pRb ' s Role in Cell Fate , Lineage Commitment , and Tumorigenesis by

Mutation of the RB-I and p53 tumor suppressors is associated with the development of human osteosarcoma. With the goal of generating a mouse model of this disease, we used conditional and transgenic mouse strains to inactivate Rb and/or p53 specifically in osteoblast precursors. The resulting Rbp53 double mutant (DKO) animals are viable but develop early onset osteosarcomas with complete penetrance. These tumors display many of the characteristics of human osteosarcomas, including being highly metastatic. We established cell lines from the DKO osteosarcomas to further investigate their properties. These immortalized cell lines are highly proliferative and they retain their tumorigenic potential, as judged by their ability to form metastatic tumors in immunocompromised mice. Moreover, they can be induced to differentiate and, depending on the inductive signal, will adopt either the osteogenic or adipogenic fate. Consistent with this multipotency, a significant portion of these tumor cells express Sca-1, a marker that is typically associated with stem cells/uncommitted progenitors. By assaying sorted cells in transplant assays, we demonstrate that the tumorigenicity of the osteosarcoma cell lines correlates with the presence of the Sca-1 marker. Finally, we show that loss of Rb and p53 in Sca-1 positive mesenchymal stem/progenitor cells is sufficient to yield transformed cells that can initiate osteosarcoma formation in vivo.

Leydig cell tumors are the most frequent interstitial neoplasms of the testis with increased incidence in recent years. They are hormonally active and are considered one of the steroid-secreting tumors. Although usually benign, the malignant phenotype responds poorly to conventional chemotherapy or radiation, highlighting the need to identify new therapeutic targets for treatment. Here, we identified a novel glucocorticoid-mediated mechanism that controls cell growth in Leydig cell tumors. We found that a synthetic glucocorticoid receptor agonist, dexamethasone, reduces cell proliferation in rat Leydig tumor cells by decreasing the expression and the enzymatic activity of the estrogen-producing enzyme aromatase. This inhibitory effect relies on the ability of activated glucocorticoid receptor to regulate the aromatase gene transcriptional activity through the recruitment of nuclear receptor corepressor protein and silencing mediator of retinoid and thyroid hormone receptors to a newly identified putative glucocorticoid responsive element within the aromatase promoter II. Our in vivo studies reveal a reduction of tumor growth, after dexamethasone treatment, in animal xenografts. Tumors from dexamethasone-treated mice exhibit a decrease in the expression of the proliferation marker Ki-67 and the aromatase enzyme. Our data demonstrate that activated glucocorticoid receptor, decreasing aromatase expression, induces Leydig tumor regression both in vitro and in vivo, suggesting that glucocorticoid receptor might be a potential target for the therapy of Leydig cell tumors. (Am J Pathol 2016, 186: 1328e1339; http://dx.doi.org/ 10.1016/j.ajpath.2015. 12.024) Leydig cell tumors (LCTs) are the most common tumors of the gonadal stroma and represent about 3% of all testicular neoplasms. 1 Several observations on both rodents and humans suggest that local estrogen synthesis plays a significant role in sustaining Leydig cell tumorigenesis. Transgenic mice overexpressing aromatase, the enzyme responsible for the conversion of androgens to estrogens, and exhibiting an enhancement of circulating 17b-estradiol concentrations show Leydig cell hyperplasia and tumors. 2,3 In a previous study, we observed that the rat Leydig tumor (R2C) cells release a conspicuous amount of 17b-estradiol, as a consequence of aromatase overexpression that stimulates a short autocrine loop that determines cell proliferation. 4 In humans, elevated aromatase expression with subsequent high plasma estradiol concentrations has been reported in patients with testicular LCTs, 5e8 further supporting the crucial role played by the aromatase enzyme on the pathogenesis of leydigiomas. All these experimental and clinical observations suggest that a reduction of local estrogen production by inhibiting aromatase expression may open new opportunities for therapeutic intervention in LCTs.
In this context, we have previously shown that a decrease of aromatase expression induced by farnesoid X receptor plays an important role in inhibiting Leydig tumor cell growth. 9,10 Particularly, we have demonstrated that farnesoid X receptor is able to compete with the steroidogenic factor 1 in binding to a common nuclear response element within the aromatase promoter II (PII), interfering negatively with its activity. 9 More recently, we also have identified the existence of a functional cross talk between the androgen receptor, the orphan nuclear receptor DAX-1, and aromatase involved in the inhibition of the estrogen-dependent Leydig cancer cell proliferation. 11 Glucocorticoid receptor (GR), another member of the nuclear receptor superfamily, highly expressed in Leydig cells 12e14 plays a physiologic role in the control of male sexual maturation and adult reproductive functions that modulate gonadal steroid synthesis and spermatogenesis. 15 It is well recognized that elevated glucocorticoid concentrations that result from diverse stressful conditions, both physical and psychological, lead to suppression of serum testosterone concentrations. 16 Conversely, reduction of endogenous corticosterone concentrations (the main glucocorticoid in rodents) leads to increased testosterone production by Leydig cells, thus supporting the repressive role of glucocorticoids on testosterone production. 17 These observations well fit with the evidences that activation of GR suppresses, in Leydig cells, expression of several steroidogenic enzyme-encoding genes, including Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, and Cyp19a1. 18e24 In addition to the repression of gene expression, glucocorticoids also induce apoptosis in rat Leydig cells. 25 However, information about the role of GR in Leydig tumor cells is still lacking.
Here, we investigated in the R2C cells whether GR activation by the specific agonist dexamethasone (DEXA) may modulate aromatase expression and thus inhibit testicular tumor growth. We report the identification of a novel glucocorticoid-mediated mechanism that controls the expression of aromatase in Leydig tumor cells by negatively regulating aromatase transcript and protein levels. Transcriptional repression of aromatase gene by GR appears to be consequent to the recruitment of the nuclear receptor corepressor protein (NCoR) and the silencing mediator of retinoid and thyroid hormone receptor (SMRT) corepressors to the glucocorticoid response element (GRE)-containing region of the aromatase promoter.

