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. 2014 Apr 11;289(15):10488-501.
doi: 10.1074/jbc.M113.534263. Epub 2014 Feb 22.

Ovarian Cancer Cell Heparan Sulfate 6-O-sulfotransferases Regulate an Angiogenic Program Induced by Heparin-Binding Epidermal Growth Factor (EGF)-like Growth factor/EGF Receptor Signaling

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Ovarian Cancer Cell Heparan Sulfate 6-O-sulfotransferases Regulate an Angiogenic Program Induced by Heparin-Binding Epidermal Growth Factor (EGF)-like Growth factor/EGF Receptor Signaling

Claire L Cole et al. J Biol Chem. .
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Abstract

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30-50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.

Keywords: Angiogenesis; Epidermal Growth Factor Receptor (EGFR); HB-EGF; Heparan Sulfate; IL-6; IL-8; Sulfotransferase; Tumor Microenvironment.

Figures

FIGURE 1.
FIGURE 1.
HS6ST-1 and HS6ST-2 expression in ovarian cancer. A and B, real time PCR analysis of HS6ST-1 (A) and HS6ST-2 (B) gene expression in 40 ovarian tumor samples of serous (32 samples), mucinous (1 sample), endometrioid (5 samples), clear cell (1 sample), and unknown histological type (1 sample) adenocarcinomas of disease stage I–IV and 8 normal (N) ovarian tissue samples. Gene expression in 8 normal ovarian tissue samples is shown as the mean, which is expressed as 1 (± S.E.). HS6ST-1 and HS6ST-2 mRNA levels in each tumor sample is shown as a value relative to the mean of mRNA levels in normal ovaries (n = 8). C and D, mRNA levels of HS6ST-1 (C) and HS6ST-2 (D) in ovarian cancer cell lines and IOSE cells were analyzed by real time PCR. Expression of HS6ST-1 and HS6ST-2 is shown as a ratio between the levels in each cancer cell line and an average expression value in both IOSE cell lines, which is expressed as 1. The numbers in parentheses indicate the number of samples. E, expression of Sulf-1 and Sulf-2 in ovarian cancer cell lines. mRNA levels of Sulf-1 and Sulf-2 were evaluated by RT-PCR. ES2 cell line shows no expression of Sulfs, OVCAR-5 and OVCAR-3 cell lines express both Sulf isoforms, and OAW42 cells express one Sulf isoform (Sulf-2). F and G, expression of HS6ST-1 in ES2 cells (F) and OVCAR-5 cells (G) transduced with nonspecific shRNA (NS) and shRNAs targeting HS6ST-1 (sh6ST1-1 and sh6ST1-2). β-Actin was used as RT-PCR control. H and I, down-regulation of HS6ST-1 or HS6ST-2 in OVCAR-3 (H) and OAW42 (I) cells was tested by RT-PCR. Two retroviral shRNA plasmids were used to down-regulate either HS6ST-1 (sh6ST1-1 and sh6ST1-2) or HS6ST-2 (sh6ST2-1 and sh6ST2-2). β-Actin was used as RT-PCR control.
FIGURE 2.
FIGURE 2.
Reduction of HS6ST-1 or HS6ST-2 expression in ovarian cancer cells impacts endothelial cell migration and tubule formation. A, proliferation of ovarian cancer cell lines. The effect of down-regulation of HS6STs is expressed as a percentage of cell proliferation at day 5 compared with the control cell proliferation (NS), which is expressed as 100%. The data represent the means ± S.D. (n = 2). B, confluent HUVEC monolayers were serum-starved and wounded. CM generated by ES2, OAW42, OVCAR-3, and OVCAR-5 cells expressing nonspecific shRNA (NS) or shRNAs targeting HS6ST-1 (sh6ST1-1 combined with sh6ST1-2) or HS6ST-2 (sh6ST2-1 combined with sh6ST2-2) were added to stimulate cell migration into the wound, which was measured at baseline and after 24 h. Repopulated wound