Biochemical and Biophysical Research Communications 285, 340 –347 (2001) doi:10.1006/bbrc.2001.5158, available online at http://www.idealibrary.com on
Expression, Function, and Clinical Implications of the Estrogen Receptor  in Human Lung Cancers Yoko Omoto,* ,† Yasuhito Kobayashi,‡ Kazunori Nishida,‡ Eiju Tsuchiya,* Hidetaka Eguchi,* Ken Nakagawa,§ Yuichi Ishikawa, ¶ Takao Yamori,㛳 Hirotaka Iwase,† Yoshitaka Fujii,† Margaret Warner,** ,†† Jan-Åke Gustafsson,** ,†† and Shin-ichi Hayashi* ,1 *Division of Endocrinology, Saitama Cancer Center Research Institute and ‡Department of Clinical Pathology, Saitama Cancer Center Hospital, Komuro, Ina, Kitaadachi-gun, Saitama 362-0806, Japan; †Department of Surgery II, Nagoya City University Medical School, 1 Kawasumi, Mizuho-ku, Nagoya 467-8601, Japan; §Department of Chest Surgery, Cancer Institute Hospital, ¶Department of Pathology and 㛳Department of Molecular Pharmacology, Cancer Chemotherapy Center, Cancer Institute, 1-37-1, Kamiikebukuro, Toshima-ku, Tokyo 170-8455, Japan; and **Department of Medical Nutrition and ††Department of Biosciences, Karolinska Institute, Huddinge, Sweden
Received May 28, 2001
The higher frequency of human lung adenocarcinoma in females than in males, strongly suggests the involvement of gender dependent factors in the etiology of this disease. This is the first investigation of estrogen receptor (ER)  in human lung. Immunohistochemical staining revealed ER expression in normal lung and in atypical adenomatous hyperplasia (AAH), considered as a precancerous lesion for adenocarcinomas. Adenocarcinomas showed significantly higher expression of ER than squamous cell carcinomas. On the contrary, ER␣ expression was not detected in all cases. The functional integrity of ER such as the binding ability to estrogen responsive element (ERE) and transcriptional activity was confirmed using a human lung cancer cell line, RERF-LC-OK. Colony formation of this cell was significantly reduced in the presence of pure antiestrogen. We conclude that ER, but not ER␣, is present in lung tissues with an important physiological function in normal lung. Furthermore, ER may play a role in growth and development of adenocarcinomas. © 2001 Academic Press Abbreviations used: 4OHT, 4-hydroxytamoxifen; AAH, atypical adenomatous hyperplasia; AP, alkaline phosphatase; DCC-FCS, FCS stripped of steroids by absorption with dextran-coated charcoal; EMSA, electrophoretic mobility shift assay; ER, estrogen receptor; ERE, estrogen response element; FCS, fetal calf serum; ICI, ICI 182,780; LBD, ligand binding domain; NSCLC, nonsmall cell lung carcinoma; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; PLSD, protected least significant difference; PRF-RPMI, phenol-red free RPMI1640; RT, reverse transcription; TBE, tris/borate/EDTA; tk, thymidine kinase. 1 To whom correspondence should be addressed at Shin-ichi Hayashi, Division of Endocrinology, Saitama Cancer Center Research Institute, 818 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan. Fax: ⫹81-48-722-1739. E-mail:
[email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Key Words: estrogen receptor ; lung cancer; adenocarcinoma.
