Stromal Cell-Derived Factor 1, a Novel Target of Estrogen Receptor Action, Mediates the Mitogenic Effects of Estradiol in Ovarian and Breast Cancer Cells
Julie M. Hall and
Kenneth S. Korach
Receptor Biology Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Kenneth S. Korach, Receptor Biology Section, National Institute of Environmental Health Sciences, P.O. Box 12233, MD B3-02, Research Triangle Park, North Carolina 27709. E-mail: korach{at}niehs.nih.gov.
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ABSTRACT
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Recent clinical studies estimate that 6070% of human ovarian and breast cancers overexpress the estrogen receptor (ER). However, despite the established mitogenic effects of estrogen in these tumors, proliferative markers of hormone action are limited. In the current study, we report that the growth stimulatory cytokine stromal cell-derived factor 1 (SDF-1) is a bona fide target of estrogen action in ER
-positive human ovarian and breast cancer cells. Notably, estradiol treatment of BG-1 (ovarian carcinoma) and MCF-7 (breast carcinoma) cells leads to rapid and robust induction of the SDF-1
and ß isoforms. This response is blocked by the pure ER antagonist ICI 182,780 and is not apparent in ER-negative ovarian cells, indicating that SDF-1 regulation is ER
mediated. Treatment with the protein synthesis inhibitor cycloheximide had no effect on estradiol induction of induction of SDF-1 mRNA levels mRNA levels, demonstrating that SDF-1 is a direct target of ER
. SDF-1 protein levels, although undetectable under basal conditions, were strikingly increased by hormone both intracellularly and in the media of cultured BG-1 and MCF-7 cells. In cell proliferation assays, the mitogenic effects of estradiol were neutralized by addition of an SDF-1 antibody and mimicked by the addition of exogenous SDF-1 protein, indicating that SDF-1 mediates the proliferative actions of hormone. Furthermore, activation of the SDF-1 receptor CXCR4 stimulated BG-1 and MCF-7 cell proliferation in a manner comparable to estradiol. Taken together, these results demonstrate a novel estrogen-mediated paracrine pathway for inducing cancer cell proliferation and suggest that SDF-1 and CXCR4 may represent novel therapeutic targets in ER
-positive ovarian and breast tumors.
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INTRODUCTION
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OVARIAN MALIGNANCIES REPRESENT a significant concern to womens health, as their associated mortality rate has not changed significantly over the past two decades.
Ovarian cancer is the fourth leading cause of cancer deaths in Western countries and the most fatal gynecological cancer (1). The ovary is the main site of sex-steroid hormone production in females, and recent studies indicate a role for estrogen in ovarian cancer. Ovarian surface epithelial cells, the site of 90% of malignancies, show a marked proliferative response to estrogens (2). Furthermore, recent clinical studies indicate that postmenopausal women receiving estrogen replacement therapy demonstrate an increased risk of ovarian cancer and mortality (3, 4). Analysis of primary tumor samples has revealed that as many as 70% of ovarian cancers express estrogen receptors (ERs), which confers estrogen responsiveness to the tissue (5, 6). Tumors with high levels of ER expression demonstrate decreased apoptotic activity and increased proliferation in areas of high receptor density (6). Collectively, these observations have suggested a role for estrogen signaling through the ER in the progression of ovarian cancers.
Much of what is known about the mitogenic actions of estrogen in ER-positive tumors has come from the study of hormone action in human breast cancer cells. The observation that greater than 60% of primary breast tumors express epithelial ER (7) suggests there could be parallels between estrogen action in ovarian and breast cancer cells. The ERs (ER
and ERß) belong to the nuclear receptor family and function as hormone-inducible transcription factors in target cells (8). In breast cancer cells, ER
is thought to mediate the mitogenic actions of estrogen by inducing the expression of genes involved in cell proliferation. Indeed, several estrogen-induced proteins have been identified from ER
-positive breast cancer cell lines including the progesterone receptor (PR), cathepsin D, c-fos, and pS2 (9, 10). In primary tumors, these markers have been useful prognostic factors for predicting whether a tumor is estrogen sensitive and will respond to antiestrogen therapy (7, 10). In the ovary, however, there is a notable lack of regulation of classical estrogen-responsive genes (PR, c-fos, pS2). Currently, fibulin-1, an extracellular matrix protein, is the only well-characterized ER-inducible protein in ovarian cancer cells (11, 12, 13). Thus, although significant advances have been made in our understanding of ER action in breast cancer, the molecular pathways involved in hormone-stimulated proliferation in the ovary remain elusive. Clearly, definition of the ovarian ER pathways that mediate growth in response to hormone will clarify the role of the receptor and target genes in the onset and progression of ER-positive tumors and provide diagnostic markers for clinical use, which are severely lacking. Such markers would also be important for evaluating whether the mitogenic actions of estrogen in the ovary and breast occur through common mechanisms and may provide avenues for developing ER-targeted therapeutics.