areas are expressed as a percentage of wound area that was repopulated by HUVEC stimulated with control (NS) cell CM (expressed as 100%). Two independent experiments were performed. Each experiment was performed in triplicate. Change in the repopulated area is shown as the mean ± S.D. (n = 2). *, p < 0.01. C, endothelial tubule formation in three-dimensional fibrin gels in the presence of CM generated by OVCAR-3 NS cells and OVCAR-3 cells with down-regulated HS6ST-1 or HS6ST-2. The average number of tubes per carrier bead was quantified in a control experiment where HUVEC were stimulated with NS CM (expressed as 100%). The change in the number of tubes per bead when stimulated with CM from OVCAR-3 cells expressing sh6ST1-1, sh6ST1-2, sh6ST2-1, and sh6ST2-2 is shown as a percentage of the control. Three independent experiments were performed. Each experiment was performed in triplicate. The values are expressed as means ± S.E. (n = 3). *, p < 0.005. D, bright field images of mouse aortic rings embedded in fibrin gels and maintained in ES2 NS and sh6ST1-1/sh6ST1-2 cell CM (upper images) or OAW42 NS, sh6ST1-1/sh6ST1-2, and sh6ST2-1/sh6ST2-2 cell CM (lower images) for 6 days. Scale bars represent 100 μm. E, quantification of the outgrowth of sprouts from the aortic rings was performed using MetaMorph software. Sprouting area when stimulated with ES2 NS or OAW42 NS CM is expressed as 100% (control). The change in the sprouting area when stimulated with CM generated by cells with down-regulated HS6ST-1 or HS6ST-2 is expressed as a percentage of the control area. The means ± S.D. (n = 2) are shown. *, p < 0.005. F, formation of HUVEC endothelial tubule structures in ovarian cancer cell and PaSMC co-cultures. After 5 days in culture, endothelial tubules were visualized by staining with the antibody against human CD31 (green). When cultured with OVCAR-3 cells expressing shRNAs targeting HS6ST-1 (shST1–1) or HS6ST-2 (shST2–1), endothelial tubule formation was absent (lower images). Scale bars, 100 μm. G, endothelial tubule area that was visualized by anti-CD31 staining of HUVEC co-cultures with ES2 NS or sh6ST1-1 cells and PaSMC as shown in F was evaluated using ImageJ software. Control, which is expressed as 100%, represents the tube area formed in the presence of ES2 NS cells. Two independent experiments were performed, and the data are expressed as means ± S.D. *, p < 0.005.
FIGURE 3.
FIGURE 3.
Down-regulation of HS6ST-1 or HS6ST-2 reduces activation of FGF receptor and downstream signaling pathways in endothelial cells. A, serum-starved HUVEC were treated for 10 min with CM generated by ovarian cancer cells expressing NS shRNA or shRNAs targeting HS6ST-1 or HS6ST-2. Phosphorylated FRS2, ERK1/2, AKT, MEK1, and p38 were detected by Western blotting using specific antibodies. Equal protein loading was monitored by probing with the anti-GAPDH antibody. The images shown are representative of three independent experiments. B, CM derived from ES2 cells with down-regulated HS6ST-1 (sh6ST1-2) maintained in the presence of 10 μg/ml of HS for 24 h restored the activation of ERK1/2 in serum-starved HUVEC. C–F, FGF2 levels as determined by ELISA in the cell lysates of ovarian cancer cell lines expressing nonspecific shRNA (NS) or shRNAs targeting HS6ST-1 (sh6ST1-1/sh6ST1-2) or HS6ST-2 (sh6ST2-1). The data represent the means ± S.D. derived from two independent experiments. *, p < 0.0025, ‡, p < 0.05. G, FGF2 protein levels in conditioned media (CM) of ovarian cancer cell lines with down-regulated HS6ST-1 or HS6ST-2. The data are expressed as percentages of FGF2 levels in CM of NS control cells (100%) and represent the means ± S.D. derived from two independent experiments. ‡, p < 0.05.
