Estrogen and Tamoxifen Induce Cytoskeletal Remodeling and Migration in Endometrial Cancer Cells
http://www.100md.com
《内分泌学杂志》
Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Abstract
Much research effort has been directed toward understanding how estrogen [17-estradiol (E2)] regulates cell proliferation and motility through the rapid, direct activation of cytoplasmic signaling cascades (i.e. nongenomic signaling). Cell migration is critical to cancer cell invasion and metastasis and involves dynamic filamentous actin cytoskeletal remodeling and disassembly of focal adhesion sites. Although estrogen is recognized to induce cell migration in some model systems, very little information is available regarding the underlying pathways and potential influence of selective estrogen receptor modulators such as 4-hydroxytamoxifen on these processes. Using the human endometrial cancer cell lines Hec 1A and Hec 1B as model systems, we have investigated the effects of E2 and Tam on endometrial nongenomic signaling, cytoskeletal remodeling, and cell motility. Results indicate that both E2 and Tam triggered rapid activation of ERK1/2, c-Src, and focal adhesion kinase signaling pathways and filamentous actin cytoskeletal changes. These changes included dissolution of stress fibers, dynamic actin accumulation at the cell periphery, and formation of lamellipodia, filopodia, and membrane spikes. Longer treatments with either agent induced cell migration in wound healing and Boyden chamber assays. Agent-induced cytoskeletal remodeling and cell migration were blocked by a Src inhibitor. These findings define cytoskeletal remodeling and cell migration as processes regulated by E2 and 4-hydroxytamoxifen nongenomic signaling in endometrial cancer. This new information may serve as the foundation for the development of new clinical therapeutic strategies.
Introduction
ESTROGEN RECEPTOR (ER) is a ligand-activated transcription factor that belongs to the steroid hormone family of the nuclear receptor superfamily. Studies of the molecular mechanism induced by 17-estradiol (E2)-dependent activation of ER have led to the paradigm of classical (i.e. genomic) and nonclassical (i.e. nongenomic) mechanisms of ER function. The former is defined by the ability of the ligand-activated receptor to regulate gene transcription through direct binding to DNA and interaction with specific coactivators or corepressors, whereas the latter consists of rapid ER-mediated signaling responses to E2 binding (1).
Nongenomic, E2-evoked responses include the rapid activation of several signaling pathways in E2-sensitive cells, including phospholipase C/protein kinase C, p38/MAPK, Janus kinase/signal transducer and activator of transcription, Pak1, casein kinase I-2, and sphingosine kinase (2, 3, 4, 5, 6, 7, 8), some of which could be cell type specific. However, the E2-dependent activation of the ERK/MAPK pathway is conserved among different cell lines, and ER can interact with Shc and Src, leading to Shc/c-Src/Ras/ERK activation (9, 10, 11).
Although E2 has been previously recognized to affect cell migration (12, 13, 14, 15), the underlying mechanisms of this effect and cell type specificity remain to be established definitively. Cell migration is a coordinated physiological process that results from a complex interplay among the site of cell attachment to the extracellular matrix, the proteins within the cell focal adhesion complexes, and the dynamics of filamentous actin (F-actin) stress fibers. Cell migration is achieved through the development of focal contacts from the focal complexes, dynamic F-actin cytoskeleton remodeling, and the disassembly of cell adhesion sites. Eventually, these events lead to the generation of membrane protrusions (i.e. lamellipodia and filopodia) and traction forces that allow the cell to move (16, 17, 18).
In pathological conditions such as cancer, cell migration is also a critical process, because it is required for cancer cell spreading, invasion, and metastasis (16, 17, 18). E2 can affect adhesion, migration, and chemoinvasion, mainly through inducing the remodeling of both the F-actin and the intermediate filament cell cytoskeletons (19, 20, 21), but several reports also indicated a negative E2-dependent regulation of cell migration (12, 13, 14, 15, 22, 23).
The selective ER modulator (SERM) 4-hydroxytamoxifen (Tam) is currently used clinically for the prevention and treatment of breast cancer, although in some cases, patients develop Tam resistance and tumor recurrence (24). Tam acts as an estrogen antagonist in some tissues, such as the mammary gland, and an estrogen agonist in others, such as the endometrium (25), where Tam mimics E2 action by inducing cell proliferation (26). However, at present, there is currently very little or no information available on the potential ability of Tam to regulate cell migration.
Cell migration-specific stimuli (e.g. integrins engagement of extracellular matrix, growth factor stimulation, and mechanical stimuli) are commonly transduced within the focal complex through the activation of specific biochemical pathways (16, 17, 18). Focal adhesion kinase (FAK) and c-Src are nonreceptor tyrosine kinases that play key roles in modulating cell migration and invasion. The dual-activated FAK:c-Src complex regulates the assembly and disassembly of the focal contacts, F-actin cytoskeleton remodeling, and the formation of lamellipodia and filopodia through the activation of specific downstream cytoskeleton-associated signaling pathways (16, 17, 27). c-Src expression and kinase activity generally increase with tumor progression, and cells with elevated c-Src kinase activity display altered cell-cell adhesion and higher invasiveness. Moreover, v-Src-transformed cells acquire a phenotype with increased cell motility (16, 17, 27). Several lines of evidence have also correlated FAK overexpression with advanced human malignant and metastatic cancer, the invasive potential of tumors, and poor patient prognosis (16, 17, 27). However, to date, the potential rapid effects of E2 on FAK activation and the ability of SERMs such as Tam to trigger nongenomic signals are poorly understood.
To address these questions, we analyzed whether rapid nongenomic signaling induced by E2 and Tam might occur and be involved in the regulation of both endometrial cells cytoskeleton changes and migration. To fulfill this purpose, two endometrial adenocarcinoma cell lines, Hec 1A and Hec 1B, were used as model systems with which to compare the agonistic effects of either E2 or Tam. We report that both agents induce rapid activation of c-Src and FAK signaling pathways and that c-Src is involved in the agent-mediated rapid actin cytoskeleton remodeling, including dissolution of actin stress fibers, accumulation of dynamic actin at the cell periphery, and formation of lamellipodia and actin spikes that may precede increased cell motility. The data presented here define cell migration as a process regulated by the nongenomic signaling of E2 and Tam.
Materials and Methods
Cell culture and reagents
The human endometrial cancer cell line Hec 1A and Hec 1B and the breast cancer cell lines MCF-7 and MDA-231 were obtained from the American Type Culture Collection (Manassas, VA). All cells were grown in DMEM/Ham’s F-12 nutrient mixture (1:1; with or without phenol red) supplemented with 10% fetal calf serum. Dextran-coated, charcoal-treated fetal calf serum (DCC), Tam, E2, and stock chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Specific antibodies against the following antigens were used: actin and vinculin (Sigma-Aldrich Corp.); ER (Chemicon International, Temecula, CA); phospho-ER Ser118, polyclonal phospho-ER Ser167, phospho-p42/p44 ERK/MAPK, and phospho-Src Tyr416 (Cell Signaling Technology, Inc., Beverly, MA); ERK1, ERK2, FAK, and c-Src (Santa Cruz Biotechnology, Santa Cruz, CA); and phospho-FAK Tyr397 (BioSource International, Camarillo, CA). The pure antiestrogen ICI 182,780 was purchased from TOCRIS (Ellisville, MO). The c-Src-specific inhibitor PP2 was purchased from Calbiochem (San Diego, CA). In all experiments, analytical or reagent grade products were used without additional purification.
