Estrogen regulation of thrombospondin-1 in human breast cancer cells - PubMed (original) (raw)

Estrogen regulation of thrombospondin-1 in human breast cancer cells

Salman M Hyder et al. Int J Cancer. 2009.

Abstract

Expression of thrombospondin-1 (TSP-1), a large extracellular matrix protein, has been associated with modulation of angiogenesis and tumor growth. Both pro and antiangiogenic properties of TSP-1 have been described, and the role of TSP-1 expression in the growth and progression of human breast cancer is not clear. Because estrogens cause progression of many breast cancers, and estradiol (E2) downregulates a TSP-1 receptor, we examined whether TSP-1 is regulated by estrogen and involved in tumor progression. E2 induced TSP-1 expression in T47-D and MCF-7 breast cancer cells in vitro within 3 to 6 hr; the induction was blocked by the anti-estrogen ICI 182,780, indicating that estrogen receptors (ER) are necessary for this effect. Furthermore, E2 caused the production of TSP-1 protein from tumor cells in an ER-alpha-dependent manner. The E2-mediated TSP-1 RNA induction was dose-dependent and blocked by actinomycin D, indicating that the response to E2 was at least partly transcriptional. Transfection studies with deletion constructs of the TSP-1 promoter identified an estrogen-responsive region in the human TSP-1 promoter, located between -2,200 and -1,792 bp upstream of the transcription start site. An antibody against TSP-1 restricted the proliferation of E2-dependent MCF-7 cells in vitro and in vivo. A panel of breast cancer cells proliferated in the presence of low concentrations of exogenous TSP-1, whereas higher concentrations inhibited proliferation. A real-time PCR analysis showed that E2 also induced TSP-1 mRNA in the normal mammary glands of immature ovariectomized mice in an ER-dependent manner. In summary, we report the novel observation that TSP-1 production is directly controlled by estrogens in ER-positive breast cancer cells, and the released protein has pro-growth regulatory functions. Consequently, we propose that TSP-1 could be a therapeutic target for anti-tumor therapy in early-stage tumors. (c) 2009 UICC.

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Figures

Figure 1

Figure 1. E2 induces TSP-1 message and protein in breast cancer cells

T47-D and MCF-7 cells were cultured and total RNA prepared and assessed for the expression of TSP-1 mRNA by Northern blot analysis. The blots were then stripped and probed for 28S RNA as a loading control. (A) T47-D cells were treated with 10 nM E2 for 1, 3, 6, or 12 h, as indicated. The TSP-1 band is marked with an arrow, and the locations of 28S and 18S ribosomal RNA are shown as reference points. (B) Cells were treated with 10 nM E2 for 6 h in the presence of actinomycin D (Act D; 2 μg/ml) or puromycin (PuRo; 10 μg/ml). (C) Cells were treated for 6 h with 10 nM E2, 10 nM E2 in the presence of 1 μM ICI 182,780 (E2 + ICI), or 1 μM ICI 182,780 alone (ICI). (D) Cells were incubated for 6 h with the indicated dose of E2, and TSP-1 message was visualized as shown in panel B. Autoradiograms were scanned and the densitometric values for the TSP-1 bands were normalized to the densitometric values for 28S in each lane. Values are mean + SEM (n = 3 per dose). * Significantly different from control (p < 0.05; Student t-test). (E) Western blot analysis of serum free media collected after treating T47-D cells with estradiol (E2) in the absence (control) and presence or absence of ICI 182,780 (ICI). Treatment of media prior to western blot analysis is described in Methods.

Figure 1

Figure 1. E2 induces TSP-1 message and protein in breast cancer cells

T47-D and MCF-7 cells were cultured and total RNA prepared and assessed for the expression of TSP-1 mRNA by Northern blot analysis. The blots were then stripped and probed for 28S RNA as a loading control. (A) T47-D cells were treated with 10 nM E2 for 1, 3, 6, or 12 h, as indicated. The TSP-1 band is marked with an arrow, and the locations of 28S and 18S ribosomal RNA are shown as reference points. (B) Cells were treated with 10 nM E2 for 6 h in the presence of actinomycin D (Act D; 2 μg/ml) or puromycin (PuRo; 10 μg/ml). (C) Cells were treated for 6 h with 10 nM E2, 10 nM E2 in the presence of 1 μM ICI 182,780 (E2 + ICI), or 1 μM ICI 182,780 alone (ICI). (D) Cells were incubated for 6 h with the indicated dose of E2, and TSP-1 message was visualized as shown in panel B. Autoradiograms were scanned and the densitometric values for the TSP-1 bands were normalized to the densitometric values for 28S in each lane. Values are mean + SEM (n = 3 per dose). * Significantly different from control (p < 0.05; Student t-test). (E) Western blot analysis of serum free media collected after treating T47-D cells with estradiol (E2) in the absence (control) and presence or absence of ICI 182,780 (ICI). Treatment of media prior to western blot analysis is described in Methods.

Figure 2

Figure 2. The TSP-1 promoter contains an estrogen-responsive region

Reporter gene constructs are shown schematically in the upper part of the figure. The DNA sequence coordinates of the human TSP-1 promoter are indicated; +1 indicates the transcription start site of the TSP-1 gene. Cells were treated with 10 nM E2 ± 1 μM ICI 182,780 (I) for 18 h. Cells were harvested and luciferase activity was quantified. pGL3, empty vector; ERE, estrogen response element from the vitellogenin gene; * significantly different from control; ** Significantly different than E2 treatment in the absence of ICI 182,780 (p < 0.05, Student _t_-test, n = 3 per treatment).

