Potential Role of Estrogen Receptor Beta as a Tumor Suppressor of Epithelial Ovarian Cancer

<div><p>Ovarian cancer is the gynecological cancer exhibiting the highest morbidity and improvement of treatments is still required. Previous studies have shown that Estrogen-receptor beta (ERβ) levels decreased along with ovarian carcinogenesis. Here, we present evidence that reintroduction of ERβ in BG-1 epithelial ovarian cancer cells, which express ERα, leads <em>in vitro</em> to a decrease of basal and estradiol-promoted cell proliferation. ERβ reduced the frequency of cells in S phase and increased the one of cells in G2/M phase. At the molecular level, we found that ERβ downregulated total retinoblastoma (Rb), phosphorylated Rb and phospho-AKT cellular content as well as cyclins D1 and A2. In addition, ERβ had a direct effect on ERα, by strongly inhibiting its expression and activity, which could explain part of the anti-proliferative action of ERβ. By developing a novel preclinical model of ovarian cancer based on a luminescent orthotopic xenograft in athymic Nude mice, we further revealed that ERβ expression reduces tumor growth and the presence of tumor cells in sites of metastasis, hence resulting in improved survival of mice. Altogether, these findings unveil a potential tumor-suppressor role of ERβ in ovarian carcinogenesis, which could be of potential clinical relevance for the selection of the most appropriate treatment for patients.</p> </div

ERβ inhibits tumor growth in an orthotopic xenograft mouse model. A.

<p>BG-1-Luc cells infected with Ad5 or Adβ adenoviruses were injected in the left ovary of Nude mice. Luc activity was monitored for 35 days as described in Fig. 1C. Results are expressed in photons/s (Ph/s) and represent one representative experiment corresponding to the average ± SD of at least 5 animals per group. Mann-Whitney test was used for comparison. * p<0.05 and ** p<0.01. A representative image of day 35 is shown. <b>B.</b> Representative pictures of whole animals and genital tract of animals euthanized at day 35 are displayed.</p

ERβ is a negative regulator of BG- cell growth. A.

<p>The growth of BG-1 cells, expressing or not ERβ, was monitored <i>in vitro</i> using a cell counter. BG-1 cells were plated in 24-well plates and cultured in the presence of vehicle or E2 (10⁻⁸M). Proliferation is expressed as % of control cells grown at day 0. Data represent the mean ± SD from triplicates. Measurements of Ad5+E2 and Adβ+E2 groups were compared by unpaired Student's <i>t</i> test. ** p<0.001. <b>B.</b> ERβ inhibits tumor growth in a bioluminescent subcutaneous mouse model. BG-1 cells stably expressing Luc and infected with Ad5 or Adβ adenoviruses were injected subcutaneously in ovariectomized female Nude mice. Luciferase activity was monitored for 25 days. Results are expressed in photons/s (Ph/s) and represent the mean ± SD from 8 animals. Measurements of Ad5+E2 and Adβ+E2 groups were compared by unpaired Student's <i>t</i> test. * p<0.05.</p

Expression levels and transcriptional activity of ERβ in BG-1. A.

<p>BG-1 cells were infected with Ad5 or Adβ adenoviruses and treated for 24h with control vehicle ethanol (Control) or E2 10⁻⁸M (E2). The expression of ERβ and rS9 reference gene was measured by real-time PCR. Results represent the mean ± SD of ERβ expression normalized by rS9 of 3 independent experiments. Measurements of Adβ and Adβ+E2 groups were compared by unpaired Student's <i>t</i> test. ** p<0.001. <b>B.</b> Proteins were extracted from cells infected in the same conditions as in A and treated for 0, 3, 6 or 24h with E2 10⁻⁸M (E2). Levels of ERα and ERβ were analyzed by western blot. Actin was used as a loading control. The upper band of ERβ blot labeled with a star corresponds to aspecific staining. <b>C.</b> Transcriptional activity of ERβ. BG-1 cells were infected with Ad5 or Adβ adenoviruses and transfected with ERE2-TK-Luc reporter along with β-galactosidase reporter. The cells were treated with ethanol as vehicle (Control) or E2 (10−8M) for 24h. Results show relative Luc activities (% of values of Ad5-infected cells without E2) ± SD after normalization with β-gal activity (3 independent experiments). Measurements of Ad5+E2 and Adβ+E2 groups were compared by unpaired Student's <i>t</i> test. * p<0.05. <b>D.</b> PEO14 cells were infected with Ad5, Adα, Adβ or the combination of Adα and Adβ adenovirus and transfected with ERE2-TK-LUC reporter along with β-galactosidase reporter. Cells were treated with control vehicle (Control) or E2 (10−8M) for 24h. Results show relative luciferase activities (% of values of Ad5 infected cells without E2) ± SD after normalization with β-gal activity (3 independent experiments). Measurements of Adα, Adβ and Adα+β groups were compared by unpaired Student's <i>t</i> test. *** p<0.001. NS: non significant. <b>E.</b> Proteins from PEO14 cells infected in the same conditions as in D and treated with vehicle ethanol (Control) or E2 10⁻⁸M (E2) for 3, 6 or 24h were analyzed by western blot with antibodies against ERα and ERβ. Actin was used as a loading control. The upper band labeled with a star in right panel corresponds to aspecific staining.</p

ERβ disturbs cell cycle of BG-1 cells and regulators. A.