Lung cancer is one of the main causes of cancer death in the world (1) and its mortality rate has been increasing in Japan since 1950 (2). Histologically, lung cancer is classified into small cell and nonsmall cell lung carcinoma (NSCLC) categories, the latter including squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and adeno-squamous cell carcinoma. While squamous cell carcinoma has a very strong association with smoking and is frequently found in males (3), adenocarcinoma is less clearly linked to tobacco and tends to occur in females and juveniles (4). According to recent statistics, along with a decline of smoking population, the incidence of squamous cell carcinoma is decreasing in many countries, whereas the incidence of adenocarcinoma is increasing rapidly. Generally, adenocarcinoma constitutes about one third of primary lung cancer cases among males and about three fourths of those among females (5–7). We have been studying the relationship between genetic alterations and clinicopathological characteristics of NSCLCs (8 –16), and have recently revealed that different subtypes of adenocarcinoma may be caused by different etiological factors (17). For instance, genetic mutations of p53 and K-ras that are commonly observed with lung tumors are rarely found in the hobnail type adenocarcinomas, seem to have a weak association with smoking, and are frequently found in females (17). On the other hand, columnar type adenocarcinomas, frequently show genetic mutations and seem to have an association with smoking. Thus, gen-
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der factors such as female sex hormones may possibly influence the genesis of lung adenocarcinoma, especially the hobnail type. Estrogens and their receptors play important roles in regulating growth and differentiation of various tissues; ER␣, a ligand activated transcription factor, which binds to the ERE, in the promoter regions of target genes (18) thereby regulates transcription of various estrogen target genes and appears to be associated with development of estrogen-dependent cancers such as breast and endometrial carcinoma. Indeed, the expression status of ER␣ is regarded as a good predictor of prognosis with endocrine treatment for these cancers. ER␣ expression has also been assessed in other cancers, such as hepatocarcinomas (19), and gastric cancers (20, 21), for which gender differences have been suggested from clinicopathological data. Although several reports have suggested the presence of ER␣ in human lung cancer (22–26), the results are somewhat inconsistent and do not seem to be important from a clinicopathological perspective. The novel ER, ER, which was identified in 1996 (27), is expressed in rat lung (28); however, expression and possible functional significance of ER in human lung tissue, including cancer, have to our knowledge not been assessed so far. We therefore undertook to study ER protein in human lung tissues by immunohistochemistry and in lung cancer cell lines by immunoblotting and, furthermore, examine the role of ER in cell growth. MATERIALS AND METHODS Patients. The subjects were thirty primary lung cancer patients who underwent surgery at the Department of Chest Surgery, Cancer Institute Hospital (Tokyo, Japan). Clinical information is listed in Table 1. Four cases of AAH were obtained at the Department of Surgery, Saitama Cancer Center Hospital (Saitama, Japan). Five normal lung specimens were obtained at surgery for metastatic lung cancer at the Department of Surgery II, Nagoya City University Medical School (Nagoya, Japan). Immunohistochemical analysis. Representative blocks of paraffin-embedded tissues were cut at 4-m thickness. The sections were autoclaved for 15 min at 120°C, and blocked for endogenous peroxidase activity with hydrogen peroxidase. After prevention of nonspecific reactions with Block-ace solution (Dai-nippon Pharmaceutical, Osaka, Japan), the primary antibodies, anti-ER chicken IgY polyclonal antibody (29) or anti-ER␣ rabbit monoclonal antibody 1D5 (Dako, Kyoto, Japan) were applied with incubation overnight at 