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Tumor Biology |
and ß and Show Biological Responses to Estrogen1
Department of Pharmacology, University of Pittsburgh [L. P. S., A. L. G. D., C. T. G., T. M. H., J. M. S.], Lung Cancer Program, University of Pittsburgh Cancer Institute [L. P. S., J. D. L., N. C., J. M. S.], Department of Surgery, University of Pittsburgh [J. D. L., N. C.], and Department of Pathology, University of Pittsburgh [S. F.], Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) and ß (ERß) in cultured human non-small cell lung cancer cells, cultured lung fibroblasts, and primary cultures of normal bronchial epithelium. Western analysis of ER suggested that the main protein expressed in lung tumor cells is a variant, probably attributable to alternative splicing. Protein for ERß was found to be a mixture of full-length as well as alternatively spliced variants. ß-Estradiol produced a proliferative response in vitro in both normal lung fibroblasts and cultured non-small cell lung tumor cells. This effect was also observed in vivo. In this regard, ß-estradiol stimulated growth of the non-small cell lung tumor line, H23, grown as tumor xenografts in SCID mice. This effect was blocked by fluvestrant (ICI 182,780). In paraffin sections of non-small cell lung tumors, ERß immunoreactivity was localized to the nucleus, whereas ER immunoreactivity was mainly localized to the cytoplasm, suggesting that both nuclear and cytoplasmic signaling may be involved in estrogenic responses in the lung. To show that the ERs found in the lung are functional, we demonstrated that ß-estradiol stimulated transcription of an estrogen response element-luciferase construct transfected in non-small cell lung tumor cell lines. Antiestrogens blocked this effect. Treatment of lung fibroblasts with ß-estradiol also increased secretion of hepatocyte growth factor by 2-fold. These results suggest that estrogen signaling plays a biological role in both the epithelium and the mesenchyme in the lung and that estrogens could potentially promote lung cancer, either through direct actions on preneoplastic or neoplastic cells or through indirect actions on lung fibroblasts. Additionally, it is possible that antiestrogens may have therapeutic value to treat or prevent lung cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A number of studies have suggested that women are more susceptible to tobacco carcinogenesis than men (2, 3, 4, 5) , taking into account baseline exposure, body weight, height, and body mass index (2) . The OR3 for adenocarcinoma of the lung in females was found to be 6.8 at 1–19 pack-years of tobacco exposure, compared with 2.4 for men, and 32.7 at 50 pack-years of exposure, compared with 13.8 for men. This same relationship was found for squamous cell carcinoma (2) . Not all studies have demonstrated this relationship (6, 7, 8) . For example, in a recent case-control study, Kruezer et al. (8) did not find increased ORs for female smokers compared with male smokers. However, there was also a large disproportion of female-to-male cases in the never-smoking group (286 versus 88); thus, there may be differences in baseline exposure (such as ETS exposure) that may confound the interpretation of these results.
There also is a difference in the relative distribution of lung cancer histological features between men and women that is not explained by differences in smoking patterns. Women who smoke appear to be at higher risk of developing small cell lung cancer, which has neuroendocrine features, than squamous cell lung cancer, whereas men who smoke have a similar risk for the two histological conditions (4)
. Furthermore, women smokers are more likely to develop adenocarcinoma of the lung, which is a secretory tumor. Nonsmokers with lung cancer (predominantly diagnosed with adenocarcinoma) are also 
2.5 times more likely to be female than male (9, 10, 11, 12, 13)
. Estrogens may play a causative role in this phenomenon of secretory types of differentiation being more prominent in female lung cancer.
Several genetic factors have been suggested to contribute to the higher ORs in women compared with men. GRPR is a receptor for the hormone gastrin-releasing peptide, which mediates cell proliferation in the lung. Gastrin-releasing peptide plays a role in lung development and possibly in lung wound healing. A sex difference in expression of GRPR, an X-linked gene, has been found, which may be caused by the ability of the GRPR gene to escape X inactivation (5) . Women were found to express this gene in their airway cells frequently without smoking, whereas men were found not to express the gene in airway cells unless they smoked (5) . Other factors that might increase the carcinogenic effects of cigarette smoke in women include increased expression of cytochrome P450 1A1 isozyme in the lungs of females compared with males (14) and a higher frequency of G-to-T transversions in the p53 gene in females compared with males (15) . Additionally, women were found to be three times more likely than men to carry the K-ras gene mutation, which is a marker for aggressive lung cancer found only in smokers or former smokers (16) . In a separate study, never-smoking women exposed to ETS who developed lung cancer were two times more likely than never-smoking women without ETS exposure who developed lung cancer to have a defective glutathione-S-transferase M1 gene, which normally deactivates carcinogens in tobacco smoke (17) . Reduced DNA repair capacity is also associated with an increased risk of lung cancer, and females were reported to have lower DNA repair capacity than males (18) . Whether regulation of cellular processes by estrogens can influence these cancer-related events is unknown.
Estrogen status is a recognized factor in lung cancer risk in women, as it is in the development of adenocarcinoma of the breast, endometrium, and ovary (19) . Taioli and Wynder (20) presented evidence that exogenous and endogenous estrogens may play a role in the development of lung cancer, particularly, adenocarcinoma, among women. Using case-control data, they demonstrated that (a) early age at menopause (40 years or younger) is associated with a reduced risk of adenocarcinoma of the lung (OR, 0.3); (b) the use of estrogen replacement therapy is associated with a higher risk of adenocarcinoma of the lung (OR, 1.7); and (c) there is a positive interaction between estrogen replacement therapy, smoking, and the development of adenocarcinoma of the lung (OR, 32.4). A recent study using a mouse model also confirmed the role of estrogen in lung tumorigenesis. In this regard, diethylstilbestrol was shown to increase the number of lung tumors when administered in conjunction with urethane (21) .
Estrogens may act directly as carcinogens through metabolism to catechol estrogens and the formation of DNA adducts (22
, 23) . Alternatively, estrogens could act as tumor promoters through a receptor-mediated mechanism. There have been conflicting reports of the presence of ERs in lung tumors (24, 25, 26, 27, 28, 29, 30)
, based mainly on immunohistochemical detection of the classical ER, now termed ER. Lung tumors from females were reported in one study to express ERs more frequently than those from males (26)
, and the ER-related protein, p29, has been reported to be expressed in lung tumors from females, where it is a negative prognostic indicator (31)
. However, other studies reported little or no ER in most lung tumors as evidenced by immunohistochemistry (25
, 27, 28, 29, 30)
. There have been few quantitative studies of ER mRNA expression in the lung, using gene probes, or protein expression, using immunoblotting, and the biological significance of ER expression in lung tumors has not been determined.
