Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism

Abstract

Selective estrogen receptor modulators (SERM) including tamoxifen are known to inhibit angiogenesis. However, the underlying mechanism, which is independent of their action on the estrogen receptor (ER), has remained largely unknown. In the present study, we found that tamoxifen and other SERM inhibited cholesterol trafficking in endothelial cells, causing a hyper-accumulation of cholesterol in late endosomes/lysosomes. Inhibition of cholesterol trafficking by tamoxifen was accompanied by abnormal subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR2) and inhibition of the terminal glycosylation of the receptor. Tamoxifen also caused perinuclear positioning of lysosomes, which in turn trapped the mammalian target of rapamycin (mTOR) in the perinuclear region of endothelial cells. Abnormal distribution of VEGFR2 and mTOR and inhibition of VEGFR2 and mTOR activities by tamoxifen were significantly reversed by addition of cholesterol-cyclodextrin complex to the culture media of endothelial cells. Moreover, high concentrations of tamoxifen inhibited endothelial and breast cancer cell proliferation in a cholesterol-dependent, but ER-independent, manner. Together, these results unraveled a previously unrecognized mechanism of angiogenesis inhibition by tamoxifen and other SERM, implicating cholesterol trafficking as an attractive therapeutic target for cancer treatment.

Keywords: Angiogenesis; Cholesterol trafficking; Selective estrogen receptor modulator; Tamoxifen.

Figure 1

Effect of SERM on cholesterol trafficking in HUVEC. (A) HUVEC were treated with tamoxifen (TMX), toremifene (TRM), clomifene (CLM) and raloxifene (RLX) for 24 h, and intracellular cholesterol was visualized by filipin. (B) HUVEC were treated with 1 μM tamoxifen (TMX, upper panel) or 1 μM toremifene (TRM, lower panel) in the presence or absence of cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24 h, and intracellular cholesterol was visualized by filipin. Representative confocal images from four independent experiments are shown.

Figure 2

Effects of SERM and cholesterol on the subcellular localization of VEGFR2 and mTOR in HUVEC. (A) HUVEC were treated with or without 1 μM tamoxifen (TMX) for 24 h and subcellular localization of VEGFR2 was assessed under a confocal microscope. Arrows indicate VEGFR2. (B) HUVEC were treated with or without 1 μM tamoxifen (TMX) in the presence or absence of cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24 h and subcellular localization of VEGFR2 and Golgi (GM130) was assessed. (C) HUVEC were treated with or without 1 μM tamoxifen (TMX) for 24 h and subcellular localization of cholesterol (Filipin), lysosomes (LAMP1), and mTOR was assessed. (D) HUVEC were treated with 1 μM tamoxifen (TMX) in the presence or absence of cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24 h and subcellular localization of mTOR and actin was assessed. Arrows indicate that tamoxifen induced a change in mTOR localization from peripheral cytoplasm to perinuclear region and this was reversed by cholesterol-cyclodextrin complex. Representative confocal images from four independent experiments are shown.

Figure 3

Effect of SERM on VEGFR2 glycosylation and mTORC1 pathway in HUVEC. (A) HUVEC were treated with SERM including tamoxifen (TMX, 5 μM), toremifene (TRM, 5 μM) and clomifene (CLM, 5 μM) for 24 h and VEGFR2 glycosylation was assessed by Western blotting. Glycosylation inhibitors including deoxymannojirimycin (dMM, 500 μM) and tunicamycin (TUM, 2 μg/ml), and a cholesterol trafficking inhibitor itraconazole (ITRA, 1 μM) were used as positive controls. Three different glycosylated forms of VEGFR2 (a: 230 kD hyper-glycosylated form, b: 200 kD intermediate glycosylated form and c: 180 kD unglycosylated form) are shown. (B), (C), and (D) Effect of various concentrations of SERM on VEGFR2 glycosylation and mTORC1 pathway – indicated by the level of phosphorylated S6K (pS6K) – are shown. Representative Western blot images from three independent experiments are shown.

