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. 2015 Jan 27;14(1):15.
doi: 10.1186/s12943-014-0279-8.

Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

Hooi Ching Lim  1   2 , Hinke A B Multhaupt  3 , John R Couchman  4
Affiliations

Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

Hooi Ching Lim et al. Mol Cancer. .

Abstract

Background: Cell surface proteoglycans interact with numerous regulators of cell behavior through their glycosaminoglycan chains. The syndecan family of transmembrane proteoglycans are virtually ubiquitous cell surface receptors that are implicated in the progression of some tumors, including breast carcinoma. This may derive from their regulation of cell adhesion, but roles for specific syndecans are unresolved.

Methods: The MDA-MB231 human breast carcinoma cell line was exposed to exogenous glycosaminoglycans and changes in cell behavior monitored by western blotting, immunocytochemistry, invasion and collagen degradation assays. Selected receptors including PAR-1 and syndecans were depleted by siRNA treatments to assess cell morphology and behavior. Immunohistochemistry for syndecan-2 and its interacting partner, caveolin-2 was performed on human breast tumor tissue arrays. Two-tailed paired t-test and one-way ANOVA with Tukey's post-hoc test were used in the analysis of data.

Results: MDA-MB231 cells were shown to be highly sensitive to exogenous heparan sulfate or heparin, promoting increased spreading, focal adhesion and adherens junction formation with concomitantly reduced invasion and matrix degradation. The molecular basis for this effect was revealed to have two components. First, thrombin inhibition contributed to enhanced cell adhesion and reduced invasion. Second, a specific loss of cell surface syndecan-2 was noted. The ensuing junction formation was dependent on syndecan-4, whose role in promoting actin cytoskeletal organization is known. Syndecan-2 interacts with, and may regulate, caveolin-2. Depletion of either molecule had the same adhesion-promoting influence, along with reduced invasion, confirming a role for this complex in maintaining the invasive phenotype of mammary carcinoma cells. Finally, both syndecan-2 and caveolin-2 were upregulated in tissue arrays from breast cancer patients compared to normal mammary tissue. Moreover their expression levels were correlated in triple negative breast cancers.

Conclusion: Cell surface proteoglycans, notably syndecan-2, may be important regulators of breast carcinoma progression through regulation of cytoskeleton, cell adhesion and invasion.