Cell extracts and immunoblotting
Cells were grown in 1% DCC medium for 48 h and then stimulated with E2 (10 nM) or Tam (1 μM). When indicated, different concentrations of the c-Src inhibitor PP2 or the ER inhibitor ICI 182,780 (1 μM) were added 1 h before agent stimulation. To prepare cell extracts, cells were washed three times with PBS, then lysed in Nonidet P-40 lysis buffer [50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 50 mM sodium fluoride, 1x protease inhibitor mixture (Roche, Indianapolis, IN), and 1 mM sodium vanadate] for 15 min on ice. The lysates were centrifuged in an Eppendorf centrifuge at 4 C for 15 min. Cell lysates containing equal amounts of protein (100 μg) were resolved on sodium dodecyl sulfate-polyacrylamide gels (8–10% acrylamide), transferred to nitrocellulose membranes, probed with the appropriate antibodies, and developed using the enhanced chemiluminescence method (Amersham Biosciences, Piscataway, NJ).
Immunofluorescent labeling and confocal microscopy
The cellular localization of proteins of interest was accomplished by indirect immunofluorescence. Briefly, HEC 1A or HEC 1B cells were plated on sterile glass cover slips in six-well plates and allowed to attach overnight. After the appropriate experimental treatments, cells were rinsed twice in PBS, fixed in 4% phosphate-buffered paraformaldehyde for 15 min, and permeabilized in acetone at –20 C for 4 min. After permeabilization, cells were blocked in 5% normal goat serum-PBS for 30 min, incubated with primary antibodies for 1 h at room temperature, washed three times in PBS, and then incubated with goat antimouse or goat antirabbit secondary antibodies conjugated with Alexa 546 (red) or Alexa 488 (green) from Molecular Probes (Eugene, OR). The DNA dye Topro-3 (Molecular Probes) was used for nuclear localization (blue). Microscopic analyses were performed using an Olympus (New Hyde Park, NY) FV300 laser scanning confocal microscope in accordance with established methods, using sequential laser excitation to minimize the possibility of fluorescence emission bleed-through. Each image is a three-dimensional reconstructed stack of serial z sections at the same cellular level and magnification.
Migration and wound healing assays
To measure cell migration potential, Hec 1A and Hec 1B cells were serum starved (0% DCC) in phenol red-free medium for 48 h. Cell were trypsinized for collection, washed in PBS, and then resuspended in phenol red-free medium in the presence of 0.1% BSA and loaded on the upper well of a uncoated Boyden chamber at a concentration of 10,000 cells/well. The agents E2 (10 nM) and Tam (1 μM) were diluted in the cell medium before plating, as described for the individual experiments. When indicated, PP2 (20 μM) was added to the cell medium in both the presence and absence of agents. The lower side of the separating filter was filled with conditioned medium of NIH-3T3 fibroblasts grown in DMEM/F-12 medium with 0.1% BSA. The number of cells that successfully migrated through the filter was counted. Experiments were performed in triplicate, and results are expressed as arbitrary units of the mean values (±SD) of the migrated cells relative to untreated controls. Data were analyzed using PRISM software (GraphPad, Inc., San Diego, CA). Analyses included using the Kruskal-Wallis test for overall significant differences, followed by Dunn’s multiple comparison test for pairwise analyses of differences within an experiment. Significance was accepted at P < 0.05.
Cell migration potential was also assessed using an established wound healing assay as previously described (28). Briefly, Hec 1A or Hec 1B cells were plated in 60-mm dishes in 10% fetal calf serum-DMEM. When cells were 80–90% confluent, they were rinsed twice in PBS, then cultured in serum-free DCC medium for 24 h. The confluent monolayer of cells was then wounded by scraping a narrow 200-μl tip across the plate in six parallel lines. Cells were rinsed twice in PBS, then grown in 0% DCC medium or medium supplemented with the agents E2 (10 nM) and Tam (1 μM). After an additional 24 h, each plate was examined by phase contrast microscopy for the amount of wound closure by measuring the physical separation remaining between the original wound widths using Axiovision 3.1 software (Zeiss, New York, NY). Ten separate measurements were made per plate, and each experiment was performed in triplicate. Data represent the mean ± SE of three experiments and were analyzed as described above.
Results
Establishment of Hec 1A and Hec 1B as E2- and Tam-responsive cells
The presence of ER in Hec cells has been debated in the literature (29, 30, 31, 32); therefore, ER expression in these cell lines was evaluated. Western blot analysis revealed a single 66-kDa band corresponding to ER in both Hec 1A and Hec 1B cells (Fig. 1A). The ER expression levels in Hec cells were comparable to those in MCF-7 mammary carcinoma cells. As expected (33), no ER expression was detected in the ER-devoid mammary carcinoma cell line MDA-MB-231 (Fig. 1A). Although HEC 1A and HEC 1B expressed similar levels of ER by Western blot, marked differences were noted in the subcellular distribution of this protein, with HEC 1A displaying both nuclear and cytoplasmic ER, whereas HEC 1B showed mostly cytoplasmic staining (Fig. 1B).
Next, we analyzed ligand-induced ER turnover (e.g. receptor degradation) and ER phosphorylation status (e.g. Ser118 and Ser167 phosphorylation), both of which have been considered markers of ER functionality (34, 35). Overnight treatment with E2 induced a reduction in cellular ER content, whereas Tam administration stabilized ER levels in both Hec 1A and Hec 1B cells (Fig. 1C). These results are in agreement with previous data reported for MCF-7 cells (36) and suggest a functional regulation of ER turnover by E2 and Tam in Hec 1 cells. A time-course analysis revealed that E2 treatment induced a rapid and sustained increase in ER Ser118 phosphorylation after 2 min until 30 min of hormone administration in Hec 1A cells (Fig. 1D). E2 also induced a rapid and transient peak of ER Ser167 phosphorylation after 10 min of treatment. In contrast, in Hec 1B cells, a persistent increase (2–30 min) in both ER Ser118 phosphorylation and ER Ser167 phosphorylation was detectable after E2 administration (Fig. 1E). No changes in total ER content were observed under the conditions used when the same membrane was reprobed for total ER (Fig. 1, D and E). These data indicate that Hec 1A and Hec 1B cells express a functional ER and raise the possibility that estrogen and antiestrogens may activate rapid nongenomic signaling pathways in these cell lines.
To test this hypothesis, E2-induced activation of the ERK/MAPK pathway was examined in Hec 1A and Hec 1B cells. As shown in Fig. 2A, E2 induced a dose-dependent increase in ERK phosphorylation in Hec 1A cells, whereas in Hec 1B cells, E2 administration resulted in a bell-shaped dose-response curve of ERK phosphorylation. In contrast, Tam increased ERK phosphorylation linearly with the dose used in both cell lines (Fig. 2B). Thus, in the following experiments, we used the optimal E2 concentration (i.e. 10 nM) and the suboptimal concentration of Tam (i.e. 1 μM) to avoid potential toxic effects. In these settings, E2 treatment resulted in the rapid phosphorylation of ERK after 15 min, which was sustained at 30 min (Fig. 2C). In contrast, Tam treatment induced a rapid (15 min) peak in ERK/MAPK phosphorylation that was reduced to the basal level after 30 min (Fig. 2D). The ligand-induced ERK/MAPK activation was not due to a direct effect of E2 on total ERK content, because no changes in the total ERK expression level was detected after reprobing the membranes with a total ERK1/2 antibody. These results indicate that E2 and Tam activate nongenomic signaling in Hec 1A and Hec 1B cells.