Cell Cultures
R2C cells were acquired in 2011 from ATCC (LGC Standards, Tedding-ton Middlesex, UK) where they were authenticated, stored according to supplier's instructions, and used within 4 months after frozen aliquots were resuscitated. R2C cells were cultured in Ham's F-12 supplemented with 15% horse serum, 2.5% FBS, and antibiotics. Mouse normal Leydig (TM3) cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 5% horse serum, 2.5% FBS, and antibiotics.

Immunoblot Analysis
R2C and TM3 cells were lyzed in 500 mL of 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2 mmol/L sodium fluoride, 2 mmol/L EDTA, 0.1% SDS, containing a mixture of protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride, and sodium orthovanadate) for protein extraction. Protein extracts from tumor tissues were homogenized in lysis buffer supplemented with 10% glycerol. Equal amounts PAGE, as described. 26 Immunofluorescence R2C cells were seeded on glass coverslips, washed with phosphate-buffered saline (PBS), and then fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. Next, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 minutes, blocked with 5% bovine serum albumin for 30 minutes, and incubated overnight with anti-GR antibody (dilution 1:100) in PBS at 4 C. The day after the cells were washed three times with PBS and incubated with the secondary antibody anti-rabbit IgG-fluorescein isothiocyanate (dilution 1:200) for 1 hour at room temperature.
To check the specificity of immunolabeling the primary antibody was replaced by normal rabbit serum (negative control). Fluorescence was photographed with Olympus BX51 microscope (Tokyo, Japan) with a 40Â objective.

Total RNA Extraction and RT-PCR Assay
Total RNA was extracted from R2C cells with the use of TRIzol reagent, and evaluation of gene expression was

Aromatase Activity Assay
The aromatase activity in subconfluent R2C cell culture medium was measured by the tritiated water release assay with the use of 0.5 mmol/L [1b-3H]androst-4-ene-3,17-dione as substrate. 10 The incubations were performed at 37 C for 2 hours under an air/CO 2 (5%) atmosphere. The results obtained were expressed as picomole/h and were normalized to mg of protein (pmol/h per mg of protein).