4°C. For ER staining, peroxidase-conjugated anti-chicken IgY rabbit antibody (Cosmobio, Tokyo, Japan) was then applied to the sections for 30 min at room temperature. For ER␣ staining, Envision solution (Dako) was applied for 45 min at room temperature. Peroxidase activity was visualized with 3,3⬘-diaminobenzidine, and 0.03 mol/l hydrogen peroxide for 5 min. The sections were lightly counterstained with hematoxylin for microscopy. As a negative control, duplicate sections were immunostained without exposure to primary antibodies. The specificity of the ER antibody has been described previously (29). Immunohistochemistry samples were evaluated blindly, by a pathologist [E.T.]. Nuclei of normal bronchiolar epithelial cells, AAH cells, and carcinoma cells stained brown with ER
TABLE 1
Clinicopathological Characteristics of Primary Lung Cancers Characteristics
No. of cases (%)
Total Age (years) Mean ⫾ SD Range Sex Male Female Histology Adenocarcinoma Hobnail Columnar Squamous cell carcinoma Differentiation Well Moderately Poorly Unknown Pathological stage I II III IV
30 60.9 ⫾ 8.8 48–75 22 (73) 8 (27) 20 (67) 10 (33) 10 (33) 10 (33) 5 (17) 18 (60) 6 (20) 1 (3) 13 (43) 5 (17) 10 (33) 1 (3)
antibody, but those of stromal cells did not show any staining. For normal bronchiolar epithelium, if brown nuclei were observed in more than one-fourth of epithelial cells of a bronchiole, the epithelium was considered positive. For AAHs and carcinomas, if cells with nuclei which showed equal or stronger staining compared with those of normal bronchiolar epithelial cells occupied more than one-fourth of the tumor, it was considered positive. Fisher’s exact probability or chi-square tests were used for the statistical analysis of correlation between ER immunostaining and histopathological status. Differences were considered significant when a P value less than 5% was obtained. Cells and culture. Human lung cancer cell lines (A549, DMS114, DMS273, EBC-1, NCI-H23, NCI-H460, NCI-H522, VMRP-LCP, LK-2, RERF-LC-MA, RERF-LC-MS, RERF-LC-OK, PC-3, PC-6, PC-9, and PC-10) and a human breast cancer cell line (MDA-MB231) were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS), a human breast cancer cell line (MCF-7) was in RPMI 1640 medium with 10% FCS, and a human osteosarcoma cell line (SaOS2) was in McCoy’s 5A medium with 15% FCS, at 37°C in a humidified atmosphere of 5% CO 2 in air. All culture media contained 2 mM L-glutamine, 5 units/ml penicillin, and 5 g/ml streptomycin (Sigma, Tokyo, Japan). For experiments evaluating the effect of 17-estradiol, FCS stripped of steroids by absorption with dextran-coated charcoal (DCC-FCS) was substituted for FCS. Immunoblotting. Western blot analysis was performed as previously described (30) with a slight modification. Briefly, total cell lysates were prepared in RIPA buffer (1 ⫻ phosphate buffered saline (PBS), 1% Igepal CA-630, 0.5% sodium deoxycholate, and 0.1% SDS) and the protein concentrations were determined with BCA Protein Assay Reagent (Pierce, Rockford, IL). Aliquots of 50 g of cell extract were subjected to 10% SDS–PAGE and proteins were electrically transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA), incubated with primary anti-ER ligand binding domain (LBD) rabbit polyclonal or anti-ER␣ rabbit polyclonal G-20 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, at 1/3000 dilution in both cases. The secondary antibody was alkaline phosphatase (AP) conjugated anti-rabbit antibody (Bio-Rad Laboratories,
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Hercules, CA) at 1/3000 dilution. Detection of ER␣ and ER was performed using Immun-Star Substrate (Bio-Rad) and a Fuji Luminoimage Analyzer LAS-1000 system (Fuji Film, Tokyo, Japan). Plasmids. An estrogen-responsive reporter plasmid, pEREluciferase, was constructed by insertion of ERE (upper strand, 5⬘AGC TAG GTC AGG ATG ACC TAG CTA C-3⬘; lower strand, 5⬘-AGC TGT AGC TAG GTC ATC CTG ACC T-3⬘) into the HindIII site of the thymidine kinase (tk)-luciferase plasmid (31). The full-length cDNA for human ER (32), kindly supplied by Dr. M. Muramatsu, was inserted into