Figure 4

Effect of tamoxifen on the phosphorylation of mTOR and the glycosylation of receptor tyrosine kinases in HUVEC. (A) HUVEC were treated with tamoxifen (TMX) or rapamycin (Rapa) at indicated concentrations for 24 h and the phosphorylation of mTOR at Ser2448 as well as phosphorylated S6K (pS6K) and total S6K were analyzed. (B) HUVEC were treated with tamoxifen (TMX), sorafenib (Soraf) or deoxymannojirimycin (dMM) at indicated concentrations for 24 h. The terminal glycosylation of the receptor tyrosine kinases was assessed by Western blotting using specific antibodies against each receptor tyrosine kinase in the presence of the known glycosylation inhibitor dMM. Representative Western blot images from three independent experiments are shown.

Figure 5

Reversal effect of cholesterol on the inhibition of VEGFR2 and mTOR activities by SERM. (A) and (B) HUVEC were treated with tamoxifen (TMX) or toremifene (TRM) with or without cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24 h, and VEGFR2 glycosylation was assessed by Western blotting. (C) HUVEC were grown in low serum media (0.1% FBS without additional growth factor supplements) and treated with drugs including tamoxifen (TMX) and sunitinib (Sunit, 100 nM) for 24 h in the presence or absence of cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD). Cells were then stimulated with 50 ng/ml of VEGF-165 for 5 min and the levels of total and phosphorylated VEGFR2 were assessed by Western blotting. (D) HUVEC were treated with tamoxifen (TMX, 5 μM) or toremifene (TRM, 5 μM) with or without cholesterol (Chol, 5 μg/ml) and cyclodextrin (CD, 0.1%) for 24 h, and mTOR activity was assessed by Western blotting of phosphorylated S6- kinase (pS6K). Representative Western blot images from three independent experiments are shown.

Figure 6

Effects of SERM and cholesterol on HUVEC proliferation. (A) HUVEC were treated with various concentrations of tamoxifen, toremifene or raloxifene for 24 h and cell proliferation was assessed through the [3]H-thymidine uptake assay. (B) HUVEC were treated with tamoxifen (TMX, 2 μM) or toremifene (TRM, 4 μM) for 24 h and were observed under a phase contrast microscope. Cholesterol (Chol, 5 μg/ml) and/or cyclodextrin (CD, 0.1%) were added together to assess reversibility on the anti-proliferative effect of SERM. NT denotes Not Treated with Chol/CD. Representative phase-contrast images from four independent experiments are shown. The cell viability was quantified by AlamarBlue staining and was plotted using the GraphPad Prism 5.0 software (right panel). Date represent mean ± standard deviation (SD) from four independent experiments. **P< 0.01 between two indicated groups.

Figure 7

Effects of SERM and cholesterol on ER-positive or ER-negative breast cancer cell proliferation. (A) and (B) MCF-7 (ER-positive) or MDA-MB-231 (ER-negative) cells were treated with various concentrations of tamoxifen, toremifene or raloxifene for 24 h and cell proliferation was assessed through the [3]H-thymidine uptake assay. SERM showed a biphasic growth inhibition in MCF-7 cells. Dotted arrows indicate concentration ranges that show marginal cell growth inhibition, whereas solid arrows represent concentration ranges with strong cell growth inhibition. (C) MCF-7 or MDA-MB-231 cells were treated with 10 μM tamoxifen (TMX) in the presence or absence of cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24 h and intracellular cholesterol was labeled with filipin staining. Representative confocal images from four independent experiments are shown. (D) MCF-7 or MDA-MB-231 cells were treated with 10 μM tamoxifen (TMX) in the presence or absence of cholesterol (Chol, 5 μg/ml)/cyclodextrin (CD, 0.1%) complex (Chol/CD) for 24 h. The cell viability was quantified by AlamarBlue staining and was plotted using the GraphPad Prism 5.0 software. Date represent mean ± standard deviation (SD) from four independent experiments. **P< 0.01 between two indicated groups.

Figure 8

Effects of SERM and cholesterol on HUVEC tube formation. (A) HUVEC were seeded onto a Matrigel-coated plate to promote tube formation. Cells were treated with 5 μM tamoxifen (TMX) or 5 μM toremifene (TRM) in the presence or absence (NT) of cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 18 h. The tube formation was visualized by staining with Calcein-AM under a fluorescence microscope. Representative fluorescence images from six independent experiments are shown. (B) Total tube lengths, sizes and number of junctions from the fluorescence images from six experiments were quantified using the AngioQuant software. **P< 0.01 between two indicated groups.