Cancer Research Annual Meeting 2010 Protein Translation and Cancer
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Cancer Research 68, 3476, May 1, 2008. doi: 10.1158/0008-5472.CAN-07-3046
© 2008 American Association for Cancer Research
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Endocrinology

The Estrogen Receptor Pathway in Rhabdomyosarcoma: A Role for Estrogen Receptor-β in Proliferation and Response to the Antiestrogen 4'OH-Tamoxifen

Joshua A. Greenberg1,3, Stig Somme2,6, Hege E. Russnes7, Adam D. Durbin1,5 and David Malkin1,3,4,5

1 Division of Hematology/Oncology and 2 General Surgery, The Hospital for Sick Children; and 3 The Institute of Medical Science and Departments of 4 Pediatrics, 5 Medical Biophysics, and 6 Surgery, University of Toronto, Toronto, Ontario, Canada; and 7 The Department of Pathology, The Norwegian Radiumhospital, Oslo, Norway

Requests for reprints: David Malkin, Division of Haematology/Oncology, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Phone: 416-813-5348; Fax: 416-813-5327; E-mail: david.malkin{at}sickkids.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Highly malignant, RMS frequently fails to respond to conventional aggressive multimodal radiation, surgery, and chemotherapy treatment protocols that also cause significant sequelae in the growing child. Other tumors of mesenchymal origin, such as locally aggressive fibromatoses and desmoid tumors, have been successfully treated with a selective estrogen receptor (ER) modulator, tamoxifen. In an effort to identify new targets for RMS therapy, our group investigated the previously uncharacterized ER pathway in RMS cell culture and primary tumors. We detected ER isoform β (ERβ), but not isoform {alpha}, RNA, and protein in five RMS cell lines. Immunohistochemical staining of primary RMS tumor sections confirmed high levels of ERβ but not ER{alpha} protein. RMS cell growth was dramatically inhibited in steroid-free conditions, and this growth inhibition was rescued with 17-β-estradiol (E2) supplementation. Exposure of RMS cells to 4'OH-tamoxifen (4OHT) decreased cell viability and inhibited colony formation as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony-forming assays. 4OHT also induced apoptotic signaling in RMS cells as detected by cleavage of caspase-3 and poly(ADP)ribose polymerase. This effect increased 3- to 8-fold in steroid-deprived conditions but was rescued by supplementation with E2. Immunofluorescence studies detected a change in the subcellular localization of ERβ in response to 4OHT. Together, these data suggest an active ERβ-mediated signal transduction pathway in RMS. The ability of 4OHT to induce apoptotic signaling and disrupt estradiol-mediated proliferation provides a rationale to explore a role for selective ER modulators in the treatment of RMS. [Cancer Res 2008;68(9):3476–85]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, accounting for ~10% of all solid tumors in children (1). RMS is thought to arise from immature mesenchymal cells already committed to the skeletal muscle lineage, and has two major histologic subtypes, embryonal (ERMS) and alveolar (ARMS), both demonstrating metastatic potential (2, 3). ERMS, the most common RMS subtype, generally has an earlier age of onset and is associated with a more favorable prognosis (2, 3). ARMS, characterized by either t(1;13) or t(2;13) translocations, is often resistant to current multimodal treatment protocols, providing strong rationale to explore new therapeutic targets. Although much is understood about paracrine and autocrine growth factor signaling pathways in RMS (4–11), very little is known about the influence of endocrine hormone activity on the development and growth of this sarcoma. Here, we investigate the role of the previously unexplored estrogen receptor (ER) signaling pathway in both histologic subtypes of RMS.

For >30 years, the clinical observations that up to 80% of aggressive fibromatoses and desmoid tumors, two locally aggressive mesenchymal tumors, occur in women in their child-bearing years has led to postulations of a role for elevated estrogens and ER signaling in the development and growth of these tumors (12, 13). Furthermore, multiple reports of remission of desmoid tumors in response to treatment with a selective ER modulator, tamoxifen (12, 14, 15), have resulted in more commonplace use of the antiestrogenic agent in the treatment of these fibroblastic neoplasms. Although the clinical evidence for an active ER pathway in these tumors has recently been bolstered by findings of strong ER expression profiles in desmoid tumors (16, 17), little other experimental or mechanistic evidence exists to support the current hypothesis of estrogen sensitivity.

The ER Family is a subset of the type I Steroid Receptor Superfamily of nuclear receptors, which also includes receptors for progesterone, glucocorticoid, mineralocorticoid, and androgens (18, 19). The two major ER isoforms, ER{alpha} and ERβ (595 and 530 amino acids, respectively), are encoded by genes located on different chromosomes (6q and 14q, respectively). Shown to be distinct receptors with similar DNA and ligand-binding patterns, ER isoforms {alpha} and β exhibit different transcriptional activation profiles in response to many agonists and antagonists (18, 20, 21). The ER pathway is stimulated by estrogens and inhibited by selective ER modulators, such as tamoxifen, opening up new therapeutic options in ER-positive tumors. In this study, we explore the potential existence of an active ER pathway in biologically aggressive, mesenchyme-derived RMS, and test the hypothesis that selective ER modulators have therapeutic potential for the treatment of RMS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture and cell lines. RMS cell lines were provided by Dr. A. Thomas Look (Dana-Farber Cancer Institute, Boston, MA) and are derived from alveolar (RH4, RH18, RH28, and RH30) and embryonal (RD) tumors. MCF-7 and T47D breast cancer cell lines were grown in the same medium conditions as described below. Cells were maintained and cultured in DMEM (Invitrogen) + 10% fetal bovine serum (FBS; Sigma) with 1% penicillin/streptomycin (Invitrogen) and 0.1% Fungizone (Invitrogen). For experiments performed in steroid-free conditions, phenol red–free (prf)DMEM + 10% charcoal stripped serum (CSS; Wisent, Inc.) supplemented with 2 mmol/L L-Glutamine (Invitrogen) was used. 17-β-estradiol (E2) was used as an ER agonist, whereas selective ER modulator 4'OH-tamoxifen (4OHT) was used as an ER antagonist, both obtained from Sigma-Aldrich Canada and dissolved in ethanol. For Western blot experiments, either 4.0 x 105 cells per well (RH28), 2.5 x 105 cells per well (RH4, RH18, RH30, and MCF7), or 2.0 x 105 cells per well (RD) were plated overnight in 6-well plates before treatment. For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assays, either 7 x 103 cells per well (RH28), 2.5 x 103 cells per well (RH18, RH30, and MCF7), or 2 x 103 cells per well (RH4 and RD) were either plated in 96-well plates in the appropriately treated medium or treated after 24 h.

