Progesterone receptors upregulate Wnt-1 to induce epidermal growth factor receptor transactivation and c-Src-dependent sustained activation of Erk1/2 mitogen-activated protein kinase in breast cancer cells

Abstract

Progesterone receptor (PR) ligand binding induces rapid and transient (5- to 10-min) activation of cytosolic c-Src-Ras-Erk1/2 mitogen-activated protein kinase (MAPK) signaling that is independent of PR functioning as transcription factors. Here, we have explored the integration of PR-dependent transcription and rapid signaling events in breast cancer cells. PR-B, but not PR-A, induced robust and sustained (6- to 72-h) Erk1/2 activation that was required for elevated cyclin D1 protein but not mRNA levels. Sustained Erk1/2 activation in response to progestins occurred via a novel mechanism distinct from rapid signaling initiated by PR/c-Src interactions and required the PR-B DNA-binding domain (DBD). PR/progestin upregulated epidermal growth factor receptor (EGFR) and Wnt-1. In response to PR-induced Wnt-1 signaling, matrix metalloprotease (MMP)-mediated membrane-proximal shedding of EGFR ligands transactivated EGFR and induced persistent downstream c-Src and Erk1/2 activities. T47D cell anchorage-independent growth was stimulated by progestins and blocked by inhibition of Erk1/2, c-Src, EGFR, or RNA interference of Wnt-1. Similarly, cell growth in soft agar required the PR DBD but was sensitive to disruption of PR/c-Src interactions, suggesting that both PR-B-induced rapid signaling events and nuclear actions contribute to this response. Our discovery that progestins are capable of robust autocrine activation of EGFR and sustained Erk1/2 signaling provides further support for the physiological linkage of growth factor and steroid hormone signaling. PR-B-induced sustained MAPK signaling may provide prosurvival or proliferative advantages to early breast cancer lesions.

FIG. 1.

Progestin treatment of T47D-YB cells stimulates PR-dependent sustained activation of Erk1/2 MAPKs. (A) T47D cells stably expressing wt PR-B were treated with EtOH vehicle control (−) or 10 nM R5020 (+) for the indicated times (m, minutes). Cell lysates were Western blotted for expression of cyclin D1, PR, or activated (phospho) forms of Erk1/2 MAPKs. Actin blotting was included as a protein-loading control; total MAPK did not change in the same lysates (not shown) (see the legend to panel C). (B) Time course of Erk1/2 phosphorylation as measured using flow cytometry following EtOH control (dashed lines) or R5020 (solid line) stimulation. The lines represent the mean fluorescence intensities (MFI) of triplicate measures versus time; the error bars represent the standard errors of the mean. Histogram overlays (below) at each time point represent fluorescence versus the percentage of maximum for EtOH (gray line) compared to R5020 (black line) treatment. The specificity of the assay was demonstrated using the MEK 1/2 inhibitor U0126 to block R5020-dependent increase in phospho-Erk1/2 (data not shown). The experiments were repeated three times with similar results. (C) T47D parental cells that endogenously expressed both PR-A and PR-B, or sublines engineered to stably express either PR-A or PR-B, were treated with or without R5020 for 18, 24, or 48 h; the cell lysates were Western blotted for phospho- (p) or total (t) Erk1/2 MAPK and total PR. Three independent experiments yielded identical results.

FIG. 2.

Progestin/PR-induced cyclin D1 protein, but not mRNA upregulation, requires MAPK activity. (A) Cyclin D1 mRNA levels were measured using real-time quantitative RT-PCR. The bars indicate the mean (plus standard deviation) of cyclin D1 mRNA, normalized to β-actin, measured in triplicate cultures of T47D-YB cells treated for 3 or 18 h with R5020 in the presence of DMSO or U0126; values (n-fold) over the EtOH control are indicated above the bars. (B) Whole-cell lysates from T47D-YB cells treated as in panel A were Western blotted for cyclin D1 and phospho- (p) and total (t) Erk1/2. The experiments were repeated three (A) or four (B) times with similar results.

FIG. 3.

