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.
Figures
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. 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. 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. 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. 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. 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. 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. 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. 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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