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Clinical Cancer Research Vol. 11, 6495-6504, September 15, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Estrogen Inhibits Cell Proliferation through In situ Production in Human Thymoma

Hironori Ishibashi1,2,4, Takashi Suzuki1, Satoshi Suzuki2, Takuya Moriya1, Chika Kaneko1, Taisuke Nakata5, Makoto Sunamori4, Masashi Handa3, Takashi Kondo2 and Hironobu Sasano1

Authors' Affiliations: 1 Department of Pathology, Tohoku University School of Medicine; 2 Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan; 3 Department of Thoracic Surgery, Iwate General Hospital, Morioka, Japan; 4 Thoracic Cardiovascular Surgery, Graduate School, Tokyo Medical and Dental University; and 5 Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan

Requests for reprints: Hironori Ishibashi, Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi Aoba-ku, Sendai, Miyagi Prefecture 980-0875, Japan. Phone: 81-22-717-8521; Fax: 81-22-717-8526; E-mail: hishiba{at}kf6.so-net.ne.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We showed previously estrogen receptor (ER) {alpha} as an independent prognostic marker in human thymoma. Estrogen sulfotransferase (EST), steroid sulfatase (STS), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and aromatase are considered to play important roles in hormone metabolism of estrogen-dependent tumors.

Experimental Design: We examined estrogen production using primary cultures of human thymoma epithelial cells (TEC), intratumoral estradiol (E2) concentrations, and status of these enzymes above using immunohistochemistry or semiquantitative reverse transcription-PCR. We then correlated these findings with clinicopathologic variables and/or clinical outcome in 132 patients.

Results: E2 inhibited cell proliferation via ER{alpha} in TEC, which synthesized estrone and E2. Intratumoral E2 concentrations were inversely correlated with EST, positively correlated with STS or 17ß-HSD type 1, and significantly higher in lower-grade or early-stage thymoma. EST status was positively correlated with tumor size, clinical stage, histologic differentiation, and Ki-67 labeling index and significantly associated with adverse clinical outcome and turned out to be a potent independent prognostic factor. STS and/or 17ß-HSD type 1 status was inversely correlated with Ki-67 labeling index and associated with lower histologic grade or early clinical stages.

Conclusions: E2 inhibits proliferation of TEC through ER{alpha}, which suggests that E2 may be effective in treatment of thymoma, especially inoperable tumor, possibly through suppressing its cell proliferation activity. EST status is a potent prognostic factor in thymoma through inactivating estrogens. In situ estrogen synthesis through intracrine mechanism therefore may play important roles in tumorigenesis and/or development of thymoma through regulation of cell proliferation in an intracrine manner.

Estrogen regulates cell proliferation and/or other biological functions in various neoplasms derived from hormone-dependent tissues, such as breast and endometrial cancer (1). In situ estrogen levels are also considered to play very important roles in the pathogenesis of these neoplasms (2). We reported previously that estrogens play an important role via binding to estrogen receptor (ER) {alpha} in the development of human thymomas (3).

A major circulating form of plasma estrogen is estrone sulfate (E1-S), a biologically inactive form of estrogen. E1-S has a relatively long half-life in the peripheral blood (4), in which serum levels of E1-S are 10-fold higher than those of unconjugated E1 or estradiol (E2; ref. 5). E1-S is transformed into a biologically active form, E1, by steroid sulfatase (STS; refs. 6, 7), whereas aromatase catalyzes circulating androgen androstenedione into E1 (8). E1 is subsequently converted to E2 by 17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 1 in several estrogen target tissues, including breast cancer cells (9). E2 acts on breast cancer cells primarily via its binding to ER{alpha} and/or ERß. STS, aromatase, and 17ß-HSD type 1 have been all considered to be involved in in situ production of estrogen in neoplastic tissues, such as breast cancer. Estrogen sulfotransferase (EST), SULT1E1 or STE gene, is a member of the superfamily of cytosolic steroid sulfotransferases (10) and catalyzes E1 into biologically inactive E1-S.