In the current study, we have investigated mechanisms by which estrogen promotes cell proliferation by the identification of estrogen-regulated genes in BG-1 ovarian cancer cells. BG-1 is an epithelial cell line derived approximately 20 yr ago from the solid tumor tissue of a patient with stage III human ovarian adenocarcinoma (14). This line expresses clinically significant levels of endogenous ER
and PR, but not ERß, consistent with known receptor expression patterns from primary ovarian tumors. Furthermore, the ER
and PR present in this cell line possess characteristics consistent with those found in known steroid-responsive tissues with regards to capacity, hormone binding affinity, and ability to activate transfected reporter vectors containing ER and PR response elements (14, 15). Using BG-1 cells as a model for ovarian cancer, microarray studies led to identification of a series of estrogen-regulated genes, providing potential markers for ER action in ovarian cancer cells. This report describes studies characterizing the regulation and function of one such gene, stromal cell-derived factor 1 (SDF-1), in estrogen signaling and proliferative pathways in ovarian and breast cancer cells.
SDF-1 is an autocrine and paracrine acting factor that signals through a cell surface G protein-coupled receptor, CXCR4 (16, 17, 18, 19). SDF-1 functions as a proliferative and chemotactic factor for B and T lymphocytes and endothelial cells, stimulating cell migration through inducing reorganization of the actin cytoskeleton (20, 21). Both SDF-1 and CXCR4 are essential components of organogenesis, hematopoiesis, and immune responses in mammals (22). In addition to its roles in normal physiology, recent studies indicate that SDF-1 is overexpressed in malignant ovarian and breast cancer cells and is involved in tumor cell migration and possibly metastasis (20, 23). However, hormonal regulation of SDF-1 has not been previously described. Our detection of SDF-1 as an estrogen-regulated gene, together with these latter observations, prompted us to study hormonal regulation of SDF-1 and to determine whether the cytokine could be a mediator of estrogens proliferative actions in the ovary.
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RESULTS
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Estradiol Regulation of SDF-1
and -1ß Isoforms in BG-1 Ovarian Carcinoma Cells
During a screen to identify novel estrogen-regulated genes in the ovary, secondary analysis of candidate genes by ribonuclease protection assay (RPA) analysis revealed that the growth-stimulatory cytokine SDF-1 is a bona fide target of estrogen action. Treatment of BG-1 cells with physiological concentrations of estradiol (1 nM) led to a rapid and robust of SDF-1ß RNA levels, reaching 15-fold at 24 h (Fig. 1A
).

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Figure 1. SDF-1 Is Rapidly and Robustly Induced by Estradiol in BG-1 Ovarian Carcinoma Cells
A, BG-1 cells were treated with vehicle (veh) or 1 nM 17ß-estradiol (E) for 2, 6, or 24 h (2 h, 6 h, 24 h). RNA was harvested from triplicate samples for each hormone condition, and 10 µg of each sample were analyzed by RPA using a riboprobe corresponding to a 352-bp region (9011252 bp) of the human SDF-1ß cDNA. A riboprobe for human cyclophilin (Cyc) was used for normalization. Shown is a representative RPA experiment; graphical data represent the average of three independent samples from a representative experiment. Values for fold induction by E are shown above each bar. B, An RPA was developed to detect the SDF-1 and ß isoforms using a 450-bp riboprobe containing a 300-bp fragment from the hSDF-1ß cDNA (198498 bp), which corresponds to the last 150 bp of the coding sequence of both hSDF-1 and -1ß and 150 bp unique to hSDF-1ß within the 3'-untranslated region. Shown is RPA analysis of SDF-1 and ß expression in 10 µg RNA harvested from three independent cultures of BG-1 cells treated with 1 nM E for 24 h. A riboprobe for human Cyc was used for normalization. C, To examine estrogen regulation of SDF-1 and ß, BG-1 cells were treated with veh or 1 nM E for 6 h. RNA was harvested and 10 µg of each sample was analyzed by RPA using the SDF-1 and ß riboprobe and a Cyc riboprobe for normalization.
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SDF-1 exists as two isoforms (
and ß) that arise from alternative splicing, resulting in an extra four amino acids at the carboxyl terminus of the ß isoform (
and ß-88 and 93 amino acids, respectively; (24, 25). Because SDF-1
and ß are functionally similar but can display nonidentical expression patterns (26, 27), we next investigated whether estrogen could induce the expression of both isoforms. An RPA was developed to detect both SDF-1
and ß within a single RNA population by synthesizing a riboprobe that recognized the last 150 bp of the coding sequence of both human (h)SDF-1
and -1ß and 150 bp unique to hSDF-1ß within the 3'-untranslated region. The use of this probe to analyze RNA from BG-1 cells treated with estradiol yielded 150-bp and 300-bp protected fragments in an RPA, corresponding to the two isoforms (Fig. 1B
). Analysis of RNA from BG-1 cells treated with vehicle or estradiol revealed that there is hormonal induction of both SDF-1
and ß at similar levels (Fig. 1C
).