Reverse transcription PCR. Total RNA was extracted from five RMS and two breast cancer cell lines using the Qiagen RNeasy mini kit according to manufacturer's protocols. The Superscript II First Strand DNA kit (Invitrogen) was used for reverse transcription to generate cDNA according to manufacturer's instructions. One microlitre of reverse transcription reaction was used as a template for PCR with Amplitaq Gold DNA Polymerase (Applied Biosystems) using 35 cycles (ER{alpha}) or 33 cycles (ERβ). All thermal cycling was performed using the GeneAmp PCR System 9700 (Applied Biosystems). For ER{alpha}, an initial activation step of 10 min at 94°C and 35 cycles of 45 s at 94°C, 45 s at 60°C, and 1 min at 72°C was followed by 7 min at 72°C. For ERβ, the initial activation step of 10 min at 94°C and 33 cycles of 1 min at 94°C, 45 s at 55.5°C, and 1 min at 72°C was followed by 10 min at 72°C. Specific primers were designed for ER{alpha} (sense, 5'-AAGAGCTGCCAGGCCTGCC-3' and antisense, 5'-TTGGCAGCTCTCATGTCTCC-3') and ERβ (sense, 5'-TAGTGGTCCATCGCCAGTTAT-3' and antisense, 5'-GGGAGCCACACTTCACCAT-3'), and were used for the PCR reaction along with a no template control tube for each series. Glyceraldehyde-3-phosphate dehydrogenase was used as a control gene for normalization of input cDNA.

Antibodies. For immunohistochemistry, primary antibodies specific to ER{alpha} and ERβ were purchased from Novocastra and Abcam Ltd, respectively. For Western blotting, primary antibodies specific to ER{alpha} and ERβ were purchased from Santa Cruz Biotechnology and Novocastra, respectively. Primary antibodies specific to cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (cPARP), phospho-extracellular signal-regulated kinase (pERK), and total ERK were purchased from Cell Signalling Technology. Primary antibody specific to loading control vinculin was purchased from Upstate Biotechnology, whereas primary antibody specific to ERβ for the purpose of immunofluorescence was purchased from Gentex. Goat anti-Mouse IRDye 800 cw and Goat anti-Rabbit IRDye 680 cw (LI-COR Biosciences) were used as secondary antibodies for all Western blots developed using the LI-COR Odyssey Infrared Imaging System.

Western blot protein analysis. Whole cell lysates from 5 RMS cell lines and 2 breast cancer cell lines were harvested using Lysis Buffer C [50 mmol/L Tris (pH 7.4), 1% v/v Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA (pH 7.4)] + Complete Mini Protease Inhibitor Tablets (Roche Applied Science), and concentration of each lysate was determined using the Bradford assay. For each experiment, equal protein samples (50–70 µg) were run on a 15% SDS-PAGE mini gel under reducing conditions. Protein was transferred to a PolyScreen PVDF Hybridization Transfer Membrane (Perkin-Elmer), which was blocked for 1 h in Odyssey Blocking Buffer (LI-COR Biosciences), and then probed at 4°C overnight for the presence of ER{alpha}, ERβ, cleaved caspase-3, cPARP, or pERK using target-specific primary antibodies. Vinculin was used as a loading control for normalization of results. Membranes were then incubated for 30 min at room temperature in anti-rabbit and anti-mouse secondary antibodies conjugated to 680 and 780 nm IR dyes, respectively, both purchased from LI-COR Biosciences. Blots were scanned at wavelengths of 700 and 800 nm using the Odyssey Infrared Imaging System from LI-COR Biosciences. Densitometry was performed using the Odyssey Application Software Version 2.1 (LI-COR Biosciences). For pERK Western blots, after scanning the membrane for pERK-1, the membrane was stripped with a polyvinylidene difluoride–stripping buffer [25 mmol/L glycine (pH 2.0) and 1.5% SDS] and reprobed for total ERK.

Immunohistochemistry. Formalin-fixed, paraffin-embedded tumor tissue from 12 patients diagnosed with RMS—four diagnosed as ARMS, four as ERMS, two as pleomorphic RMS, and two RMS not otherwise specified—was cut into 3-µm sections and mounted onto slides. Immunohistochemistry was performed using the EnVision+ peroxidase system (DAKO). Deparaffinized sections were microwaved in citrate buffer then treated with hydrogen peroxide to block endogenous peroxidase. Sections were incubated with either mouse monoclonal antibodies against ER{alpha} (clone 6F11; diluted 1:100) or ERβ (clone 14C8; diluted 1:300), stained with 3,3-diaminobenzidine tetrahydrochloride (DAB), and counterstained with hematoxylin. Both series included positive controls of both benign and malignant breast tissue. Using a microscope, fields were then scored based on the percentage of cells staining positive for the target protein, as well as for the relative strength of stain in each cell.

Cell growth and proliferation assays. Cells from one breast cancer line and five RMS cell lines were plated in 96-well plates in either DMEM or phenol red–free DMEM containing the appropriate treatment. At 24, 48, 72, or 96 h, the TACS MTT Reagent (R&D Systems), a tetrazolium dye that is metabolized by cells to produce punctate, colored crystals, was added to each well. After 4 h, a detergent reagent was added to the wells, and the following day, the plate was read at a wavelength of 570 nm and a control wavelength of 650 nm in the SpectraMAX 250 colorimetric plate reader (Molecular Devices Corporation). Statistical analysis of all results was performed using a two-sample Student's t test, assuming equal variances, with an {alpha} of 0.01. Results are presented as means ± SE.