In this study, we examined the expression and biological significance of EST, STS, 17ß-HSD type 1, and aromatase in human thymoma to further elucidate the status of in situ estrogen production in thymoma. We first studied effects of estrogens on cell proliferation and in situ E1 or E2 production from E1-S using primary cultures of thymoma epithelial cells (TEC). We then determined E2 concentrations and immunolocalized EST, STS, and 17ß-HSD type 1 in human thymoma. In addition, mRNA levels of EST, STS, 17ß-HSD type 1, and aromatase were examined using semiquantitative reverse transcription-PCR in human thymoma. We then correlated EST, STS, or 17ß-HSD type 1 immunoreactivity with clinicopathologic factors and clinical outcome in 132 patients with thymomas to establish clinical significance of these findings.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture analysis
Establishment of thymoma-derived neoplastic epithelial cellsin primary culture. Fresh thymoma tissues (five thymoma cases: two cases for type A, two cases for type B2, and one case for type B3), which expressed ER{alpha}, STS, or 17ß-HSD type 1 but not ERß were repeatedly washed in PBS and carefully minced and digested in a collagenase-containing mixture composed of collagenase type I (1 mg/mL; Wako, Osaka, Japan), DMEM, 10% fetal bovine serum (FBS), and antibiotics (penicillin and streptomycin) for 8 hours at 37°C (11). These minced and digested thymoma tissues were subsequently filtrated through three layers of sterile gauze and cultured by a 10-day culture in DMEM supplemented with 10% FBS and antibiotics but without any growth factors. On confluence, cells were mechanically dislodged by 0.25% trypsin and 0.02% EDTA and subjected to secondary cultures. Cells were subsequently characterized by their morphology and immunoreactivity of cytokeratin using immunocytostaining. Coexisting fibroblasts were eliminated by anti-fibroblast microbeads (MACS, Miltenyi Biotec, Gladbach, Germany) according to the manufacturer's instructions. The purity of the TEC, which expressed ER{alpha}, STS, or 17ß-HSD type 1 but not ERß, was examined by flow cytometry and the proportion of cytokeratin-positive cells was nearly always >97% (data not shown). TEC was grown to confluence in DMEM in the presence of 10% FCS and antibiotics under standard concentrations.

Cell proliferation assay. TEC was seeded at a density of 5 x 103 per mL into 24-well plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) in a final volume of 1 mL phenol red–free DMEM with 5% charcoal-stripped FBS. Medium was then replaced with phenol red–free DMEM without FBS to arrest the growth in 24 hours after seeding. The medium was replaced again with phenol red–free DMEM with 5% charcoal-stripped FBS together with either a vehicle (0.1%), increasing concentration of E2 (0.1, 1, 10, 100, and 1,000 nmol/L), and/or 100 nmol/L ICI 182,780 in 24 hours later. Following 72-hour incubation with these agents, the cells were trypsinized and resuspended. E2 was purchased from Sigma (St. Louis, MO) and ICI 182,780 from Tocris Cookson Ltd. (Ellisville, MO). Estrogens and anti-estrogens were dissolved in absolute ethanol (Sigma) and added to the medium daily. Cell cultures that were not treated with estrogenic compounds received absolute ethanol as a vehicle control. Total additive ethanol concentrations never exceeded 0.2% throughout the culture period. The cells were refed with freshly prepared medium every other day. We then used a Cell Counting Kit-8 system (Dojindo Technologies, Kumamoto, Japan) to determine the cell number. Then, 100 µL from each sample was aliquoted into a 96-well microtiter plate with 10 µL of the working solution containing WST-8 and then incubated for an additional 2 hours in a CO2 incubator at 37°C. The absorbance of each well was measured at 450 nm with a reference wavelength at 650 nm with a M-UVmax microscope reader (Molecular Devices Corp., Menlo Park, CA). An aliquot was taken from the medium to count the number of cells with a Burker-Turk counter (Nitirin, Tokyo, Japan).

Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling analysis. Visualization of apoptotic cells in chamber slides was done using the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling method (Apoptosis In situ Detection kit, Wako; ref. 12). As a negative control, fixed and permeabilized cells incubated without terminal deoxynucleotidyl transferase, but with secondary antibodies and 3,3'-diaminobenzidine, were used. As a positive control, permeabilized cells were incubated with bovine pancreatic DNase I before terminal deoxynucleotidyl transferase treatment to induce DNA fragmentation. The presence of terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling positivity was determined by visual inspection of cultures using light microscopy.