Estrogen Regulation of SDF-1 Is Mediated through ER
as a Primary Response
ER
is expressed in the epithelial cells of the normal ovary and in 70% of ovarian tumors. However, due to lack of known estrogen-responsive genes in the ovary, the role of the ER is not yet clear. To determine whether estrogen regulation of SDF-1 is mediated through ER
, the ability of the ER antagonist ICI 182,780 to block the observed estrogen induction was tested. Treatment of BG-1 cells with estradiol led to a significant induction of SDF-1
and ß levels (15-fold), whereas administration of ICI 182,780 completely attenuated the hormonal response (Fig. 2A
). Notably, ICI 182,780 alone or in combination with estradiol decreased basal levels of SDF-1 expression to 30% or 40% of vehicle, respectively (Fig. 2A
), consistent with its known inverse agonist activity (28). Estrogen induction of SDF-1 was absent in ER-negative UCI 107 ovarian cancer cells (not shown), further suggesting that ER
is involved in its regulation.
To determine whether SDF-1 is a direct target of ER
signaling, the effect of the protein synthesis inhibitor cycloheximide (CHX) on estrogen induction of SDF-1 expression was examined. Notably, treatment of BG-1 cells with CHX at a concentration known to block greater than 95% of protein synthesis (29) did not decrease estrogen induction of SDF-1
and ß (Fig. 2B
). In contrast, the expression of both isoforms was enhanced by CHX in the presence of hormone, likely due to message stabilization as reported by others using this agent to study gene expression (30). Control experiments demonstrated that within the same RNA samples, CHX significantly diminished hormone induction of the vitamin D receptor, a secondary estrogen-responsive gene (data not shown). Taken together, these results indicate that SDF-1 is a primary ER
-regulated gene in ovarian cancer cells.
SDF-1 Is a Target of Estrogen Action in ER
-Positive Breast Cancer Cells
Although ER
is expressed in at least 60% of ovarian and breast tumors, it is not yet clear whether estrogen plays a similar role in each tumor type, as hormone-responsive genes common to both tissues have not been identified. To determine whether SDF-1 could be a shared target of hormone action, the ability of estrogen to stimulate SDF-1 expression in MCF-7 cells, a human epithelial breast cancer line, was next examined (Fig. 3
). RT-PCR analysis verified that the MCF-7 cells used expressed endogenous ER
but not ERß (data not shown). Significant levels of SDF-1 RNAs were detected in the absence of hormone; however, estradiol administration led to a 7-fold induction of SDF1
and ß (Fig. 3
). As seen with the BG-1 cells (Fig. 2A
), administration of ICI 182,780 completely attenuated the hormone response and decreased basal levels of SDF-1 expression to 3040% (Fig. 3
). These results indicate that hormone regulation is mediated through ER
, and constitutive SDF-1 expression in MCF-7 cells is likely due to ligand-independent receptor activity. Thus, SDF-1 represents a common component of estrogen-ER
signaling in both ovarian and breast cancer cells.
To determine whether estrogen induction of SDF-1 occurs in other ER
-positive cancer cells, SDF-1 expression was analyzed in RNA from Ishikawa endometrial carcinoma cells that, like the BG-1 and MCF-7 lines studied, express endogenous ER
but not ERß. Notably, SDF-1 expression was undetectable both under basal conditions and when cells were administered 10 nM estradiol (data not shown). Under the same conditions, however, the known estrogen-responsive lactoferrin RNA was up-regulated by estradiol, indicating that the ER
signaling pathway was functional in the Ishikawa cell line studied. These results suggest that estrogen regulation of SDF-1 displays tissue specificity, and ER
may not function in activated gene expression in an equivalent manner in different types of cancers.
Estrogen Induces Production and Enhances Extracellular Levels of SDF-1 Protein in BG-1 and MCF-7 Cells
To determine whether estradiol enhances SDF-1 protein production, whole cell extracts from BG-1 and MCF-7 cells were analyzed for intracellular SDF-1 content by Western blotting (Fig. 4A
). For these experiments, a human SDF-1 antibody capable of recognizing both the
and ß isoforms was used. SDF-1 protein was undetectable in cell extracts under basal conditions (Fig. 4A
). However, cells administered estradiol produced significant levels of a 10-kDa protein at the correct predicted size. The substantial signal detected was comparable to that of 250 ng of a fragment of the purified SDF-1
protein (8 kDa), the positive control in the assay. Notably, coadministration of ICI 182,780 with estradiol (E + I) completely blocked protein expression, indicating that intracellular SDF-1 RNA levels correlate with protein production (Figs. 2A
, 3
, and 4A
).

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Figure 4. Estrogen Induces Production and Enhances Extracellular Levels of SDF-1 Protein in BG-1 and MCF-7 Cells
A, BG-1 and MCF-7 cells were treated with vehicle (veh), 10 nM 17ß-estradiol (E), or E + 1 µM ICI 182,780 (E + I) for 24 h. Cells were harvested, and protein content was analyzed by electrophoresis and Western blotting using a human anti-SDF-1 antibody. Shown is a representative Western blot experiment. A partial fragment of purified SDF-1 (68 amino acids) was used as a positive control (SDF-1 ). SDF-1 was detected at the predicted molecular mass of 10 kDa in the E-treated samples. B, BG-1 and MCF-7 cells were treated with veh, 10 nM E, or E + 1 µM ICI 182,780 (E + I) for 24 h. Total spent cell media were collected, and protein content was analyzed by electrophoresis and Western blotting using a human SDF-1 antibody. Shown is a representative Western blot experiment. A partial fragment of purified SDF-1 (68 amino acids; 8 kDa) was used as a positive control (SDF-1 ). A duplicate media sample from estradiol-treated MCF-7 cells was incubated with SDF-1 antibody that was pretreated with a blocking peptide (E + P).