A novel ER referred to as ERß was cloned and characterized in human tissues and found to be localized to chromosome 14 (32
, 33)
. The ERß protein has similarities to the classical ER in terms of structure and function. The tissue distribution of these two receptors is not identical, but it appears to overlap in some cases (33)
. ER and ERß also differ in their effect on transcription at AP-1 sites (34)
. Whereas ER liganded to estradiol activates transcription at AP-1 sites, ERß liganded to estradiol inhibits transcription at AP-1 sites. In addition, several antiestrogens are transcriptional activators with ERß at AP-1 sites, whereas the same compounds are inhibitors with ER at this same site. This evidence suggests that ER and ERß may be regulated by distinct mechanisms and play different roles in gene regulation although they share functional characteristics. Although ERß shows some expression in the mammary gland, relative levels of ERß mRNA are highest in human granulosa cells, endothelial cells, ovary, and lung (33
, 35)
. Recently, ERß has been shown to be expressed in both normal lung as well as lung tumors and to have biological function (30)
. In the present study, we demonstrate that both ER and ERß mRNA and protein are expressed in normal bronchial epithelial cells, cultured lung fibroblasts, and lung tumor cells and that these receptors play a biological role in the lung. Additionally, we provide evidence that antiestrogens inhibit tumor growth and may be a useful strategy for lung cancer therapy.
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MATERIALS AND METHODS |
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RNA Isolation and RT-PCR.
RNA was isolated from cells by the guanidinium thiocyanate method (37)
. Oligo(dT)12–18 (Life Technologies, Inc., Grand Island, NY) was annealed to 1 µg of total RNA and then reverse transcribed with Superscript II (Life Technologies). The reaction contained 1 µg of total RNA, 500 ng of oligo(dT)12–18, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each deoxynucleotide triphosphate and 200 units of Superscript. Briefly, total RNA and oligo(dT)12–18 were incubated at 70°C for 10 min and then placed on ice; the remaining components were then added, and the reactions were incubated at 42°C for 50 min and 70°C for 10 min. The cDNA generated was used as a template for PCR with primers specific for ER and ERß. PCR amplification was performed in a 20-µl reaction containing 2 µl of the reverse transcription reaction, Taq DNA polymerase (Perkin-Elmer, Foster City, CA), 1x PCR buffer, 1.5 mM MgCl2, 1 mM each deoxynucleotide triphosphate, and 1 µM each primer. PCR was carried out in a Perkin-Elmer 9600 Thermocycler, and the conditions were initial denaturation at 95°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55 or 56°C (ER or ERß, respectively) for 30 s, and 72°C for 30 s. Final extension was at 72°C for 10 min. Ten µl of each reaction were run on a 1% agarose 1x Tris-borate-EDTA gel.
The ER forward and reverse primers were 5'-GGAGACATGAGAGCTGCCAA-3' and 5'-CACCACGTTCTTGCACTTCA-3', respectively, and were located in exon 4 and exon 8, respectively. The expected size product for ER was 752 bp. The ERß forward and reverse primers were 5'-CTGTTACTGGTCCAGGTTCA-3' and 5'-CCAGCTGATCATGTGTACCA-3', respectively. These primers are located in exons 2 and 4 of ERß and produce a 529-bp fragment. The RNA from each cell line was also analyzed for GAPDH as a control for RNA integrity, using the forward primer 5'-GTCAACGGATTTGGTCTGTATT-3' and reverse primer 5'-AGTCTTCTGGGTGGCAGTGAT-3'. These primers produce a 520-bp fragment of GAPDH.
Protein Extraction and Western Analysis.
Cells were grown to 90% confluency in three 75-cm2 flasks per cell line. The medium was removed from the cells, which were then rinsed with 1x PBS at room temperature. Protein was extracted by adding 2 ml of ice-cold RIPA buffer [1x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml phenylmethylsulfonyl fluoride, 0.045 mg/ml aprotinin (Sigma Chemical Co., St. Louis, MO), and 1 mM sodium orthovanadate] to each flask. Cells were scraped, and the lysate was passed through a 21-gauge needle to shear the DNA. Twenty µl of 10 mg/ml phenylmethylsulfonyl fluoride stock were added to each lysate and incubated on ice for 30–60 min. The cell lysate was centrifuged at 10,000 x g for 10 min at 4°C. The protein concentration was measured in the supernatant using the Pierce BCA-200 Protein Assay Kit.
Protein aliquots (25 µg of all lung cells and 10 µg of MCF-7 breast cancer cells) were separated by size on a 10% SDS-tricine-polyacrylamide gel (Novex/Invitrogen, San Diego, CA) and transferred to nitrocellulose membrane. Nonspecific binding sites were blocked by incubation in 1x TBS-T (0.2 M Tris, 0.14 M NaCl, 0.1% Tween 20) containing 5% milk for 1 h at room temperature, followed by incubation overnight at 4°C with either a 1:3000 dilution of rabbit polyclonal anti-ER antibody (HC-20; Santa Cruz Biotechnology, Santa Cruz, CA) or a 1:1000 dilution of rabbit polyclonal anti-ERß antibody (PanVera, Madison, WI) in 1x TBS-T containing 1% dry milk. After cells were washed three times in 1x TBS-T (10 min each at room temperature), horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham, Piscataway, NJ) was added at a 1:2000 dilution and incubated for 2 h at room temperature. After three more washes with 1x TBS-T, the immunoreactive peptide was detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL), followed by exposure to autoradiography film. Recombinant human ER (0.5 ng) and ERß (2 ng; PanVera) were used as standards. SeeBlue Plus 2 prestained protein standards (Invitrogen, Carlsbad, CA) were also included in each gel.
Cell Proliferation Assay.
Incorporation of BrdUrd was performed in duplicate wells in chamber slides (Nunc, Naperville, IL) as described previously (38)
using the BrdUrd Labeling and Detection Kit II (Boehringer-Mannheim, Indianapolis, IN). Normal lung fibroblasts (935NLFB) and lung tumor cells (H23) were plated at a density of 1 x 104 cells/well in DMEM or RPMI, respectively, plus 10% FBS and were allowed to reach 50% confluency. After the cells were rinsed twice with 1x PBS, they were exposed to phenol red-free medium without serum for 24 h to deprive the cells of estrogen. Fresh phenol red-free medium plus 10% charcoal-stripped serum with or without (0.1–10 nM) ß-estradiol was placed on the cells for 48 h. BrdUrd (10 µM) was added for the last 5 h of the incubation. Cells were washed, fixed, and exposed to a 1:250 dilution of the anti-BrdUrd working solution provided in the kit. The binding of the anti-BrdUrd antibody was detected by a 1:200 dilution of the antimouse immunoglobulin alkaline phosphatase working solution. A total of 1000 nuclei in each duplicate chamber were scored for BrdUrd labeling by light microscopy. The chambers were coded to prevent bias on the part of the observer. In addition to a saline control, a no-BrdUrd control was used to determine nonspecific background reaction. The no-BrdUrd control value was subtracted from all other values to normalize samples.