FIGURE 4.
FIGURE 4.
Down-regulation of HS6ST-1 or HS6ST-2 in ovarian cancer cells causes a reduction in protein and mRNA levels of angiogenic cytokines. A, cytokine levels in OVCAR-3 and OVCAR-5 cell CM without and with HS6ST-1 or HS6ST-2 down-regulation. CM collected from control NS cells and cells expressing shRNAs targeting HS6ST-1 or HS6ST-2 were incubated with RayBio® human cytokine antibody array 3, and the levels of cytokines were visualized by chemiluminescence. B and C, IL-8 (B) and IL-6 (C) protein levels were determined by ELISA in cell lysates (CL) and CM of OVCAR-3 and OVCAR-5 NS, sh6ST1-1, or sh6ST2-1 cells. The data are expressed as pg of IL-8 and IL-6 normalized to 1 mg of total protein and represent the means ± S.D. (n = 2). *, p < 0.0025, ‡, p < 0.05. D, FGF2, IL-6, and IL-8 mRNA levels as determined by real time PCR in OVCAR-3 cells with down-regulated HS6ST-1 or HS6ST-2. The expression in NS cells represents 1. Two independent experiments were performed. Each experiment was performed in triplicate. The data are shown as the means ± S.D.
FIGURE 5.
FIGURE 5.
Neutralization of FGF2, IL-6, and IL-8 causes a reduction in HUVEC tubule formation in co-culture with NHDF. A, HUVEC endothelial tube formation as visualized by staining with the antibody recognizing human CD31. The experiment was performed using CM generated by OVCAR-3 nonspecific (NS) cells containing neutralizing antibodies against FGF2 (5 μg/ml), IL-6 (0.5 μg/ml), and IL-8 (0.5 μg/ml) and CM from OVCAR-3 sh6ST1-1 or sh6ST2-1 cells. Normal goat serum was used as a control for antibodies against IL-6 and anti-IL-8 and purified mouse IgG1 as a control for the antibody against FGF2. Scale bars, 100 μm. B, quantification of the tubule area in A. The data are expressed as percentages of the area of anti-CD31 staining per field in NS CM (100%) treatment and represent the means ± S.E. (n = 3). *, p < 0.0025; ‡, p < 0.05.
FIGURE 6.
FIGURE 6.
Inhibition of HB-EGF/EGFR signaling in ovarian cancer cells reduces IL-8 and IL-6 expression and inhibits endothelial tubule formation. A, levels of phosphorylated EGFR (pEGFR) in OVCAR-3 NS, sh6ST1-1/sh6ST1-2, and sh6ST2-1/sh6ST2-2 cell lysates as demonstrated by receptor tyrosine kinase phosphorylation antibody arrays. Densitometric examination showed a reduction in phosphorylated EGFR in OVCAR-3 cells with down-regulated HS6ST-1 and HS6ST-2 by 26 and 32%, respectively. B, HB-EGF levels in CM generated by OVCAR-3 NS cells, OVCAR-3 NS cells treated with AG1478 (1 μm), OVCAR-3 NS cells treated with neutralizing anti-HB-EGF antibody (1 μg/ml), and OVCAR-3 sh6ST2-1 cells were analyzed by ELISA. HB-EGF levels are expressed as pg/ml and represent the means ± S.D. derived from two independent experiments, each performed in duplicate. *, p < 0.03. C, pEGFR levels were analyzed by immunoprecipitating (IP) EGFR from untreated OVCAR-3 NS cells, OVCAR-3 NS cells treated with EGFR inhibitor AG1478 (1 μm) or neutralizing anti-HB-EGF antibody (1 μg/ml) and OVCAR-3 cells with down-regulated HS6ST-2 (sh6ST2-1). Western blotting was performed using the antibody against phosphorylated EGFR (Y1173). D, IL-8 concentrations in CM generated by OVCAR-3 and OVCAR-5 NS cells treated