Soft agar assays. MCF7 (5 x 103) or RMS (RD, RH4, RH18, and RH30) cells were suspended in a solution of 0.35% sterile agarose (Invitrogen) in OPTIMEM I (Invitrogen), supplemented with 10% FBS and vehicle (sterile ethanol), 5, or 10 µmol/L 4OHT. Cultures were incubated for 3 wk, and once weekly, 500 µL of OPTIMEM I supplemented with vehicle (sterile ethanol), 5, or 10 µmol/L 4OHT were added to the surface of the assays. The number of colonies formed was counted using the average of two fields at x4 magnification, using a Leica Leitz DM IL phase-contrast microscope (Leica Microsystems Canada).

Colony forming assays. MCF7 (5 x 103) or RMS (RD, RH4, RH18, and RH30) cells were plated in 100-mm2 dishes, in DMEM supplemented with 10% FBS and vehicle (sterile ethanol), 5, or 10 µmol/L 4OHT. Cultures were incubated for 2 wk with one medium change and stained with 0.0025% crystal violet at room temperature for 30 min. Colonies were counted manually.

Statistical analysis of soft agar and colony forming assays. All data for soft agar and colony-forming assays was analyzed using Graphpad Prism 3.0 (Graphpad Software, Inc.). Results are presented as means ± SE. All statistical analyses were performed with two-way ANOVA with post hoc Tukey tests. A P value of <0.05 was considered significant.

Immunofluorescence. Cells from one breast cancer line and two RMS cell lines were plated on Poly-L-lysine–coated coverslips (BD Biosciences; VWR) in 6-well plates. Twenty-four hours after plating, medium containing the appropriate treatment was added to each well. After another 24 h, cells were fixed using 4% formaldehyde and then permeabilized with 0.2% Triton X-100 in PBS. Cells were incubated in primary antibody against ERβ, incubated with a CY-3–tagged secondary antibody (Jackson ImmunoResearch), and then stained with 4',6-diamidino-2-phenylindole (DAPI) nuclear stain (Molecular Probes). Cover slips were mounted on slides, and images were captured by CCD camera coupled to a Leica DMRA2 compound microscope equipped with standard epifluorescence filters and Nomarski optics at x630 and x1,000 magnification.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RMS primary tumors and cell lines express ERβ but not ER{alpha} isoform. To establish whether there is an active ER signaling pathway in RMS, we first tested for the expression status of both ER isoforms—ER{alpha} and ERβ—in five RMS cell lines. Two breast cancer cell lines were selected as experimental controls. The MCF-7 cell line has been shown to express both ER isoforms (22, 23), whereas T47D expresses ER{alpha} but not ERβ (24). Total mRNA was extracted from all cell lines, and reverse transcription-PCR was performed to selectively amplify the gene of interest using primer pairs specific to either ER{alpha} or ERβ. All five RMS cell lines expressed ERβ but not ER{alpha} mRNA (Fig. 1A ). We then measured ER expression at the protein level, using whole cell protein lysates from the same cell lines. Western blot analysis confirmed the same expression profiles for both ER{alpha} and ERβ in all cell lines tested (Fig. 1B).


Figure 1
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  		Figure 1. Messenger RNA and protein expression levels of ER isoforms {alpha} and β in RMS. Five RMS (RH4, RH18, RH28, RH30, and RD) and two breast cancer (T47D and MCF7) cell lines were studied for relative ER expression levels. A, total mRNA was isolated and amplified with primers specific to ER{alpha} or ERβ. B, whole cell lysates were harvested, and 50 µg of protein from each cell line were separated by SDS-PAGE and assessed by Western blot analysis using specific antibodies against ER{alpha} or ERβ. Twelve primary RMS samples were prepared for immunohistochemistry by incubation with specific antibodies against ER{alpha} (C) and ERβ (D) proteins, stained with DAB, and counterstained with hematoxylin. E and F, ER{alpha} and ERβ-positive control (breast tumor epithelial tissue). ERβ-negative control represented by nontumor cells demonstrating lack of immunostaining (internal tissue control). Cells were scored as <10% tumor cells with nuclear stain (+), 10% to 50% tumor cells with nuclear stain (++), above 50% tumor cells with nuclear stain (+++), or no tumor cells with nuclear stain (–; ref. 48).

 
To test for clinical relevance of our in vitro findings, we analyzed 12 primary RMS tumor samples by immunohistochemistry for the relative expression of both ER{alpha} (Fig. 1C) and ERβ (Fig. 1D). These 12 samples included both RMS histologic subtypes, ERMS (samples 1–4) and ARMS (samples 5–8), pleomorphic (samples 9 and 10) and, other (samples 11 and 12). All 12 primary tumors stained strongly positive for ERβ, with 10 exhibiting expression in >50% of the cells within any given field of view. In contrast, only 2 of the 12 samples stained positive for ER{alpha}. In these two cases, ER{alpha} staining was weak, with only 10% to 30% of cells within any given field of view expressing the protein. Tumor samples staining positive for ERβ exhibited predominantly nuclear localization of the protein.

ER agonist estradiol stimulates cell proliferation in RMS cell lines. With ERβ expression established in RMS cell lines, we next examined whether the ER agonist (E2) could stimulate cell proliferation. When RMS cells were grown in steroid-free conditions (prfDMEM/10% CSS), a highly significant decrease in cell proliferation of 50% to 90% (P < 0.005) was observed at 48 hours (Fig. 2A ). When steroid-free medium was supplemented with 100 nmol/L E2, which would represent a physiologic equivalent level of circulating E2 (24), a significant 50% to 550% rescue of cell proliferation (compared with steroid-free conditions) was observed in all five RMS lines (Fig. 2A).


Figure 2
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  		Figure 2. Impact of estradiol and 4OHT on cell proliferation in RMS cell lines. A, cells from five RMS lines were plated for MTT assays in control (DMEM/10% FBS), steroid-free (prfDMEM/10% CSS), or steroid-free + estradiol (prfDMEM/10% CSS + 100 nmol/L E2) conditions; cell proliferation was measured at 48 h; n = 3. B, cells from five RMS lines and one breast cancer control line were plated for MTT assays in DMEM/10% FBS; new medium-containing vehicle (sterile ethanol), 5, 7.5, or 10 µmol/L 4OHT was added at 24 h, and cell proliferation was measured at 72 h. Representative comparison shown is between DMEM control and 7.5 µmol/l 4OHT; n = 3. All data are normalized to the appropriate DMEM control group. *, P < 0.01; **, P < 0.005; ***, P < 0.001).