Estrone and estradiol production from thymoma epithelial cells. E1-S was added after a 48-hour culture in phenol red and FCS–free DMEM (final concentration, 0.5, 2.5, and 12.5 nmol/L). Medium (2 mL) was extracted twice with diethyl ether. Extracts were evaporated to dryness under nitrogen and reconstituted in 100 µL dextran-coated, charcoal-treated FBS. E1 and E2 levels of the extracts were then determined by RIA using an assay kit (Diagnostic Products, Tokyo, Japan). The sensitivity of the assay was 3 pmol/L.

Estradiol concentrations in human thymoma and thymus. E2 concentrations were examined in 20 cases of fresh frozen thymoma. These specimens were carefully dissected to eliminate all adjacent adipose and connective tissues. Cytosol and nuclear fractions were prepared by centrifugation of the homogenates (1.0 g specimens in 10 mL cold physiologic saline) at 4°C for 60 minutes at 15,000 x g in an ultracentrifuge. Tissue E2 concentrations were determined according to the methods described above.

Patients and tissue specimens. Thymoma cases (n = 132) were retrieved from surgical pathology files at Sendai Kousei Hospital (Sendai, Japan) and Tokyo Medical and Dental University (Tokyo, Japan). All specimens were fixed for 24 hours in 10% formalin at room temperature and embedded in paraffin wax. Relevant clinical findings, including patient age, sex, menopausal status, presence or absence of myasthenia gravis, tumor size, clinical stage (13), and WHO histologic classification (14) are summarized in Table 1.


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  		Table 1. Summary of clinical data in 132 patients and in 20 patients with thymoma examined in this study

 
Freshly frozen specimens were also available for reverse transcription-PCR analysis, real-time PCR, and E2 concentration analysis in 20 cases (Table 1) among these 132 cases of thymoma examined for immunohistochemistry. Specimens for RNA isolation were immediately frozen in liquid nitrogen and stored at –80°C until used for RNA isolation. RNA was extracted within 2 weeks following surgery. Informed consent was obtained from all the patients. The research protocols used in this study were approved by the Ethics Committee at Tohoku University School of Medicine, Sendai Kousei Hospital and Tokyo Medical and Dental University School of Medicine.

Antibodies. Rabbit polyclonal antibody for EST was raised against the synthetic NH2-terminal peptide of human EST corresponding to amino acids 1 to 13 (PV-P2237, Medical Biological Laboratory, Nagoya, Japan; 1:750 dilution). STS antibody was raised against the enzyme purified from human placenta, which recognizes the STS peptide corresponding to amino acids 420 to 428 (KM1049, kindly provided by Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan; 1:3,000 dilution) and has been reported previously in the evaluation of human breast cancer (15). 17ß-HSD type 1 antibody was a rabbit polyclonal antibody against the enzyme purified from human placenta (kindly provided by Dr. Van Luu-The, Laboratory of Molecular Endocrinology, CHUL Research Center, Quebec, Quebec, Canada; ref. 16).

The antibodies used were mouse monoclonal ER{alpha} (NCLER-6F11, Novocastra Lab, Newcastle, United Kingdom; 1:50 dilution), progesterone receptor-B (PR-B; hPRa2, NeoMarkers Co. Ltd., Fremont, CA; 1:200 dilution), and Ki-67 (MIB-1, Immunotech, Marseilles, France; 1:50 dilution) described in our previous study (3).