the HindIII site of a pRc/CMV expression plasmid (Invitrogen, Carlsbad, CA). The sequences of these plasmids were confirmed using a BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Tokyo, Japan) and an ABI PRISM 310 genetic analyzer (Perkin-Elmer, Foster City, CA). Electrophoretic mobility shift assay. Double-stranded oligonucleotides, containing a vitellogenin ERE (upper strand, 5⬘-GGT TTG GCA AGG GTC ACA ATG ACC TCA ACA-3⬘; lower strand, 5⬘-GGT GTT GAG GTC ATT GTG ACC CTT GCC AAA-3⬘) were labeled with [␣- 32P]dCTP using the Klenow fragment of DNA polymerase I (Takara, Tokyo, Japan). Nuclear extracts of RERF-LC-OK and MCF-7 cells were prepared by Dignam’s method (33). Prepared nuclear extracts were stored at ⫺150°C until use, and electrophoretic mobility shift assay (EMSA) reactions were carried out as described previously (34). Increasing amounts (20 ng, 200 ng) of nonlabeled competitor ERE, unspecific competitor AP-1 (120 ng) and Sp-1 (120 ng), or anti-ER rabbit polyclonal antibody, prepared against synthesized peptides of the N-terminal region of ER (SLNSPSSYNC), were added to the incubation as indicated. Two nanograms of radiolabeled ERE probe (10 4 c.p.m.) were added to the reaction mixture and incubated on ice for 20 min. The reaction mixture was then separated on a 5% polyacrylamide gel run for 3.5 h at 4°C in 0.5 ⫻ tris/borate/EDTA (TBE) buffer. Transient transfection and luciferase assays. Transient transfection was performed essentially as described previously (35) with some slight modifications. Briefly, after one week’s culture of RERFLC-OK cells in PRF-RPMI medium with DCC-FCS, 5 ⫻ 10 5 cells were plated on 6 cm diameter plastic culture plates in the same medium and incubated for 24 h. One microgram of tk-ERE-Luc plasmid, with or without 0.1 g of pRc/CMV-ER expression plasmid, was mixed with 5 l TransIT LT-1 reagent (Takara) in 1 ml of serum-free medium and subjected to transfection according to the manufacturer’s instructions. After culturing the cells for a further 24 h in the presence of ethanol or the indicated concentrations of 17-estradiol, 4-hydroxytamoxifen (4OHT), or pure anti-estrogen ICI 182,780 (ICI) (supplied from AstraZeneca, London, UK), the cells were lysed in lysis buffer and luciferase activity was measured in triplicate using a Luciferase Assay System (Promega, Madison, WI). The luciferase activity was normalized with each protein concentration. The Fisher’s protected least significant difference (PLSD) test was used for comparing luciferase activity between control and experimental groups. Differences were considered significant when a P value less than 5% was obtained. Colony formation assay. After a week’s culture of the RERFLC-OK lung cancer cells in phenol-red free (PRF) RPMI medium with 5% DCC-FCS, they were split at 5 ⫻ 10 5, and plated in duplicate in 6 cm diameter plastic culture plates with the same medium and incubated for 24 h. One microgram of pRc/CMV expression plasmid, harboring the neomycin gene was mixed with TransIT LT-1 reagent (Takara), and added to the culture, following the manufacturer’s instructions. After 4 h of incubation, the medium was replaced with fresh medium containing 1 mg/ml Geneticin (G418) (Sigma) in the presence of the indicated additives, with incubation for 2 weeks with medium changed twice a week. Cells were then fixed with 3.8% formaldehyde and stained with 0.1% (w/v) crystal violet. The number of colonies larger than 100 m in diameter were counted per dish.
Anchorage independent colony formation assay. After a week’s culture of the RERF-LC-OK lung cancer cells in PRF-RPMI medium with 5% DCC-FCS, 2 ⫻ 10 4 cells were suspended in 1.5 ml of 0.33% Difco’s noble agar in PRF-RPMI with 10% DCC-FCS in the presence of the indicated additives and layered over 4 ml of 0.66% agarmedium basal layer in triplicate in 6 cm diameter plastic culture plates. Cells were then fed with PRF-RPMI medium with DCC-FCS in the presence of the indicated additives and incubated for 21 days with medium changed every 3 days. The number of colonies larger than 50 m in diameter were then counted per dish.