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SDF-1 protein is functional after it is secreted from cells, an event that enables it to signal through its cell surface receptor CXCR4 (17, 19). Thus, we were next interested in determining whether estrogen could enhance extracellular SDF-1 protein levels. For these analyses, BG-1 and MCF-7 cells were plated at 60% confluency in 96-well plates and induced with 17ß-estradiol for 24 h. Media were aspirated from individual wells and analyzed for SDF-1 protein expression by Western blotting (Fig. 4B
). Extracellular SDF-1 was undetectable in vehicle-treated cells, yet strikingly increased in the media of cells administered estradiol. This induction was attenuated with ICI, indicating a role for ER
in elevation of both intracellular and extracellular SDF-1 protein levels (Fig. 4
, A and B). No signal was detected in a media sample from estradiol-treated MCF-7 cells when the SDF-1 antibody was pretreated with a blocking peptide, demonstrating antibody specificity (Fig. 4B
, E + P). Taken together, these results indicate that estradiol-activated ER
induces production and enhances extracellular levels of SDF-1 protein in BG-1 and MCF-7 cells, and that SDF-1 expression is tightly regulated by estradiol.
SDF-1 Mimics the Proliferative Effects of Estrogen in a Dose-Dependent Manner
Because estrogen can function as a mitogen in ER
-positive tumors, the observed hormonal regulation of SDF-1 suggested a role in cancer cell proliferation. To test this hypothesis, the effect of SDF-1 on cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium salt (MTS) compound in a colorimetric assay, in which MTS is bioreduced in metabolically active cells to a soluble formazan product that is quantitated spectrophotometrically at 490 nm. The quantity of formazan product is directly proportional to the number of viable cells in culture (31). To establish linearity between cell number and absorbance in our assays, BG-1 and MCF-7 cells were plated in 96-well plates at various densities, incubated with MTS solution for 1 h, and absorbance values at 490 nm were measured and corrected for absorbance values read from blank controls containing media with no cells. Graphical representative of the data demonstrated a linear relationship between cell number and absorbance when concentrations ranging from 5 x 10480 x 104 cells/well were used in the assay (Fig. 5A
); at higher cell concentrations, saturation was observed (data not shown). These results validated the assay as an approach to quantitate BG-1 and MCF-7 cell proliferation.

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Figure 5. SDF-1 Mimics the Proliferative Effects of Estrogen in a Dose-Dependent Manner
A, To demonstrate linearity between cell number and absorbance reading in the cell proliferation assay, BG-1 and MCF-7 cells were seeded in 96-well plates at different concentrations and incubated with MTS solution for 1 h. Absorbance values at 490 nm were measured, and all values were corrected for absorbance values read from blank controls containing media with no cells. Shown is the graphical relationship between cell number and absorbance using linear regression analysis. Each data point represents the average of quadruplicate samples, and results were reproducible in independent experiments. B, BG-1 and MCF-7 cells were seeded in 96-well plates and treated with veh, 10 nM 17ß-estradiol (E), or increasing concentrations of purified SDF-1ß (5, 10, 50, 100, and 200 ng/ml, respectively). After 48 h, cells were incubated for 1 h with MTS solution and the absorbance was read at 490 nm. All values were corrected for absorbance readings from blank control wells containing media with no cells. Graphical data show the average of quadruplicate samples for each condition in a representative experiment. Results were highly reproducible in three independent experiments.
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Using this assay, the effect of estradiol and SDF-1 on cell proliferation was next assessed (Fig. 5B
). BG-1 and MCF-7 cells were seeded in 96-well plates (at 5 x 104 or 10 x 104 cells/well; within the linear range of the assay), allowed to adhere, and treated for 48 h with vehicle, estradiol, or increasing concentration of purified SDF-1ß protein. A physiological dose of estradiol (10 nM) caused an approximate doubling of cell number, indicating that the hormone was proliferative in both BG-1 and MCF-7 cells. Strikingly, low concentrations of SDF-1ß (5 ng/ml) stimulated cell growth, and SDF-1ß mimicked the effects of estradiol in a dose-dependent manner (Fig. 5B
), surpassing estradiol at known biologically active levels (100200 ng/ml; Ref. 32). Likewise, purified SDF-1
also induced BG-1 and MCF-7 cell proliferation, indicating functional similarity between the two isoforms (data not shown). Similar results were seen when cells were treated as above, stained with Trypan Blue, and the number of viable cells under each condition was quantitated (data not shown).