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Figures

Figure 1
Figure 1
Heparinoids promote adhesion and reduced invasive activity. MDA-MB231 cultures were treated with 20 μg/ml heparan sulfate (HS), chondroitin sulfate (CS), heparin (Hep) or untreated (Ctrl) for 24 h after which cells were fixed and assessed for (A) F-actin (red) to detect microfilament bundles and paxillin (green) to detect focal adhesions (arrows), (B) spread cell areas (n ≥ 50 per condition), (C) adherens junction (arrows) components cadherin-11 (green) and p120-catenin (red). (D) Sensitivity of microfilament organisation to 30 μM of Rho kinase inhibitor, Y-27632 added for 30 min before fixation and staining for F-actin, Bars = 25 μm in A, C, D. (E) Representative western blot of diphosphorylated (Thr18/Ser19) myosin light chain (ppMLC) and total MLC from cells treated with glycosaminoglycans or untreated. Quantitation for ppMLC and total MLC levels relative to β-tubulin is shown under the blots. Similar results were obtained in three independent experiments. (F) F-actin organization in cells transfected with cDNAs encoding wild type MLC (pEGFP-MLC), phosphomimetic MLC (pEGFP-MLC Thr18Asp/Ser19Asp) or control vector (pEGFP). Phalloidin-stained cultures of the same areas are shown in the inserts. Bar, 25 μm. (G) Cells were plated onto type I collagen gel coated transwells in the presence or absence of glycosaminoglycans. After 24 h, invading cells were fixed, stained with DAPI and counted. (H) Cells were cultured on native type I collagen coated plates in the presence or absence of glycosaminoglycans for 48 h. After this period, cells were removed by trypsin and degraded areas were detected as clear zones by Coomassie Blue staining. Images at higher magnification are shown on the lower panels. Quantification of matrix degradation images is shown. Bar = 100 μm. Error bars = s.e.m. from three independent experiments. **p < 0.01, ***p < 0.001, n.s.: not significant. Significance was tested by one-way ANOVA with Tukey’s post-hoc test.
Figure 2
Figure 2
Effects of heparinoids partly results from inhibition of thrombin. MDA-MB231 cells were transfected with PAR-1 or control siRNA. (A) After 48 h, RNAs were extracted and subjected to qRT-PCR analysis to ascertain PAR-1 mRNA expression levels. siCtrl – control siRNA treatment. (B) PAR-1 depleted and control cells were incubated with glycosaminoglycans for 24 h, fixed and stained for F-actin. (C) Cell spreading induced by PAR-1 depletion and addition of glycosaminoglycans to PAR-1 depleted cells was quantified, n ≥ 50 cells per condition. (D) PAR-1 depleted and control cells were stained for paxillin, cadherin-11 and p120-catenin. Small focal adhesions are arrowed. (E). Levels of phosphomyosin light chain (ppMLC) are unchanged by PAR-1 depeletion by siRNA. Total myosin light chain (MLC) and β-tubulin are shown. (F) Quantification of PAR-1 and control siRNA treated cells that invaded type I collagen gels after 24 h. (G) Images and quantification of type I collagen degradation by control and PAR-1 depleted cells. Images at higher magnification are shown in the lower panels. Bars = 25 μm in B and D. Bar = 100 μm in G. Error bars = s.e.m. from three independent experiments. **p < 0.01, ***p < 0.001, n.s.: not significant. Significance was tested by one-way ANOVA with Tukey’s post-hoc test (C) or two-tailed paired t-test (A, F and G).
Figure 3
Figure 3
MDA-MB231 cell collagen gel invasion requires MMP14 and is slowed by antithrombin III treatment. (A) Various concentrations of antithrombin III (ATIII) were used to treat MDA-MB231 cells for 24 h followed by fixation and staining for F-actin. The representative images shown were cells treated with 3 μg/ml of ATIII. Bar = 25 μm. (B) Quantification of spread cell areas in response to ATIII treatment, n ≥ 50 cells per condition. (C-F) Cell invasion after 24 h (C, E) or degradation (D, F) of type I collagen gels after 48 h in the presence or absence of 3 μg/ml antithrombin III (C, D) or 50 μM MMP inhibitor GM6001 (E, F). Higher magnification images are shown in the lower panels of D and F. Bars =100 μm. (G) Cell lysates were prepared 48 h after transfection with siRNA targeting MMP14 or control siRNA. Western blotting confirmed the siRNA-mediated MMP14 depletion in the treated cells. (H, I) MMP14 depleted cells were subjected to type I collagen cell invasion and degradation assays. Higher magnification images are shown in the lower panel (I). (J) Relative cell surface expression of MMP14 in PAR-1 depleted cells was assessed by flow cytometry. Error bars = s.e.m. from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.; not significant by two-tailed paired t-test.
Figure 4
Figure 4
Syndecan-2 is a regulator of MDA-MB231 cytoskeleton and behaviour. (A) Specific knockdown of mRNA levels after syndecan-1, −2 or −4 siRNA treatments. mRNA was extracted from cells 48 h after transfection with syndecan-1, syndecan-2, syndecan-4 or control siRNAs. Levels of other syndecan mRNAs were not affected by syndecan-2 knockdown (left). (B) Flow cytometry analysis confirmed loss of cell surface syndecan-2 after silencing syndecan-2 with siRNA. (C) Syndecan-2 depleted cells were stained for F-actin, which showed extensive microfilament bundle formation. Bar = 25 μm. (D) Spread cell areas were increased by syndecan-2 depletion, n ≥ 50 cells per condition. (E) Cadherin-11 containing adherens junctions were visible after syndecan-2 depletion. Bar = 25 μm. (F, G) Type I collagen invasion (F) and degradation (G) were reduced in syndecan-2 depleted cells. Higher magnification images are shown on the lower panels of G. Bar = 100 μm. (H) Cells depleted of each syndecan individually or in combination, by siRNA treatment were stained for focal adhesions (arrows) with a paxillin antibody. Quantification of focal adhesion (FA) size and number was done by one-way ANOVA with Tukey’s post-hoc test. Bar = 25 μm. Error bars = s.e.m. from three independent experiments. ***p < 0.001, n.s.; not significant by two-tailed paired t-test.