 
ER antagonist 4OHT inhibits RMS cell proliferation in vitro. We next explored whether treatment with 4OHT, a selective ER modulator and ER antagonist, could inhibit cell proliferation in RMS. The breast cancer cell line MCF-7 was selected as a positive control because inhibition of cell proliferation in response to clinically therapeutic dose levels of 4OHT has previously been shown (25). Forty-eight hours after treatment of all RMS cell lines with 7.5 µmol/L 4OHT, a significant decrease in cell proliferation of 20% to 90% (P < 0.005) was observed compared with control groups (Fig. 2B). Similar and significant trends were seen using treatment with both 5 and 10 µmol/L 4OHT (data not shown).

4OHT treatment inhibits RMS colony formation and number in vitro. To examine the biological significance of 4OHT-induced inhibition of RMS cell growth and proliferation, we performed colony formation assays in both treated soft agar and treated culture medium. RMS cell lines RH4, RH18, RH30, and RD were tested. For low numbers of RMS cells (5 x 103) after 3 weeks of growth in 0.35% agar treated with OPTIMEM I (10% FBS) medium containing vehicle (sterile ethanol), 5, or 10 µmol/L 4OHT, a significant and dramatic reduction in the number of colonies formed by 4OHT-treated RMS cell lines was observed (Fig. 3A ). These reductions in colony number ranged from 30% to 70% in 5 µmol/L 4OHT and up to 75% to 95% in 10 µmol/L 4OHT. For low numbers of the same RMS cell lines (5 x 103) after 2 weeks of growth in DMEM (10% FBS) medium containing vehicle (sterile ethanol), 5, or 10 µmol/L 4OHT, a dramatic reduction in the number of colonies formed by 4OHT-treated RMS cells was shown (Fig. 3B). These reductions in colony number ranged from a 35% to 90% in 5 µmol/L 4OHT and up to 75% to 99% reductions in 10 µmol/L 4OHT when compared with the vehicle control. MCF-7 was used as a positive control for both experiments. RMS cell line RH28 was not tested due to its poor ability to form colonies when plated in small numbers.


Figure 3
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  		Figure 3. 4OHT reduces colony formation and number in biological assays of RMS growth. A, cells from four RMS cell lines (RH4, RH18, RH30, and RD) and one breast cancer control cell line (MCF7) were plated in 0.35% agarose in OPTIMEM I supplemented with 10% FBS and vehicle (sterile ethanol), 5, or 10 µmol/L 4OHT. Colony formation was assayed 3 wk postplating, and colonies larger than 10 cells were counted; n = 4. B, cells from four RMS cell lines (RH4, RH18, RH30, and RD) and one breast cancer control cell line (MCF7) were plated for 2 wk in colony forming assays in 10-mm2 dishes in DMEM supplemented with 10% FBS and vehicle (sterile ethanol), 5, or 10 µmol/L 4OHT. Cells were fixed and colony numbers were counted. n = 3. All data are normalized to the vehicle-treated control group. *, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
Treatment with 4OHT activates the apoptotic pathway in RMS cells. To test the hypothesis that the dramatic decreases observed in RMS cell proliferation and colony formation in response to 4OHT treatment were due to induction of apoptosis, we treated all five RMS lines with 4OHT and performed Western blot analysis to determine whether apoptotic markers were induced. Cleavage of caspase-3 was used as an indicator of early apoptotic signaling, and relative activation of the protein was measured in response to treatment of cells with 4OHT. Increases (1.8–5-fold) in cleaved caspase-3 were detected in a dose-dependent response to treatment with 4OHT in the MCF-7–positive control and in all five RMS cell lines (Fig. 4A ).


Figure 4
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  		Figure 4. 4OHT-induced cleavage of apoptotic markers caspase-3 (casp-3) and PARP in RMS cell lines. Cells from five RMS lines and one breast cancer control line were plated in DMEM/10% FBS; new medium containing vehicle (sterile ethanol), 5, 7.5, or 10 µmol/L 4OHT was added at 24 h, and whole cell protein lysates were harvested at 48 h. A, fold increase in cleaved caspase-3 was measured by Western blot using target-specific antibodies. B, fold increase in cPARP was measured by Western blot using target-specific antibodies. All fold increase data are normalized to the appropriate DMEM control group. This experiment is representative of at least three independent assays.

 
We next verified these findings by examining the effect of 4OHT on a second indicator of apoptotic signaling, cPARP. In this experiment, it was possible to discern differences in apoptotic induction between additional concentrations of 4OHT, likely due to the fact that PARP cleavage is a late apoptotic event, and because it is further downstream in the apoptotic cascade, may be subject to some degree of signal amplification. Increases (1.5–8-fold) in PARP cleavage were observed in a dose-dependent curve according to the concentration of 4OHT added (Fig. 4B).

Treatment with 4OHT in steroid-free conditions induces high levels of apoptotic signaling that can be rescued with E2 supplementation in RMS cell lines. To explore the effect of removing steroids from the growth medium on activation of the apoptotic pathway in RMS cell lines, both in the presence and absence of antiestrogens, Western blot analysis of the apoptotic marker cPARP was performed. All results were normalized against a DMEM/10% FBS control set to one, and plotted on a log order graph (Fig. 5A ). Removal of steroids from the medium resulted in 2- to 2.5-fold increase in cPARP in all RMS lines. When 4OHT was added to the steroid-free medium, a dose-dependent 6- to 50-fold increase in cPARP was observed for all cell lines. On average, the dose-dependent curve resulting from 4OHT exposure was 3 to 6 times greater in steroid-free conditions (Fig. 5A) than had been observed in DMEM/10% FBS medium (Fig. 4B). Similar trends were observed in apoptotic marker cleaved caspase-3 in both DMEM control and steroid-free conditions (data not shown).


