A local paracrine and endocrine network involving TGFβ, Cox-2, ROS, and estrogen receptor β influences reactive stromal cell regulation of prostate cancer cell motility

Abstract

The tumor microenvironment plays a critical role in supporting cancer cells particularly as they disengage from limitations on their growth and motility imposed by surrounding nonreactive stromal cells. We show here that stromal-derived androgenic precursors are metabolized by DU145 human prostate cancer (PCa) cells to generate ligands for estrogen receptor-β, which act to limit their motility through transcriptional regulation of E-cadherin. Although primary human PCa-associated fibroblasts and the human WPMY-1-reactive prostate stromal cell line maintain this inherent estrogen receptor (ER)β-dependent motility inhibitor activity, they are subverted by TGF-β1 pro-oxidant signals derived from cocultured DU145 PCa cells. Specifically, stromal-produced H(2)O(2), which requires Cox-2, acts as a second paracrine factor to inhibit ERβ activity in adjacent DU145 cells. Chromatin immunoprecipitation analysis reveals that ERβ recruitment to the E-cadherin promoter is inhibited when H(2)O(2) is present. Both neutralization of H(2)O(2) with catalase and prevention of its production by silencing Cox-2 expression in stromal cells restore the motility-suppression activity of stromal-derived ERβ ligand precursors. These data suggest that reactive stromal cells may still have a capacity to limit cancer cell motility through a local endocrine network but must be protected from pro-oxidant signals triggered by cancer cell-derived TGF-β1 to exhibit this cancer-suppressive function.

Fig. 1.

Paracrine signaling modulates cancer cell motility. A, In indirect coculture, DU145 motility is unaffected by the presence of CAF or WPMY-1 cells, but is significantly reduced by PS30 cells. Data represent the mean ± sem from three independent experiments, each repeated in technical triplicate. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05 compared with respective DU145 control. B, WPMY-1 and PS30 cells were transiently cotransfected with the 3TP-lux-luciferase (luc) and Renilla-luc reporter plasmids overnight, and then subjected to a 6-h TGF-β1 treatment the following day (0, 1, 2, 5, and 10 ng/ml). Data represent the mean ± sem of three independent experiments, each performed in triplicate. A two-way ANOVA followed by Bonferroni posttest was performed. ***, P < 0.001. C, WPMY-1 cells express higher levels of Smad 3 and Smad 4. A representative blot is shown, and a graphic display of densitometric analysis representing the mean ± sem of three independent experiments is presented. A two-way ANOVA followed by a Bonferroni posttest was performed. *, P < 0.05. D, The left panel is representative images from the modified wound-healing assay; the right panel is a graphic display of all replicates. Data represent the mean ± sem from four independent experiments, each repeated in technical triplicate. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05 compared with respective DU145 control. **, P < 0.01 relative to DU145 treated with TGFβ neutralizing antibody. E, Naïve DU145 cells were wounded and media replaced with WPMY-1 CM. Wound closure over 24 h was determined. Data represent the mean ± sem from three independent experiments, each repeated in technical triplicate. A one-way ANOVA followed by Tukey's multiple comparison test was performed. **, P < 0.01 compared with control media. F, DU145 cells were treated for 24 h with either 1% serum-containing media (control) or WPMY-1 CM, followed by Western blot analysis for E-cadherin expression. The left panel shows an image from three independent samples; the right panel is a graphic representation of the normalized densitometry. A t test was performed for pairwise comparison. *, P < 0.05. Ab, Antibody.

Fig. 2.

TGF-β1 treatment triggers H₂O₂ production in reactive stromal cells, which can limit SMIF activity. A, In response to exogenous TGF-β1, WPMY-1 cells produce increased levels of H₂O₂ as measured by an endpoint Amplex Red assay. WPMY-1 cells were serum starved for 90 min before addition of TGF-β1 in fresh serum-free media. Cells were incubated with the TGFβ for 3 h, Amplex Red was added to cell cultures, and an endpoint reading was recorded at 1 h. Data represent mean from three biological replicates ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05; **, P < 0.01 relative to untreated WPMY-1 control. B, Addition of catalase to cocultures reverses the permissive role of CAF and WPMY-1 cells on DU145 motility. The modified wound-healing assay was performed with the addition of 1500 U of catalase per 1 ml of media. Data are representative of three independent experiments ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05 comparing control coculture with coculture with the addition of catalase.

Fig. 3.

TGFβ-inducible Cox-2 in stromal cells generates the H₂O₂ necessary to limit SMIF activity and permit PCa cell motility in cocultures. A, A transwell insert containing DU145 cells was placed into a chamber containing serum-starved WPMY-1 cells, and the media were replaced with fresh 1% serum-containing RPMI −/+ a TGF-β1 neutralizing antibody. The cells were cocultured for 24 h, and the Cox-2 levels in WPMY-1 cells were determined by qRT-PCR. Data represent the mean ± sem from three independent experiments. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05. B, A lentiviral vector expressing shRNA either scrambled (Scr) or directed against Cox-2 (SH4) was used to stably infect WPMY-1 cells. The resulting lines were subjected to a 24-h treatment with TGF-β1 in serum-free media (5 ng/ml), and Cox-2 mRNA levels were determined by qRT-PCR. Data represent three independent experiments ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05. C, SH4 cells were serum starved for 90 min before addition of TGF-β1 in fresh serum-free media. Cells were incubated with the TGFβ for 3 h, Amplex Red was added to cocultures, and an endpoint reading was recorded at 1 h. Data represent mean from three biological replicates ± sem. D, The modified wound-healing assay was performed with the addition of 1500 U of catalase per 1 ml of media. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05 relative to appropriate DU145 control. Ab, Antibody; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 4.