In Vivo Tumor Xenograft Model.
17-ß-Estradiol pellets (1.7 mg; 60-day release; Innovative Research of America, Sarasota, FL) or placebo pellets were implanted s.c. into 20 female SCID mice (10 estrogen-treated and 10 placebo-treated controls; 5 weeks of age; Harlan Sprague Dawley, Indianapolis, IN). Three days after pellet implantation, H23 lung tumor cells were injected s.c. at two sites per mouse (4 x 106 cells/site). The mice were divided into four treatment groups (five animals per group): (a) estrogen only; (b) estrogen + ICI 182,780 (fluvestrant); (c) no estrogen + vehicle control; and (d) no estrogen + ICI 182,780. ICI 182,780 (30 mg/kg of mouse weight; Tocris, Ballwin, MO) or vehicle control (peanut oil) was injected s.c. twice a week for 5 weeks. Tumor size was measured before each weekly injection and reported as an average relative tumor volume, calculated as: (l x w x h x 
)/2 (mm3), where l is the length, w is the width, and h is the height of the tumor measured with calipers. At the end of the 5-week period, the animals were sacrificed, and the tumors were removed. One-third of the tumor was harvested for protein analysis, one-third for RNA analysis, and the other third was fixed in 10% formalin for immunohistochemical analysis. Animal care was in strict compliance with the institutional guidelines established by the University of Pittsburgh.
Immunohistochemical Staining.
Tissue samples were fixed in 10% formalin for 15–30 min at room temperature and stored in 100% ethanol. Tissues were then paraffin embedded, sliced, and mounted on slides. Paraffin was removed from the slides with xylenes, and slides were stained according to standard procedures. Primary antibodies used were rabbit polyclonal anti-ER antibody (HC-20) or rabbit polyclonal anti-ERß antibody. The secondary antibody was a biotinylated IgG specific for the primary antibody. Brown staining was considered positive. The positive control for both ER and ERß was breast adenocarcinoma tissue. The negative control staining was done without the addition of primary antibody or with neutralization of the antibody with a blocking peptide.
Transient Transfection and Luciferase Assay.
Cells were plated in phenol red-free medium containing 10% supplemented calf serum at 1 x 105 cells/well in 6-well plates. The next day, the medium was changed to one containing 10% charcoal-stripped serum to deprive the cells of estrogen and then transfected the following day. Transfections were performed using Lipofectamine (Life Technologies) or DC-Chol (synthesized by L. Huang, University of Pittsburgh, Pittsburgh, PA) for MCF-7 and H23 cells, respectively, according to the manufacturers’ instructions. The conditions for transfection in these different cell lines had been optimized previously. All plasmids used for transfection were purified using the Qiagen EndoFree kit to remove harmful endotoxins. Cells were washed twice with 1x PBS and transiently cotransfected with reporter plasmid (pERE-TK-LUC; a gift from M. Nichols, University of Pittsburgh, Pittsburgh, PA) and the pRL-CMV (Promega, Madison, WI) control to correct for transfection efficiency. Cells were incubated with 0–10 nM estrogen with or without 1 µM ICI 182,780 or tamoxifen for 24 h and then harvested using passive lysis buffer (Promega). Luciferase activity was measured using the Dual-Luciferase System (Promega) and a TD 20/20 luminometer. Values were corrected for protein concentration and are presented as the mean ± SE of three independent experiments, each of which had two samples per treatment.
Isolation of Conditioned Medium and Detection of Secreted HGF.
A total of 6 x 105 normal lung fibroblasts (999NLFB) were plated on 100-mm plates in phenol red-free DMEM containing 10% supplemented calf serum and allowed to attach to the plate overnight. The medium was then replaced with one containing 10% charcoal-stripped serum to deprive cells of estrogen. Twenty-four h later, estrogen (0.1–10 nM) or vehicle control (ethanol) was added. After 48 h in the presence of estrogen, conditioned medium was isolated to measure secreted HGF protein. Briefly, the medium was removed from the plates, centrifuged for 10 min at 3000 x g, acidified with 0.1% acetic acid, and concentrated using a Sep-Pak C18 cartridge (Waters, Milford, MA). The concentrate was eluted with 2 ml of elution buffer (60% acetonitrile, 0.1% acetic acid, 2 mM pefabloc), lyophilized, and resuspended in 500 µl of sample buffer [2% SDS, 10% sucrose, 10 mM DTT, 60 mM Tris (pH 6.8)]. Five µg of protein from each sample were separated on a 10–20% SDS-tricine-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was immunoblotted with a 1:1000 dilution of monoclonal antihuman HGF antibody (R&D Systems, Minneapolis, MN), followed by a 1:1000 dilution of a horseradish peroxidase-conjugated secondary antibody (Amersham). Immunoreactive bands were visualized by SuperSignal West Pico chemiluminescent substrate (Pierce), followed by exposure to autoradiography film. Signals were quantified by ImageQuant analysis.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß mRNA Expression in Normal and Tumor Lung Cells.. We examined both cultured normal lung fibroblast cell lines and normal bronchial epithelial cells for the presence of ER and ERß, using RT-PCR (Fig. 1A)
. A RT-PCR product for ER was present in all of the normal lung fibroblast cell lines and cultured normal bronchial epithelial cells that we examined. Two major bands for ER were amplified from most cell lines. The upper band was the expected 752-bp fragment that represents the full-length sequence between exons 4 and 8. The DNA from this band was isolated and sequenced to confirm the product and was found to be the predicted ER sequence. The lower band was 568 bp and corresponded to an exon 7 deletion (ER
7), which was also confirmed by sequencing. This deletion is the most widespread deletion present in most human breast, uterus, and endometrial tumors (42, 43, 44)
. In some samples (i.e., 851NLFB) a small band slightly above ER7 was observed, which might correspond to ER5 or ER6, which should be 613 and 618 bp, respectively, with this primer set. We could also observe any double or triple exon deletions in exons 5–7 with this assay, which may account for some of the smaller bands that we observed. However, this RT-PCR assay for ER would not detect deletions in exons 2, 3, or 4.
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3, which is 413 bp and may be present in sample 1022. All of the normal bronchial epithelial cells that we examined also expressed ERß. To verify the integrity of the RNA in the samples, we also analyzed GAPDH expression in all of the cell lines. All samples expressed GAPDH at similar levels.
