Estrogen stimulation of cell migration involves multiple signaling pathway interactions

Abstract

Hormone-dependent breast cancers respond to inhibitors of estrogen synthesis or action with tumor regression and with a reduction of new metastases. The mechanisms underlying the effects of estrogen on metastasis likely differ from those on tumor regression. Cell migration is a key first step in the metastatic process. Based on our prior work and other published data, we designed and tested a working model that suggested that estrogen receptor α, epidermal growth factor receptor, focal adhesion kinase (FAK), paxillin, phosphatidylinositol 3 kinase, p60 Src tyrosine kinase (c-Src), c-Jun N-terminal kinase, and MAPK interact to facilitate estradiol (E(2))-induced cell migration. Accordingly, we examined the effect of E(2) on activation of these pathways and demonstrated mechanistic effects by blocking each component and assessing cell migration as a biologic endpoint. Initial studies validated a robust cell migration assay characterized by highly reproducible, dose-dependent responses to E(2). Examining various mechanisms involved in migration, we showed that E(2) induced activation of c-Src, FAK, and paxillin with early peaks within 5-30 min and later peaks at 24 h. ERK and protein kinase B phosphorylation exhibited only early peaks. Blockade of various steps in these signaling pathways with use of small interfering RNA or specific inhibitors demonstrated mechanistic effects of these signaling molecules on cell migration. Our results suggest that the effects of E(2) on cell migration involve multiple, interacting signaling pathways. Important effects are mediated by the MAPK, phosphatidylinositol 3 kinase, and c-Jun N-terminal kinase pathways and use FAK, paxillin, and c-Src for activation. Each pathway represents a potential target for blocking cell migration and metastasis of breast cancer cells.

Figure 1

Working model of the effects of E₂ on cell migration. This model is a hypothetical construct based on a comprehensive review of the literature and was used as a means of designing and interpreting our experiments. Components of the cell migration signaling pathways are divided into an input phase, a nodal point, and an output phase. This model provides us with a means of integrating the multiple signaling pathways involved in cell migration. In the diagram, we have highlighted the molecules examined in this study in red. Molecules highlighted in blue are upstream kinases or downstream mediators of cell migration not studied in this paper. In this model, E₂ transactivates the EGF-R, perhaps through a Gα13 mediated GTP-coupled protein, and at the same time causes the phosphorylation and activation of c-Src. FAK and paxillin form a complex that acts as a nodal point to initiate downstream effects on the Rac-1, PAK-1, and Rho proteins responsible for migration. c-Src plays a central role in phosphorylating paxillin, in transactivation of the EGF-R, and in binding to FAK (9). MAPK (ERK1/2) is one of the kinases that phosphorylates and activates paxillin. Downstream, PI-3 kinase activates Akt and VAV2, a guanosine triphosphatase protein exchange factor that is involved in activation of RAC-1. P-130CAS is activated by FAK, which results in the formation of a complex consisting of CRK II, DOCK180, and ELMO. This complex activates RAC-1, leading to activation of PAK-1. MAPK is involved in the regulation of RHO. RAF (MAPK kinase kinase) and MEK (MAPK kinase) are upstream kinases that activate MAPK.

Figure 2

A, Photomicrographs to illustrate the effect of E₂ on migration. In each panel, the vertical line (arrow) represents the starting line for migration, and the right hand side of the panel is the area initially scraped free of cells. T47D cells have been exposed to vehicle or to 1 nm E₂ for 24 h. The cells crossing the starting line were counted. B, E₂ induced cell migration as a function of dose in T47D cells. The specific step down culture conditions are described in Materials and Methods. Vehicle as well as E₂ concentrations from 0.001 to 100 nm are present. C, Effect of the antiestrogen fulvestrant (ICI) at a concentration of 100 nm on the migration of cells stimulated by 10 and 1 nm E₂. Results are expressed as mean ± sem. *, P < 0.05 compared with vehicle controls by Student’s t test.

Figure 3

Time course of E₂ induced phosphorylation of c-Src (A), FAK (B), and paxillin (C). T47D cells grown in 60-mm dishes were stepped down to RPMI 1640 medium containing 5% DCC-FBS for 48 h before addition of E₂ (1 nm). Whole-cell lysates (50 μg proteins) were used for Western blot analysis using specific antibodies against phosphor-c-Src (Y416), phospho-FAK (Y576/577), and phospho-paxillin (Y118). The densitometry of phosphorylated molecules were quantified and normalized by the total protein. The experiments were repeated three times with similar results. Shown in the figure is a representative of three experiments.