Immunohistochemistry. Immunohistochemical procedures employed in this study have been described previously in detail (2, 17, 18). Briefly, immunohistochemical staining was done by the streptavidin-biotin method with a Histofine kit (Nichirei Co. Ltd., Tokyo, Japan). The antigen-antibody complex was then visualized with 3,3'-diaminobenzidine solution and counterstained with hematoxylin. Antigen retrieval for ER{alpha}, PR-B, and Ki-67 immunostaining was done by heating the slides in an autoclave at 120°C for 5 minutes in citric acid buffer. Similarly, antigen retrieval for EST immunostaining was done by heating the slides in a microwave (500 W) for 15 minutes in citric acid buffer. No antigen retrieval was done for STS and 17ß-HSD type 1 immunostaining. As a positive control, normal liver was used for EST (18), normal full-term placenta for STS (18) and 17ß-HSD type 1 (19), breast cancer for ER{alpha}, and endometrium for PR-B (3). Normal rabbit and mouse IgG was used instead of the primary antibody as a negative control. No specific immunoreactivity was detected in these tissue sections.

Scoring of immunoreactivity. Immunoreactivity of EST, STS, and 17ß-HSD type 1 was analyzed according to a previously described method (20). After completely reviewing all the slides of immunostained sections for each thymoma, two of the authors (H.I. and T.S.) independently and blindly divided the thymomas into the following three groups: ++, >50% immunopositive cells; +, 1% to 50% immunopositive cells; and –, no immunoreactivity. Cases with discordant results among the observers were simultaneously reevaluated using a multiheaded microscope. Semiquantitative analysis of immunoreactivity of ER{alpha} and PR-B H-score and Ki-67 labeling index (LI) were done according to a previous report (3, 21, 22). Cases associated with a H-score of >50 were regarded as steroid receptor–positive thymomas (23).

Reverse transcription-PCR
RNA extraction and cDNA synthesis. Total RNA was extracted by homogenizing frozen tissue samples in 1 mL TRIzol reagent (Life Technologies, Inc., Grand Island, NY) followed by a phenol/chloroform phase extraction and isopropanol precipitation. The SuperScript Preamplification System RT kit (Life Technologies) was employed in the synthesis and amplification of cDNA according to the manufacturer's instructions.

Real-time reverse transcription-PCR. The Light Cycler System (Roche Diagnostics GmbH, Mannheim, Germany) was used to semiquantify the level of STS, EST, 17ß-HSD type 1, and aromatase mRNA expression in 20 cases of thymoma using real-time PCR (24). Settings for the PCR thermal profile were as follows: initial denaturing step of 95°C for 1 minute followed by 40 cycles, respectively, of 95°C for 0 second, 15-second annealing at 58°C (EST) and 60°C (glyceraldehyde-3-phosphate dehydrogenase, STS, and aromatase), and extension for 15 seconds at 72°C. The primer sequences used in this study are as follows: EST (NM005420; forward 5-AGAGGAGCTTGTGGACAGGA-3 and reverse 5-GGCGACAATTTCTGGTTCAT-3; ref. 25), STS (M16505; forward 5-AGGGTCTGGGTGTGTCTGTC-3 and reverse 5-ACTGCAACGCCTACTTAAATG-3; ref. 26), 17ß-HSD type 1 (XM 012644; forward 5'-AGGGCCGCGTGGACGTGCTGGTGTGTAAC-3' and reverse 5'-CCATCAATCCTCCCACGCTCCCGG-3'; ref. 17), aromatase (X13589; forward 5'-GTGAAAAAGGGGACAAACAT-3' and reverse 5'-TGGAATCGTCTCAGAAGTGT-3'; ref. 17), and glyceraldehyde-3-phosphate dehydrogenase (M33197; forward 5'-TGAACGGGAAGCTCACTGG-3' and reverse 5'-TCCACCACCCTGTTGCTGTA-3'; ref. 3). Liver (25) cells were used as a positive control for EST, whereas frozen tissues of placenta were used as a positive control for STS (26, 27), 17ß-HSD type 1, and aromatase (17). Negative control experiments lacked cDNA substrate to check for the presence of exogenous contaminant DNA. No amplified products were observed under these conditions and PCR products were purified and subjected to direct sequencing to verify amplification of the correct sequences as described previously. The mRNA levels for STS, EST, and aromatase in each case are summarized as a ratio of glyceraldehyde-3-phosphate dehydrogenase and evaluated as a ratio (%) compared with that of each positive control.