Progestin-induced sustained Erk1/2 MAPK signaling requires transcription and translation. (A) T47D-YB cells were treated for 20 h with or without R5020 in the presence of H₂O vehicle control, CHX, EtOH vehicle control, or DRB, and phospho- (p) and total (t) Erk1/2 MAPKs were detected by Western blotting of whole-cell lysates. (B) T47D-YB cells were pretreated for 30 min with DMSO vehicle control or U0126 (MEK inhibitor) prior to addition of R5020. After 30 to 60 min, U0126 was washed out of the cultures represented in lanes 5 and 6 (U0 w/o; the 45-min time is shown). Cell lysates were collected after 18 h and Western blotted for phospho- or total Erk1/2 MAPKs and PR. The experiments were repeated three times with similar results.

FIG. 4.

Transcriptional and signaling activities of DNA-binding (mDBD) and c-Src binding (mPro) mutant PRs relative to wt PR-B. (A) Wt and mPro PR-B, but not mDBD PR-B, stimulate transcription of a transiently cotransfected PRE-luciferase reporter construct in HeLa cells. The bars indicate the averages of triplicate measurments of firefly luciferase normalized to Renilla luciferase (± standard deviations) for each condition; similar results were obtained in T47D cells (not shown). (B) Rapid Erk1/2 activation by wt PR-B and mDBD PR-B, but not mPro PR-B. Erk1/2 activity was measured using flow cytometry following 5-min R5020 treatment (heavy black line, unfilled) or treatment with EtOH vehicle control (gray-filled line). The data are represented as fluorescence (x axis) versus percent maximum (Max) (y axis).The experiments were repeated three times, in triplicate, with similar results.

FIG. 5.

Contributions of rapid PR signaling and PR transcriptional events to sustained MAPK activation and anchorage-independent growth. (A) Duplicate cultures of T47D cells stably overexpressing wt PR-B or the c-Src binding mutant, PR-B mPro, were treated with EtOH vehicle control (−) or R5020 (+) for 18 h or 10 min (bottom), and whole-cell lysates were Western blotted for phospho-Erk1/2 (p-Erk1/2) MAPK and total PR. The duplicate 18-h experimental time points shown, from a representative experiment, were quantified by densitometry of visible control and experimental bands on scanned images and expressed as increased Erk2 MAPK activity (n-fold) relative to vehicle controls (the bars represent means plus standard deviations). Densitometry of multiple exposures yielded similar results. Note that Western blotting confirmed the inability of PR-B mPro to rapidly activate Erk1/2 at 10 min relative to wt PR-B (bottom). The results were repeated three times. (B) T47D-YB cells were treated with EtOH, 10 nM R5020, 10 nM R5020 plus 100 nM RU486, or 100 nM RU486 alone for 18 h; the cell lysates were subjected to Western blotting with antibodies specific for phospho- (p) or total (t) Erk1/2 MAPK and total PR. Similar results were obtained for three experiments. (C) Phospho-Erk1/2 MAPK Western blotting and quantification by densitometry of wt-PR-B-expressing T47D-YB cells and two clones (19 and 5) stably expressing the PR-DBD mutant, C587A (mDBD). The cells were treated as described above with vehicle control (−) or R5020 (+) for 18 h, and whole-cell lysates were Western blotted for phospho- or total Erk1/2 and PR-B. PR blotting indicated roughly equivalent levels of PR protein in stable cell lines. The bars represent the R5020-stimulated activation (n-fold; plus standard deviation) of Erk2 MAPK for duplicate measurments performed within the same experiment relative to controls, using stable PR-DBD mutant C587A clone 19 (mDBD). Densitometry of multiple exposures yielded similar results. The experiments were repeated three times with similar results. (D) Progestin-stimulated soft-agar colony formation of T47D cells stably expressing either wt PR-B or PR-B mutants, mPro or mDBD. Cells were plated as described in Materials and Methods, and the bars represent the increase (n-fold) (plus standard deviation) in colony numbers for each stable cell line relative to EtOH controls. The inset shows colony numbers (n-fold) for R5020-treated T47D-YB (PR-B) cells relative to cells stably expressing PR-A (T47D-YA; PR-A). The asterisks denote significance (P < 0.01) determined by an unpaired Student's t test between EtOH- and R5020-treated conditions. The results were confirmed in two independent experiments.