Cox-2-dependent H₂O₂ generation in WPMY-1 cells inhibits DU145 cell response to SMIF. A, CM loses its inhibitory effect on DU145 motility at concentrations of H₂O₂ greater than or equal to 10 μm. CM was generated as previously described from WPMY-1 cells. Naïve DU145 cells were wounded, and the media were replaced with CM to which varying amounts of H₂O₂ had been added. Data represent the mean of six independent experiments ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. **, P < 0.01 relative to appropriate control media. B, Addition of H₂O₂ (10 μm) to the DU145/SH4 coculture reverses the motility inhibition observed under basal conditions. A modified wound-healing assay as previously described was carried out with and without the addition of H₂O₂. Data represent the results of four independent experiments ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05 relative to appropriate DU145 control. C, CM was incubated with 10 μm H₂O₂ for 3 h. After this pretreatment, a portion of the media was then treated with catalase (1500 U/ml), and the two different treatment groups were added to wounded naïve DU145 cells. Data represent three independent experiments ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05 compared with all other conditions.

Fig. 5.

SMIF from WPMY-1 CM blocks DU145 motility in an ERβ-dependent manner. A, WPMY-1 CM was fractionated into high- and low-molecular mass fractions, and each fraction was tested independently for biological activity. The low molecular mass fraction (pore size <5 kDa) retains inhibitory effects on DU145 motility. Data are representative of four independent biological replicates ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed; ***, P < 0.001 compared with both DU145 control and the high-molecular mass fraction. B, Addition of exogenous 3β-adiol (10⁻⁶ m) is able to significantly inhibit DU145 motility. This is reversed when 10 μm H₂O₂ is added to the media. Data are representative of four independent biological replicates ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. **, P < 0.01 compared with all other conditions. C, Naïve DU145 cells were wounded and treated with either control or WPMY-1 CM with 4-hydroxytamoxifen (10⁻⁷ m) or PHTPP (0.1 μm). Results are representative of four independent biological replicates ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. ***, P < 0.001 compared with conditions containing control media; *, P < 0.05 compared with CM. D, Naïve DU145 cells were treated for 18 h with either control or WPMY-1 CM, −/+ 10 μm H₂O₂. The cells were harvested in Trizol and subjected to qRT-PCR for E-cadherin transcript levels. Results are indicative of three independent samples and are displayed ± sem. A one-way ANOVA followed by Tukey's multiple comparison test was performed. *, P < 0.05 relative to all other conditions.

Fig. 6.

Oxidized ERβ exhibits less DNA binding at the E-cadherin promoter.A, Nuclear and cytoplasmic lysates were prepared from DU145 cells and subjected to Western blot analysis for ERβ protein. Results are displayed as densitometric analysis from three independent blots. A two-way ANOVA followed by a Bonferroni posttest was performed. *, P < 0.05. B, DU145 cells were grown in either control or CM −/+ H₂O₂ (10 μm) for 18 h. ChIP using ERβ antibodies was performed, and qRT-PCR analysis for previously defined ERβ-binding sites within the E-cadherin promoter proximal region (Table 2) revealed a significant increase in promoter occupancy in the presence of CM. This increase was reversed when H₂O₂ was present in CM. Results are representative of three independent experiments. A one-way ANOVA followed by Tukey multiple comparison test was performed. *, P < 0.05 compared with all other conditions at that promoter site.

Fig. 7.

SMIF is an androgenic precursor metabolized by the DU145 cells to an ERβ ligand. A, FA, but not dutasteride, blocks the inhibitory activity of DHEA. Wound-healing assays were performed on DU145 cells treated with either and DHEA (100 nm), FA (50 μm), or dutasteride (1 μm) where indicated. Results are representative of four independent experiments, each done in technical quadruplicate. A one-way ANOVA followed by Dunnett's multiple comparison test using untreated DU145 cells as the control column was performed. **, P < 0.01. B, FA inhibits SMIF activity in CM. Wound-healing assays were performed on DU145 cells treated with CM ± FA (50 μm). Results are representative of four independent experiments, each done in technical quadruplicate. A one-way AONVA followed by Tukey's multiple comparison test was performed. *, P < 0.05. C, In a normal fibroblast, DHEA is constitutively produced and secreted. It is metabolized by 17βHSD enzymes in the DU145 cells into potent ERβ ligands, which then act to limit motility. D, In reactive myofibroblasts, locally produced TGF-β1 stimulates the production of ROS, which is secreted in the form of H₂O₂. The H₂O₂ acts as a second paracrine factor to limit the transcriptional activity of ERβ, thus alleviating its inhibitory effect on DU145 motility.