Antiestrogen effects on ER ligand signaling
To examine the role of ER as a mediator of E2-triggered rapid ERK/MAPK activation, we used the pure antiestrogen ICI 182,780 as a competitive inhibitor of ER-mediated signaling. Exponentially growing Hec 1A and Hec 1B cells were treated with ICI (1 μM) for 1 h, then the phosphorylation status of ERK1/2 was assayed by Western blot. As shown in Fig. 3A, treatment with ICI barely affected ERK/MAPK phosphorylation in Hec 1A cells, whereas it significantly reduced ERK/MAPK phosphorylation in Hec 1B cells. However, ICI treatment induced the degradation of ER in both cells lines. These data suggest that the effect of E2 on ERK activation might be mediated by an ER-independent mechanism in Hec 1A cells and by an ER-dependent mechanism in Hec 1B. Examination of ER and F-actin localization in exponentially growing Hec 1A and Hec 1B cells showed dynamic actin structures and ER expression in both cell lines (Fig. 3B). Interestingly, treatment of exponentially growing cells with ICI for 16 h barely affected the phenotype of HEC 1A cells, but did decrease ER staining (Fig. 3C), in agreement with the biochemical data.
Agent-dependent cytoskeletal changes
The observation of ICI-induced cytoskeletal alterations implicated ER and/or its ligands in HEC cell cytoskeletal control. To examine whether E2 or Tam could induce cytoskeletal rearrangements and possibly affect cell phenotypes, HEC 1A and HEC 1B cells were maintained in steroid-free, low serum medium for 3 d, then were stimulated with either E2 or Tam for 20 or 60 min. Before stimulation, both cell lines displayed extensive actin stress fiber networks and smooth, regular cell borders (Fig. 4).
Both agents induced rapid dissolution of actin stress fibers and formation of motile cell structures, but marked phenotypic differences were noted depending on the agent and cell line. In HEC 1A cells, E2 induced mostly membrane ruffling at the cell periphery and few lamellipodia, filopodia, or membrane spikes. Tam, in contrast, induced all of these features of motile cells to varying degrees in HEC 1A cells, with early predominant filopodia and lamellipodia resolving into peripheral actin accumulation and extensive membrane ruffles (Fig. 4). However, distinct agent-dependent morphological changes were observed in HEC 1B cells. E2 again induced the rapid breakdown of actin stress fibers, but caused filopodia and long cytoplasmic extensions as opposed to the predominant membrane ruffling observed in the HEC 1A cells.
Tam had similar effects in HEC 1A and 1B, but the changes were even more extreme in 1B cells. After 20 min of Tam, actin stress fibers were not observed; instead, frequent filopodia and lamellipodia were present in addition to what appeared to be pseudopods extending from clustered cells (Fig. 4). These motile features were also observed after 60 min (Fig. 4) and 16 h (data not shown) of Tam treatment.
Migration and migration-related signaling pathways in Hec 1A and Hec 1B cells
To delineate weather the phenotypic changes observed with E2 and Tam on the actin cytoskeleton correlated with a functional physiological process, we assessed the migration of Hec 1A and Hec 1B cells under stimulation with the two agents using a noncoated Boyden chamber and an established wound healing assay. Serum-starved cells showed a basal migration behavior in the Boyden chamber assay that was higher in Hec 1B cells than in Hec 1A. Overnight treatment with E2 resulted in a significant increase in cell migration in both cell lines, although the magnitude of change was greater in Hec 1A cells. Conversely, Tam induced a stronger increase in the Hec 1B cell migration compared with that achieved in Hec 1A cells (Fig. 5A).
Similar results were obtained when E2 or Tam-dependent migration was assessed by wound healing assays (Fig. 5B). In Hec 1A cells, both agents induced significantly greater migration than that in the control group. Basal migration was higher in Hec 1B cells, but these cells still responded to both E2 and Tam treatment with significantly greater wound closure. Thus, in both Hec 1A and Hec 1B endometrial cancer cells, the ER ligand E2 and the SERM Tam can induce cell migration.
The nonreceptor tyrosine kinase c-Src and FAK have been previously linked to cell motility and cell migration (16, 27). Therefore, we evaluated whether E2 and Tam affected the activation status of those signaling kinases in Hec 1A and Hec 1B cells. Dose-response experiments on E2- and Tam-dependent effects of c-Src and FAK phosphorylation are shown in Fig. 6. In both Hec 1A and Hec 1B cells, E2 induced a biphasic phosphorylation of c-Src and FAK, with the maximum extent of activation at 10 nM (Fig. 6A). Similarly, Tam treatment produced an increase in c-Src and FAK phosphorylation in a dose-dependent manner in both Hec 1A and Hec 1B cells (Fig. 6B). In addition, time-course analyses revealed that in either cell line, E2 induced c-Src and FAK phosphorylation with similar kinetics. In particular, in Hec 1A cells, the E2-evoked c-Src phosphorylation reached a maximum after 15 min of hormone treatment, was reduced at 30 min, and decreased to basal levels after 60 min. However, in Hec 1B cells, c-Src phosphorylation was slightly induced by E2 after 15 min of treatment and reached a peak at 30 min (Fig. 7A, upper panels). In contrast, E2-triggered FAK autophosphorylation at Tyr397 was detectable after 15 min, with an intense peak at 30 min (Fig. 7A, lower panels). Conversely, Tam increased c-Src phosphorylation as well as FAK autophosphorylation at Tyr397 in both Hec 1A and Hec 1B cells in a biphasic manner; stimulation was evident after 15 min of Tam administration and decreased toward the basal level within 30 min. The second wave of c-Src and FAK phosphorylation peaked later at 60 min (Fig. 7B), suggesting a cyclic nature for Tam-induced signal transduction. The total amounts of c-Src and FAK protein did not change, as detected when the same membrane was reprobed with the respective total antibody. These data strongly suggest a role for agent-induced c-Src and FAK activation in the migration of Hec 1A and Hec 1B cells.
ER agent-induced changes in phosphorylated FAK and focal adhesion complexes
We next sought to determine whether the agent-induced changes in c-Src and FAK activation were translated into dynamic cell morphological alterations indicative of a more motile phenotype. Hec 1A and 1B cells were maintained in DCC medium for 48 h, then were treated with either E2 or Tam for 20 and 60 min. Cells were fixed and immunofluorescently labeled for phosphorylated FAK (pFAK) Tyr397 to examine focal adhesion size, pattern, and dynamics. Basal pFAK was present in both cell lines (Fig. 8, left panels). Hec 1A cell showed pFAK accumulation along the cell periphery in long outlining bands, whereas Hec 1B showed a more dynamic pFAK pattern, with small clusters spread throughout the cell area. As demonstrated by Western blot, E2 induced rapid increases in pFAK, with HEC 1A cells showing dissolution of longitudinal patterns and formation of outward projecting spikes and smaller pFAK clusters, whereas Hec 1B cells showed a more generalized increase in immunofluorescence and some peripheral accumulation (Fig. 8). These patterns were generally repeated with Tam treatment in both cell lines, although the magnitude of the response was amplified compared with the response to E2 treatment. Accepting that increased pFAK is indicative of more dynamic focal adhesion structures (17), these data are in agreement with both the cell migration and Western blot signaling data presented in Fig. 7.