FIG. 6.

Progestin-induced EGFR transactivation mediates sustained Erk1/2 MAPK signaling. (A) T47D-YB cells were treated without (−) or with (+) R5020 and/or RU486 for 18 h and then Western blotted for total (t) EGFR or total Erk1/2 MAPK as a loading control. (B) T47D-YB cells were treated for 18 h without (−) or with (+) R5020 in the presence of control IgG or with ligand-blocking antibodies (Ab) against EGFR. EGFR was then immunoprecipitated, and immune complexes were subjected to Western blotting to detect (active) phospho-Tyr1173 EGFR. The membranes were stripped and reprobed to detect the total EGFR immunoprecipitated. Monoclonal IgG served as a negative IP control, and lysate (10% of the IP input) demonstrated upregulation of total EGFR by 18-h R5020 treatment. The increase (n-fold) in EGFR activation was determined by densitometric analysis of immunoblots, where the pY-1173 signal was normalized to total EGFR in the immunoprecipitation. Densitometry of multiple exposures yielded similar results. (C) Concentrated culture medium from T47D-YB cells treated for 18 h with EtOH (Et) or R5020, or PMA as a positive control, was separated by a nondenaturing gelatin zymogram as described in Materials and Methods and stained with Coomassie blue. The clear regions indicate areas of protease activity. The results were confirmed in two independent experiments. (D) T47D-YB cells were pretreated for 30 min with DMSO vehicle control, the EGFR inhibitor AG1478 or PD168393, or the broad-range MMP inhibitor GM6001 and then treated without or with R5020 for 18 h as described above. Whole-cell lysates were Western blotted for phospho- or total Erk1/2 MAPK. (E) To control for the selectivity of the MMP inhibitor GM6001, T47D-YB cells were pretreated for 30 min with DMSO (D) or GM6001 (G), followed by a 5-min (m) exposure to 20 ng/ml EGF. The lysates were Western blotted for phospho- or total Erk1/2 MAPK. (F) T47D-YB cells were preincubated for 30 min with either control IgG or ligand-blocking antibodies against the EGFR or IGF-1R prior to 18-h R5020 treatment. Western blotting was performed as described above for phospho- or total Erk1/2. Except where noted, all experiments were repeated three or four times with similar results.

FIG. 7.

Persistent c-Src activation is required for progestin-induced sustained Erk1/2 activity. (A) T47D-YB cells were pretreated for 30 min with DMSO vehicle control, U0126, the c-Src family inhibitor PP2 or SU6656 (SU), or the PI3-K inhibitor, LY294002 (LY), followed by 18 h of R5020 treatment, and whole-cell lysates were Western blotted for phospho- (p) and total (t) Erk1/2 MAPKs. The specificity and activity of each c-Src inhibitor, and the LY294002 and SB203580 compounds, were confirmed by Western blotting for activated (phospho) forms of c-Src, Akt and p38, respectively (not shown). (B) T47D-YB cells were treated for 18 h with R5020 or vehicle control or 5 min (m) with EGF as a positive control for c-Src activation. Tyrosine-phosphorylated proteins were immunoprecipitated using 4G10 antibody-conjugated agarose beads (p-Tyr) or IP IgG control and Western blotted with c-Src-specific antibodies. Western blotting of c-Src in whole-cell lysates (10% of IP input) indicated no effect of vehicle control or R5020 treatments on total c-Src levels. The results were confirmed in two independent experiments. (C) T47D-YB cells were pretreated (pre) for 30 min with DMSO (lanes 1 and 2, 5 and 6) or PP2 (lanes 3 and 4, 7 and 8) prior to the addition of R5020 (+) or EtOH vehicle control (−) for 10 min or 18 h. In lanes 9 and 10, PP2 was added at the beginning of the 12th hour (post) of R5020 treatment. Cell lysates were Western blotted for phospho- or total Erk1/2 MAPK, cyclin D1, or total PR. (D) Progestin-induced persistent c-Src activity downstream of EGFR signaling to sustained activation Erk1/2 MAPK and cyclin D1 upregulation. (Inset) c-Src functions downstream of EGFR. T47D-YB cells pretreated for 30 min with DMSO, PP2, or SU6656 were treated for 18 h with R5020. EGFR was immunoprecipitated using specific antibodies or control IgG, and immune complexes were subjected to Western blotting to detect active (phospho-Tyr1173) EGFR. The membranes were stripped and reprobed to detect the total EGFR pulled down in each IP reaction. Whole-cell lysates indicate 10% of IP input. (E) T47D-YB cells were plated in soft agar as described in Materials and Methods and treated without (−) or with (+) R5020 in the presence of DMSO, U0126 (U0), PP2, or PD168393 (PD). The bars represent the increase (n-fold) in colony numbers (plus standard deviation) for cell cultures treated with progestin over EtOH DMSO controls, and the asterisk denotes statistical significance (P < 0.01) determined by an unpaired Student's t test. Except where noted, experiments were repeated three times with similar results.