We next looked at mRNA levels in lung tumor cell lines from both males and females (Fig. 1B)
. We examined samples from both males and females to diminish the possibility that a female’s lung tumor had been mistakenly diagnosed and was actually a breast tumor that had metastasized to the lung. These samples represented adenocarcinomas (91T, 784T, H23, and Calu-6), squamous cell lung tumors (54T and 128-88T), and bronchioloalveolar (A549) tumor cells. MCF-7 breast cancer cells were used as a positive control. ER mRNA was found in high levels in the 91T, 784T, and 54T cell lines and low levels in the 128-88T, H23, A549, and Calu-6 cell lines. Thus, ER is not confined to adenocarcinomas, but can also be found in squamous cell tumors. In H23 cells, the predominant band was ER7 and what could possibly be a double exon deletion of 450 bp. The expected sizes of 5 + 6, 6 + 7, and 5 + 7 are 479, 434, and 429 bp, respectively. Additionally, a larger product was present in MCF-7 and 54T cells. This could possibly represent a variant of ER that contained an in-frame duplication of certain exons. This idea is not without precedence. For example, a duplication of exons 6 and 7 has been described in breast cancer cells (45)
. The expected size of this variant would be 1025 bp with this primer set. ERß mRNA was present in all tumor cell lines examined. A possible ERß3 band was observed in H23 and MCF-7 cells.
The MCF-7 positive control showed 2–40-fold higher levels of ER mRNA than in lung tumors. On the other hand, similar levels of ERß were observed in both MCF-7 cells and lung cancer cell lines. GAPDH mRNA levels were similar in all samples. There appeared to be no correlation between ER levels and whether the cells were derived from males or females. Additionally, the same bands were present in both normal lung cells (Fig. 1A)
and tumor cells (Fig. 1B)
and at similar ratios. One trend that was noticeable was that the lung tumor lines that expressed high ER mRNA (784T and 54T) also expressed low ERß. On the other hand, those that expressed low ER mRNA (128-88T, H23, A549, and 91T) expressed high levels of ERß.
ER and ERß Protein Levels in Both Normal and Tumor Lung Cells.
We next sought to examine protein levels of the receptor in the lung cells. We analyzed the same cell lines that were used for the RNA analysis. Using an antibody to the COOH terminus (last 20 amino acids, encoded by exon 8) of ER, we would not detect any alternatively spliced variants that produced a frameshift and truncated protein attributable to a premature stop codon. Deletions in ER exons 2, 5, 6, and 7 resulted in frameshifts that produced premature stop codons at nucleotides 1008, 1613, 1941, and 1943, respectively. The normal ER stop codon is located in exon 8 at nucleotide 2146. We could detect variants that contained deletions in exons 3 or 4 with this antibody. These alternatively spliced variants use the normal stop codon and thus would be recognized by the antibody used in this assay.
The full-length ER 66-kDa protein was observed in MCF-7 cells and the recombinant human ER standard control, but little or no full-length ER protein was present in the lung cancer cell lines that we examined (Fig. 2A)
. Cell lines H23 and 91T showed a small amount of full-length protein. We observed a band that was smaller than the full-length ER band and could possibly represent an exon 4 deletion, which should be 53.6 kDa. This band was present in all of the lung tumor lines as well as the breast cancer cell line. We did not observe the exon 3 deletion protein, which would be 61.7 kDa. We also observed a larger band in all of the cell extracts, which could possibly represent an ER variant that contains an in-frame duplication of certain exons. The expected size of the previously reported exon 6 and 7 duplication form of the protein is 80 kDa. Alternatively, this larger band may represent a novel form of ER that may use a unique translation initiation start site further upstream. The less intense bands observed may represent other variants or degradation products. There was no difference in pattern of expression between cell lines that were derived from males versus females. In addition, the same bands were observed in adenocarcinomas and squamous and bronchioloalveolar tumor cells. We obtained the same data using a polyclonal antibody to the same epitope from a different source (data not shown).
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66-kDa band, the 53.6-kDa putative exon 4 variant, and the larger 80-kDa band were completely blocked in all of the cell extracts (data not shown). The band that is slightly smaller than the 66-kDa band was not blocked, suggesting that all three of the intense bands are indeed specific for ER. Thus, the full-length ER is not present at all or is present in a very small amount in the various lung tumor cell lines that we examined; however, it is the most abundant form in breast cancer cells. This suggests that there may be tissue-specific differences in the expression of ER full-length versus alternatively spliced variants.
We also examined ER in normal lung fibroblasts. The full-length ER was also not observed in these normal cells (data not shown). Thus, there is not a difference in ER variants between normal lung fibroblasts and tumor cells. The fact that we do not observe a full-length ER protein but do see the full-length ER mRNA encompassing exons 4–8 suggests that there must be other deletions in exons 2 or 3 that the RT-PCR assay does not recognize.
ERß immunoblots of the same cell lines showed the full-length ERß 59-kDa protein as well as several smaller bands (Fig. 2B)
. The ERß antibody recognizes the COOH terminus of the protein from amino acids 512–530. As was the case with the ER antibody, we would not observe any variants that produced a frameshift that resulted in a premature stop codon and truncation of the protein. The variants that we could detect with this antibody were ERß3 and ERß4, which would result in proteins of 54.9 and 48.1 kDa, respectively. We have not further elucidated which variants the observed bands represent. The same bands that we observed in the lung tumor extracts were present in extracts from MCF-7 cells. When the antibody was neutralized with a peptide specific to the region that the antibody recognized, the full-length protein band and the recombinant ERß protein were completely blocked (data not shown). The smaller bands were only partially blocked and thus may not be specific for ERß. As an additional control, an antiactin antibody was used to show that equal amounts of protein from each sample were loaded on these gels and that the protein was intact (data not shown).
ß-Estradiol Increases Cellular Proliferation of Normal Lung Fibroblasts and Lung Tumor Cell Lines in Vitro.
If estrogen influences lung tumor development, a stimulatory effect attributable to estrogen on the growth of lung tumor cells would be expected. We investigated this in several lung tumor cell lines. Cell proliferation was assessed by measurement of BrdUrd incorporation during DNA synthesis in proliferating cells. Fig. 3
shows the effect of ß-estradiol on the growth of normal lung fibroblasts (935NLFB; Fig. 3A
) and lung tumor cells (H23 cells; Fig. 3B
).
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In Vivo Tumor Growth in Immunocompromised Mice.