Statistical analysis. Values for patient age, tumor size, Ki-67 LI, H-scores of ER{alpha} or PR-B, and mRNA levels for EST, STS, 17ß-HSD type 1, or aromatase were summarized as a mean ± 95% confidence interval. Statistical analyses between E2 tissue concentration and aromatase mRNA level, ER{alpha} H-score, or PR-B H-score were done using a correlation coefficient (r) and regression equation. An association between immunoreactivity for EST, STS, or 17ß-HSD type 1 and these variables were evaluated using Kruskal-Wallis tests. Statistical differences between immunoreactivity of EST, STS, or 17ß-HSD type 1 and sex, status of myasthenia gravis, clinical stage, or WHO histologic classification were evaluated in a cross-table using the {chi}2 test. Overall survival curves were generated according to the Kaplan-Meier method, and the statistical significance was calculated using the log-rank test. Univariate and multivariate analyses were evaluated by a proportional hazard model (Cox) using PROC PHREG in SAS software. P < 0.05 was considered significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cell proliferation assay. E2 inhibits cell proliferation of TEC in proportion to the concentration of E2 (10, 100, and 1,000 nmol/L; P < 0.001; Fig. 1A). ICI 182,780, which is a steroidal anti-estrogen with no agonist activities, blocks inhibition of cell proliferation exerted by E2 (Fig. 1B; ref. 28). Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling analysis showed few apoptotic cells after treatment of E2 (data not shown).



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  		Fig. 1. Effects of E2 and anti-estrogen on cell growth of TEC. Cells (5 x 103/mL) were plated in triplicate well onto a 24-well plates. After 24 hours for cell attachment, cells were treated for 73 hours with E2 at various concentrations with or without anti-estrogen, ICI 182,780. Cells treated with vehicle (absolute ethanol) were used as control. Columns, mean (n = 5); bars, SD. *, P < 0.001, compared with control.

 
Estrone and estradiol synthesis in primary culture of thymoma-derived neoplastic epithelial cells. E1 and E2 production in primary cultures of TEC are summarized in Fig. 2. E1 and E2 concentrations are detected in proportion to the duration and concentration of E1-S. Concentrations of E1 and E2 in the medium of TEC treated with E1-S (2.5 and 12.5 nmol/L) were significantly higher than those with E1-S of 0.5 nmol/L after 8 and 24 hours (P < 0.05). E1 and E2 concentrations in phenol red and FBS–free DMEM were below the limits of detection.



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  		Fig. 2. Time course of E1 (A) and E2 (B) concentration produced from E1-S in primary culture of TEC. E1 and E2 concentrations were significantly increased in proportion to the duration and concentration of E1-S. Points, mean of three determinations; bars, SE. *, P < 0.05.

 
Correlation among estradiol concentrations and estrogen sulfotransferase, steroid sulfatase, or 17ß-hydroxysteroid dehydrogenase type 1 immunoreactivity, aromatase mRNA level, estrogen receptor {alpha} and progesterone receptor-B H-score, and clinical stage in 20 human thymomas. Results are summarized in Table 2. E2 concentrations in thymoma were significantly higher than those of normal thymus (59.3 ± 9.5 versus 19.2 ± 3.6; P < 0.05), inversely correlated with that of EST immunoreactivity (P = 0.013), and positively correlated with that of STS (P = 0.008), 17ß-HSD type 1 (P = 0.007), aromatase mRNA level (r = 0.601; P = 0.002), ER{alpha} H-score (r = 0.774; P < 0.001), and PR-B H-score (r = 0.749; P = 0.001). E2 concentrations were significantly higher in cases with earlier clinical stage (P = 0.006).


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  		Table 2. Correlation between E2 tissue concentrations and other variables in 20 human thymomas

 
Immunolocalization of estrogen sulfotransferase, steroid sulfatase, and 17ß-hydroxysteroid dehydrogenase type 1 in human thymoma. EST immunoreactivity was detected predominantly in the cytoplasm of epithelial cells of thymoma (Fig. 3A) but not in lymphocytes (Fig. 3B). The number of the cases positive for EST in 132 human thymomas was summarized as follows: ++, n = 22 (16.6%); +, n = 55 (41.7%); and –, n = 55 (41.7%).