To ensure that the results from these assays reflected enhanced proliferation rather than an antiapoptotic effect, BG-1 and MCF-7 cells were treated with vehicle, hormone, SDF-1ß, or the known apoptotic agent TNF
, and stained for annexin V, an indicator of apoptosis. Flow cytometric analysis revealed no effect of either estradiol or SDF-1ß on annexin V staining, in contrast to a substantial proapoptotic influence observed with TNF
administration (data not shown). Taken together, the results indicate that, at known biologically active concentrations, SDF-1 stimulates cancer cell proliferation in a manner that is comparable to estradiol.
SDF-1 Mediates the Proliferative Effects of Estrogen in Human Ovarian and Breast Cancer Cells
To determine whether SDF-1 is required for the mitogenic action of estrogen, the effect of an SDF-1 antibody on hormone-induced cell proliferation was tested (Fig. 6A
). As observed in Fig. 5B
, estradiol and SDF-1 were effective mitogenic factors in BG-1 and MCF-7 cells (Fig. 6A
). No additive effect was noted when the two agents were coadministered, suggesting that they function in the same pathway and/or a maximal proliferative response had been achieved. Strikingly, addition of SDF-1 antibody completely neutralized the stimulatory effect of estradiol and attenuated the proliferative effects of exogenously administered SDF-1 (Fig. 6A
). The inhibitory effects of the SDF-1 antibody on both SDF-1 and estradiol-induced proliferation were overcome with the addition of increasing concentrations of SDF-1, demonstrating specificity of the antibody (not shown). Additional control experiments revealed no effect of an equivalent quantity of an antibody to the yeast GAL4 transcription factor on SDF-1 or estrogen-induced proliferation, suggesting that the observed results were not caused by a cytotoxic effect of antibody administration (not shown). Cumulatively, these results indicate that SDF-1 mediates the proliferative effects of estrogen in BG-1 and MCF-7 cells.

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Figure 6. SDF-1 and CXCR4 Mediate the Proliferative Effects of Estrogen in Human Ovarian and Breast Cancer Cells
A, BG-1 and MCF-7 cells were seeded in 96-well plates and treated with vehicle, 10 nM 17ß-estradiol (E), 50 ng/ml purified SDF-1ß, E + SDF1ß, 10 ng/ml human SDF-1 antibody ( SDF-1), or E + SDF-1. After 48 h, cells were incubated for 1 h with MTS solution and the absorbance was read at 490 nm. All values were corrected for absorbance values read from blank controls containing media with no cells. Graphical data show the average of quadruplicate samples for each condition in a representative experiment. Results were highly reproducible in three independent experiments. B, RT-PCR analysis of CXCR4 expression in BG-1 and MCF-7 cells. RNA was harvested from cells and used in RT-PCRs that included primers corresponding to the 5' and 3' ends of the CXCR4 cDNA. Shown is the gel electrophoresis analysis: DNA ladder (lane 1), and 5 µl of the RT-PCRs from the negative control (no RNA), BG-1 RNA, and MCF-7 RNA reactions (lanes 24, respectively). The amplified cDNA products (lanes 3 and 4) migrate at the correct size for CXCR4 (1.1 kb). C, BG-1 and MCF-7 cells were seeded in 96-well plates and treated with vehicle, 10 nM E, 50 ng/ml purified SDF-1ß, 10 ng/ml human CXCR4 antibody ( CXCR4), SDF-1ß + CXCR4, E + CXCR4, or CXCR4 preincubated with a blocking peptide. After 48 h, cells were incubated for 1 h with MTS solution and the absorbance was read at 490 nm. All values were corrected for absorbance values read from blank controls containing media with no cells. Graphical data show the average of quadruplicate samples for each condition in a representative experiment. Results were highly reproducible in three independent experiments.
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To verify that the SDF-1 signaling pathway was functional in BG-1 and MCF-7 cells, the role of CXCR4 activation in cell proliferation was next examined. RT-PCR analysis revealed that both cell lines express CXCR4 (Fig. 6B
). An analysis of CXCR4 in the estrogen signaling pathway proved challenging due to the lack of readily available CXCR4 antagonists and the apparent shared downstream effectors of CXCR4 and nongenomic estrogen signaling pathways such as G proteins and MAPK (33, 34, 35, 36, 37). Thus, we elected to study the role of CXCR4 in cell proliferation using an antibody to CXCR4. Consistent with the known activating effect of antibodies on their respective cytokine receptors, addition of CXCR4 antibody to BG-1 and MCF-7 stimulated proliferation approximately 2-fold after 48 h in a manner comparable to estradiol (Fig. 6C
). This effect was not augmented by coadministration of hormone or SDF1ß (Fig. 6C
) but was elevated 30% when the antibody concentration was doubled (20 ng/ml; data not shown). Antibody specificity was demonstrated with the use of a competitor peptide, which completely attenuated the stimulatory effects (Fig. 6C
). Thus, activation of the SDF-1 receptor CXCR4 stimulates proliferation of BG-1 and MCF-7 cells. Taken together, these studies indicate that the mitogenic effects of estrogen in human ovarian and breast cancer cells can occur through hormonal elevation of extracellular SDF-1 levels, which in turn enhances activation of the CXCR4 signaling pathway, resulting in cancer cell proliferation.