Figure 4

A, Effect of the c-Src inhibitor PP2 on cell migration (top) and paxillin phosphorylation at Tyr118 (bottom). T47D cells were pretreated with PP2 (5 μm) for 1 h before the addition of 1 nm E₂. Incubation time with E₂ is 24 h for cell migration assay and 1 h for Western blot analysis. Tyr118 phosphoryated paxillin was quantified by densitometric scanning and normalized by total paxillin. B, Effect of posttreatment of PP2 on cell migration. T47D cells were exposed to E₂ for 1 h, and then PP2 (5 μm) was added. Cell migration was assessed at 24 h of E₂ treatment. C, Effects of siRNA targeted to paxillin on paxillin protein and E₂-induced paxillin phorphorylation (top) and migration (bottom). β-Actin served as a loading control and off target protein. Results from scrambled, nontarget siRNA were compared with siRNA directed against paxillin. Data represent the relative migration of cells expressed as a percent of control values. Data shown are means ± sem. *, P < 0.05 compared with the nontarget siRNA; and †, P < 0.05 compared with E₂ + paxillin siRNA with Student’s t test. Cont, Vehicle control.

Figure 5

A, Western blot analysis of cells exposed to 1 nm E₂ for 5 min through 24 h with an antibody against phospho-ERK1/2 at the Thr202/Tyr204 positions. Total ERK1/2 is blotted for loading controls. Bottom panel, Densitometric quantification of the Western blot analyses shown in the top panel. B, Western blot analysis of cells exposed to 1 nm E₂ for 1 h and blotted for phosphorylated ERK1/2. E₂ and the ERK inhibitors U0126 (10 μm) and PP2 (5 μm) were added alone and together as shown. The lower lanes show β-actin as the loading controls. Bottom panel, Densitometric quantification of phospho-ERK1/2 determined from the Western blot analyses shown in the top panel. C, Effect of MEK inhibitor U0126 on E₂-induced migration. Data represent the relative migration of cells expressed as a percent of control values. Cells are grown in the presence of vehicle, 1 nm E₂ with or without U0126 (10 μm) for 24 h. D, Effect of posttreatment with U0126 on E₂ induced cell migration. T47D cells were exposed to E₂ for 1 h, and then U0126 (10 μm) was added. Cell migration was assessed at 24 h of E₂ treatment. Data are means ± sem. *, P < 0.05 compared with vehicle control; and †, P < 0.05 compared with the E₂-stimulated group by Student’s t test.

Figure 6

Effect of the EGF-R inhibitor, AG 1478, on the migration of cells exposed to 1 nm E₂ alone or E₂ plus AG 1478 (5 μm). *, P < 0.05 compared with vehicle control; and †, P < 0.05 compared with the E₂-stimulated group by Student’s t test.

Figure 7

A, Western blot analysis of cells exposed to 1 nm E₂ for various time points and blotted for Ser473-phosphorylated Akt. The lower lanes represent total Akt. Bottom panel, Densitometric analysis of the Western blot analyses shown above. B, Western blot analyses for Ser473 phosphorylated Akt in cells exposed to 1 nm E₂ or vehicle and combinations of LY 294002 (10 μm) to block PI-3-kinase or PP2 to block c-Src. Bottom panel, Densitometric analysis of the Western blot analyses shown in the top panel. C, Effect of the PI-3-kinase inhibitor LY 294002 on the migration of cells exposed to 1 nm E2. Dimethyl sulfoxide and LY 294002 controls are shown. *, P < 0.05 compared with vehicle control; and †, P < 0.05 compared with the E₂-stimulated group by Student’s t test.

Figure 8

A, Western blot analyses for phosphorylated JNK in cells exposed to 1 nm E₂ for various times. Total JNK is used in the lower lanes as loading controls. Bottom panel, Densitometric analysis of the Western blot analyses shown in the top panels. B, Effect of the JNK inhibitor SP 600125 (10 μm) on the migration of cells exposed to 1 nm E₂ or dimethyl sulfoxide as vehicle. Data are expressed as mean ± sem. *, P < 0.05 compared with vehicle control; and †, P < 0.05 compared with the E₂-stimulated group by Student’s t test.