Plasmids, Transfections, and Luciferase Reporter Assays
R2C cells were transiently transfected with FuGENE HD reagent with the plasmids that contained different segments of the rat aromatase PII sequence ligated to a luciferase reporter gene À1037/þ94 (p-1037), À688/þ94 (p-688), À475/þ94 (p-475), À183/þ94 (p-183) previously described. 27 After transfection, R2C cells were treated with DEXA (0.01, 0.1, 1 mmol/L) for 24 hours. Thymidine kinase Renilla luciferase plasmid was used to normalize the efficiency of the transfection. Firefly and Renilla luciferase activities were measured by the Dual Luciferase kit. The firefly luciferase data for each sample were normalized on the basis of transfection efficiency measured by Renilla luciferase activity.

ChIP and Re-ChIP Assay
R2C cells were treated with 0.1 and 1 mmol/L DEXA for 1 hour and then cross-linked with 1% formaldehyde and sonicated. Supernatant fluids were immunocleared with salmon sperm DNA/protein A-agarose for 1 hour at 4 C. For chromatin immunoprecipitation (ChIP), the precleared chromatin was immunoprecipitated with specific anti-GR or anti-polymerase II antibodies and re-immunoprecipitated with anti-NCoR or anti-SMRT antibodies. A normal mouse serum IgG was used as negative control. Pellets were washed as reported, eluted with elution buffer (1% SDS, 0.1 mol/L NaHCO 3 ), and digested with proteinase K. DNA was obtained by phenol/chloroform/isoamyl alcohol extractions and precipitated with ethanol. A 5-mL volume of each sample and input were used for real-time PCR with the use of the primers flanking the GRE sequence in the P450 aromatase PII region: 5 0 -GTAGAAGGGTACAGTTCT-CGG-3 0 and 5 0 -CCTAGGACACACATGCTCAC-3 0 . PCR were performed in the iCycler iQ Detection System (Bio-Rad), with the use of 0.1 mmol/L of each primer, in a total volume of 30 mL of reaction mixture according to the manufacturer's recommendations. SYBR Green Universal PCR Master Mix with the dissociation protocol was used for gene amplification. Negative controls contained water instead of DNA. Final results were calculated with the DDCt method as previously reported, 4 using input Ct values. The basal sample was used as calibrator.
Cell Viability and Proliferation Assays MTT Assay Cell viability was determined by using the MTT assay as previously described. 28 Results are expressed as fold change AE SD relative to vehicle-treated cells and are representative of three different experiments, each performed in triplicate.
Soft Agar Anchorage-Independent Growth Assays Cells (10 4 /well) were plated in 2 mL of 0.5% agarose with 5% charcoal stripped-FBS in phenol red-free media, with a 0.7% agarose base in 6-well plates. Two days after plating, media that contained vehicle or treatments as indicated were added to the top layer and replaced every 2 days. After 14 days, colonies were counted as described. 29 Data shown are the mean colony numbers of three plates and are representative of three independent experiments, each performed in triplicate.

DNA Flow Cytometry
To determine cell cycle distribution analysis, cells were harvested by trypsinization, fixed, and stained with 100 mg/mL propidium iodide after treatment with 20 mg/mL RNase A. The DNA content was measured with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and the data acquired with CellQuest software version 3.3. Cell cycle profiles were determined with ModFit LT.

In Vivo Experiments
The in vivo experiments were performed in 35-day-old male nude mice (nu/nu Swiss; Charles River, Milan, Italy). At day 0, mice were inoculated with 1.0 Â 10 5 R2C cells/ mouse into the intrascapular region. DEXA treatment was started at day 14 later and delivered daily to the animals by i.p. injection. Tumor growth was monitored as described. 30 At the time of sacrificing, 30 days after injection, tumors were dissected from the neighboring connective tissue, frozen in nitrogen, and stored at À80 C. All animals were maintained and handled in accordance with the recommendation of the Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care Committee of University of Calabria.

Histopathologic Analysis
Tumor, testis, livers, spleen, and kidneys were fixed in 4% formalin, sectioned at 5 mm, and stained with hematoxylin and eosin, as suggested by the manufacturer (Bio-Optica, Milan, Italy). Hematoxylin and eosin was photographed with Olympus BX51 microscope with a 20Â objective.