FIG. 8.

Wnt-1 expression links PR transcriptional activity to EGFR transactivation and sustained MAPK signaling. (A) The time course of R5020-treated (+) T47D-YB cells demonstrates specific upregulation of Wnt-1 mRNA by RT-PCR. The results are representative of four independent experiments. (B) Duplicate cultures of untreated T47D-YB parental (YB) cells or cells stably expressing Neg shRNA control (N2) or Wnt-1 shRNA (Wnt-1) were treated for 18 h with R5020, and knockdown of Wnt-1 mRNA expression was measured by RT-PCR. (C) EGFR from T47D-YB cells stably expressing control shRNA (Neg) or Wnt-1 shRNA (Wnt-1) was immunoprecipitated using specific antibodies or control IgG and Western blotted using antibodies specific for phospho-Y1173 (p-Y1173) (activated) EGFR and total (t) EGFR. Lysates indicate 10% IP input. Normalization of phospho-Y1173 to total EGFR in immunoprecipitates, as measured by densitometry, indicated EGFR activity was increased 1.9-fold in R5020-treated control (Neg) cells, whereas cells expressing Wnt-1 shRNA exhibited no increase in EGFR activity (0.9-fold compared to the EtOH signal); densitometry of multiple exposures yielded similar results. The results were confirmed in two independent experiments. (D) Duplicate cultures of wt T47D-YB (YB) cells or cells stably expressing either control (Neg) or Wnt-1 shRNA (Wnt-1) were treated without (−) or with (+) R5020 for 18 h, and whole-cell lysates were Western blotted for cyclin D1, EGFR, and phospho- and total Erk1/2 MAPKs. Duplicate measurments of active Erk2 MAPK and cyclin D1 protein bands were quantified by densitometry and are represented as bars in the lower graphs (± standard deviations). (E) Triplicate cultures of T47D-YB (YB parent) cells or cells stably expressing either control shRNA (Neg) or Wnt-1 shRNA (Wnt-1) were plated in soft agar as described in Materials and Methods and treated without (EtOH) or with (black bars) R5020 for 18 h, and soft-agar colonies were counted after 3 weeks. The average colony numbers are presented as n-fold over the EtOH control for the T47D-YB parent, shRNA Neg, or Wnt-1 (± standard errors of the mean). The asterisks denote statistical significance (P < 0.01) determined by an unpaired Student's t test. The experiments were repeated twice with similar results.

FIG. 9.

Model showing mechanism(s) of progestin/PR-mediated sustained MAPK signaling. Integration of PR rapid signaling events and PR-nuclear actions induce the transcriptional upregulation of EGFR, cyclin D1, and Wnt-1, followed by activation of MMP and transactivation of EGFR. Persistent c-Src signaling downstream of activated EGFR induces sustained Erk1/2 MAPK activity and serves as a direct input to cyclin D1 protein expression and anchorage-independent cell growth. We previously showed the MAPK dependence of progestin regulation of the proximal cyclin D1 promoter and a requirement for PR/c-Src interactions for S-phase entry of cultured T47D cells (62).