Figure 5
Figure 5
Heparan sulfate treatment causes loss of syndecan-2 and gain of syndecan-4 on the cell surface. MDA-MB231 cells were analysed at 2, 16 and 24 h after treatment with 20 μg/ml heparan sulfate for cell surface levels of syndecans-1, −2 and −4. Control cells were untreated. FACS analysis showed decreased syndecan-2 to near background, while levels of syndecan-4 increased. Syndecan-1 levels were unchanged throughout the treatment.
Figure 6
Figure 6
Caveolin-2 interacts with syndecan-2 and regulates cell adhesion in MDA-MB231 cells. (A) Syndecan-2, but not syndecan-4, was co-immunoprecipitated with caveolin-2 from cell lysates. Co-immunoprecipitation was not affected by ectopic glycosaminoglycan treatment of the cells (UT; untreated, CS; chondroitin sulfate, HS; heparan sulfate). (B) Confocal laser scanning microscopy and profile of the line scanning (white arrow on image) confirmed partial co-localization of syndecan-2 (green) and caveolin-2 (red). (C) Caveolin-2 levels were reduced where syndecan-2 was depleted by siRNA, compared with control siRNA. (D) Western blotting verified the knockdown efficiency of siRNA targeting caveolin-1 and −2 compared to control siRNA. Downregulation of caveolin-1 reduced the expression of caveolin-2 by around 30%, but knockdown of caveolin-2 had no impact on caveolin-1 levels. (E) F-actin containing microfilament bundles were abundant after caveolin-2, but not caveolin-1 depletion. Bar = 25 μm. (F) Spread cell areas were measured in caveolin-2 depleted cells and control cells, n ≥ 50 per condition. (G) Adherens junctions and focal adhesions (arrows) were characteristic of caveolin-2 depleted cells, shown by cadherin-11 and p120-catenin, or paxillin distributions. Bar = 25 μm. (H) Microfilament bundles formed in response to either syndecan-2 or caveolin-2 knockdown were sensitive to 30 μM Rho kinase inhibitor, Y-27632. Bar = 25 μm. (I) Diphosphorylated myosin light chain (ppMLC; Thr18/Ser19) was enhanced in both syndecan-2 and caveolin-2 depleted cells. Densitometry analysis of western blots for ppMLC and total MLC were normalised to β-tubulin. Similar results were obtained in three independent experiments. (J, K) Caveolin-2 depleted cells had reduced ability to invade or degrade type I collagen gels. Higher magnification images are shown in the lower panels. Caveolin-1 depletion also reduced collagen gel invasion. Bar = 100 μm. Error bars = s.e.m. **p < 0.01, ***p < 0.001, n.s.; not significant by two-tailed paired t-test.
Figure 7
Figure 7
Exogenous heparan sulfate influences subcellular localisation of caveolin-2. (A) Detergent-resistant membrane (DRM) preparations were made from MDA-MB231 lysates, some treated with 20 μg/ml heparan sulfate for 24 h. Nine fractions were collected from each centrifuge tube and western blotted for flotillin as a marker of DRM pools, transferrin receptor as a marker of detergent soluble membrane proteins (non-DRM), and caveolin-2. The caveolin-2 is almost exclusively in the DRM pool of control cells, while it is displaced to the non-DRM pool when cells are treated with heparan sulfate. Syndecan-2 is reduced after heparan sulfate treatment (B) Caveolin-2 staining of untreated (Ctrl) MDA-MB231 cells and cells treated with 20 μg/ml heparan sulfate (HS) for 24 h before fixation. While the HS-treated cells are more spread than control cells, there is no obvious alteration in the localisation of caveolin-2. Bar = 25 μm.
Figure 8
Figure 8
Syndecan-2 expression correlates with metastatic status in patient. (A) Normal breast tissues (Normal) and breast cancer patient tissues (Cancer) from tissue microarrays were stained for syndecan-2 or caveolin-2 on serial sections. Magnified images are shown in lower panels, bars = 100 μm. (B) Intensity per area for syndecan-2 or caveolin-2 staining was quantified for each core in tissue microarray to compare normal breast tissue and patient breast tissue of different tumour grades, n = 161 for syndecan-2 and n = 165 for caveolin-2. Bar graph was plotted in ln scale. (C) Breast tissue from patients diagnosed with Grade II carcinoma and metastatic lymph node tissue from the same patient were stained for syndecan-2 and caveolin-2. Higher magnification images are shown in lower panels, bars = 100 μm. (D) Quantification data of intensity per area of syndecan-2 or caveolin-2 staining from (C), n = 26 for syndecan-2 cases and n = 28 for caveolin-2 cases. Bar graph was plotted in ln scale. Error bars = s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant, tested by one-way ANOVA with Bonferroni post-hoc test. (E) Levels of syndecan-2 and caveolin-2 staining from serial sections of the 47 triple negative breast carcinoma cases were significantly correlated. r: Spearman’s correlation coefficient = 0.328, p = 0.024.
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