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DISCUSSION
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Identification of a Novel Marker of Estrogen Action in Ovarian Cancer Cells
Due to the lack of known estrogen-responsive genes in the ovary, the role of estrogen signaling through ER in cancer cell proliferation has remained elusive. In ovarian tumors, receptor-positivity is associated with increased proliferation and a less favorable prognosis (6, 38). These observations suggest that ER could be involved in mitogenic pathways in the ovary, perhaps by inducing genes involved in cell proliferation. The identification of ER target genes in human tumors and animal models has remained a challenge because the ovary is the major site of estrogen production, making it difficult to alter the hormonal environment to study genomic responses to ER activation. Thus, the use of cultured ovarian cells as a model to study ER gene regulation provides a system where hormonal manipulations can be easily achieved.
In the current study, using BG-1 cells as a model for ovarian cancer, we have identified SDF-1 as a novel target of ER action. Notable in these studies was the strict hormonal regulation of SDF-1 expression, as there was an absence of detectable protein under basal conditions, whereas estradiol administration led to a rapid and robust induction of SDF-1 protein. Not surprisingly, therefore, we found that SDF-1 is a direct target of ER. This result is supported by the existence of sequences resembling estrogen response elements in the 2-kb genomic region upstream from the transcription start site in the SDF-1 gene (our unpublished observations). Thus, SDF-1 is a novel upstream marker for estrogen action and ER signaling in ovarian cancer cells. Analysis of primary ovarian tumor samples for ER status and SDF-1 expression will provide insight into whether SDF-1 could represent a new clinical marker for hormone responsiveness in ovarian cancer.
SDF-1 Illustrates a New Mechanism by which Estrogen Promotes Ovarian Cancer Cell Proliferation
A significant finding in our studies was a role for SDF-1 in mediating the mitogenic effects of estradiol in ovarian cancer cells. Notably, extracellular SDF-1 protein was a potent stimulator of BG-1 cell proliferation. Furthermore, the mitogenic effects of estradiol were attenuated when SDF-1 was neutralized with antibody, indicating that SDF-1 is a required component of estrogen action in these cells. Based on these observations, we propose a new mechanism by which estrogen promotes ovarian cancer cell proliferation (Fig. 7
). Estradiol-activated ER induces the production and secretion of SDF-1, which binds the cell surface receptor CXCR4 in a potentially autocrine or paracrine manner. As shown previously, SDF-1 binding activates CXCR4, initiating downstream signaling events (21, 39). Then, as apparent from our studies, stimulation of CXCR4 signaling in ovarian cancer cells promotes cell proliferation. Consistent with this model is the identification of MAPK, an established component in mitogenesis, as a downstream effector of SDF-1 and CXCR4 (33).

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Figure 7. A New Mechanism by which Estrogen Promotes Proliferation of ER-Positive Ovarian Epithelial Cancer Cells
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SDF-1, a Common Mitogenic Pathway in Ovarian and Breast Cancer Cells
The description of estrogen-regulated genes such as c-fos, pS2, PR, and cathepsin D in breast cancer cells and tumors has provided insight into the mechanisms contributing to the mitogenic effects of hormone in the breast (9, 10, 40). However, the observation that these genes lack estrogen responsiveness in ER-positive ovarian cells suggested that the mitogenic actions of hormone in ovarian and breast tumors occur through different mechanisms. It was interesting, therefore, to discover that SDF-1 represents a point of convergence between the estrogen signaling pathways in the two tissues. Clearly this is not a mechanism common to all ER-expressing cells, as an absence of SDF-1 regulation was noted in ER-positive Ishikawa endometrial cells and in the mouse uterus (our unpublished observations), contexts where estrogen displays known mitogenic effects. It is possible that tissue-specific factors contribute to differential ER activation of growth regulatory proteins such as SDF-1 in different cancer cells. Regardless, the identification of SDF-1 as a common ER target and mediator of estrogens proliferative effects in ovarian and breast cancer cells suggests that SDF-1 and CXCR4 could represent therapeutic targets in ER-positive ovarian and breast tumors.
A New Role for SDF-1 in Hormone-Stimulated Cell Proliferation
Recent studies have demonstrated a critical role for SDF-1 in cell proliferation and trafficking during embryonic development. Mice that lack SDF-1 die perinatally, displaying severe deficits in B-cell lymphopoiesis, angiogenesis, bone marrow myelopoiesis, neuronal migration, and cerebral cortex formation, and cardiogenesis (16, 41, 42). In normal physiology, SDF-1 is required for immune responses and organ vascularization, functioning as a growth and chemotactic factor for lymphocytes, monocytes, and endothelial cells. Furthermore, in disease models, SDF-1 mediates the inflammatory events in cardiovascular infarction, and notably, CXCR4 functions as a coreceptor for strains of HIV (17, 18, 26).