Figure 5
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  		Figure 5. 4OHT-induced cleavage of apoptotic marker PARP in steroid-free and estradiol-supplemented conditions. A, cells from five RMS lines were plated in DMEM/10% FBS; steroid-free medium (prfDMEM/10% CSS) containing vehicle (sterile ethanol), 5, 7.5, or 10 µmol/L 4OHT was added at 24 h, and whole cell protein lysate was harvested at 48 h. Lysates were analyzed by Western blot for relative levels of cPARP. B, cells from RMS line RD were plated in DMEM/10% FBS; steroid-free medium with or without estradiol (prfDMEM/10% CSS ± 100 nmol/L E2) and containing vehicle (sterile ethanol), 5, 7.5, or 10 µmol/L 4OHT was added at 24 h, and whole cell protein lysate was harvested at 48 h. Relative levels of cPARP were analyzed by Western blot. Similar trends were observed for RMS cell lines RH30 and RH28 (other RMS lines not tested). All fold-increase data are normalized to the appropriate DMEM control (CTRL) group. This experiment is representative of three independent assays.

 
We next tested whether this dramatic increase in activation of the apoptotic pathway in steroid-free conditions could be rescued back to the levels observed in RMS cells grown in regular growth medium simply by resupplementing cells with E2. The relative levels of PARP cleavage were investigated under both steroid-free and E2-supplemented conditions (Fig. 5B). When steroids were removed from the medium, PARP cleavage increased 4- to 10-fold at higher 4OHT concentrations, but when cells were supplemented with E2, this increase in activation of the apoptotic pathway was rescued back to the levels observed for the DMEM control groups (data shown is for the RD cell line). This trend was also observed in RMS cell lines RH30 and RH28, and a similar trend was observed for the cleavage of caspase-3 in all cell lines tested (data not shown).

Treatment with 4OHT changes the subcellular localization of ERβ in RMS cell lines. To explore possible mechanism by which 4OHT treatment affects RMS cell lines, we performed immunofluorescence to investigate whether subcellular ERβ localization is affected by exposure to 4OHT. In untreated cultures growing in DMEM with 10% FBS, ERβ localization was observed to be predominantly nuclear (with some cytoplasmic signal) in RH30 cells (Fig. 6 ). These findings were in keeping with immunohistochemical observations made in the 12 primary RMS samples (Fig. 1D). However, in RH30 cells treated with 4OHT for 24 hours, ERβ was primarily perinuclear in location. 4OHT-treated RH30 cells were photographed at x1,000 due to a decrease in cell number per field of view, resulting from exposure to 4OHT. Similar results were observed with the RH4 line, and at concentrations of 5 and 10 µmol/L 4OHT (data not shown).


Figure 6
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  		Figure 6. Impact of 4OHT on the subcellular localization of ERβ in RMS cell lines. Cells from RMS line RH30 were plated on poly-L-lysine–coated coverslips in DMEM/10% FBS; at 24 h, new medium containing vehicle (sterile ethanol), 5, 7.5, or 10 µmol/L 4OHT was added; at 48 h, cells were fixed, permeabilized, and incubated with a specific antibody against ERβ. Cells were then incubated with Cy-3–tagged secondary antibodies and a DAPI nuclear stain, mounted on slides, and images at x630 magnification were captured. Endogenous ERβ localization (1), DAPI nuclear stain (2), a merge of ERβ and DAPI (3), and ERβ localization after treatment with 7.5 µmol/L 4OHT taken at x1,000 (4) are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although paracrine and autocrine growth factor signaling—including pathways activated by epidermal growth factor (6), insulin-like growth factor (4, 5, 8, 10, 11), vascular endothelial growth factor (7), and basic fibroblast growth factor (9)—have been well-described in RMS, little is known about the function of endocrine hormone signaling in these tumors. Although Ishizuka et al. (16) have recently identified ER{alpha} and ERβ expression in desmoid tumors, the activity of ER signaling in tumors of mesenchymal origin, including the sarcomas, has yet to be elucidated. Furthermore, ERβ may play an important role in a variety of cancers with recent reports of significant ERβ expression profiles being shown in human bladder cancer (26), prostate cancer (27, 28), salivary gland adenocarcinoma (29), astrocytic cancers (30), and colonic adenocarcinoma (31).

Our experiments have shown, for the first time, the presence of ERβ and lack of ER{alpha} expression in RMS primary tumor samples and cell lines, as well as a functional, estrogen-sensitive signaling pathway. This pathway is also shown to be responsive to both ER agonists and antagonists, and is capable of inducing proliferation or activating apoptotic signaling in RMS cell lines under both normal and steroid-free conditions. A recent report by Gruvberger-Saal et al. (32) demonstrating that for patients with ER{alpha}-negative breast carcinomas, ERβ expression is a predictive marker for tamoxifen response and an independent marker for favorable prognosis after adjuvant tamoxifen treatment, further highlights the clinical relevance of these findings.

It should be noted that no differences were observed between ARMS and ERMS cell lines with respect to their responses to treatment with E2 and 4OHT, and that the effects observed did not seem to be related to the relative level of ERβ expression in each line.

Additional studies are necessary to determine if ERβ-mediated regulation of tumor cell growth and survival occurs in other ERβ expressing tumors, and whether clinical testing for ERβ expression may be a useful tool for determining whether adjuvant antiestrogenic therapy could be relevant for the treatment of previously unconsidered tumor types.

A role for ERβ in RMS cell growth. Here, we show, for the first time, that multiple RMS tumor samples and cell lines express an active ER. Although Zacharin et al. (33) previously speculated about the importance of hormones, including estrogen, on an RMS tumor that recurred after 25 years, directly following the patient's commencement of hormone therapy, this is the first study to show a functional and estrogen-sensitive signaling pathway in both ARMS and ERMS models in vitro.

Our data shows that removal of steroids from growth medium results in a dramatic decrease in RMS cell growth and proliferation, and a 2-2.5 fold increase in markers of apoptotic induction. This effect can be significantly rescued by E2 supplementation. It is important to note that the remaining discrepancy in cell growth observed in several RMS cell lines between those groups partially rescued from steroid-free conditions with E2 supplementation, and those grown in regular growth medium (Fig. 2A) is suggestive of a role for steroid hormones other than estrogens in the growth of RMS cells. Such factors likely account for the only partial rescue of cell growth in RH4, RH18, and RD and would be in keeping with the recent findings of Ishizuka et al. (16), which suggest that desmoid tumors, of similar ontogeny to RMS, express a variety of steroid hormone receptors, including progesterone and androgen receptors.