Involvement of c-Src activation in E2- and Tam-induced cell migration
To determine weather c-Src is involved in agent-induced cell migration, we used the commercially available c-Src inhibitor PP2. First, we verified the ability of PP2 to effectively block c-Src activation. As shown in Fig. 9, 1-h pretreatment with PP2 (20 μM) was able to inhibit E2-induced c-Src phosphorylation without affecting the expression level of c-Src. There was no effect of PP2 treatment on cell viability during the treatment period (data not shown). Based on these results, we used this inhibitor as a tool to evaluate the involvement of c-Src activation in cell migration.
To address these questions, the agent-induced cell migration was assessed in the presence of the c-Src inhibitor PP2 (20 μM). Boyden chamber assays revealed that PP2 treatment was able to significantly prevent both E2- and the Tam-induced migration in Hec 1A and Hec 1B cells (Fig. 10). Notably, PP2 alone did not significantly modify the basal level of migration detected in either cell line (Fig. 10). These data indicate that c-Src is involved in E2-induced cell migration and suggest that Tam-dependent migration may be regulated by at least two different pathways.
Discussion
The overall goal of these studies was to determine the viability of Hec 1A and Hec 1B cells as model systems with which to study nongenomic ER ligand-induced signaling in endometrial cancer. This was necessary because of the known stimulatory effects of E2 and SERMs on endometrial cells in vitro and in vivo, but the paucity of information on the contributions of nongenomic signaling and dynamic cytoskeletal and migratory phenotypes in these processes.
We have now established that both Hec 1A and Hec 1B cells express ER at similar levels, although there are marked differences in the subcellular distribution of this receptor and in E2-induced increases in ER activation. Both cell lines showed significant cytoplasmic ER under exponentially growing conditions, which may contribute to their ability to activate nongenomic signaling pathways. Rapid E2-induced activation of cytoplasmic signaling cascades has been shown to be independent from ER transcriptional activity both in vitro (37) and in vivo (38), suggesting that E2 interacts with ER located in close proximity of the cell plasma membrane.
The mechanism for ER plasma membrane localization is indeed highly debated. Recently, it has been reported that plasma membrane localization of ER occurs through palmitoylation (39) that, in turn, allows receptor association with specific membrane proteins (e.g. caveolin-1) (40, 41). However, confocal microscopic analyses have shown that upon E2 binding, ER relocalizes in close proximity of the plasma membrane in association with both striatin and growth factor receptors (i.e. epidermal growth factor and IGF-I receptors) (39, 42, 43, 44). Although distinct membrane localization of ER was not examined in this report, rapid movement of ER to the plasma membrane cannot be excluded in Hec cells and may be the subject of future investigations. Furthermore, the differential sensitivity of Hec 1A and Hec 1B cells to antiestrogen (i.e. ICI 182,780) suggests the existence of a functional nonclassical membrane ER (e.g. GPR30) (45) in addition to the nuclear ER (41, 46, 47, 48). Thus, these cell lines may serve as valuable models for membrane ER studies.
We also demonstrate that nongenomic signaling, including ERK1/2, c-Src, and FAK, are rapidly and dose-dependently activated after E2 or Tam treatment of Hec 1A and Hec 1B cells. FAK overexpression is often linked with increased phosphorylation of tyrosine residue 397 (Tyr397). Autophosphorylation of Tyr397 occurs with different stimuli and creates a conformational change that allows the association of c-Src with FAK. The binding of c-Src to FAK leads to the conformational activation of c-Src (i.e. c-Src Tyr416 phosphorylation), which, in turn, phosphorylates FAK on Tyr576 and Tyr577 residues, thus resulting in FAK maximal catalytic activity (16, 17). Nongenomic, ER-mediated c-Src activation after E2 treatment has been described previously (9, 10). This is the first demonstration of ER ligand-induced changes in FAK activation in endometrial cells. We also show that the effects of E2 on ERK activation might be mediated by an ER-independent mechanism in Hec 1A cells and by an ER-dependent mechanism in Hec 1B cells, as previously reported (31). In addition, these ligand-induced dynamic changes in cytoplasmic signaling cascades were translated into F-actin cytoskeletal rearrangements, adoption of motile cell phenotypes, and increased ability of stimulated cells to migrate.
Contrasting data have been reported about the regulation of these processes by E2 and Tam. E2 is thought to affect adhesion, migration, and chemoinvasion mainly through inducing the remodeling of both the F-actin and the intermediate filament cell cytoskeletons (19, 20, 21). Nonetheless, contradictory information is available on the role of E2 in regulating cell migration. Although E2 induces cell motility in MCF-7 cells (12, 15), in some endometrial cell lines (13, 14), and in aortic endothelial cells (22), recent studies found that E2 can also inhibit migration in vascular smooth muscle cells (22, 23). Together, these data suggest cell type-specific, E2-dependent mechanisms for the modulation of cytoskeletal remodeling and migration.
Tam has also been shown to inhibit cell migration and motility in vascular endothelial growth factor-stimulated endothelial cells (49), in mammary carcinoma cell lines (e.g. MCF-7) (15, 50), and in vascular smooth muscle cells (23), most likely through ER interaction. However, an ER-independent inhibitory effect of Tam on cell migration has been documented in follicular thyroid cancer cells (51). In contrast, Tam has been reported to increase cell migration in Tam-resistant glioma cell lines and to affect cell shape and cytoskeletal arrangements (e.g. alteration of F-actin localization), including cytoplasmic protrusion and ruffling membranes, in a pattern reminiscent of that seen with E2 stimulation of MCF-7 cells (19, 52).
In addition to the effects of cytoskeletal remodeling on cell motility, many other critical cell processes depend upon and are regulated or impacted by dynamic changes in F-actin structure. These targets include cell-cell adhesion, endocytosis, intracellular trafficking, organelle function, cell survival, gene expression, and cell division (17, 53). Thus, because the cytoskeleton plays a central role in cell functions, the rapid effects of E2 and Tam on F-actin structure may have a more broad impact on the cell physiology of endometrial cancer cells.
Our data demonstrate that ligand-induced c-Src activity is involved in the regulation of the dynamic F-actin cytoskeletal rearrangement, most likely through focal adhesion complexes, adoption of motile cell phenotypes, and migration, thus indicating that cytoplasmic signaling is relevant to the biology of endometrial cancers. In addition, the ability of both an ER agonist (E2) and an antagonist (Tam) to induce cytoskeletal changes and movement suggest that the effects of SERMs on cell motility may eventually lead to the development of aggressive endometrial cancers. Together, these new findings may provide insight into potential new routes for intervention and into the mechanistic causes and treatment of aggressive endometrial cancer.
Footnotes
This work was supported by National Institutes of Health Grant CA-109379 (to R.K.).
F.A., C.J.B., and R.K. have nothing to declare.
First Published Online December 8, 2005
1 F.A. and C.J.B. contributed equally to this work.
Abbreviations: DCC, Dextran-coated, charcoal-treated fetal calf serum; E2, 17-estradiol; ER, estrogen receptor; F-actin, filamentous actin; FAK, focal adhesion kinase; pFAK, phosphorylated FAK; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; SERM, selective ER modulator; Tam, 4-hydroxytamoxifen.