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  		Fig. 3. Immunohistochemistry for EST (A and B), STS (C and D), and 17ß-HSD type 1 (E and F) in tissue specimens of human thymoma. Immunoreactivity for EST, STS, and 17ß-HSD type 1 was detected in the cytoplasm of TEC but not in lymphocytes. A, C, and E, type A by WHO classification (same field); B, D, and F, type B3 (same field). Original magnification, x400. Bar, 25 µm.

 
STS immunoreactivity was also detected predominantly in the cytoplasm of epithelial cells of thymoma (Fig. 3C) but not in lymphocytes (Fig. 3D). The number of cases positive for STS immunoreactivity was summarized as follows: ++, n = 20 (15.2%); +, n = 57 (43.2%); and –, n = 55 (41.6%).

17ß-HSD type 1 immunoreactivity was detected predominantly in the cytoplasm of epithelial cells of thymoma (Fig. 3E) but not in lymphocytes (Fig. 3F). The number of cases positive for 17ß-HSD type 1 immunoreactivity in these cases was summarized as follows: ++, n = 27 (20.5%); +, n = 46 (34.8%); and –, n = 59 (44.7%).

Real-time PCR analysis. mRNA expression of EST, STS, 17ß-HSD type 1, aromatase, and glyceraldehyde-3-phosphate dehydrogenase was identified as a specific single band at 114, 290, 201, 215, and 307 bp, respectively (data not shown). There were significant positive correlations between EST immunoreactivity and its mRNA level (P = 0.001), STS immunoreactivity and its mRNA level (P = 0.010), and 17ß-HSD type 1 immunoreactivity and its mRNA level (P = 0.007; data not shown).

Correlation between estrogen sulfotransferase, steroid sulfatase, and 17 ß-hydroxysteroid dehydrogenase type 1 immunoreactivity and clinicopathologic variables. An association between EST, STS, or 17ß-HSD type 1 immunoreactivity and clinicopathologic factors in thymoma patients were summarized in Table 3. EST immunoreactivity was positively correlated with tumor size (P = 0.013), clinical stage of the patients (P < 0.001), Ki-67 LI (P = 0.045), and mRNA level of aromatase (P = 0.029). EST immunoreactivity was inversely correlated with ER{alpha} H-score (P = 0.009) and PR-B H-score (P = 0.015).


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  		Table 3. Correlation between EST, STS, and 17ß-HSD type 1 immunoreactivity and pathologic variables in 132 human thymomas

 
There were significant inverse correlations between STS immunoreactivity and clinical stage of the patients (P = 0.036) or Ki-67 LI (P = 0.011). There were significant positive correlations between STS immunoreactivity and ER{alpha} H-score (P = 0.005) or PR-B H-score (P = 0.006).

There were significant inverse correlations between 17ß-HSD type 1 immunoreactivity and clinical stage of the patients (P = 0.008) or Ki-67 LI (P = 0.005) and positive correlations between 17ß-HSD type 1 immunoreactivity and ER{alpha} H-score (P = 0.001) or PR-B H-score (P = 0.001).

Correlation between WHO classification and other variables. An association between WHO classification and clinicopathologic factors in thymoma patients was summarized in Table 4. E2 concentrations were significantly higher in type A thymoma than type B thymoma (P = 0.005). There was a significant positive correlation between WHO classification and EST immunoreactivity (P < 0.001) and inverse correlations between WHO classification and STS immunoreactivity (P < 0.001), ER{alpha} H-score (P = 0.015), or PR-B H-score (P = 0.028).


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  		Table 4. Correlation between WHO classification and other variables in human thymomas

 
Correlation between estrogen sulfotransferase, steroid sulfatase, and 17ß-hydroxysteroid dehydrogenase type 1 immunoreactivity and overall survival of patients with thymoma. There was a significant positive correlation between EST immunoreactivity and clinical outcome of the patients (P = 0.0001; Fig. 4A). No significant correlations were detected between STS or 17ß-HSD type 1 immunoreactivity and clinical outcome in patients with thymoma (Fig. 4B and C).