Growth experiments were also conducted in vivo to assess whether estrogen could stimulate tumor growth and also whether antiestrogens could inhibit growth in immunocompromised mice. The H23 cell line that was used in this experiment showed expression of both ER and ERß protein. In addition, because the cells were derived from a male, there is very little possibility that H23 could be derived from a breast tumor that metastasized to the lung rather than a primary lung tumor. Fig. 4
shows a representative experiment with five animals per treatment group. As shown in Fig. 4
, estrogen stimulated tumor growth by 1.2-fold compared with placebo-treated controls. This suggests that estrogens do indeed affect lung tumorigenesis, although the increase is not statistically significant. The mice used in this experiment were female and were not ovariectomized, so there were background circulating levels of estrogen present in the placebo-treated controls. In addition, there may be differences in these background estrogen levels that may account for the large variation of tumor size that we observed in this particular group (range of tumor volumes, 42–1304 mm3). In verification of this model, ICI 182,780 treatment in combination with estrogen treatment (ß-estradiol + ICI versus ß-estradiol) inhibited tumor growth by 44%. ICI 182,780 treatment alone inhibited growth by 41% (ICI alone versus vehicle control, P < 0.05). This experiment was repeated two additional times with similar results. Thus, the use of antiestrogens may be a valuable treatment for lung cancer. Although we achieved <50% tumor growth inhibition, antiestrogens may be useful in combination with other available treatments to achieve further inhibition of tumor growth.
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and ERß were present in these tumors (data not shown). Immunohistochemical staining of these tumors also demonstrated expression of ER (Fig. 5)
and ERß (data not shown). Curiously, in these tumors, ER was localized mainly in the cytoplasm with very few nuclei stained (Fig. 5C)
. On the other hand, ERß was located primarily in the nucleus (data not shown). The tumors that were treated with estrogen pellets did not show any more ER nuclear staining than those that received the placebo pellet (Fig. 5, C versus D)
, suggesting that the presence of estrogen does not noticeably change the subcellular distribution of ER. The staining was somewhat more intense in the estrogen-treated tumor versus the placebo-treated tumor. There may be physiological significance for this subcellular distribution in the cytoplasm. Because we observed little or no full-length ER in Western blots (Fig. 2A)
, it is possible that the major ER protein in lung cells is a variant protein that functions in the cytoplasm. Control staining of breast carcinoma tissue showed both ER (Fig. 5A) and ERß (data not shown) in the nucleus. Without addition of the ER primary antibody, there was very little background staining (Fig. 5B)
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and ERß in paraffin sections of lung tumor sections from patients. Once again, we observed ER in the cytoplasm and ERß in the nucleus (Fig. 6)
. Fig. 6A
shows nuclear ER staining in a breast adenocarcinoma. Lymphocytes are negative in this section, indicating that the staining is specific. Conversely, in a lung squamous cell dysplasia, very few nuclei stained positive for ER and most of the staining was cytoplasmic (Fig. 6B)
. ERß, on the other hand, was found mainly in the nucleus of both tissue sections from a lung squamous cell dysplasia (Fig. 6C)
as well as from a lung squamous cell carcinoma (Fig. 6D)
.
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or ERß.
ß-Estradiol Increases Transcription from an ERE in Lung Cancer Cells.
To show that the ER is functional in lung cells, we used a gene reporter assay with a single vitellogenin ERE upstream of a minimal thymidine kinase promoter and the firefly luciferase gene (pERE-TK-LUC). In addition, we cotransfected a control plasmid, pRL-CMV, to control for transfection efficiency from plate to plate. The control plasmid contains the CMV immediate-early enhancer/promoter region, which provides strong expression of the Renilla luciferase cDNA. Thus, we were able to measure both firefly and Renilla luciferase levels in one assay based on dissimilar enzyme structures and substrate requirements of the two luciferases.
The results of three independent experiments in a lung cancer cell line, H23, are shown in Fig. 7A
. MCF-7 cells were used as a positive control (Fig. 7B)
. Estrogen increased transcription in a dose-dependent manner in both MCF-7 and H23 cell lines. The effect of estrogen was greater in MCF-7 cells than it was in the lung cancer cell line that we examined. The addition of 10 nM ß-estradiol resulted in an 4-fold increase in transcription in MCF-7 cells (P < 0.05; Fig. 7B
). The presence of either ICI 182,780 or tamoxifen inhibited the stimulation of transcription attributable to estrogen back to basal levels. In H23 cells (Fig. 7A)
, transcription was stimulated 1.5–2-fold in the presence of estrogen (P < 0.01). ICI 182,780 and tamoxifen blocked this stimulation. These results suggest that at least some forms of the ERs present in the lung cancer cell line that we examined are functioning in the expected manner, i.e., they can bind to an ERE and activate gene transcription. We observed similar results in 784T lung cancer cells (data not shown). The cell lines that we examined expressed both ER and ERß. The fact that estrogen does not stimulate transcription to the same extent in lung cancer cells compared with breast cancer cells may reflect the fact that the lung cells do not contain full-length ER and that the ER variants present are mainly cytoplasmic. Thus most, if not all, of the stimulation may be attributed to ERß, which is found in the nucleus. These results also suggest that the stimulation of transcription by estrogen is ER dependent.
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and ERß (Fig. 8)
. These results demonstrate that estrogens can alter the biology of lung fibroblasts to produce more of a growth factor known to promote lung cancer growth, invasion, and progression.
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DISCUSSION |
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and ERß are indeed present in normal lung cells as well as in lung tumors. Additionally, we believe that little or no full-length ER is expressed in these cells, but rather that the lung expresses several variants of ER, including those lacking the nuclear localization signal in exon 4 and possibly a novel protein with a longer NH2 terminus because of a unique translation start site. This is the opposite of what is observed in breast tissue, which is that the ER variants in total generally represent a minority of the ER mRNA compared with the full-length form. The expression of these variants in lung cells at levels detectable by Western analysis suggests a possible physiological role for the variants. We also present several lines of evidence that the ERs are functional in the lung: (a) Estrogen induces cell proliferation in the lung in vitro as well as in vivo. Antiestrogens can block this effect. (b) Estrogen can activate transcription from an ERE in lung cancer cells. (c) Estrogen can stimulate secretion of a growth factor thought to be involved in lung tumorigenesis. Collectively, these data demonstrate that ERs play a biological role in the lung.
Past studies of ER expression in lung tissues used reagents optimized to identify the 66-kDa full-length ER protein. Antibodies raised against epitopes in the deleted exons of ER or recognizing a conformation of the protein specific for breast tissue might not detect the cytoplasmic form of ER found in the lung. ERß protein might also not be recognized. The conclusion might have been that lung tumors are ER negative. We have performed a direct comparison between monoclonal versus polyclonal antibodies to the COOH terminus of ER. Our results show that only the full-length 66-kDa ER protein was detected with the monoclonal antibody, whereas two additional ER-specific bands were detected with the polyclonal antibody. Previous studies have used both biochemical binding assays (39, 40, 41)
and more specific immunohistochemical assays (24, 25, 26, 27, 28, 29, 30)
. The immunohistochemical reports that showed no or little reactivity to ER used an antibody to the NH2 terminus. The study that reported 96.8% positivity did not identify the source of antibody used (24)
.