Accepted for publication November 29, 2005.
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Abstract
Much research effort has been directed toward understanding how estrogen [17-estradiol (E2)] regulates cell proliferation and motility through the rapid, direct activation of cytoplasmic signaling cascades (i.e. nongenomic signaling). Cell migration is critical to cancer cell invasion and metastasis and involves dynamic filamentous actin cytoskeletal remodeling and disassembly of focal adhesion sites. Although estrogen is recognized to induce cell migration in some model systems, very little information is available regarding the underlying pathways and potential influence of selective estrogen receptor modulators such as 4-hydroxytamoxifen on these processes. Using the human endometrial cancer cell lines Hec 1A and Hec 1B as model systems, we have investigated the effects of E2 and Tam on endometrial nongenomic signaling, cytoskeletal remodeling, and cell motility. Results indicate that both E2 and Tam triggered rapid activation of ERK1/2, c-Src, and focal adhesion kinase signaling pathways and filamentous actin cytoskeletal changes. These changes included dissolution of stress fibers, dynamic actin accumulation at the cell periphery, and formation of lamellipodia, filopodia, and membrane spikes. Longer treatments with either agent induced cell migration in wound healing and Boyden chamber assays. Agent-induced cytoskeletal remodeling and cell migration were blocked by a Src inhibitor. These findings define cytoskeletal remodeling and cell migration as processes regulated by E2 and 4-hydroxytamoxifen nongenomic signaling in endometrial cancer. This new information may serve as the foundation for the development of new clinical therapeutic strategies.
Introduction
ESTROGEN RECEPTOR (ER) is a ligand-activated transcription factor that belongs to the steroid hormone family of the nuclear receptor superfamily. Studies of the molecular mechanism induced by 17-estradiol (E2)-dependent activation of ER have led to the paradigm of classical (i.e. genomic) and nonclassical (i.e. nongenomic) mechanisms of ER function. The former is defined by the ability of the ligand-activated receptor to regulate gene transcription through direct binding to DNA and interaction with specific coactivators or corepressors, whereas the latter consists of rapid ER-mediated signaling responses to E2 binding (1).
Nongenomic, E2-evoked responses include the rapid activation of several signaling pathways in E2-sensitive cells, including phospholipase C/protein kinase C, p38/MAPK, Janus kinase/signal transducer and activator of transcription, Pak1, casein kinase I-2, and sphingosine kinase (2, 3, 4, 5, 6, 7, 8), some of which could be cell type specific. However, the E2-dependent activation of the ERK/MAPK pathway is conserved among different cell lines, and ER can interact with Shc and Src, leading to Shc/c-Src/Ras/ERK activation (9, 10, 11).
Although E2 has been previously recognized to affect cell migration (12, 13, 14, 15), the underlying mechanisms of this effect and cell type specificity remain to be established definitively. Cell migration is a coordinated physiological process that results from a complex interplay among the site of cell attachment to the extracellular matrix, the proteins within the cell focal adhesion complexes, and the dynamics of filamentous actin (F-actin) stress fibers. Cell migration is achieved through the development of focal contacts from the focal complexes, dynamic F-actin cytoskeleton remodeling, and the disassembly of cell adhesion sites. Eventually, these events lead to the generation of membrane protrusions (i.e. lamellipodia and filopodia) and traction forces that allow the cell to move (16, 17, 18).
In pathological conditions such as cancer, cell migration is also a critical process, because it is required for cancer cell spreading, invasion, and metastasis (16, 17, 18). E2 can affect adhesion, migration, and chemoinvasion, mainly through inducing the remodeling of both the F-actin and the intermediate filament cell cytoskeletons (19, 20, 21), but several reports also indicated a negative E2-dependent regulation of cell migration (12, 13, 14, 15, 22, 23).
The selective ER modulator (SERM) 4-hydroxytamoxifen (Tam) is currently used clinically for the prevention and treatment of breast cancer, although in some cases, patients develop Tam resistance and tumor recurrence (24). Tam acts as an estrogen antagonist in some tissues, such as the mammary gland, and an estrogen agonist in others, such as the endometrium (25), where Tam mimics E2 action by inducing cell proliferation (26). However, at present, there is currently very little or no information available on the potential ability of Tam to regulate cell migration.
Cell migration-specific stimuli (e.g. integrins engagement of extracellular matrix, growth factor stimulation, and mechanical stimuli) are commonly transduced within the focal complex through the activation of specific biochemical pathways (16, 17, 18). Focal adhesion kinase (FAK) and c-Src are nonreceptor tyrosine kinases that play key roles in modulating cell migration and invasion. The dual-activated FAK:c-Src complex regulates the assembly and disassembly of the focal contacts, F-actin cytoskeleton remodeling, and the formation of lamellipodia and filopodia through the activation of specific downstream cytoskeleton-associated signaling pathways (16, 17, 27). c-Src expression and kinase activity generally increase with tumor progression, and cells with elevated c-Src kinase activity display altered cell-cell adhesion and higher invasiveness. Moreover, v-Src-transformed cells acquire a phenotype with increased cell motility (16, 17, 27). Several lines of evidence have also correlated FAK overexpression with advanced human malignant and metastatic cancer, the invasive potential of tumors, and poor patient prognosis (16, 17, 27). However, to date, the potential rapid effects of E2 on FAK activation and the ability of SERMs such as Tam to trigger nongenomic signals are poorly understood.
To address these questions, we analyzed whether rapid nongenomic signaling induced by E2 and Tam might occur and be involved in the regulation of both endometrial cells cytoskeleton changes and migration. To fulfill this purpose, two endometrial adenocarcinoma cell lines, Hec 1A and Hec 1B, were used as model systems with which to compare the agonistic effects of either E2 or Tam. We report that both agents induce rapid activation of c-Src and FAK signaling pathways and that c-Src is involved in the agent-mediated rapid actin cytoskeleton remodeling, including dissolution of actin stress fibers, accumulation of dynamic actin at the cell periphery, and formation of lamellipodia and actin spikes that may precede increased cell motility. The data presented here define cell migration as a process regulated by the nongenomic signaling of E2 and Tam.
Materials and Methods
Cell culture and reagents
The human endometrial cancer cell line Hec 1A and Hec 1B and the breast cancer cell lines MCF-7 and MDA-231 were obtained from the American Type Culture Collection (Manassas, VA). All cells were grown in DMEM/Ham’s F-12 nutrient mixture (1:1; with or without phenol red) supplemented with 10% fetal calf serum. Dextran-coated, charcoal-treated fetal calf serum (DCC), Tam, E2, and stock chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Specific antibodies against the following antigens were used: actin and vinculin (Sigma-Aldrich Corp.); ER (Chemicon International, Temecula, CA); phospho-ER Ser118, polyclonal phospho-ER Ser167, phospho-p42/p44 ERK/MAPK, and phospho-Src Tyr416 (Cell Signaling Technology, Inc., Beverly, MA); ERK1, ERK2, FAK, and c-Src (Santa Cruz Biotechnology, Santa Cruz, CA); and phospho-FAK Tyr397 (BioSource International, Camarillo, CA). The pure antiestrogen ICI 182,780 was purchased from TOCRIS (Ellisville, MO). The c-Src-specific inhibitor PP2 was purchased from Calbiochem (San Diego, CA). In all experiments, analytical or reagent grade products were used without additional purification.