Immunohistochemical Analysis
Paraffin-embedded section, 5 mm thick, were mounted on slides precoated with polylysine, and then they were deparaffinized and dehydrated (seven to eight serial sections). Immunohistochemical experiments were performed as described, 31 using rabbit polyclonal Ki-67 and mouse monoclonal anti-aromatase primary antibody at 4 C overnight. For the detection of GR in human LCT, formalinfixed, paraffin-embedded testicular tissue, obtained from one male patient (aged 33 years) provided by the Pathological Anatomy Unit (Annunziata Hospital, Cosenza, Italy), was incubated with rabbit polyclonal GR. Then, a biotinylated goateanti-rabbit and goateanti-mouse IgG was applied for 1 hour at room temperature, followed by the avidin biotin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA). Immunoreactivity was visualized by using the diaminobenzidine chromogen (Sigma-Aldrich, St. Louis, MO) method. The primary antibody was replaced by normal rabbit and normal mouse serum negative control section. Immunohistochemical experiments were photographed with Olympus BX51 microscope with a 40Â objective.

Statistical Analysis
Each datum point represents the means AE SD of three different experiments. Data were analyzed by Student's t-test with the use of the GraphPad Prism 4 software program (GraphPad Inc., San Diego, CA). P < 0.05 was considered statistically significant.

GR Is Expressed in R2C Cells
We first evaluated, by immunoblot analysis, the expression of GR in the R2C cell line, a well-documented experimental model for leydigioma. 4,9e11,27 Indeed, it has been shown that these cells, similar to human LCTs, exhibit high aromatase expression and activity, leading to a consequent excess of in situ estradiol production that sustains tumor cell survival and proliferation. 4e8, 27 We showed the presence of a GR-immunoreactive protein band whose levels were comparable with those observed in TM3 cells, an immortalized Leydig cell line derived from normal mouse testis (Supplemental Figure S1A). These results were further confirmed by immunofluorescence analysis that detected GR immunoreactivity in the cytoplasm region of R2C cells.
No fluorescence was noticed in the cells processed without primary antibody (NC) (Supplemental Figure S1B). Moreover, a positive GR immunostaining was revealed in neoplastic cells from human Leydig tumors (Supplemental Figure S1C), indicating that human LCTs do express GR.

Inhibitory Effects of the GR Agonist DEXA on Aromatase Expression in R2C Cells
On the basis of previous data to indicate that GR activation down-regulates the aromatase enzyme in testes, 24 we explored the possibility that the GR agonist DEXA might modulate aromatase gene expression in R2C cells. Treatment with 0.01, 0.1, and 1 mmol/L DEXA was able to reduce the cellular content of the enzyme, in a concentration-dependent manner, at both mRNA and protein levels, as determined by RT-PCR and immunoblot analyses (Figure 1, A and B). The reduction of aromatase expression on DEXA administration was also reflected by a change in its enzymatic activity, as evaluated by the tritiated water release assay ( Figure 1C). A direct involvement of GR in modulating aromatase expression was provided by the evaluation of aromatase mRNA levels, protein content, and enzymatic activity after administration of the GR inhibitor RU-486 in R2C cells treated with DEXA (Figure 1, DeF). The addition of RU-486 completely reversed the down-regulatory effects induced by DEXA, suggesting that activated GR may directly regulate aromatase expression in R2C cells.

Ligand-Activated GR Decreases the Transcriptional Activity of Aromatase Proximal PII
Next, we tested whether the down-regulatory effects of DEXA on aromatase expression could be due to a negative influence on aromatase gene transcriptional activity. Aromatase activity is regulated primarily at the level of gene expression by tissuespecific promoters and is present in testicular somatic cells and along the maturative phases of male germ cells. 32,33 Specifically, a promoter proximal to the translation start site, PII, regulates aromatase expression in fetal and adult testis, R2C and H540 rat Leydig tumor cells, and in purified preparations of rat Leydig, Sertoli, and germ cells. 34,35 Thus, R2C cells were transiently transfected with a luciferase reporter construct containing the PII-aromatase promoter sequence (À1037/þ94, p-1037) and treated with increasing concentrations of DEXA. We found a significant dose-dependent inhibition of aromatase promoter activity after treatment with DEXA ( Figure 2A). To identify the region within the aromatase promoter that is functionally

