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

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

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