RESULTS ER and ER␣ Protein Expression in Normal Bronchiolar Epithelium, AAHs, and Carcinomas We first carried out immunohistochemical staining of lung specimens with specific anti-ER and -ER␣ antibodies. Normal bronchiolar epithelium in all 35 cases stained positively for ER (Fig. 1A). All four cases of AAH showed ER staining (Fig. 1B), the intensity being much stronger than that of bronchiolar epithelium. Out of 30 lung cancers, 20 (67%) were positive (Fig. 1C), and 10 (33%) were negative (Fig. 1D) (Table 2). A significant difference in ER expression was observed between adenocarcinomas and squamous cell carcinomas: 17 out of 20 (85%) adenocarcinomas were positive, but this was the case for only 3 out of 10 (30%) squamous cell carcinomas (Table 2) (P ⫽ 0.0048, Fisher’s exact probability test). When we considered the subtypes of adenocarcinoma, all of 10 (100%) hobnail type and whereas 7 of 10 (70%) columnar type were positive (Table 2). Differences between these three groups were statistically significant (P ⫽ 0.0039, chi-square test). No expression of ER␣ was evident in bronchiolar epithelium, AAHs, or primary lung cancer samples (data not shown). Western Blotting Analysis of ER in Lung Cancer Cell Lines Next, we studied the expression of ER in human lung cancer cell lines using Western blot analysis. Four of sixteen lung cancer cell lines, RERF-LC-OK, PC-3, DMS114 and PC-6, showed ER protein expression (Fig. 2). The expected results for SaOS2 osteosarcoma and MDA-MB-231 breast cancer cells, both of which are known to be ER positive, and the ER negative MCF-7 breast cancer cells are also shown in Fig. 2. We used RERF-LC-OK cells for our further studies because ER expression in this case was the highest of all lung cancer cell lines we tested. ER␣ protein was not detected in any of the examined samples, except for MCF-7 cells (data not shown). ER Binding Activity to ERE in the RERF-LC-OK Lung Cancer Cell Line To test whether ER, observed in lung cancer cells on Western blot analysis, possessed ERE binding activity,
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FIG. 1. Immunohistochemical staining of ER in human lung tissues. Blocks of paraffin-embedded tissues were cut at 4-m thickness and anti-ER chicken IgY polyclonal antibody was applied. Magnification, ⫻400. (A) Normal bronchiolar epithelium. Nuclei of epithelial cells are stained brown with ER antibody. (B) Atypical adenomatous hyperplasia. Nuclei of AAH show strongly positive staining. (C) Hobnail type adenocarcinoma. Nuclei of carcinoma cells are positive. (D) Columnar type adenocarcinoma. Nuclei of carcinoma cells show negative staining.
we performed an electrophoretic mobility shift assay (EMSA). Nuclear extracts from RERF-LC-OK cells demonstrated faster migrating DNA binding complexes (Fig. 3, lane 2) than those from MCF-7 cells (Fig. 3, lane 1). This complex formation was blocked by excess amounts
of unlabeled ERE but not AP-1 and Sp-1 oligonucleotides (Fig. 3, lanes 3– 6). Furthermore, the intensity of the complex was decreased on addition of anti-ER antibodies (Fig. 3, lane 7), strongly indicating ER–ERE complex formation with ER, expressed in RERF-LC-OK cells.
TABLE 2
Association of ER Staining with Histology for the 30 Primary Lung Cancers No. of cases (%) ER immunostained
Total Histology Squamous cell carcinoma Adenocarcinoma Hobnail Columnar
⫹
⫺
20 (67)
10 (33)
3 (30) 17 (85) 10 (100) 7 (70)
7 (70) 3 (15) 0 (0) 3 (30)
* P, Fisher’s exact probability test. ⫹ P, chi-square test. 343
*P ⫽ 0.0048 ⫹
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FIG. 2. Western blot analysis of ER protein in various human cancer cell lines. Results for six lung cancers, one osteosarcoma, and two breast cancer cell lines are presented. Fifty micrograms of total cell lysates were subjected to 10% SDS–PAGE, transferred onto a PVDF membrane, and anti-ER LBD polyclonal antibody was applied for immunostaining. M, protein marker.