In this study, the discovery of a role for SDF-1 and CXCR4 in estrogen signaling was interesting, as hormonal regulation of SDF-1 expression has not been reported, nor has the ER been implicated in promoting cellular chemotaxis. Our interest in SDF-1 as an ER target was further motivated by recent studies indicating that SDF-1 and CXCR4 are overexpressed in malignant ovarian and breast tumors (20, 23). Muller et al. (23) demonstrated that SDF-1 signaling through CXCR4 could promote tumor metastasis by stimulating the migration of breast cancer cells to lymph nodes. Thus, we initially hypothesized that ER regulation of SDF-1 could provide a common mechanism by which estrogens could promote ovarian and breast cancer metastasis. However, using phase contrast microscopy and dual-chamber migration assays, we observed an inhibitory effect of estradiol on BG-1 and MCF-7 cell migration (our unpublished observations). This result, although surprising, is consistent with previous reports on the counteractive effects of estrogen on ovarian and breast cancer cell invasion and motility (43). Thus, as proposed by Rochefort et al. (40, 43), estrogens in ER-positive tumors may have a dual role in their ability to stimulate cell proliferation yet inhibit cell motility and metastasis.
Our studies describe a new role of SDF-1 in mediating proliferative actions of estrogen in ovarian and breast cancer cells. Although this is paradoxical, considering that the known chemotactic functions of SDF-1 suggest a role in cellular motility rather than in proliferation. However, it is possible that the mitogenic effects of SDF-1 are more manifest in ER-positive cancer cells. A similar story has emerged in the study of cathepsin D, an estrogen-regulated protease in ovarian and breast cancer cells, which is overexpressed in metastatic tumors. Proteases are known to promote metastasis by breaking down basement membranes, enabling cancer cells to migrate through the extracellular matrix. Rochefort et al. (40) have demonstrated that cathepsin D is more involved in cell proliferation than in motility in ER-positive breast and ovarian cancer cells. Based on these observations, they proposed that different signal transducing pathways are involved in the mitogenic and antimotility responses to estrogen, and other proteases may be more involved in the latter pathway. Likewise, downstream effectors of estrogen signaling may neutralize the motility function of SDF-1, while still enabling the mitogenic effects to be manifest. In support of this hypothesis, the chemoattractant properties of SDF-1 are inhibited by the cell surface ephrin receptor (44), whose activating ligand ephrin B is inducible by estradiol in microarray analysis of BG-1 cells (our unpublished results). On the other hand, SDF-1 signaling results in activation of the MAPK pathway, which is known to enhance ER-regulated gene expression (45), providing a positive feedback pathway for mitogenesis.
In summary, we have identified SDF-1 as a novel ER-regulated gene and mediator of the mitogenic effects of estrogen in ovarian and breast cancer cells. Significantly, hormonal regulation of SDF-1 provides an estrogen-mediated autocrine/paracrine pathway for inducing cancer cell proliferation and exists as a point of convergence between the ER-mediated proliferative pathways in the ovary and breast. Based on this newly ascribed role in mitogenesis, SDF-1 can now be considered a proliferative marker of estrogen action in ovarian and breast cancer cells. Thus, SDF-1 and CXCR4 may represent novel therapeutic targets in ER-positive ovarian and breast tumors.
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MATERIALS AND METHODS
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Riboprobe Templates
Full-length human SDF-1
and ß were cloned by RT-PCR. The riboprobe for human SDF-1ß was generated by amplifying a 352-bp region (9011252 bp of the cDNA) contained within the 3'-untranslated region of the transcript. The sequences of the primers for PCR were 5'-gggcagcagggctaccctgagctg and 3'-gggggaacagtccatgcatcaagac. The riboprobe for detecting both human SDF-1
and ß was generated by amplifying a 300-bp fragment from the hSDF-1ß cDNA (198498 bp). This region corresponds to the last 150 bp of the coding sequence of both hSDF-1
and -1ß, and 150 bp unique to hSDF-1ß within the 3'-untranslated region. The sequences of the primers for PCR were 5'-gccagagccaacgtcaagcatctc and 3'-ggcaaagtgtccaaaacaaagccc. PCR products were cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA) in the antisense orientation with respect to the Sp6 promoter. All PCR products were verified by sequencing. Riboprobe templates were prepared for in vitro transcription by digesting 10 µg of each plasmid with BamHI (for pCRII-hSDF-1ß) or XbaI (for pCRII-hSDF1
ß and pCRII-mSDF-1
ß). Linearized templates were incubated for 1 h at 65 C with 10 µl proteinase K (10 mg/ml) and 5 µl 20% sodium dodecyl sulfate in 100 µl final volume, and purified by phenol:chloroform extraction and ethanol precipitation. The human cyclophilin template was purchased from Ambion, Inc. (Austin, TX).
RNA Isolation and Analysis
RNA was isolated from cultured BG-1 and MCF-7 cells using the RNeasy Midi Kit (QIAGEN, Valencia, CA). Riboprobes were synthesized with the MAXIscript in vitro Transcription SP6/T7 Kit (Ambion, Inc.), using the Sp6 RNA polymerase. RPAs were performed on 10 µg RNA using the HybSpeed RPA Kit (Ambion, Inc.). Each 10-µg RNA sample was hybridized with 50,000 cpm of riboprobe. Reactions were run on 6% Tris-borate-EDTA Urea Novex gels (Invitrogen) for 70 min at 180 V. Gels were fixed in 10% acetic acid/0.5% glycerol for 1 h, dried, and analyzed by autoradiography. Data were quantitated by phosphoimager (Amersham Biosciences, Piscataway, NJ) analysis with STORM Scanner Control and ImageQuant 5.2 software. Triplicate samples were used for each hormone condition in each experiment, and all experiments were repeated three times.
