Serglycin is implicated in the promotion of aggressive phenotype of breast cancer cells

Abstract

Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients' material. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ~ 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (~ 87%), 6-sulfated (~ 10%) and non-sulfated (~ 3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion.

Figure 1. Immunohistochemical reactivity of serglycin in normal breast and breast carcinoma.

Normal epithelial breast cells in mammary glands were moderately reactive with the polyclonal antibody against serglycin and the reactivity was cytoplasmic (A). Breast cancer cells were stained strongly for serglycin in grade 2 breast carcinomas (B–D). The serglycin reactivity was mainly cytoplasmic, although in some cases cell-surface associated staining was detected in breast cancer cells (D, arrowheads and insert). Bars, 50 µm.

Figure 2. Serglycin is highly expressed in aggressive breast cancer cell lines.

(A) Expression of serglycin across the 51 individual breast cancer cell lines grouped in the basal A (red), basal B (grey) and luminal (blue) subgroups. (B) Detection of serglycin gene transcripts in low aggressive MCF-7 (luminal), medium aggressive MDA-MB-468 (basal A) and high aggressive MDA-MB-231 (basal B) breast cancer cells (bold and underlined in figure 2A) cultured in the presence or absence of serum by RT-PCR. The PCR products were analyzed on 1% agarose gels stained with GelRed. (C). Subcellular distribution of serglycin in breast cancer cells. Immunofluorescence staining was done for serglycin (green), nuclei (blue) and F-actin (red) in high aggressive MDA-MB-231 and low aggressive MCF-7 breast cancer cells after permeabilization. MCF-7 cells stained faintly for serglycin in the cytoplasm. Serglycin was found in the perinuclear regions and juxtanuclear area typical of the Golgi complex in MDA-MB-231 cells (arrowheads), and in the cytoplasm and at the cell periphery (arrows). Serglycin distribution was observed in vesicular (V) and vesiculotubular structures (VST) in the cytoplasm (insert). Bars, 25 µm.

Figure 3. Serglycin is secreted to the culture medium by aggressive breast cancer cells.

Equal amounts of protein from concentrated cell culture supernatants of breast cancer cell lines correspond to known number of cells were treated with chondroitinase ABC and subjected to Western blot analysis for serglycin using chondroitinase ABC digested standard serglycin (SG) as positive control (A). (B) Various amounts of standard SG measured as protein from the CAG myeloma cell line and known amounts of protein from concentrated cell culture supernatants from MDA-MB-231 breast cancer cells correspond to known number of cells (two independent experiments) were treated with chondroitinase ABC and were simultaneously analyzed for the presence of SG by Western blotting. (C) A standard curve was created each time by plotting protein band density against the amount of SG subjected to Western blot analysis. A linearity in the standard curve was obtained for amounts up to 24 ng of loaded SG protein (R² = 0.998), and the curve was used for the quantification of SG present in the culture medium of MDA-MB-231 cells.

Figure 4. Fractionation of PGs secreted from the MDA-MB-231 breast cancer cell line by anion-exchange and gel permeation chromatographies.

One liter of conditioned medium was fractionated on a DEAE-Sephacel column (40 ml bed volume). The column was eluted stepwise with 3 volumes of the formamide buffer described under “Experimental Procedures” containing 0.2 M NaCl and 10 volumes of a NaCl linear gradient ranging from 0.2 to 1.0 M NaCl. Fractions of 6.7 ml were collected, and aliquots were precipitated by the addition of ethanol in the presence of potassium acetate. Precipitates were dissolved in distilled water, analyzed for their GAG content by the DMMB method (A), and subjected to SDS-PAGE analysis using a 4% stacking - 10% separating gel (B). Gels were stained with toluidine blue, followed by staining with coomassie blue. Serglycin was detected throughout the dissolved aliquots by Western blotting analysis after chondroitinase ABC digestion using a polyclonal antibody against serglycin (C) (SG, standard of serglycin used as positive control). A major PG peak was eluted from 0.50 to 0.7 M NaCl and pooled as indicated by the bar. Pooled serglycin was further fractionated by gel permeation chromatography (D). The pooled fractions were chromatographed on a Sepharose CL-4B column. The column was eluted with 4 M guanidine hydrochloride, 50 mM sodium acetate buffer, pH 5.8. Fractions were collected, and PGs were monitored by the DMMB method (D), were subjected to SDS-PAGE following toluidine blue and coomassie staining (E), and were analyzed by Western blotting after chondroitinase ABC digestion (F) (SG, standard of serglycin used as positive control).

Figure 5. Serglycin inhibits the classical and lectin pathways of complement and binds to complement components C1q and MBL.

(A) Serglycin inhibits the classical pathway. NHS was pre-incubated with increasing concentrations of total glycanated serglycin isolated from MDA-MB-231 breast cancer cells and the CAG myeloma cell line (positive control) and mixed with Ab-sensitized sheep erythrocytes. No impact on hemolysis was observed when BSA was used (negative control). Erythrocyte lysis was evaluated after 1 h by measuring the amount of released hemoglobin at 405 nm. (B) Inhibition of the alternative pathway was measured by subjecting rabbit erythrocytes to NHS pre-incubated with serglycin isolated from MDA-MB-231 breast cancer cells and the CAG myeloma cell line and erythrocyte lysis was determined as above. For the alternative pathway, Factor H (FH) and BSA were used as positive and negative controls, respectively. (B and C) To examine the inhibition of the lectin pathway, NHS was pre-incubated with increasing concentrations of total glycanated serglycin isolated from MDA-MB-231 breast cancer cells, D-mannose (positive control) or BSA (negative control) and added to mannan-coated wells. Deposition of C4b (E) and C3b (F) was measured by ELISA. Data were normalized by setting the obtained absorbance for NHS without inhibitor to 1. All data are given as means and SD of three independent assays. Statistical significance of variances compared to negative control was calculated using using a two-way ANOVA test and a Bonferroni post-test (*p<0.05). Wells coated with total glycanated serglycin isolated from MDA-MB-231 breast cancer cells or BSA (negative control) were incubated with increasing concentrations of purified C1q (E) and MBL (F) for 1 h at RT, and bound C1q and MBL were detected with ELISA. Data are given as means and SD of three independent experiments. Scatchard-type plots representing the binding of serglycin isolated from breast cancer to C1q (G) and MBL (H). Data are given as means and SD of three independent experiments.