Human ER mRNA is very heterogeneous. Both ER and ERß undergo alternative splicing to generate transcripts that have deletions in various combinations of the eight exons in ER. There have been at least 20 ER alternatively spliced mRNA transcripts created by single, double, or multiple deletions reported using RNase protection assays and RT-PCR (42
, 47, 48, 49, 50)
. ERß splice variants have been identified in human breast tumors and in ovarian, uterine, and mammary tissues (51)
. Deletions in ERß exons 3, 5, 6, and 5 + 6 have been characterized. It has not been until recently that a variant protein was actually shown to be produced from an alternatively spliced transcript. In this regard, it has been reported that a 52-kDa truncated ER protein, ER7P, is the predominant variant of ER in MCF-7 breast cancer cells (52)
. At this time it is not clear whether these ER variants are involved in tumorigenesis or whether they have any physiological role at all. For example, the use of ER7P in a yeast transfection assay resulted in dominant-negative activity (53)
. This result, however, was not observed in a transfection assay in mammalian cells (54)
. Conflicting results of functionality have also been reported for ER5P (55
, 56)
.
The classical model for the mechanism of action of steroid hormone receptors is that they undergo translocation to the nucleus when bound to steroids. Some evidence suggests that unbound receptors are in equilibrium between the nucleus and the cytoplasm (57)
. On the basis of immunohistochemistry results, the forms of ER expressed in the lung were located primarily in the cytoplasm and ERß in the nucleus in several of the lung cancer samples and normal lung cells that we have examined. There are two possible reasons that this may occur. One reason is that there may be enough ER in the nucleus in lung cells to act as a transcription factor and any excess ER in the cytoplasm has no physiological function. An alternative possibility is that there is another mechanism for ER in the lung that is distinct from that of ER in breast and other tissues. This may involve a variant protein of ER that has a physiological function different from that of wild-type ER. This idea is not without precedence. For example, Pasqualini et al. (58)
found a differential subcellular distribution of the different ER variants. In this regard, full-length ER was predominantly nuclear and ER3 and ER4 were present in both the cytoplasm and nucleus. ER3 + 4 was predominantly cytoplasmic. Upon hormone treatment, the locations of ER3 and ER3 + 4 were unaffected. If indeed lung tumors contain mainly variant ER proteins, it is possible that these variants play a unique role in the cytoplasm. The cellular location of ER in the lung tumors may be attributable to lack of ligand because the tissue samples studied were from postmenopausal women. Alternatively, this may be a novel mechanism for ER regulation in lung cancer cells. The fact that ERß was found in the nucleus when the same specimens were used suggests that lack of ligand may not be a factor. We suggest that lung cancer cells have little or no full-length ER and that the variant that is present is mainly cytoplasmic. This would be the case if an exon 4 deletion were the predominant protein because the nuclear localization signal is located in exon 4.
We were also interested in determining whether the ERs in the lung played any biological role. We observed an increase in transcriptional activation from an ERE in response to ß-estradiol. These increases were not as large as those observed with MCF-7 cells. This could be because all of the effects are attributable to nuclear ERß only and not to ER, which is found only in the cytoplasm. In addition, it is possible that the ER variants have an altered estrogen-binding pocket and thus may not be responsive to estrogen.
Estrogens are known to cause transcriptional activation of several growth factor genes, among them transforming growth factor , epidermal growth factor, and insulin-like growth factor-1 (59
, 60) . All of these growth factors are known to mediate mitogenesis in lung tumors. We have found that lung fibroblasts can be induced to secrete more HGF, a paracrine growth factor in the lung, after estrogen exposure. This result also suggests that the stromal cells are affected by estrogen, not just the epithelium. Recently, the stromal cell component in reproductive tissues has been shown to be the site of the majority of ER expression, compared with the epithelial component (61)
. In addition, activation of the stromal ER is responsible for estrogen-induced epithelial cell proliferation; it is believed that estrogen binding to its receptor in stromal cells causes release of paracrine growth factors that induce epithelial cell growth, whereas the proliferation induced by estrogen binding to the epithelial cell receptor itself is not significant (61)
. This suggests that in normal lung tissue, lung fibroblasts may be the source of much of the receptor expression and response to estrogens. Our results show expression of ER mRNA in lung fibroblasts from most individuals. Proliferation of lung fibroblasts can lead to thickening of the airways and lung obstruction. Among smokers, those with obstructive lung disease have the greatest risk for developing lung cancer (62
, 63)
, and there is a growing body of literature showing that women are more susceptible to obstruction induced by smoking than men (64, 65, 66)
.
ER-mediated events may be responsible for at least some of the increased risk of women to lung cancer compared with men because women have higher circulating levels of estrogen. However, in men, tissue levels of estrogen may be high enough to show biological effects because testosterone can be converted to estrogen locally in tissues through the action of aromatase. If ER is indeed involved in lung tumorigenesis, inhibiting lung tumor growth may be possible with antiestrogens, and antiestrogen therapy could be used in combination with other available therapies. At present, lung cancer patients have few therapeutic options. Potentially, these studies provide the basis for a new type of adjuvant therapy in selected lung cancer patients. Antiestrogens used at present for prevention of breast cancer may also have utility to prevent lung cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Pittsburgh Foundation (to J. M. S.) and by Grants CA 79882 and CA 90440 (SPORE in Lung Cancer; awarded to J. M. S.) from the National Cancer Institute. L. P. S. was supported by a fellowship from institutional National Research Service Award T32-HL07 from the National Heart, Lung, and Blood Institute. 
2 To whom requests for reprints should be addressed, at Department of Pharmacology, University of Pittsburgh, E1340 Biomedical Science Tower, Pittsburgh, PA 15261. Phone: (412) 648-1942; Fax: (412) 648-1945; E-mail: siegfrie{at}server.pharm.pitt.edu
3 The abbreviations used are: OR, odds ratio; ETS, environmental tobacco smoke; GRPR, gastrin-releasing peptide receptor; ER and ERß, estrogen receptor and ß; AP-1, activator protein; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TBS-T, Tris-buffered saline-Tween; BrdUrd, bromodeoxyuridine; HGF, hepatocyte growth factor; ERE, estrogen response element.
Received 9/18/01. Accepted 1/22/02.