ER Transcriptional Activity in RERF-LC-OK Lung Cancer Cell Line To investigate whether ER in lung cancer cells may mediate transcriptional activity via conventional ERE, we performed a transient transfection experiment using the ERE-Luc reporter plasmid in RERF-LC-OK cells, expressing ER, but lacking ER␣. ER/EREmediated transactivation was weakly enhanced by addition of 17-estradiol (Fig. 4A), indicating endogenous lung ER may mediate ERE-transcription activity. Furthermore, RERF-LC-OK cells cotransfected with the ER-expression plasmid (pRc/CMV-ER) exhibited highly stimulated transcription activity by 17-estradiol (Fig. 4B). The pure anti-estrogen, ICI 182,780, strongly inhibited this ER mediated ERE-transcription activity, in both endogenous and ectopic expression systems in a dose dependent manner. Colony Formation Assay To investigate the influence of ER expression on growth of lung cancer cells we performed anchorageindependent colony formation assay in soft agar as described under Materials and Methods. RERF-LC-OK cells, transfected with pRc/CMV plasmid, showed a reduced number of colonies with pure anti-estrogen ICI in a dose dependent manner (ICI 1 M, P ⫽ 0.0049; ICI 10 M, P ⫽ 0.0003; Fisher’s PLSD test) (Fig. 5). Anchorage-independent growth of RERF-LC-OK cells was also decreased in soft agar (P ⫽ 0.0058, Fisher’s PLSD test) (Fig. 6). No significant effect on cell growth was observed with 17-estradiol (Figs. 5 and 6). Similar results were obtained when the RERF-LC-OK cells were transfected with the pRc/CMV-ER expression
FIG. 3. Electrophoretic mobility shift assay (EMSA). Nuclear extracts from MCF-7 (lane 1) and RERF-LC-OK (lanes 2–7) cells were used to test whether the two types of ER possessed [␣- 32P]labeled ERE binding ability. Extracts from RERF-LC-OK cells showed a faster migrating DNA complex (lane 2, white arrow), compared with those from MCF-7 (lane 1, black arrow). Specific binding of ER was confirmed by a competition experiment using increasing amounts (20 ng, 200 ng) of cold ERE (lanes 3 and 4), and unspecific competitors, AP-1 (120 ng) (lane 5), and Sp-1 (120 ng) (lane 6) oligonucleotides. Binding complexes were decreased upon addition of specific anti-ER antibody (lane 7).
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FIG. 4. Transcriptional activity of ER in the RERF-LC-OK lung cancer cell line assessed using the tk-ERE-Luc reporter plasmid. (A) Cells were transfected with 1 g of tk-ERE-Luc plasmid, and cultured for a further 24 h in the presence of ethanol or the indicated concentrations of 17-estradiol, and pure anti-estrogen ICI182,780. Luciferase activity was measured in triplicate and normalized for the protein concentration. (B) Cells were cotransfected with 1 g of the tk-ERE-Luc plasmid and 0.1 g of the pRc/CMV-ER expression plasmid. Columns, means; bars, SD; *P ⬍ 0.0001; #P ⬍ 0.01; ⫹P ⬍ 0.05; (Fisher’s PLSD test); compared with control.
plasmid (data not shown). The data strongly indicate that growth of RERF-LC-OK cells can be suppressed by the pure anti-estrogen ICI. DISCUSSION Several studies have been performed to assess expression of ER in human lung cancers in order to explain gender differences in incidence, histology, and prognosis of human NSCLCs between females and
FIG. 5. Colony formation assay. RERF-LC-OK cells were transfected with the pRc-CMV plasmid and incubated with 1 mg/ml G418 for 2 weeks in the presence of ethanol or the indicated concentration of 17-oestradiol or pure anti-estrogen ICI182,780. After 2 weeks culture, cells were fixed with 3.8% formaldehyde and stained with 0.1% (w/v) crystal violet. The number of colonies larger than 100 m in diameter were counted per dish. Percentage of colonies relative to the control; Columns, means; ⫹P ⬍ 0.05; P ⬍ 0.001; (Fisher’s PLSD test); compared with control.