GR Inhibits Leydig Tumor Growth
The American Journal of Pathologyajp.amjpathol.org important for transcriptional regulation by DEXA, transient transfection experiments were performed by using constructs that contained different 5 0 -deleted regions of the rat PII-aromatase promoter ( Figure 2B). Similarly to what was observed for the PII-aromatase promoter (À1037/þ94), the transcriptional activity of the PII-aromatase promoter constructs (À688/þ94, p-688 and À475/þ94, p-475) was decreased in response to DEXA stimulation; whereas, in the presence of the PII-aromatase promoter construct encoding the sequence from À183 to þ94 (p-183), we evidenced the loss of the DEXA-mediated inhibitory effect ( Figure 2B). This latter result highlights that the region from À475 to À183 was essential for the down-regulation induced by DEXA on aromatase promoter activity. The nucleotide sequence analysis of this region revealed a putative GRE binding motif located at position À470/À460, suggesting that P450 aromatase regulation by DEXA may require GRE motif.

GR Reduces Aromatase Promoter Activity at GRE Site through Specific Corepressor Recruitment
The role of GR in regulating the aromatase gene promoter activity was further investigated by ChIP assay. With the use of specific antibody against GR and RNA-Pol II, protein-chromatin complexes were immunoprecipitated from R2C cells cultured with or without DEXA at 0.1-and 1-mmol/L doses. GR occupancy to the GRE site-containing sequence of aromatase promoter was induced in a liganddependent manner by DEXA ( Figure 2C). The negative transcriptional role of GR on aromatase expression is evidenced by the dynamic of RNA Pol II recruitment onto the aromatase promoter that appears to be drastically reduced on DEXA treatment ( Figure 2C). To assess whether the decrease in aromatase promoter transcriptional activity might be caused by the cooperative interaction between GR and negative transcriptional regulators, we investigated the involvement of NCoR and SMRT, which interact with GR and function as negative coregulators. 36 Re-ChIP assays demonstrated increased NCoR and SMRT occupancy of the GRE-containing aromatase promoter region after DEXA exposure ( Figure 2D).

DEXA Inhibits R2C Cell Proliferation through GR Activation
We have previously demonstrated that local estradiol production by highly expressed aromatase represents a major feature involved in the positive control of R2C cell proliferation. 4 Thus, the effect of increasing concentrations of DEXA on R2C cell viability was evaluated by MTT assay. Treatment with DEXA at 0.01, 0.1, and 1 mmol/L doses for 24 and 48 hours significantly decreased R2C cell viability in a dosedependent manner ( Figure 3A). The inhibitory effects exerted by DEXA were completely reversed in the presence of the GR inhibitor RU-486 ( Figure 3B). In addition, we examined the effects of activated GR on cell growth with the use Figure 2 DEXA decreases transcriptional activity of aromatase proximal PII. A and B: Schematic map of the P450 aromatase proximal PII constructs. All of the promoter constructs contain the same 3 0 boundary (þ94). The 5 0 boundaries of the promoter fragments varied from À1037 to À183 (p-1037, p-688, p-475, p-183). R2C cells were transiently transfected with the reported constructs and treated for 24 hours with vehicle (-) or 0.01, 0.1, and 1 mmol/L DEXA. C: R2C cells were treated in the presence of vehicle (-) or 0.1 and 1 mmol/L DEXA for 1 hour, then cross-linked with formaldehyde, and lyzed. The precleared chromatin was immunoprecipitated with anti-GR, and anti-RNA Pol II antibodies. D: Chromatin immunoprecipitated with the anti-GR antibody was re-immunoprecipitated with anti-NCoR and SMRT antibodies. The PII sequence, including the GRE site, was detected by real-time PCR with specific primers, as described in Material and Methods. Data are expressed as means AE SD. n Z 3 different experiments performed in triplicate. *P < 0.05. DEXA, dexamethasone; GR, glucocorticoid receptor; GRE, glucocorticoid response element; IP, immunoprecipitated; PII, promoter II; NCoR, nuclear receptor corepressor protein; R2C, rat Leydig tumor; SMRT, silencing mediator of retinoid and thyroid hormone receptors.
of anchorage-independent soft agar assays, which better reflect in vivo three-dimensional tumor growth ( Figure 3C). Consistent with data from MTT assays, DEXA treatment significantly reduced colony formation in R2C cells. To extend our results, we also investigated the effects of DEXA in affecting growth of TM3 cell line. DEXA exposure did not elicit any significant inhibitory effects in TM3 cells at all of the doses tested (Supplemental Figure S2). This may be due to the different expressions of aromatase between normal and tumor cell lines, because, as previously reported, 4 TM3 cells show undetectable levels of aromatase protein.
Moreover, to investigate the effects of DEXA on cell cycle progression, flow cytometric analysis was performed in R2C cells. DEXA treatment caused a cell cycle arrest in G 0 /G 1 phase concomitant with a reduced fraction of cells in S-phase compared with untreated cells ( Figure 3D). Accordingly, the expression of cell cycle regulator genes was modulated by DEXA administration as revealed by p53, p27 Kip1 , p21 WAF1/CIP1 up-regulation and Cyclin D1 down-regulation ( Figure 3E).