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  		Fig. 4. Overall survival of 132 patients with thymoma with respect to EST (A), STS (B), and 17ß-HSD type 1 (C) immunoreactivity (Kaplan-Meier method). EST immunoreactivity was significantly associated with an improved overall survival (P = 0.0001) but STS and 17ß-HSD type 1 immunopositive thymoma was relatively associated with better clinical outcome, although this association did not reach statistical significance.

 
Following a univariate analysis (Table 5), clinical stage (P = 0.0017), ER{alpha} immunoreactivity (P = 0.0021), EST immunoreactivity (P = 0.0023), and tumor size (P = 0.0024) all turned out to be significant prognostic factors of overall survival in 132 thymoma patients. A subsequent multivariate analysis revealed that only clinical stage (P = 0.0239), ER{alpha} immunoreactivity (P = 0.0355), and EST immunoreactivity (P = 0.0436) were independent prognostic factors with relative risks of >1.0 in our series of 132 thymoma patients.


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  		Table 5. Univariate and multivariate analyses of overall survival in 132 thymoma patients

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Estrogens have been considered to play important roles in biological features of thymoma. However, this is the first study to show an inhibition of thymic epithelial cell proliferation by estrogens, the presence of in situ production of estrogens in human thymoma using primary cell culture, and a correlation between the status of estrogen metabolizing or producing enzymes and clinicopathologic variables or endogenous estrogen concentrations in human thymus and thymoma.

In our present study, TEC proliferation was inhibited by E2 via ER{alpha}. This finding is consistent with our previous study, which suggests that E2 or E2-like agents may be effective in treatment of thymoma, especially in cases of inoperable or disseminated tumor possibly through suppressing its cell proliferation activity (3). In this study, E1 and E2 were both produced from E1-S in ER{alpha}, STS, and 17ß-HSD type 1 positive TEC in proportion to the duration and concentration of E1-S administered. In addition, E2 tissue concentrations were inversely correlated with the status of EST immunoreactivity, positively correlated with the status of STS or 17ß-HSD type 1 immunoreactivity, and significantly higher in earlier clinical stages and lower histologic grades. These findings showed that lower histologic grade tumors or well-differentiated thymoma cells were associated with in situ estrogen biosynthesis and estrogenic actions via binding to ER{alpha}, which result in E2-dependent inhibition of tumor growth.

EST immunoreactivity was also detected in TEC and positively correlated with Ki-67 LI or tumor size and inversely correlated with H-score of ER{alpha} or PR-B. In addition, EST-positive thymoma patients tended to be associated with advanced clinical stages with higher histologic grades and significantly adverse clinical outcome than EST-negative thymoma. In addition, EST immunoreactivity was shown to be an independent prognostic factor for overall survival as well as ER{alpha} and clinical stage following multivariate analysis. Tissue E2 concentrations were also inversely correlated with EST immunoreactivity. Therefore, EST-negative thymoma is considered to result in an increased in situ concentration of biologically active estrogens, which may contribute to inhibition of cell proliferation in thymoma through ER{alpha}. Qian et al. showed that MCF-7 breast cancer cells transfected with EST expressed EST at levels similar to those observed in normal human mammary epithelial cells and are associated with much lower estrogen-stimulated DNA synthesis or cell proliferation than MCF-7 cells not associated with EST expression (29). EST-negative breast cancer is therefore considered to be associated with an increased in situ estrogen concentration, which results in an increased incidence of tumor recurrence and subsequent poor clinical outcome for the patients diagnosed with breast cancer (20, 30). EST is more frequently detected in TEC in higher histologic grades or advanced clinical stages, which are consistent with decreased expression of STS or 17ß-HSD type 1. All of these are considered to result in decreased levels of E2 in these thymoma cases. STS and 17ß-HSD type 1 immunoreactivity was also positively correlated with the H-score of ER{alpha} and PR-B and inversely correlated with Ki-67 LI. STS-positive and 17ß-HSD type 1–positive thymoma cases were significantly correlated with earlier clinical stages and lower histologic grades. In addition, 17ß-HSD type 1–positive thymoma tends to be associated with favorable clinical outcome. STS catalyzes E1-S to E1 and 17ß-HSD type 1 catalyzes E1 to E2 in human thymoma, which contributes to increase in situ estrogen concentration (31, 32). Results of our present study suggest that STS and 17ß-HSD type 1 also contribute to estrogenic actions in human thymoma through in situ estrogen production. In contrast, a significant inverse correlation was reported between 17ß-HSD type 1 immunoreactivity and Ki-67 LI or histologic grade in patients with breast cancer (33, 34). 17ß-HSD type 1 immunoreactivity is considered to reflect its enzymatic activity (16), and ER immunoreactivity has been shown to be correlated with estrogen-dependent biological phenomena (35). Therefore, results of our present study showed that in situ produced E2 exerts its effects through ER{alpha} in human thymoma, which may be consistent with the relatively better clinical outcome of 17ß-HSD type 1–positive human thymoma patients. Further investigations are required for clarification.