In contrast to the increased proliferation induced by resupplementation of E2, treatment with an antiestrogen, 4OHT, resulted in decreased proliferation of RMS cells. These results were dramatic and highly significant (P = 0.0039–0.000022), ranging from 20% to 90% reductions in RMS cell growth at 24-, 48-, and 72-hour time points. In most RMS cell lines, the decrease in proliferation observed after 4OHT treatment was comparable with that shown for breast cancer cell line MCF-7, one of the first cell lines on which tamoxifen treatment was successfully tested. Previous studies by Wakeling et al. (25) have shown ~50% reductions in MCF-7 growth in vitro in response to 4OHT treatment. This effect was comparable with that observed in our experiments with MCF-7.

The in vitro studies of the ability of tamoxifen to inhibit MCF-7 proliferation (25) were subsequently confirmed in vivo in a study that showed 4OHT-induced inhibition of MCF-7 xenograft tumor growth in a mouse model (34). Our in vitro tests of the biological significance of tamoxifen-induced RMS growth inhibition showed significant and dramatic reductions in the ability of RMS cell lines to form colonies in either soft agar or growth medium. Reductions in RMS colony number ranged from 35% to 99% (depending on the cell line and tamoxifen concentration used) and confirmed the importance of the effect of tamoxifen on RMS cells in a more biologically relevant system.

Thus, based on the success of tamoxifen in the treatment of breast cancer after inhibition of MCF-7 xenograft growth in mouse models (34), our in vitro results with RMS growth suppression and inhibition of colony formation in soft agar and growth medium provide a solid rationale for future analysis of antiestrogen therapy for this type of tumor.

Tamoxifen-mediated induction of the apoptotic pathway in RMS. Our data shows that the ER pathway in RMS can be modified with the antiestrogen 4OHT, which not only inhibits cell proliferation and colony formation but also induces apoptotic signaling in both the presence and absence of steroids. Tamoxifen and its derivatives are most commonly associated with inhibition of cell growth (35), and findings of 4OHT-induced apoptosis are uncommon but not unprecedented. A recent study has shown that tamoxifen is capable of inducing dose-dependent effects on apoptosis and proliferative arrest when added to growth medium used to culture mesenchyme–derived chondrocytes in rat metatarsal bones (36). An additional study by Wu et al. (37) has shown a similar dose-dependent induction of apoptosis in another mesenchyme-derived cell type, murine osteoclasts. Cumulatively, these studies are supported by the many clinical case reports in which tamoxifen has induced rapid regression of desmoid tumors, although little mechanistic evidence is provided (12, 14, 15, 38). Here, we report the identification of a targeted therapeutic agent that decreases cellular growth and activates the apoptotic pathway in RMS tumor cells but has not been shown to induce gross cytotoxic effects in ER-negative cells even after extensive clinical use. This finding may prove to be of significant relevance for reducing off-target effects and toxicity in the future treatment of RMS.

The fact that removal of steroids from growth medium resulted in dramatic increases in the susceptibility of RMS cells to 4OHT-induced activation of apoptosis pathways suggests a possible degree of protection conferred on RMS cells by one or more steroid hormones. However, these dramatic increases in apoptotic signaling could be rescued back to control levels by E2 supplementation. This observation is likely due to competitive binding between E2 and 4OHT as explored by Barkhem et al. (39), confirming ERβ as a specific target of 4OHT in RMS cells. Changes in mitogen-activated protein kinase (MAPK) pathway activation and cellular localization of ERβ in response to 4OHT were also observed and provide potential insight into mechanistic explanations for the results obtained (data not shown).

Finally, all of these findings of 4OHT-induced activation of apoptosis pathways were shown in an ERβ-positive/ER{alpha}-negative tumor model, providing new insights into the poorly defined activity of ERβ in tumor growth.

Potential mechanisms for tamoxifen-induced proliferative inhibition and apoptosis in RMS. The nuclear to cytoplasmic change in subcellular localization of ERβ after exposure of RMS cell lines to the ER antagonist 4OHT observed here is consistent with a model in which ER antagonists disrupt genomic ER signaling (which takes place in the nucleus) and induce changes in nongenomic, ERK-mediated cascades (which occur in the cytoplasm). Several studies have previously shown that nuclear localization of ERs in response to E2 stimulation can be disrupted in ER{alpha}-expressing cells by treatment with pure ER{alpha} antagonist ICI 182,780 (40, 41). This results in an induced cytoplasmic localization of ER{alpha} in contrast to its normal and predominantly nuclear location, likely due to a relative increase in nuclear export of the receptor (40, 41). Although this effect was not observed after treatment with tamoxifen in these experiments, tamoxifen is a partial agonist of ER{alpha} (42) but a pure antagonist of ERβ (39), explaining the changes in ERβ localization observed in RMS cells. Changes in the activation of MAPK pathway kinases have also been implicated in the regulation of ER nucleocytoplasmic shuttling (41).

A tamoxifen-induced apoptotic pathway that both induces and is dependent on ERK phosphorylation has recently been described in detail for both ER{alpha} and ERβ-expressing MCF-7 and T47D breast cancer cells (43). Experiments with several RMS cell lines (RD and RH18) also showed up to 2-fold increases in ERK-1 phosphorylation at the time points at which the highest levels of tamoxifen-induced apoptosis were observed (data not shown). Further studies to determine the outcome, role, and significance of ERK-1 phosphorylation in the RMS response to both E2 and tamoxifen may help to elucidate the importance of this pathway for both RMS proliferation and apoptosis.