Cell extracts and immunoblotting
Cells were grown in 1% DCC medium for 48 h and then stimulated with E2 (10 nM) or Tam (1 μM). When indicated, different concentrations of the c-Src inhibitor PP2 or the ER inhibitor ICI 182,780 (1 μM) were added 1 h before agent stimulation. To prepare cell extracts, cells were washed three times with PBS, then lysed in Nonidet P-40 lysis buffer [50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 50 mM sodium fluoride, 1x protease inhibitor mixture (Roche, Indianapolis, IN), and 1 mM sodium vanadate] for 15 min on ice. The lysates were centrifuged in an Eppendorf centrifuge at 4 C for 15 min. Cell lysates containing equal amounts of protein (100 μg) were resolved on sodium dodecyl sulfate-polyacrylamide gels (8–10% acrylamide), transferred to nitrocellulose membranes, probed with the appropriate antibodies, and developed using the enhanced chemiluminescence method (Amersham Biosciences, Piscataway, NJ).
Immunofluorescent labeling and confocal microscopy
The cellular localization of proteins of interest was accomplished by indirect immunofluorescence. Briefly, HEC 1A or HEC 1B cells were plated on sterile glass cover slips in six-well plates and allowed to attach overnight. After the appropriate experimental treatments, cells were rinsed twice in PBS, fixed in 4% phosphate-buffered paraformaldehyde for 15 min, and permeabilized in acetone at –20 C for 4 min. After permeabilization, cells were blocked in 5% normal goat serum-PBS for 30 min, incubated with primary antibodies for 1 h at room temperature, washed three times in PBS, and then incubated with goat antimouse or goat antirabbit secondary antibodies conjugated with Alexa 546 (red) or Alexa 488 (green) from Molecular Probes (Eugene, OR). The DNA dye Topro-3 (Molecular Probes) was used for nuclear localization (blue). Microscopic analyses were performed using an Olympus (New Hyde Park, NY) FV300 laser scanning confocal microscope in accordance with established methods, using sequential laser excitation to minimize the possibility of fluorescence emission bleed-through. Each image is a three-dimensional reconstructed stack of serial z sections at the same cellular level and magnification.
Migration and wound healing assays
To measure cell migration potential, Hec 1A and Hec 1B cells were serum starved (0% DCC) in phenol red-free medium for 48 h. Cell were trypsinized for collection, washed in PBS, and then resuspended in phenol red-free medium in the presence of 0.1% BSA and loaded on the upper well of a uncoated Boyden chamber at a concentration of 10,000 cells/well. The agents E2 (10 nM) and Tam (1 μM) were diluted in the cell medium before plating, as described for the individual experiments. When indicated, PP2 (20 μM) was added to the cell medium in both the presence and absence of agents. The lower side of the separating filter was filled with conditioned medium of NIH-3T3 fibroblasts grown in DMEM/F-12 medium with 0.1% BSA. The number of cells that successfully migrated through the filter was counted. Experiments were performed in triplicate, and results are expressed as arbitrary units of the mean values (±SD) of the migrated cells relative to untreated controls. Data were analyzed using PRISM software (GraphPad, Inc., San Diego, CA). Analyses included using the Kruskal-Wallis test for overall significant differences, followed by Dunn’s multiple comparison test for pairwise analyses of differences within an experiment. Significance was accepted at P < 0.05.
Cell migration potential was also assessed using an established wound healing assay as previously described (28). Briefly, Hec 1A or Hec 1B cells were plated in 60-mm dishes in 10% fetal calf serum-DMEM. When cells were 80–90% confluent, they were rinsed twice in PBS, then cultured in serum-free DCC medium for 24 h. The confluent monolayer of cells was then wounded by scraping a narrow 200-μl tip across the plate in six parallel lines. Cells were rinsed twice in PBS, then grown in 0% DCC medium or medium supplemented with the agents E2 (10 nM) and Tam (1 μM). After an additional 24 h, each plate was examined by phase contrast microscopy for the amount of wound closure by measuring the physical separation remaining between the original wound widths using Axiovision 3.1 software (Zeiss, New York, NY). Ten separate measurements were made per plate, and each experiment was performed in triplicate. Data represent the mean ± SE of three experiments and were analyzed as described above.
Results
Establishment of Hec 1A and Hec 1B as E2- and Tam-responsive cells
The presence of ER in Hec cells has been debated in the literature (29, 30, 31, 32); therefore, ER expression in these cell lines was evaluated. Western blot analysis revealed a single 66-kDa band corresponding to ER in both Hec 1A and Hec 1B cells (Fig. 1A). The ER expression levels in Hec cells were comparable to those in MCF-7 mammary carcinoma cells. As expected (33), no ER expression was detected in the ER-devoid mammary carcinoma cell line MDA-MB-231 (Fig. 1A). Although HEC 1A and HEC 1B expressed similar levels of ER by Western blot, marked differences were noted in the subcellular distribution of this protein, with HEC 1A displaying both nuclear and cytoplasmic ER, whereas HEC 1B showed mostly cytoplasmic staining (Fig. 1B).
Next, we analyzed ligand-induced ER turnover (e.g. receptor degradation) and ER phosphorylation status (e.g. Ser118 and Ser167 phosphorylation), both of which have been considered markers of ER functionality (34, 35). Overnight treatment with E2 induced a reduction in cellular ER content, whereas Tam administration stabilized ER levels in both Hec 1A and Hec 1B cells (Fig. 1C). These results are in agreement with previous data reported for MCF-7 cells (36) and suggest a functional regulation of ER turnover by E2 and Tam in Hec 1 cells. A time-course analysis revealed that E2 treatment induced a rapid and sustained increase in ER Ser118 phosphorylation after 2 min until 30 min of hormone administration in Hec 1A cells (Fig. 1D). E2 also induced a rapid and transient peak of ER Ser167 phosphorylation after 10 min of treatment. In contrast, in Hec 1B cells, a persistent increase (2–30 min) in both ER Ser118 phosphorylation and ER Ser167 phosphorylation was detectable after E2 administration (Fig. 1E). No changes in total ER content were observed under the conditions used when the same membrane was reprobed for total ER (Fig. 1, D and E). These data indicate that Hec 1A and Hec 1B cells express a functional ER and raise the possibility that estrogen and antiestrogens may activate rapid nongenomic signaling pathways in these cell lines.
To test this hypothesis, E2-induced activation of the ERK/MAPK pathway was examined in Hec 1A and Hec 1B cells. As shown in Fig. 2A, E2 induced a dose-dependent increase in ERK phosphorylation in Hec 1A cells, whereas in Hec 1B cells, E2 administration resulted in a bell-shaped dose-response curve of ERK phosphorylation. In contrast, Tam increased ERK phosphorylation linearly with the dose used in both cell lines (Fig. 2B). Thus, in the following experiments, we used the optimal E2 concentration (i.e. 10 nM) and the suboptimal concentration of Tam (i.e. 1 μM) to avoid potential toxic effects. In these settings, E2 treatment resulted in the rapid phosphorylation of ERK after 15 min, which was sustained at 30 min (Fig. 2C). In contrast, Tam treatment induced a rapid (15 min) peak in ERK/MAPK phosphorylation that was reduced to the basal level after 30 min (Fig. 2D). The ligand-induced ERK/MAPK activation was not due to a direct effect of E2 on total ERK content, because no changes in the total ERK expression level was detected after reprobing the membranes with a total ERK1/2 antibody. These results indicate that E2 and Tam activate nongenomic signaling in Hec 1A and Hec 1B cells.