<p>BG-1 cells were collected 24h after infection with Ad5 or Adβ adenoviruses and analyzed for cell cycle distribution. Results represent the mean ± SEM of 3 experiments. Ad5 and Adβ groups were compared by unpaired Student's <i>t</i> test. ** p<0.001. <b>B.</b> BG-1 cells were infected with Ad5 or Adβ viruses. After infection, cells were treated for 3, 6 or 24h with vehicle (ethanol, C) or 10⁻⁸M E2. 30 µg of protein extracts were used for Western blot. β-actin was used as a loading control. Unless specified, the ratio of target proteins over β-actin is indicated below the gels. Representative of 2 experiments.</p

Polycyclic Aromatic Hydrocarbons Reciprocally Regulate IL-22 and IL-17 Cytokines in Peripheral Blood Mononuclear Cells from Both Healthy and Asthmatic Subjects

<div><p>Pollution, including polycyclic aromatic hydrocarbons (PAH), may contribute to increased prevalence of asthma. PAH can bind to the Aryl hydrocarbon Receptor (AhR), a transcription factor involved in Th17/Th22 type polarization. These cells produce IL17A and IL-22, which allow neutrophil recruitment, airway smooth muscle proliferation and tissue repair and remodeling. Increased IL-17 and IL-22 productions have been associated with asthma. We hypothesized that PAH might affect, through their effects on AhR, IL-17 and IL-22 production in allergic asthmatics. Activated peripheral blood mononuclear cells (PBMCs) from 16 nonallergic nonasthmatic (NA) and 16 intermittent allergic asthmatic (AA) subjects were incubated with PAH, and IL-17 and IL-22 productions were assessed. At baseline, activated PBMCs from AA exhibited an increased IL-17/IL-22 profile compared with NA subjects. Diesel exhaust particle (DEP)-PAH and Benzo[a]Pyrene (B[a]P) stimulation further increased IL-22 but decreased IL-17A production in both groups. The PAH-induced IL-22 levels in asthmatic patients were significantly higher than in healthy subjects. Among PBMCs, PAH-induced IL-22 expression originated principally from single IL-22- but not from IL-17- expressing CD4 T cells. The Th17 transcription factors <i>RORA</i> and <i>RORC</i> were down regulated, whereas AhR target gene <i>CYP1A1</i> was upregulated. IL-22 induction by DEP-PAH was mainly dependent upon AhR whereas IL-22 induction by B[a]P was dependent upon activation of PI3K and JNK. Altogether, these data suggest that DEP-PAH and B[a]P may contribute to increased IL22 production in both healthy and asthmatic subjects through mechanisms involving both AhR -dependent and -independent pathways.</p></div

Transcript levels of genes involved in Th17/Th22 polarization.

<p>Activated PBMCs from nonallergic (NA) subjects (n = 8) and allergic asthmatic (AA) patients (n = 11) were incubated with or without PAH for 72hr, and gene mRNA level was assessed by Q-RT-PCR. Results are expressed as mean relative expression (RE) of 2^(-ΔCt) ± SEM, where the ΔCt value of the sample was determined by subtracting the Ct value of the target gene from the Ct value of the rs9 house keeping gene. *<i>P</i><.05,**<i>P</i><.01, ^#<i>P</i><.05 and ^##<i>P</i> <.01 AA versus NA subjects.</p

Effects of AhR antagonist on IL-22 and IL-17A secretion by peripheral blood mononuclear cells from Allergic asthmatics (AA) and nonallergic subjects (NA).

<p>Activated peripheral blood mononuclear cells from NA subjects (n = 10) and AA patients (n = 12) were incubated or not with PAH, in the presence or not of AhR antagonist CH-223191 for 72hr, and cytokine production was assessed by ELISA in the supernatants. Results are expressed as mean ± SEM. *<i>P</i><.05, **<i>P</i><.01. The dotted line is set on the level of the antagonist-treated control cells.</p

Cytokine profile of PAH-stimulated peripheral blood mononuclear cells in allergic asthmatics (AA) and nonallergic subjects (NA).

<p>IL-17A, IL-22 and IL-10 secretion by activated peripheral blood mononuclear cells from NA subjects (n = 10) and AA patients (n = 12) stimulated or not with different PAH for 72hr was determined by ELISA. Results are expressed as mean ± SEM. *<i>P</i><.05 and **<i>P</i><.01 versus control. ^#<i>P</i><.05 and ^##<i>P</i><.01 AA versus NA subjects.</p

Cytokine profile and cell expression of PAH-stimulated peripheral blood mononuclear cells cultured in Th17 conditions.

<p><b>A</b> IL-17A and IL-22 production by activated peripheral blood mononuclear cells from 6 nonallergic (NA) subjects and 6 allergic asthmatic (AA) patients stimulated or not with SRM or B[a]P for 6 days was determined by ELISA. Results are expressed as mean ± SEM. *<i>P</i><.05 versus corresponding control. ^#<i>P</i><.05 AA versus NA subjects. <b>B</b> Activated peripheral blood mononuclear cells were stimulated or not with SRM and B[a]P, stained with antibodies against cytokine and cell surface markers, and evaluated by flow cytometry. Results are expressed as mean ± SEM percentage of cytokine positive cell populations for n = 7–11 subjects. *<i>P</i><.05 versus corresponding control, ^##<i>P</i><.01, ^###<i>P</i><.001.</p