Sex steroid hormones in sarcoma development. RMS has two major histologic subtypes each with a distinct age of onset. ERMS, the embryonal version of this tumor, generally presents in children ages <6 years, with some 35% of RMS cases occurring in the first 4 years of life, and as many as 10% occurring in the neonatal period (2, 3, 44). ARMS, the more aggressive subtype, is more typically seen in early to midadolescence. Elevated levels of hormone expression associated with these age groups—maternal estrogen in fetal development and sex hormones during adolescence—might contribute to the onset of RMS through endocrine steroid signaling. Other examples of this potentially hormone-related pattern of onset in tumors of similar lineage include fibrosarcoma, with an infantile (<2 years) and adult (10–15 years) age of onset (2, 3, 45). Together, these observations may provide further rationale for exploring the influence of steroid signaling in tumors of mesenchymal origin. Additionally, the exploration of a potential role for ERs in the differentiation of normal skeletal myoblasts warrants investigation in light of findings that differential ER{alpha} and especially ERβ expression patterns have been linked to the process of differentiation in osteoblasts (46) and osteoclasts (47), both of which are derived from the same mesenchymal progenitor cells as myoblasts.

Our findings provide new insight into the relatively poorly understood mechanisms of ERβ activity in both RMS and other ER-positive tumor types. Furthermore, the evidence of estrogen sensitivity and tamoxifen-induced apoptosis in RMS cell lines constitutes a compelling rationale for further study of the ER pathway as a therapeutic target in RMS, as well as other mesenchymally derived sarcomas, as a potential synergistic or adjuvant treatment to be used in conjunction with current chemotherapy protocols.


   Acknowledgments
 
Grant support: In part by the Andrew Mizzoni Cancer Research Fund, a SickKids Restracom Studentship (J.A. Greenberg and S. Somme), Ontario Graduate Scholarship (J.A. Greenberg), and Canadian Institutes for Health Research Studentship (A.D. Durbin).

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.


   Footnotes
 
Note: J.A. Greenberg and S. Somme contributed equally to this work.