RT-PCR amplification of CXCR4 was performed using 500 ng BG-1 and MCF-7 RNA and the CXCR4 primer pair set (Biosource International, Camarillo, CA).
Cell Culture and Hormone Induction
BG-1 and MCF-7 cells were maintained in DMEM F12 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (Invitrogen). For RNA analysis, cells were plated at 60% confluency in 100-mm culture dishes in 10 ml media 24 h before treatment. For hormone induction, cells were administered 10 ml phenol red-free DMEM F12 containing 10% charcoal-stripped FCS (HyClone Laboratories, Inc., Logan, UT); media contained either ethanol (vehicle), 1 nM or 10 nM 17ß-estradiol (Sigma, St. Louis, MO), 1 µM ICI 182,780 (a gift from Dr. A. Wakeling, AstraZeneca Pharmaceuticals, Cheshire, UK), or 10 nM 17ß-estradiol and 1 µM ICI 182,780. For cell proliferation assays, cells were induced with vehicle or 10 nM 17ß-estradiol in the absence or presence of purified human SDF-1ß (Biosource International), or 10 ng/ml human polyclonal antibodies to SDF-1 or CXCR4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For protein analysis, BG-1 and MCF-7 cells were plated in 40 µl phenol red-free DMEM F12 containing 10% charcoal-stripped FCS and appropriate hormones in 96-well plates or 100-mm plates. CHX (Sigma) was used at 50 µM in the culture media where indicated. All hormone treatments were performed in triplicate or quadruplicate in each experiment.
Cell Proliferation Assays
Cell proliferation was measured using the MTS colorimetric assay. For standard assays, BG-1 and MCF-7 cells were seeded and adhered in 96-well plates at 5 x 104 and 10 x 104 cells/well, respectively. Cells were incubated in the absence or presence of hormones, purified SDF-1ß, and antibodies for 48 h in phenol red-free DMEM F12 containing 10% charcoal-stripped FCS (100 µl/well). After 48 h, 100 µl of Cell Titer 96 Aqueous One Solution MTS solution (Promega Corp., Madison, WI) was added to each well, and cells were incubated at 37 C for 1 h. The absorbance was read at 490 nm using a 96-well plate reader (Microplate Reader 550, Bio-Rad Laboratories, Inc., Hercules, CA). All values were corrected for absorbance values read from blank controls containing media with no cells. Quadruplicate samples were used for each hormone condition in each experiment, and all experiments were repeated three times.
Western Immunoblotting
BG-1 and MCF-7 cell media were analyzed for secreted SDF-1 protein as follows: BG-1 and MCF-7 cells were plated in 96-well plates (40 µl media/well), allowed to adhere, and induced with hormone as described above. After 24 h, media were collected from individual wells, and each sample was run on a Nupage 12% Bis-Tris gel (Invitrogen). For detection of intracellular SDF-1, cells were seeded in 100 mm plates, allowed to adhere, and induced with hormone for 24 h. Whole cell extracts were isolated as described previously with modifications (46). Samples (60 µg) were run on Nupage 12% Bis-Tris gels. Proteins were transferred to nitrocellulose, and membranes were blocked and analyzed in TBS containing 5% milk and 0.1% Tween 20. SDF-1 protein was detected using a human polyclonal anti-SDF-1 antibody (Santa Cruz Biotechnology, Inc.), and a horseradish peroxidase-conjugated anti-goat antibody (Sigma). A partial fragment of purified SDF-1
(68 amino acids; Biosource Technologies, Inc.) was used as a positive control. Immunocomplexes were detected by chemiluminescence with the SuperSignal West Pico Kit (Pierce Chemical Co., Rockford, IL). Triplicate samples were used for each hormone condition in each experiment, and all experiments were repeated three times.
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ACKNOWLEDGMENTS
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We would like to thank Drs. C. A. Afshari, E.K. Lobenhofer, and J. Collins in the NIEHS microarray facility for excellent assistance in the microarray analyses that preceded this study. We thank S. Curtis Hewitt and Dr. B. DeRoo for critical review of the manuscript. We thank members of the Korach group, Dr. D. P. McDonnell, and members of the McDonnell laboratory for helpful discussions throughout the course of this study.
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FOOTNOTES
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This work was supported by the Division of Intramural Research of the NIEHS (to K.S.K.). J.M.H. is supported by a Pharmacology Research Training postdoctoral fellowship from the NIH.
Abbreviations: BG-1, Ovarian carcinoma cells; CHX, cycloheximide; CXCR4, cell surface G protein-coupled receptor for SDF-1; ER, estrogen receptor; FCS, fetal calf serum; h, human; MCF-7, breast carcinoma cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium salt; PR, progesterone receptor; RPA, ribonuclease protection assay; SDF-1, stromal cell-derived factor 1.
Received for publication December 23, 2002.
Accepted for publication February 7, 2003.
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