males. However, the reported values varied widely (6.1% (22), 7.1% (23), 16.7% (24), 40% (25), and 96.9% (26) of tumors positive) and the involvement of this receptor in lung physiology and/or pathology remains controversial. The novel ER subtype, ER discovered more recently, is expressed in various tissues of the rat (28), mouse (36), and human (27). Most investigations have been performed at the mRNA expression level using reverse transcription RT-PCR analysis; very few studies, however, have applied immunohistochemistry (37). To our knowledge, this is the first report of ER protein expression in human lung. Notably, ER␣ was
FIG. 6. Anchorage independent colony formation assay. Twentythousand RERF-LC-OK cells were suspended in 0.33% Difco’s noble agar and layered over a 0.66% agar-medium basal layer in the presence of ethanol or the indicated concentrations of 17-oestradiol or pure anti-estrogen ICI182,780. After 3 weeks’ incubation, the number of colonies larger than 50 m in diameter were counted per dish in triplicate. Percentage of colonies relative to control; Columns, means; bars, SD; $P ⫽ 0.0058 (Fisher’s PLSD test); n.s., not significant; compared with the control.
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not detected in human lung tissues or cell lines in our study. A recent immunohistochemical study also failed to demonstrate ER␣ expression in lung cancers (38). From our results showing ER expression in all normal bronchiolar epithelium tested, we conclude that ER may have important physiological functions in normal lung. All AAH specimens, considered as precancerous lesions for adenocarcinoma, also showed ER staining, stronger than normal samples, and 85% of adenocarcinomas were positive for ER, compared to only 30% of squamous cell carcinomas. With the hobnail type of adenocarcinoma, not strongly associated with genetic mutations or smoking history, and frequently found in females (17), 100% were ER positive. From the present investigation, we conclude that ER is present in lung cancer, especially in hobnail type of adenocarcinoma and suggest that this receptor may play a role in growth and development of lung cancer. Of great interest, both anchorage-dependent and -independent colony formation of RERF-LC-OK lung cancer cells, expressing ER but not ER␣, was significantly inhibited by the pure anti-estrogen ICI. This observation is in line with the finding that EREmediated transcriptional activation by ER was suppressed by addition of ICI to these cells. These data suggest that the pure anti-estrogen ICI may inhibit growth of some lung cancer cells expressing ER, making it a possible agent for hormone therapy of certain types of lung cancer. Generally, ER seems to have growth suppressive effects in various tissues, sometimes apparently through the attenuation of ER␣ function (39). Such a role is consonant with the findings of proliferative tendencies in the uterus and prostate of mice in which the ER gene has been inactivated (40, 41). It is possible that the growth suppressive effect of ICI observed on ER containing lung cancer cells in the present study, may also have been mediated via ER. In line with this notion, anti-estrogens have been shown to activate ER signaling via AP-1 and Sp-1 response elements (42). One may perhaps speculate that the increased expressions of antiproliferative ER in certain lung cancer cells, constitutes a defense mechanism reflecting an attempt from the cell to counteract uncontrolled cell growth. Obviously, more studies are needed to work out the mechanisms whereby ICI inhibits lung cancer cell growth under the conditions used in this study. In conclusion, we have confirmed that human lung tissues express ER, but not ER␣, and this expression could be seen in both normal and malignant lung specimens. Enhanced expression of ER in AAH compared with normal lung tissues and a higher incidence in adenocarcinoma compared with squamous cell carcinoma, may suggest some important roles of ER in genesis of adenocarcinomas, especially in hobnail type.
Since pure anti-estrogen ICI suppressed colony formation by the lung cancer cell line RERF-LC-OK expressing ER but not ER␣, we propose that pure antiestrogen ICI may be a useful agent for hormone therapy of certain types of lung cancer. ACKNOWLEDGMENTS This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, for Scientific Research Expenses for Health and Welfare Programs and the Foundation for the Promotion of Cancer Research, and by 2nd-Term Comprehensive 10-Years Strategy for Cancer Control, as well as by a grant from the Swedish Cancer Fund.
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