Figure 6. Characterization of serglycin expression in stably transfected MCF-7 cells.

Detection of serglycin gene transcripts in MCF-7 breast cancer cells carrying empty vector (MCF-7V), vector with serglycin cDNA (MCF-7VSG) and vector coding serglycin cDNA lacking GAG attachment sites (MCF-7VSG/−GAG) by RT-PCR. The PCR products were analyzed on 1% agarose gels stained with GelRed nucleic acid gel stain (A). The ratio of serglycin (SG) to β-actin was determined following quantification of bands’ densities using Scion Image software (B). Statistical significance of variances was calculated using using a one-way ANOVA test (*p<0.05). (C) Immunofluorescence staining for serglycin (green) and nuclei (blue) in stably transfected MCF-7V, MCF-7VSG and MCF-7VSG/−GAG cells. Cells were cultured in chamber slides, then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 and incubated with a polyclonal rabbit anti-serglycin antibody. Bars, 25 µm. (D) Equal amounts of protein from concentrated cell culture supernatants from stably transfected MCF-7V, MCF-7VSG and MCF-7VSG/−GAG cells correspond to known number of cells were subjected to Western blot analysis using a polyclonal antibody against serglycin without (D) or after treatment with chondroitinase ABC (E). (D) MCF-7VSG secreted to the culture medium a high molecular mass population, which remained on the top of the separating gel and represents highly glycanated serglycin, and low glycanated sub-populations with molecular masses ranging from 170 kDa to 70 kDa. Furthermore, MCF-7VSG and MCF-7VSG/−GAG cells secreted a core protein of 48 kDa that might represent non-glycanated serglycin core containing GFP-tag. (E) When samples were treated with chondroitinase ABC, MCF-7VSG cells found to secrete a major protein core of 58 kDa (1) that might represent the core protein of highly glycanated serglycin containing oligosaccharides remaining after chondroitinase ABC treatment and the GFP-tag. Minor protein cores of 52–48 kDa (2) secreted from MCF-7VSG and a protein core of 48 kDa (arrowhead) secreted from MCF-7VSG/−GAG cells might represent the low glycanated or/and non-glycanated core proteins of serglycin containing the GFP-tag. Arrow shows the top of the separating gel.

Figure 7. Increased cell proliferation and colony formation in MCF-7 cells over-expressing serglycin.

(A) For the evaluation of cell proliferation on adhesive matrix, 1.5×10⁴ of stably transfected cells were plated in triplicates in 24 well plates and incubated for several time points in normal culture conditions. MTT was added in wells and cells were incubated for 2 h at 37°C. The absorbance was measured at 570 nm and related to the number of cells by using a standard curve. Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using using a one-way ANOVA test (*p<0.05). (B) For the colony formation assay, 5×10⁴ cells were cultured on agarose for 12 days and then stained with 0.05% crystal violet and photographed. (C) Photographs from six random fields were taken, and cell colonies were counted using Image J software. Data are given as means and SD of three independent experiments. Statistical significance of variances between MCF-7VSG and MCF-7V or MCF-7VSG/−GAG cells was calculated using a one-way ANOVA test (*p<0.05).

Figure 8. Over-expression of glycanated serglycin increases breast cancer cell migration.

Cells (5×10⁵) were plated in triplicates in 12 well plates and cultured until confluence. Then wounds were made using a sterile pipette tip, debris was removed and fresh culture medium was added. The cells were monitored at 0, 24 and 48 h and were photographed (A). Wound areas were quantified at various time intervals using Image J software (B). Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (C) Migratory properties of the cells were also evaluated by Transwell migration assay. 1×10⁵ cells were suspended in culture medium supplemented with 0.5% FBS and loaded onto the top of Transwell chambers. Cells were then maintained in Transwell chambers for 48 h with 10% FBS as chemotactic stimuli in the bottom chamber. Transmigrating cells were stained with Giemsa and counted. Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (D) In a set of experiments, cells were cultured on glass coverslips. After wounding with a sterile pipette tip, debris was removed, and cells were either immediately fixed in 3% formaldehyde in PBS (t = 0 h) or cultured for 24 h and then fixed. Immunofluorescence staining for serglycin (green), nuclei (blue) and F-actin (red) in MCF-7VSG cells was performed. Bars, 25 µm.

Figure 9. Serglycin promotes breast cancer cell invasion.

1×10⁵ cells were suspended in culture medium supplemented with 0.5% FBS and loaded onto the top of Transwell chambers equipped with matrigel-coated cell culture inserts. As chemotactic stimuli in the bottom chambers was used culture medium supplemented with 10% FBS. After 72 h of incubation, cells on the upper surface of the filter were mechanically removed with a cotton swab, and those which invaded underneath the surface were stained with Giemsa and counted. (A) Representative photos of cells invaded through matrigel and quantification (B). Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05).