as in B and C. The data represent the means ± S.D. (n = 2). *, p < 0.006; ‡, p < 0.02. E, IL-6 concentrations in CM generated by OVCAR-3 and OVCAR-5 NS cells treated as in B and C. Each of the two independent experiments was performed in duplicate. The data represent the means ± S.D. (n = 2). ‡, p < 0.05. F, staining for human CD31 shows HUVEC tubule formation in a co-culture assay with NHDF in the presence of CM generated by OVCAR-3 NS cells, OVCAR-3 NS cells treated with AG1478 (1 μg/ml) or anti-HB-EGF antibody (1 μg/ml), and OVCAR-3 sh6ST2-1 cells. Scale bars, 100 μm. G, quantification of the effect of CM treatment shown in F. The area of staining for CD31 per field as determined using ImageJ software is shown. The data show the means (± S.E.) derived from three independent experiments, each performed in duplicate. *, p < 0.006. H, HB-EGF concentrations in CM generated by ovarian cancer cell lines were determined by ELISA. Two independent experiments were performed, each in duplicate. The data are shown as the means ± S.D. (n = 2). I, competition of the binding for HB-EGF to OVCAR-3 NS cell HS with unmodified or de-6-O-sulfated porcine intestinal mucosal HS. The control, which is expressed as 100%, represents HB-EGF binding to OVCAR-3 NS HS in the absence of competitors. The data show the percentages of HB-EGF bound to OVCAR-3 HS in the presence of increasing concentrations of the competitors and represent the means (± S.D.) derived from two independent experiments.
FIGURE 7.
FIGURE 7.
Down-regulation of HS6ST-2 affects tumor growth through impaired angiogenesis. A, OVCAR-3 parental cells, OVCAR-3 cells expressing NS shRNA (NS), and OVCAR-3 cells with down-regulated HS6ST-2 (sh6ST2-1) were grown as xenografts in NSG mice, and tumor volume was measured for 110 days. The data are expressed as the means ± S.E. (n = 5 xenografts). B, OVCAR-3 xenograft sections were stained for mouse CD31 (green) to visualize the infiltration of host vasculature and Hoechst 33342 (blue) to detect nuclei. Scale bars show 100 μm. C, evaluation of blood vessel density was performed with ImageJ program where the images were analyzed for CD31 staining. The number of vessels per normalized area was calculated and expressed as the means ± S.E. (n = 5 xenografts). *, p < 0.01. D, xenograft sections were stained for mouse α-SMA (red) and CD31 (green) to visualize blood vessel coverage with mural cells. Nuclei were visualized with Hoechst 33342 (blue). White arrows show CD31-positive blood vessels that lack α-SMA staining. Scale bars represent 100 μm. E, average number of vessels positive for mouse α-SMA staining was evaluated using the ImageJ program. The means ± S.E. (n = 5) are shown. *, p < 0.05. F–H, IL-6 (F), IL-8 (G), and FGF2 (H) protein levels in OVCAR-3, OVCAR-3 NS, and OVCAR-3 sh6ST2-1 xenograft lysates as determined by ELISA. Data show pg of cytokine normalized to 1 mg of total protein and represent the means ± S.E. (n = 5). *, p < 0.02.
FIGURE 8.
FIGURE 8.
A model of an autocrine and a paracrine regulation of ovarian cancer angiogenesis by HS6STs. The data generated in this study and our previous study (26) show that HS with reduced 6-O-sulfation directly inhibits FGF2 and VEGF165 ability to signal through endothelial cell receptors and negatively impacts HB-EGF/EGFR signaling and expression of angiogenic cytokines in ovarian cancer cells. BM, basement membrane; MC, mural cells.

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