Antiestrogen effects on ER ligand signaling
To examine the role of ER as a mediator of E2-triggered rapid ERK/MAPK activation, we used the pure antiestrogen ICI 182,780 as a competitive inhibitor of ER-mediated signaling. Exponentially growing Hec 1A and Hec 1B cells were treated with ICI (1 μM) for 1 h, then the phosphorylation status of ERK1/2 was assayed by Western blot. As shown in Fig. 3A, treatment with ICI barely affected ERK/MAPK phosphorylation in Hec 1A cells, whereas it significantly reduced ERK/MAPK phosphorylation in Hec 1B cells. However, ICI treatment induced the degradation of ER in both cells lines. These data suggest that the effect of E2 on ERK activation might be mediated by an ER-independent mechanism in Hec 1A cells and by an ER-dependent mechanism in Hec 1B. Examination of ER and F-actin localization in exponentially growing Hec 1A and Hec 1B cells showed dynamic actin structures and ER expression in both cell lines (Fig. 3B). Interestingly, treatment of exponentially growing cells with ICI for 16 h barely affected the phenotype of HEC 1A cells, but did decrease ER staining (Fig. 3C), in agreement with the biochemical data.
Agent-dependent cytoskeletal changes
The observation of ICI-induced cytoskeletal alterations implicated ER and/or its ligands in HEC cell cytoskeletal control. To examine whether E2 or Tam could induce cytoskeletal rearrangements and possibly affect cell phenotypes, HEC 1A and HEC 1B cells were maintained in steroid-free, low serum medium for 3 d, then were stimulated with either E2 or Tam for 20 or 60 min. Before stimulation, both cell lines displayed extensive actin stress fiber networks and smooth, regular cell borders (Fig. 4).
Both agents induced rapid dissolution of actin stress fibers and formation of motile cell structures, but marked phenotypic differences were noted depending on the agent and cell line. In HEC 1A cells, E2 induced mostly membrane ruffling at the cell periphery and few lamellipodia, filopodia, or membrane spikes. Tam, in contrast, induced all of these features of motile cells to varying degrees in HEC 1A cells, with early predominant filopodia and lamellipodia resolving into peripheral actin accumulation and extensive membrane ruffles (Fig. 4). However, distinct agent-dependent morphological changes were observed in HEC 1B cells. E2 again induced the rapid breakdown of actin stress fibers, but caused filopodia and long cytoplasmic extensions as opposed to the predominant membrane ruffling observed in the HEC 1A cells.
Tam had similar effects in HEC 1A and 1B, but the changes were even more extreme in 1B cells. After 20 min of Tam, actin stress fibers were not observed; instead, frequent filopodia and lamellipodia were present in addition to what appeared to be pseudopods extending from clustered cells (Fig. 4). These motile features were also observed after 60 min (Fig. 4) and 16 h (data not shown) of Tam treatment.
Migration and migration-related signaling pathways in Hec 1A and Hec 1B cells
To delineate weather the phenotypic changes observed with E2 and Tam on the actin cytoskeleton correlated with a functional physiological process, we assessed the migration of Hec 1A and Hec 1B cells under stimulation with the two agents using a noncoated Boyden chamber and an established wound healing assay. Serum-starved cells showed a basal migration behavior in the Boyden chamber assay that was higher in Hec 1B cells than in Hec 1A. Overnight treatment with E2 resulted in a significant increase in cell migration in both cell lines, although the magnitude of change was greater in Hec 1A cells. Conversely, Tam induced a stronger increase in the Hec 1B cell migration compared with that achieved in Hec 1A cells (Fig. 5A).
Similar results were obtained when E2 or Tam-dependent migration was assessed by wound healing assays (Fig. 5B). In Hec 1A cells, both agents induced significantly greater migration than that in the control group. Basal migration was higher in Hec 1B cells, but these cells still responded to both E2 and Tam treatment with significantly greater wound closure. Thus, in both Hec 1A and Hec 1B endometrial cancer cells, the ER ligand E2 and the SERM Tam can induce cell migration.
The nonreceptor tyrosine kinase c-Src and FAK have been previously linked to cell motility and cell migration (16, 27). Therefore, we evaluated whether E2 and Tam affected the activation status of those signaling kinases in Hec 1A and Hec 1B cells. Dose-response experiments on E2- and Tam-dependent effects of c-Src and FAK phosphorylation are shown in Fig. 6. In both Hec 1A and Hec 1B cells, E2 induced a biphasic phosphorylation of c-Src and FAK, with the maximum extent of activation at 10 nM (Fig. 6A). Similarly, Tam treatment produced an increase in c-Src and FAK phosphorylation in a dose-dependent manner in both Hec 1A and Hec 1B cells (Fig. 6B). In addition, time-course analyses revealed that in either cell line, E2 induced c-Src and FAK phosphorylation with similar kinetics. In particular, in Hec 1A cells, the E2-evoked c-Src phosphorylation reached a maximum after 15 min of hormone treatment, was reduced at 30 min, and decreased to basal levels after 60 min. However, in Hec 1B cells, c-Src phosphorylation was slightly induced by E2 after 15 min of treatment and reached a peak at 30 min (Fig. 7A, upper panels). In contrast, E2-triggered FAK autophosphorylation at Tyr397 was detectable after 15 min, with an intense peak at 30 min (Fig. 7A, lower panels). Conversely, Tam increased c-Src phosphorylation as well as FAK autophosphorylation at Tyr397 in both Hec 1A and Hec 1B cells in a biphasic manner; stimulation was evident after 15 min of Tam administration and decreased toward the basal level within 30 min. The second wave of c-Src and FAK phosphorylation peaked later at 60 min (Fig. 7B), suggesting a cyclic nature for Tam-induced signal transduction. The total amounts of c-Src and FAK protein did not change, as detected when the same membrane was reprobed with the respective total antibody. These data strongly suggest a role for agent-induced c-Src and FAK activation in the migration of Hec 1A and Hec 1B cells.
ER agent-induced changes in phosphorylated FAK and focal adhesion complexes
We next sought to determine whether the agent-induced changes in c-Src and FAK activation were translated into dynamic cell morphological alterations indicative of a more motile phenotype. Hec 1A and 1B cells were maintained in DCC medium for 48 h, then were treated with either E2 or Tam for 20 and 60 min. Cells were fixed and immunofluorescently labeled for phosphorylated FAK (pFAK) Tyr397 to examine focal adhesion size, pattern, and dynamics. Basal pFAK was present in both cell lines (Fig. 8, left panels). Hec 1A cell showed pFAK accumulation along the cell periphery in long outlining bands, whereas Hec 1B showed a more dynamic pFAK pattern, with small clusters spread throughout the cell area. As demonstrated by Western blot, E2 induced rapid increases in pFAK, with HEC 1A cells showing dissolution of longitudinal patterns and formation of outward projecting spikes and smaller pFAK clusters, whereas Hec 1B cells showed a more generalized increase in immunofluorescence and some peripheral accumulation (Fig. 8). These patterns were generally repeated with Tam treatment in both cell lines, although the magnitude of the response was amplified compared with the response to E2 treatment. Accepting that increased pFAK is indicative of more dynamic focal adhesion structures (17), these data are in agreement with both the cell migration and Western blot signaling data presented in Fig. 7.