PR-B H-score was positively correlated with STS and 17ß-HSD type 1 immunoreactivity and inversely correlated with EST immunoreactivity in human thymoma. These findings are also consistent with the fact that expression of PR is estrogen related, because PR has been regarded as one of the markers of a functional estrogen pathway (36) and ER{alpha}-positive thymomas are generally positive for PR-B (3). The levels of immunoreactive EST and EST mRNA in Ishikawa cells, a cell line established from human endometrial adenocarcinoma, were both shown to be increased by progesterone. These results suggest that progesterone is capable of specifically inducing EST and estrogen sulfation in the human Ishikawa endometrial adenocarcinoma cell line (37). Furthermore, progestin has also been reported to induce 17ß-HSD enzyme protein in the T-47D human breast cancer cell line (38). Therefore, coexpression of EST, STS, and/or 17ß-HSD type 1 and PR detected thymoma in this study suggests that progesterone may play an important role in regulating the expression of these enzymes involved in in situ estrogen metabolism in thymoma. However, it awaits further investigating to identify the specific roles of these enzymes in metabolizing estrogens in thymoma.

Aromatase cytochrome P450 (CYP19 gene) is an enzyme located in the endoplasmic reticulum of estrogen-producing cells and a key enzyme mainly involved in the aromatization of androstenedione to E1 (29). In the present study, there was a statistically significant positive correlation between aromatase mRNA level and immunoreactivity for EST but not for STS, 17ß-HSD type 1, and aromatase mRNA levels tended to correlate with E2 tissue concentrations. These results also suggest that estrogen can still be produced via aromatase pathway in EST-positive thymoma case even if the activity of aromatase was 0.5% to 2% of STS (3).

We show the findings of in situ estrogen production in human thymoma (Fig. 5). Advanced stage or high-grade thymoma produces low level of E2, which cannot inhibit the proliferation of TEC (Fig. 5A). On the other hand, high concentration of E2 is produced in early-stage or low histologic grade thymoma and inhibits the proliferation of TEC (Fig. 5B).



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  		Fig. 5. Summary of local production of estrogens in high-grade (A) and low-grade (B) thymoma. High concentrations of circulating inactive steroids, androstenedione and E1-S, are major precursor substrates of local estrogen production in these tissues. Aromatase catalyzes androstenedione into E1, and STS hydrolyzes E1-S to E1. E1 is subsequently converted to potent E2 by 17ß-HSD type 1 and acts on TEC via ER{alpha}. EST sulfonates E1 to biologically inactive E1-S. High intratumoral E2 concentration as a result of in situ production in thymoma with early-stage or well-differentiated histologic features is therefore considered to result in inhibition of cell proliferation of TEC.

 
In summary, high intratumoral E2 concentration as a result of in situ production in thymoma with early-stage or well-differentiated histologic features is therefore considered to result in inhibition of cell proliferation of TEC.


   Acknowledgments
 
We thank Drs. Touichirou Takizawa and Takumi Akashi (Department of Pathology, Graduate School, Tokyo Medical and Dental University) and Dr. Hideki Akamatsu (Department of Thoracic Cardiovascular Surgery, Graduate School, Tokyo Medical and Dental University) for their kind efforts in retrieving the specimens of thymoma and Andrew D. Darnel (Department of Pathology, Tohoku University School of Medicine) for careful editing of this article.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/ 6/04; revised 4/26/05; accepted 5/31/05.


   References
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 Abstract
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