Activation of GR Inhibits R2C Tumor Xenograft Growth
As a final step of the current study, we used the R2C tumor xenograft model to examine the effects of DEXA on Leydig tumor growth in vivo. To this aim, we injected R2C cells into the intrascapular region of male nude mice and followed tumor growth after administration of DEXA at 1 and 10 mg/kg per day. This administration was well tolerated because no changes in body weight or in food and water consumption were observed along with no evidence of reduced motor function. In addition, no significant differences in the mean weights or histologic features of the major organs (liver, spleen, and kidney) after sacrifice were observed between vehicle-and DEXA-treated mice, indicating a lack of toxic effects at the dose given. Treatment with DEXA at both 1 and 10 mg/kg per day induced a significant regression in tumor growth ( Figure 4A). Thirty days after injection, tumor weight and tumor size were markedly smaller in animals treated with DEXA than in vehicle-treated mice ( Figure 4B). Hematoxylin and eosin staining of tumor tissues are shown in Figure 4C. In agreement with our in vitro findings, we observed in R2C xenograft tumors from mice treated with DEXA a significant decrease of aromatase expression, evaluated by both immunoblotting and immunohistochemistry analysis ( Figure 5, A and B). This was concomitant with a reduced expression of Ki-67, a well-known marker for cell proliferation, in R2C xenograft tumors from mice treated with DEXA compared with tumors from vehicle-treated mice ( Figure 5C).
It is worthwhile to underline that the histopathologic features of seminiferous tubules from DEXA-treated mice showed a normal cellularity and morphology that were similar to those of the control groups (Supplemental Figure S3).