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REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ERß at AP1 sites. Science (Wash. DC), 277: 1508-1510, 1997. and ß. Endocrinology, 138: 863-870, 1997. variant protein in cell lines and tumors. Mol. Cell. Endocrinol., 162: 167-180, 2000.[Medline]
E3, E4, and E3-4 isoforms of the rat estrogen receptor-. Mol. Endocrinol., 15: 894-908, 2001.This article has been cited by other articles:
|
|
 |
|
  A. M. Egloff, M. E. Rothstein, R. Seethala, J. M. Siegfried, J. R. Grandis, and L. P. Stabile Cross-Talk between Estrogen Receptor and Epidermal Growth Factor Receptor in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., November 1, 2009; 15(21): 6529 - 6540. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. Raso, C. Behrens, M. H. Herynk, S. Liu, L. Prudkin, N. C. Ozburn, D. M. Woods, X. Tang, R. J. Mehran, C. Moran, et al. Immunohistochemical Expression of Estrogen and Progesterone Receptors Identifies a Subset of NSCLCs and Correlates with EGFR Mutation Clin. Cancer Res., September 1, 2009; 15(17): 5359 - 5368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-C. LAI, J.-Y. WU, Y.-W. CHENG, K.-T. YEH, T.-C. WU, C.-Y. CHEN, and H. LEE O6-Methylguanine-DNA Methyltransferase Hypermethylation Modulated by 17{beta}-Estradiol in Lung Cancer Cells Anticancer Res, July 1, 2009; 29(7): 2535 - 2540. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Diaz, I. Ceja-Ochoa, I. Restrepo-Angulo, F. Larrea, E. Avila-Chavez, R. Garcia-Becerra, E. Borja-Cacho, D. Barrera, E. Ahumada, P. Gariglio, et al. Estrogens and Human Papilloma Virus Oncogenes Regulate Human Ether-a-go-go-1 Potassium Channel Expression Cancer Res., April 15, 2009; 69(8): 3300 - 3307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Cote, W. Yoo, A. S. Wenzlaff, G. M. Prysak, S. K. Santer, G. B. Claeys, A. L. Van Dyke, S. J. Land, and A. G. Schwartz Tobacco and estrogen metabolic polymorphisms and risk of non-small cell lung cancer in women Carcinogenesis, April 1, 2009; 30(4): 626 - 635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Seow, W.-P. Koh, R. Wang, H.-P. Lee, and M. C. Yu Reproductive Variables, Soy Intake, and Lung Cancer Risk among Nonsmoking Women in the Singapore Chinese Health Study Cancer Epidemiol. Biomarkers Prev., March 1, 2009; 18(3): 821 - 827. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Zhang, X. Liu, A. M. Farkas, A. V. Parwani, K. L. Lathrop, D. Lenzner, S. R. Land, and H. Srinivas Estrogen Receptor {beta} Functions through Nongenomic Mechanisms in Lung Cancer Cells Mol. Endocrinol., February 1, 2009; 23(2): 146 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Nose, K. Sugio, T. Oyama, T. Nozoe, H. Uramoto, T. Iwata, T. Onitsuka, and K. Yasumoto Association Between Estrogen Receptor-{beta} Expression and Epidermal Growth Factor Receptor Mutation in the Postoperative Prognosis of Adenocarcinoma of the Lung J. Clin. Oncol., January 20, 2009; 27(3): 411 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Niikawa, T. Suzuki, Y. Miki, S. Suzuki, S. Nagasaki, J. Akahira, S. Honma, D. B. Evans, S.-i. Hayashi, T. Kondo, et al. Intratumoral Estrogens and Estrogen Receptors in Human Non-Small Cell Lung Carcinoma Clin. Cancer Res., July 15, 2008; 14(14): 4417 - 4426. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Gallo, G. F. Zannoni, I. De Stefano, M. Mosca, C. Ferlini, E. Mantuano, and G. Scambia Soy Phytochemicals Decrease Nonsmall Cell Lung Cancer Growth In Female Athymic Mice J. Nutr., July 1, 2008; 138(7): 1360 - 1364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Hammoud, B. Tan, S. Badve, and R. M Bigsby Estrogen promotes tumor progression in a genetically defined mouse model of lung adenocarcinoma Endocr. Relat. Cancer, June 1, 2008; 15(2): 475 - 483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mahabir, M. R. Spitz, S. L. Barrera, Y. Q. Dong, C. Eastham, and M. R. Forman Dietary Boron and Hormone Replacement Therapy as Risk Factors for Lung Cancer in Women Am. J. Epidemiol., May 1, 2008; 167(9): 1070 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Schwartz, A. S. Wenzlaff, G. M. Prysak, V. Murphy, M. L. Cote, S. C. Brooks, D. F. Skafar, and F. Lonardo Reproductive Factors, Hormone Use, Estrogen Receptor Expression and Risk of Non Small-Cell Lung Cancer in Women J. Clin. Oncol., December 20, 2007; 25(36): 5785 - 5792. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Hammes and E. R. Levin Extranuclear Steroid Receptors: Nature and Actions Endocr. Rev., December 1, 2007; 28(7): 726 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Mah, D. B. Seligson, A. Li, D. C. Marquez, I. I. Wistuba, Y. Elshimali, M. C. Fishbein, D. Chia, R. J. Pietras, and L. Goodglick Aromatase Expression Predicts Survival in Women with Early-Stage Non Small Cell Lung Cancer Cancer Res., November 1, 2007; 67(21): 10484 - 10490. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Tammemagi, M. T. Freedman, T. R. Church, M. M. Oken, W. G. Hocking, P. A. Kvale, P. Hu, T. L. Riley, L. R. Ragard, P. C. Prorok, et al. Factors Associated with Human Small Aggressive Non Small Cell Lung Cancer Cancer Epidemiol. Biomarkers Prev., October 1, 2007; 16(10): 2082 - 2089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Pietras and D. C. Marquez-Garban Membrane-Associated Estrogen Receptor Signaling Pathways in Human Cancers Clin. Cancer Res., August 15, 2007; 13(16): 4672 - 4676. [Full Text] [PDF]
|
|
|
|
|
|