Received 8/ 8/07. Revised 1/23/08. Accepted 3/ 6/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pappo AS. Rhabdomyosarcoma and other soft tissue sarcomas in children. Curr Opin Oncol 1996;8:311–6.[Medline]
  2. Pizzo PA, Poplack DG. Principles and Practice of Pediatric Oncology. 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2006; p. xiv, 1780.
  3. Voute PA, Barrett A, Stevens CG, Caron HN. Cancer in Children: Clinical Management. 5th ed. Oxford University Press; 2005. p. 448.
  4. Anderson J, Gordon A, McManus A, Shipley J, Pritchard-Jones K. Disruption of imprinted genes at chromosome region 11p15.5 in paediatric rhabdomyosarcoma. Neoplasia 1999;1:340–8.[CrossRef][Medline]
  5. Baserga R, Peruzzi F, Reiss K. The IGF-1 receptor in cancer biology. Int J Cancer 2003;107:873–7.[CrossRef][Medline]
  6. De Giovanni C, Melani C, Nanni P, et al. Redundancy of autocrine loops in human rhabdomyosarcoma cells: induction of differentiation by suramin. Br J Cancer 1995;72:1224–9.[Medline]
  7. Gee MF, Tsuchida R, Eichler-Jonsson C, Das B, Baruchel S, Malkin D. Vascular endothelial growth factor acts in an autocrine manner in rhabdomyosarcoma cell lines and can be inhibited with all-trans-retinoic acid. Oncogene 2005;24:8025–37.[CrossRef][Medline]
  8. Maloney EK, McLaughlin JL, Dagdigian NE, et al. An anti-insulin-like growth factor I receptor antibody that is a potent inhibitor of cancer cell proliferation. Cancer Res 2003;63:5073–83.[Abstract/Free Full Text]
  9. Schweigerer L, Neufeld G, Mergia A, Abraham JA, Fiddes JC, Gospodarowicz D. Basic fibroblast growth factor in human rhabdomyosarcoma cells: implications for the proliferation and neovascularization of myoblast-derived tumors. Proc Natl Acad Sci U S A 1987;84:842–6.[Abstract/Free Full Text]
  10. Xia SJ, Pressey JG, Barr FG. Molecular pathogenesis of rhabdomyosarcoma. Cancer Biol Ther 2002;1:97–104.[Medline]
  11. Zhan S, Shapiro DN, Helman LJ. Activation of an imprinted allele of the insulin-like growth factor II gene implicated in rhabdomyosarcoma. J Clin Invest 1994;94:445–8.[Medline]
  12. Kinzbrunner B, Ritter S, Domingo J, Rosenthal CJ. Remission of rapidly growing desmoid tumors after tamoxifen therapy. Cancer 1983;52:2201–4.[CrossRef][Medline]
  13. McAdam WA, Goligher JC. The occurrence of desmoids in patients with familial polyposis coli. Br J Surg 1970;57:618–31.[Medline]
  14. Ohashi T, Shigematsu N, Kameyama K, Kubo A. Tamoxifen for recurrent desmoid tumor of the chest wall. Int J Clin Oncol 2006;11:150–2.[CrossRef][Medline]
  15. Sportiello DJ, Hoogerland DL. A recurrent pelvic desmoid tumor successfully treated with tamoxifen. Cancer 1991;67:1443–6.[CrossRef][Medline]
  16. Ishizuka M, Hatori M, Dohi O, et al. Expression profiles of sex steroid receptors in desmoid tumors. Tohoku J Exp Med 2006;210:189–98.[CrossRef][Medline]
  17. Deyrup AT, Tretiakova M, Montag AG. Estrogen receptor-β expression in extraabdominal fibromatoses: an analysis of 40 cases. Cancer 2006;106:208–13.[Medline]
  18. Nilsson S, Makela S, Treuter E, et al. Mechanisms of estrogen action. Physiol Rev 2001;81:1535–65.[Abstract/Free Full Text]
  19. Tsai MJ, O'Malley BW. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 1994;63:451–86.[CrossRef][Medline]
  20. Enmark E, Pelto-Huikko M, Grandien K, et al. Human estrogen receptor β-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab 1997;82:4258–65.[Abstract/Free Full Text]
  21. Gosden JR, Middleton PG, Rout D. Localization of the human oestrogen receptor gene to chromosome 6q24–q27 by in situ hybridization. Cytogenet Cell Genet 1986;43:218–20.[Medline]
  22. Dougherty SM, Mazhawidza W, Bohn AR, et al. Gender difference in the activity but not expression of estrogen receptors {alpha} and β in human lung adenocarcinoma cells. Endocr Relat Cancer 2006;13:113–34.[Abstract/Free Full Text]
  23. Martin LA, Pancholi S, Chan CM, et al. The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling. Endocr Relat Cancer 2005;12:1017–36.[Abstract/Free Full Text]
  24. Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA. Estrogen receptor β inhibits 17β-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci U S A 2004;101:1566–71.[Abstract/Free Full Text]
  25. Wakeling AE. Dukes M, Bowler J. A potent specific pure antiestrogen with clinical potential. Cancer Res 1991;51:3867–73.[Abstract/Free Full Text]
  26. Shen SS, Smith CL, Hsieh JT, et al. Expression of estrogen receptors-{alpha} and -β in bladder cancer cell lines and human bladder tumor tissue. Cancer 2006;106:2610–6.[Medline]
  27. McPherson SJ, Ellem SJ, Simpson ER, Patchev V, Fritzemeier KH, Risbridger GP. Essential role for estrogen receptor β in stromal-epithelial regulation of prostatic hyperplasia. Endocrinology 2007;148:566–74.[Abstract/Free Full Text]
  28. Takahashi Y, Perkins SN, Hursting SD, Wang TT. 17β-estradiol differentially regulates androgen-responsive genes through estrogen receptor-β-and extracellular-signal regulated kinase-dependent pathways in LNCaP human prostate cancer cells. Mol Carcinog 2007;46:117–29.[CrossRef][Medline]
  29. Ohshiro K, Rayala SK, Williams MD, Kumar R, El-Naggar AK. Biological role of estrogen receptor β in salivary gland adenocarcinoma cells. Clin Cancer Res 2006;12:5994–9.[Abstract/Free Full Text]
  30. Batistatou A, Stefanou D, Goussia A, Arkoumani E, Papavassiliou AG, Agnantis NJ. Estrogen receptor β (ERβ) is expressed in brain astrocytic tumors and declines with dedifferentiation of the neoplasm. J Cancer Res Clin Oncol 2004;130:405–10.[Medline]
  31. Campbell-Thompson M, Lynch IJ, Bhardwaj B. Expression of estrogen receptor (ER) subtypes and ERβ isoforms in colon cancer. Cancer Res 2001;61:632–40.[Abstract/Free Full Text]
  32. Gruvberger-Saal SK, Bendahl PO, Saal LH, et al. Estrogen receptor {β} expression is associated with tamoxifen response in ER{{alpha}}-negative breast carcinoma. Clin Cancer Res 2007;13:1987–94.[Abstract/Free Full Text]
  33. Zacharin M, Waters K, Chow CW, Crock P, McKelvie P. Recurrent rhabdomyosarcoma after 25 years: a possible association with estrogen and progestogen therapy. J Pediatr Hematol Oncol 1997;19:477–81.[CrossRef][Medline]
  34. Gottardis MM, Robinson SP, Jordan VC. Estradiol-stimulated growth of MCF-7 tumors implanted in athymic mice: a model to study the tumoristatic action of tamoxifen. J Steroid Biochem 1998;30:311–4.[CrossRef]
  35. Goldstein SR, Siddhanti S, Ciaccia AV, Plouffe L, Jr. A pharmacological review of selective oestrogen receptor modulators. Hum Reprod Update 2000;6:212–24.[Abstract/Free Full Text]
  36. Chagin AS, Karimian E, Zaman F, Takigawa M, Chrysis D, Savendahl L. Tamoxifen induces permanent growth arrest through selective induction of apoptosis in growth plate chondrocytes in cultured rat metatarsal bones. Bone 2007;40:1415–24.[Medline]
  37. Wu X, Ahn EY, McKenna MA, Yeo H, McDonald JM. Fas binding to calmodulin regulates apoptosis in osteoclasts. J Biol Chem 2005;280:29964–70.[Abstract/Free Full Text]
  38. Wilcken N, Tattersall MH. Endocrine therapy for desmoid tumors. Cancer 1991;68:1384–8.[CrossRef][Medline]
  39. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J, Nilsson S. Differential response of estrogen receptor {alpha} and estrogen receptor β to partial estrogen agonists/antagonists. Mol Pharmacol 1998;54:105–12.[Abstract/Free Full Text]
  40. Dauvois S, White R, Parker MG. The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. J Cell Sci 1993;106:1377–88.[Abstract]
  41. Lee H, Bai W. Regulation of estrogen receptor nuclear export by ligand-induced and p38-mediated receptor phosphorylation. Mol Cell Biol 2002;22:5835–45.[Abstract/Free Full Text]
  42. Metzger D, Ali S, Bornert JM, Chambon P. Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cells. J Biol Chem 1995;270:9535–42.[Abstract/Free Full Text]
  43. Zheng A, Kallio A, Harkonen P. Tamoxifen-induced rapid death of MCF-7 breast cancer cells is mediated via ERK signaling and can be abrogated by estrogen. Endocrinology 2007;148:2764–77.[Abstract/Free Full Text]
  44. Rodriguez-Galindo C, Hill DA, Onyekwere O, et al. Neonatal alveolar rhabdomyosarcoma with skin and brain metastases. Cancer 2001;92:1613–20.[Medline]
  45. Parham DM. Appearances may be deceiving: does aggressive histology correlate with behavior of infantile sarcomas? Pediatr Dev Pathol 2002;5:513–4.[CrossRef][Medline]
  46. Arts J, Kuiper GG, Janssen JM, et al. Differential expression of estrogen receptors {alpha} and β mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinology 1997;138:5067–70.[Abstract/Free Full Text]
  47. Sorensen MG, Henriksen K, Dziegiel MH, Tanko LB, Karsdal MA. Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts. DNA Cell Biol 2006;25:475–83.[CrossRef][Medline]
  48. Andersen J, Orntoft T, Skorvgaard Poulsen H. Semiquantitative oestrogen receptor assay in formalin-fixed paraffin sections of human breast cancer tissue using monoclonal antibodies. Br J Cancer 1986;53:691–4.[Medline]




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