Discussion
Leydigioma is a rare testicular tumor that affects males at any age with two peaks of incidence, during prepuberty between 5 and 10 years, and in adulthood between 25 and 35 years of age. 37 Although usually benign, about 10% of LCTs in adult patients reveal a malignant phenotype that metastasizes to retroperitoneal lymph nodes, liver, lungs, and bone. 38 Unfortunately, malignant LCTs respond poorly to chemotherapy or radiation, 39,40 rendering it necessary to identify new therapeutic targets for LCT treatment.
In this study, we have shown that GR is a potential new target to inhibit Leydig tumor cell proliferation. Indeed, GR is expressed in this type of cancer, and its activation is associated with a drastic reduction of cell proliferation that results from the inhibition of aromatase expression and activity.
Glucocorticoid hormones control a wide variety of biological processes, such as metabolic homeostasis, inflammation, immune response, development, and reproduction. 41 Moreover, glucocorticoids are key regulators of cell growth and proliferation in many cell types. They can induce a G 0 /G 1 cell cycle arrest and programed cell death of immature thymocytes, several leukemic cell lines, and mature peripheral T lymphocytes. 42,43 They can also inhibit the proliferation of mammary epithelial cells, 44,45 fibroblasts, 46 and hepatoma cells. 47 There are several proposed mechanisms to explain the inhibitory effects of glucocorticoids on cell growth that appear to operate in a cell-typeespecific manner. These mechanisms include activation of cyclin-dependent protein kinase inhibitors, transcriptional repression of mitogenic factors, and activation of glycogen synthase kinase-3b, resulting in the proteolytic degradation of c-Myc and Cyclin D1. 48e50 The antiproliferative effect exerted by glucocorticoids has prompted their use clinically as a part of anticancer therapy for a diverse range of dysplasias, including lymphoproliferative disorders and several solid tumors. 50e53 Here, we identified a novel glucocorticoid-mediated mechanism that controls growth in Leydig tumor cells. We found that a synthetic GR agonist, DEXA, is able to significantly reduce proliferation rate in a well-documented in vitro model for Leydig cell neoplasms, such as the R2C cell line. We demonstrated that DEXA treatment induces cell cycle arrest and modulates genes involved in the glucocorticoid-dependent regulation of cell growth. DEXA-treated mice were tested for aromatase expression by immunoblot analysis. b-Actin was used as a loading control. B and C: Representative images of aromatase (B) or Ki-67 (C) immunohistochemical staining of R2C xenograft tumors. Insets indicate negative control. Data are expressed as means AE SD. n Z 3 separate experiments in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentages of the control, which was assumed to be 100%. *P < 0.05. Scale bar Z 12.5 mm. C, control; DEXA, dexamethasone; R2C, rat Leydig tumor.
Moreover, we evidenced that the observed effects of DEXA on R2C proliferation appear to be related to a reduction of local estrogen production, which represents a major feature of R2C cells. 4 Indeed, in R2C cells, DEXA treatment negatively affects the enzyme aromatase by decreasing its expression at both mRNA and protein levels, together with the inhibition of its enzymatic activity. Our results indicate that the downregulation of aromatase expression induced by DEXA administration strictly depends on GR activation, because it was completely reversed in the presence of the GR inhibitor RU-486.
All these findings suggest that GR-mediated inhibition of aromatase involves regulation of aromatase gene transcriptional activity; thus, we focused on the molecular mechanisms by which GR mediates repression of the aromatase enzyme. Distinctive tissue-specific aromatase promoters are used to control the expression of aromatase mRNA. The promoter located immediately upstream of the transcriptional initiation site (PII) regulates aromatase expression in rat Leydig, Sertoli, and germ cells and in R2C cells. 34,35 Here, we provided evidence that activated GR is a transcriptional repressor of the aromatase gene in Leydig tumor cells. Specifically, we have demonstrated by functional studies and ChIP assays that GR-mediated inhibition of the aromatase is due to direct binding of the GR to a newly identified putative glucocorticoid responsive site within the aromatase proximal promoter. It is well known that glucocorticoids exert their effects by activating the GR, a liganddependent transcriptional regulator that transduces the hormonal signal into the nucleus to alter the expression of target genes. On glucocorticoid binding, GR undergoes conformational changes, dissociates from the heat shock proteins, homodimerizes, and translocates into the nucleus, where it binds to GREs into DNA in the promoter of target genes, resulting in stimulation or suppression of the transcription of response genes (known as transactivation or transrepression effect, respectively). 41 GR can regulate transcription by several distinct mechanisms, and its function, as shown for other corticosteroid receptors, seems to depend not only on ligand binding, which is known to regulate receptor conformation, but also on the context of the gene and associated promoter factors that contribute to create a gene-specific topography, achieving specific profiles of gene expression. Indeed, the liganded GR can interact with many components of the transcriptional machinery, including coactivators, corepressors, chromatin remodeling proteins, components of the mediator complex, and RNA polymerase II and components of the basal transcriptional machinery. 54 Data from ChIP analysis revealed that GR occupancy to the GRE-containing promoter region is concomitant with a decrease in RNA Pol II recruitment, consistent with the reduced aromatase transcriptional activity. GR-mediated repression of the aromatase gene involves the recruitment of the corepressors NCoR and SMRT, which share the same molecular architecture, interact with many of the same transcription factors, and assemble into similar corepressor complexes. 55 Indeed, NCoR and SMRT are recruited by GR to regulate the transcription of different genes. 56 The physiologic relevance of the inhibitory effects exerted by glucocorticoids on Leydig tumor cell growth is pointed out by our in vivo studies showing that DEXA significantly decreases the growth of R2C xenografts. Our results evidenced in tumor sections from DEXA-treated mice a marked reduction in the expression of the nuclear proliferation antigen Ki-67 and the estrogen-producing enzyme aromatase. Importantly, DEXA administration did not affect the normal testis structure. Indeed, no significant differences were found on the histopathologic features of seminiferous tubules between vehicle-treated and DEXAtreated mice.
In conclusion, our findings suggest the possibility that targeting the GR could be helpful in improving new molecular and pharmacologic approaches for LCT treatment.