Figure 3

Figure 3. E2 causes release of TSP-1 protein from breast cancer cells

T47-D or MCF-7 cells were incubated overnight in DMEM/F12 + 5% charcoal stripped FBS. Fresh serum-free media was added and cells were incubated for 24 h. The media was then replaced with either serum free DMEM/F12 (T47-D) or DMEM/F12 with 1% charcoal stripped serum (MCF-7) and cells treated with 10 nM E2 (E2-10), an ER-alpha–specific ligand (PPT; 10 nM), or an ER-beta–specific ligand (DPN; 10 nM) ± 1 μM ICI 182,780 (ICI) for 18 h as indicated. The media were collected, and TSP-1 was quantified by ELISA. * significantly different from control; ** significantly different from hormone treatment in the absence of ICI 182,780 (p < 0.05 ANOVA).

Figure 4

Figure 4. TSP-1-induced proliferation of breast cancer cells

(A) MCF-7 cells were cultured in DME/F12 with 5% DCC serum. The medium was changed to serum-free medium, and the cells were treated for 24 h in the presence of E2 alone, E2 + an antibody against TSP-1 (E2 + Ab), or Ab alone. BrdU was added to the media, and the cells were incubated for an additional 3 h. Cells were then harvested and BrdU was quantified by ELISA. * significantly different from control; ** significantly different from E2 treated group (p < 0.05 ANOVA). (B) MCF-7 xenografts were grown in intact nude mice supplemented with an E2 pellet as described in the Methods. On day 9, the mice were treated with an antibody against TSP-1 or an IgM control. Additional antibody injections were given on days 11, 12, and 13. * Significantly different from IgM-treated control animals (p < 0.05, student t-test).

Figure 5

Figure 5. Effect of TSP-1 on proliferation of breast cancer cells

(A) Cells were serum-starved and then incubated in fresh medium without serum with the indicated concentrations of exogenous TSP-1 for 15 h. BrdU was added to the media, and the cells were incubated for an additional 3 h. The cells were harvested, and BrdU was quantified by ELISA. (B) The effect of TSP-1 on breast cancer cell viability. Cells were seeded into 96-well plates overnight as described in Methods. The medium was removed and cells were washed once with DMEM/F12, and treated with identical concentrations shown in (A) in serum free DMEM/F12 medium for 24 hrs. Cell growth and viability were determined by SRB assay as described in Methods. (B) Specificity of TSP-1 effects on breast cancer cells. Cells were cultured as above and treated with either 0.01 or 0.1 μg/ml TSP-1 ± an antibody against TSP-1 (Ab) for 18 h. BrdU was added to the media 3 h prior to termination of the experiment (at 15h). Cells were harvested and BrdU was quantified by ELISA. * Significantly different from control; ** significantly different from hormone treatment in the absence of Ab (p < 0.05, ANOVA).

Figure 5

Figure 5. Effect of TSP-1 on proliferation of breast cancer cells

(A) Cells were serum-starved and then incubated in fresh medium without serum with the indicated concentrations of exogenous TSP-1 for 15 h. BrdU was added to the media, and the cells were incubated for an additional 3 h. The cells were harvested, and BrdU was quantified by ELISA. (B) The effect of TSP-1 on breast cancer cell viability. Cells were seeded into 96-well plates overnight as described in Methods. The medium was removed and cells were washed once with DMEM/F12, and treated with identical concentrations shown in (A) in serum free DMEM/F12 medium for 24 hrs. Cell growth and viability were determined by SRB assay as described in Methods. (B) Specificity of TSP-1 effects on breast cancer cells. Cells were cultured as above and treated with either 0.01 or 0.1 μg/ml TSP-1 ± an antibody against TSP-1 (Ab) for 18 h. BrdU was added to the media 3 h prior to termination of the experiment (at 15h). Cells were harvested and BrdU was quantified by ELISA. * Significantly different from control; ** significantly different from hormone treatment in the absence of Ab (p < 0.05, ANOVA).

Figure 5

Figure 5. Effect of TSP-1 on proliferation of breast cancer cells

(A) Cells were serum-starved and then incubated in fresh medium without serum with the indicated concentrations of exogenous TSP-1 for 15 h. BrdU was added to the media, and the cells were incubated for an additional 3 h. The cells were harvested, and BrdU was quantified by ELISA. (B) The effect of TSP-1 on breast cancer cell viability. Cells were seeded into 96-well plates overnight as described in Methods. The medium was removed and cells were washed once with DMEM/F12, and treated with identical concentrations shown in (A) in serum free DMEM/F12 medium for 24 hrs. Cell growth and viability were determined by SRB assay as described in Methods. (B) Specificity of TSP-1 effects on breast cancer cells. Cells were cultured as above and treated with either 0.01 or 0.1 μg/ml TSP-1 ± an antibody against TSP-1 (Ab) for 18 h. BrdU was added to the media 3 h prior to termination of the experiment (at 15h). Cells were harvested and BrdU was quantified by ELISA. * Significantly different from control; ** significantly different from hormone treatment in the absence of Ab (p < 0.05, ANOVA).

Figure 6

Figure 6. E2-induced TSP-1 message in normal mouse mammary gland

(A) Ovariectomized Balb/c mice (n = 3 per time point) were injected with 40 μg/kg E2 and sacrificed at the times shown. RNA was prepared as described in Methods. TSP-1 message was quantified using real-time PCR with 18S as an internal control. (B) ICI 182,780 (ICI) inhibits estradiol-induced TSP-1 in mouse mammary gland. Ovariectomized mice were injected with ICI (3 mg/kg) 30 min prior to the administration of E2. The mice were sacrificed 3 h later, and RNA was prepared using a Qiagen RNeasy kit. TSP-1 message was quantified using real-time PCR analysis with 18S as an internal control. Numbers in parentheses refer to the number of animals used. * Significantly different from control (p < 0.05; ANOVA).

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