M. Majidi, H. A. Al-Wadei, T. Takahashi, and H. M. Schuller Nongenomic {beta} Estrogen Receptors Enhance {beta}1 Adrenergic Signaling Induced by the Nicotine-Derived Carcinogen 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone in Human Small Airway Epithelial Cells Cancer Res., July 15, 2007; 67(14): 6863 - 6871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nozoe, T. Oyama, M. Takenoyama, T. Hanagiri, K. Sugio, and K. Yasumoto Significance of Immunohistochemical Expression of Estrogen Receptors {alpha} and {beta} in Squamous Cell Carcinoma of the Esophagus Clin. Cancer Res., July 15, 2007; 13(14): 4046 - 4050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Tsuchiya, M. Iwasaki, T. Otani, J.-i. Nitadori, K. Goto, Y. Nishiwaki, Y. Uchitomi, and S. Tsugane Breast Cancer in First-degree Relatives and Risk of Lung Cancer: Assessment of the Existence of Gene-Sex Interactions Jpn. J. Clin. Oncol., June 23, 2007; (2007) hym048v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Wisnivesky and E. A. Halm Sex Differences in Lung Cancer Survival: Do Tumors Behave Differently in Elderly Women? J. Clin. Oncol., May 1, 2007; 25(13): 1705 - 1712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Naff, M. L. Cote, A. S. Wenzlaff, and A. G. Schwartz Racial Differences in Cancer Risk Among Relatives of Patients With Early Onset Lung Cancer Chest, May 1, 2007; 131(5): 1289 - 1294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Subramanian and R. Govindan Lung Cancer in Never Smokers: A Review J. Clin. Oncol., February 10, 2007; 25(5): 561 - 570. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Cerfolio, A. S. Bryant, E. Scott, M. Sharma, F. Robert, S. A. Spencer, and R. I. Garver Women With Pathologic Stage I, II, and III Non-small Cell Lung Cancer Have Better Survival Than Men Chest, December 1, 2006; 130(6): 1796 - 1802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. P. Stabile, J. S. Lyker, S. R. Land, S. Dacic, B. A. Zamboni, and J. M. Siegfried Transgenic mice overexpressing hepatocyte growth factor in the airways show increased susceptibility to lung cancer Carcinogenesis, August 1, 2006; 27(8): 1547 - 1555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. A. Weber Positron Emission Tomography As an Imaging Biomarker J. Clin. Oncol., July 10, 2006; 24(20): 3282 - 3292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M Dougherty, W. Mazhawidza, A. R Bohn, K. A Robinson, K. A Mattingly, K. A Blankenship, M. O Huff, W. G McGregor, and C. M Klinge Gender difference in the activity but not expression of estrogen receptors {alpha} and {beta} in human lung adenocarcinoma cells. Endocr. Relat. Cancer, March 1, 2006; 13(1): 113 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Siegfried Hormone Replacement Therapy and Decreased Lung Cancer Survival J. Clin. Oncol., January 1, 2006; 24(1): 9 - 10. [Full Text] [PDF]
|
|
|
|
|
|
A. K. Ganti, A. E. Sahmoun, A. W. Panwalkar, K. K. Tendulkar, and A. Potti Hormone Replacement Therapy Is Associated With Decreased Survival in Women With Lung Cancer J. Clin. Oncol., January 1, 2006; 24(1): 59 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. K. Weinberg, D. C. Marquez-Garban, M. C. Fishbein, L. Goodglick, H. J. Garban, S. M. Dubinett, and R. J. Pietras Aromatase Inhibitors in Human Lung Cancer Therapy Cancer Res., December 15, 2005; 65(24): 11287 - 11291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Stelck, G. L. Baker, K. M. Sutherland, and L. S. Van Winkle ESTROUS CYCLE ALTERS NAPHTHALENE METABOLISM IN FEMALE MOUSE AIRWAYS Drug Metab. Dispos., November 1, 2005; 33(11): 1597 - 1602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Schwartz, G. M. Prysak, V. Murphy, F. Lonardo, H. Pass, J. Schwartz, and S. Brooks Nuclear Estrogen Receptor {beta} in Lung Cancer: Expression and Survival Differences by Sex Clin. Cancer Res., October 15, 2005; 11(20): 7280 - 7287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-T. Wu, Y.-L. Chang, J.-Y. Shih, and Y.-C. Lee The significance of estrogen receptor {beta} in 301 surgically treated non-small cell lung cancers J. Thorac. Cardiovasc. Surg., October 1, 2005; 130(4): 979 - 986. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Schabath, L. M. Hernandez, X. Wu, P. C. Pillow, and M. R. Spitz Dietary Phytoestrogens and Lung Cancer Risk JAMA, September 28, 2005; 294(12): 1493 - 1504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kawai, A. Ishii, K. Washiya, T. Konno, H. Kon, C. Yamaya, I. Ono, Y. Minamiya, and J. Ogawa Estrogen Receptor {alpha} and {beta} are Prognostic Factors in Non-Small Cell Lung Cancer Clin. Cancer Res., July 15, 2005; 11(14): 5084 - 5089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Thomas, L. A. Doyle, and M. J. Edelman Lung Cancer in Women: Emerging Differences in Epidemiology, Biology, and Therapy Chest, July 1, 2005; 128(1): 370 - 381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Patel Lung Cancer in Women J. Clin. Oncol., May 10, 2005; 23(14): 3212 - 3218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C S A Markides and J G Liehr Specific binding of 4-hydroxyestradiol to mouse uterine protein: evidence of a physiological role for 4-hydroxyestradiol J. Endocrinol., May 1, 2005; 185(2): 235 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Fu, T. Y. Kau, R. K. Severson, and G. P. Kalemkerian Lung Cancer in Women: Analysis of the National Surveillance, Epidemiology, and End Results Database Chest, March 1, 2005; 127(3): 768 - 777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. P. Stabile, J. S. Lyker, C. T. Gubish, W. Zhang, J. R. Grandis, and J. M. Siegfried Combined Targeting of the Estrogen Receptor and the Epidermal Growth Factor Receptor in Non-Small Cell Lung Cancer Shows Enhanced Antiproliferative Effects Cancer Res., February 15, 2005; 65(4): 1459 - 1470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Hershberger, A. C. Vasquez, B. Kanterewicz, S. Land, J. M. Siegfried, and M. Nichols Regulation of Endogenous Gene Expression in Human Non-Small Cell Lung Cancer Cells by Estrogen Receptor Ligands Cancer Res., February 15, 2005; 65(4): 1598 - 1605. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Barnes The Changing Face of Lung Cancer Chest, December 1, 2004; 126(6): 1718 - 1721. [Full Text] [PDF]
|
|
|
|
|
|
J. Olak and Y. Colson Gender differences in lung cancer: Have we really come a long way, baby? J. Thorac. Cardiovasc. Surg., September 1, 2004; 128(3): 346 - 351. [Full Text] [PDF]
|
|
|
|
|
|
J. D. Patel, P. B. Bach, and M. G. Kris Lung Cancer in US Women: A Contemporary Epidemic JAMA, April 14, 2004; 291(14): 1763 - 1768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Johnson and C. L. Arteaga Gefitinib in Recurrent Non-Small-Cell Lung Cancer: An IDEAL Trial? J. Clin. Oncol., June 15, 2003; 21(12): 2227 - 2229. [Full Text] [PDF]
|
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), 1.7 mg ß-estradiol pellet (
), 1.7 mg ß-estradiol pellet + 1 mg/week ICI 182,780 (
), or 1 mg/week ICI 182,780 (
). Each point represents the mean value for 10 tumors/group. This is a representative experiment that was repeated two additional times. Tumor volume was plotted over time. *, P < 0.05 versus vehicle control. Data were analyzed by a two-tailed Student’s t test. Bars, SE.