Involvement of c-Src activation in E2- and Tam-induced cell migration
To determine weather c-Src is involved in agent-induced cell migration, we used the commercially available c-Src inhibitor PP2. First, we verified the ability of PP2 to effectively block c-Src activation. As shown in Fig. 9, 1-h pretreatment with PP2 (20 μM) was able to inhibit E2-induced c-Src phosphorylation without affecting the expression level of c-Src. There was no effect of PP2 treatment on cell viability during the treatment period (data not shown). Based on these results, we used this inhibitor as a tool to evaluate the involvement of c-Src activation in cell migration.
To address these questions, the agent-induced cell migration was assessed in the presence of the c-Src inhibitor PP2 (20 μM). Boyden chamber assays revealed that PP2 treatment was able to significantly prevent both E2- and the Tam-induced migration in Hec 1A and Hec 1B cells (Fig. 10). Notably, PP2 alone did not significantly modify the basal level of migration detected in either cell line (Fig. 10). These data indicate that c-Src is involved in E2-induced cell migration and suggest that Tam-dependent migration may be regulated by at least two different pathways.
Discussion
The overall goal of these studies was to determine the viability of Hec 1A and Hec 1B cells as model systems with which to study nongenomic ER ligand-induced signaling in endometrial cancer. This was necessary because of the known stimulatory effects of E2 and SERMs on endometrial cells in vitro and in vivo, but the paucity of information on the contributions of nongenomic signaling and dynamic cytoskeletal and migratory phenotypes in these processes.
We have now established that both Hec 1A and Hec 1B cells express ER at similar levels, although there are marked differences in the subcellular distribution of this receptor and in E2-induced increases in ER activation. Both cell lines showed significant cytoplasmic ER under exponentially growing conditions, which may contribute to their ability to activate nongenomic signaling pathways. Rapid E2-induced activation of cytoplasmic signaling cascades has been shown to be independent from ER transcriptional activity both in vitro (37) and in vivo (38), suggesting that E2 interacts with ER located in close proximity of the cell plasma membrane.
The mechanism for ER plasma membrane localization is indeed highly debated. Recently, it has been reported that plasma membrane localization of ER occurs through palmitoylation (39) that, in turn, allows receptor association with specific membrane proteins (e.g. caveolin-1) (40, 41). However, confocal microscopic analyses have shown that upon E2 binding, ER relocalizes in close proximity of the plasma membrane in association with both striatin and growth factor receptors (i.e. epidermal growth factor and IGF-I receptors) (39, 42, 43, 44). Although distinct membrane localization of ER was not examined in this report, rapid movement of ER to the plasma membrane cannot be excluded in Hec cells and may be the subject of future investigations. Furthermore, the differential sensitivity of Hec 1A and Hec 1B cells to antiestrogen (i.e. ICI 182,780) suggests the existence of a functional nonclassical membrane ER (e.g. GPR30) (45) in addition to the nuclear ER (41, 46, 47, 48). Thus, these cell lines may serve as valuable models for membrane ER studies.
We also demonstrate that nongenomic signaling, including ERK1/2, c-Src, and FAK, are rapidly and dose-dependently activated after E2 or Tam treatment of Hec 1A and Hec 1B cells. FAK overexpression is often linked with increased phosphorylation of tyrosine residue 397 (Tyr397). Autophosphorylation of Tyr397 occurs with different stimuli and creates a conformational change that allows the association of c-Src with FAK. The binding of c-Src to FAK leads to the conformational activation of c-Src (i.e. c-Src Tyr416 phosphorylation), which, in turn, phosphorylates FAK on Tyr576 and Tyr577 residues, thus resulting in FAK maximal catalytic activity (16, 17). Nongenomic, ER-mediated c-Src activation after E2 treatment has been described previously (9, 10). This is the first demonstration of ER ligand-induced changes in FAK activation in endometrial cells. We also show that the effects of E2 on ERK activation might be mediated by an ER-independent mechanism in Hec 1A cells and by an ER-dependent mechanism in Hec 1B cells, as previously reported (31). In addition, these ligand-induced dynamic changes in cytoplasmic signaling cascades were translated into F-actin cytoskeletal rearrangements, adoption of motile cell phenotypes, and increased ability of stimulated cells to migrate.
Contrasting data have been reported about the regulation of these processes by E2 and Tam. E2 is thought to affect adhesion, migration, and chemoinvasion mainly through inducing the remodeling of both the F-actin and the intermediate filament cell cytoskeletons (19, 20, 21). Nonetheless, contradictory information is available on the role of E2 in regulating cell migration. Although E2 induces cell motility in MCF-7 cells (12, 15), in some endometrial cell lines (13, 14), and in aortic endothelial cells (22), recent studies found that E2 can also inhibit migration in vascular smooth muscle cells (22, 23). Together, these data suggest cell type-specific, E2-dependent mechanisms for the modulation of cytoskeletal remodeling and migration.
Tam has also been shown to inhibit cell migration and motility in vascular endothelial growth factor-stimulated endothelial cells (49), in mammary carcinoma cell lines (e.g. MCF-7) (15, 50), and in vascular smooth muscle cells (23), most likely through ER interaction. However, an ER-independent inhibitory effect of Tam on cell migration has been documented in follicular thyroid cancer cells (51). In contrast, Tam has been reported to increase cell migration in Tam-resistant glioma cell lines and to affect cell shape and cytoskeletal arrangements (e.g. alteration of F-actin localization), including cytoplasmic protrusion and ruffling membranes, in a pattern reminiscent of that seen with E2 stimulation of MCF-7 cells (19, 52).
In addition to the effects of cytoskeletal remodeling on cell motility, many other critical cell processes depend upon and are regulated or impacted by dynamic changes in F-actin structure. These targets include cell-cell adhesion, endocytosis, intracellular trafficking, organelle function, cell survival, gene expression, and cell division (17, 53). Thus, because the cytoskeleton plays a central role in cell functions, the rapid effects of E2 and Tam on F-actin structure may have a more broad impact on the cell physiology of endometrial cancer cells.
Our data demonstrate that ligand-induced c-Src activity is involved in the regulation of the dynamic F-actin cytoskeletal rearrangement, most likely through focal adhesion complexes, adoption of motile cell phenotypes, and migration, thus indicating that cytoplasmic signaling is relevant to the biology of endometrial cancers. In addition, the ability of both an ER agonist (E2) and an antagonist (Tam) to induce cytoskeletal changes and movement suggest that the effects of SERMs on cell motility may eventually lead to the development of aggressive endometrial cancers. Together, these new findings may provide insight into potential new routes for intervention and into the mechanistic causes and treatment of aggressive endometrial cancer.
Footnotes
This work was supported by National Institutes of Health Grant CA-109379 (to R.K.).
F.A., C.J.B., and R.K. have nothing to declare.
First Published Online December 8, 2005
1 F.A. and C.J.B. contributed equally to this work.
Abbreviations: DCC, Dextran-coated, charcoal-treated fetal calf serum; E2, 17-estradiol; ER, estrogen receptor; F-actin, filamentous actin; FAK, focal adhesion kinase; pFAK, phosphorylated FAK; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; SERM, selective ER modulator; Tam, 4-hydroxytamoxifen.
Accepted for publication November 29, 2005.
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