Shed syndecan-2 enhances tumorigenic activities of colon cancer cells.

Because earlier studies showed the cell surface heparan sulfate proteoglycan, syndecan-2, sheds from colon cancer cells in culture, the functional roles of shed syndecan-2 were assessed. A non-cleavable mutant of syndecan-2 in which the Asn148-Leu149 residues were replaced with Asn148-Ile149, had decreased shedding, less cancer-associated activities of syndecan-2 in vitro, and less syndecan-2-mediated metastasis of mouse melanoma cells in vivo, suggesting the importance of shedding on syndecan-2-mediated pro-tumorigenic functions. Indeed, shed syndecan-2 from cancer-conditioned media and recombinant shed syndecan-2 enhanced cancer-associated activities, and depletion of shed syndecan-2 abolished these effects. Similarly, shed syndecan-2 was detected from sera of patients from advanced carcinoma (625.9 ng/ml) and promoted cancer-associated activities. Furthermore, a series of syndecan-2 deletion mutants showed that the tumorigenic activity of shed syndecan-2 resided in the C-terminus of the extracellular domain and a shed syndecan-2 synthetic peptide (16 residues) was sufficient to establish subcutaneous primary growth of HT29 colon cancer cells, pulmonary metastases (B16F10 cells), and primary intrasplenic tumor growth and liver metastases (4T1 cells). Taken together, these results demonstrate that shed syndecan-2 directly enhances colon cancer progression and may be a promising therapeutic target for controlling colon cancer development.


Plasmid constructs
The vector construct encoding of Flag-tagged syndecan-2 was kindly provided by Dr. Yamaguchi Y. (Sanford-Burnham Medical Research Institute). In the current study, site-directed mutagenesis of the syndecan-2 extracellular proteolytic cleavage site [9] was performed using the QuikChange kit (Stratagene, CA, USA) according to the manufacturer's protocols. The Leucine residue at position 149 of the syndecan-2 extracellular domain was replaced with Isoleucine using the following primers that were deigned against the syndecan-2 mRNA of Rattus norvegicus (GenBank accession number NM_013082.3): forward 5′-ACGTG TACACCGAGAAACATTCAGACAATATCTTCAAGC GG-3′ and reverse 5′-CCGTCCGCTTGAACGAATTGT CTGAATGTTTCTCGGTGTAC-3′.

Flow cytometry
Colon cancer cells were treated with indicated synthetic peptide for 24 hr, then washed with PBS and released 1 mM EDTA-5% FBS in PBS. After centrifugation, cells were resuspended in PBS and washed with PBS three times. Cells were incubated with anti-syndecan-2 monoclonal antibody in 10% FBS in PBS for overnight at 4°C. Followed by washing three times with PBS containing 0.05% Tween-20, cells were incubated FITC-conjugated anti-mouse antibody in 10% FBS in PBS for 1 hr at 25°C. Syndecan-2 expression on cell surface was analyzed by flow cytometry.

Slot blotting
Either conditioned media or serum of cancer patients was slot-blotted onto a nitrocellulose membrane in a Bio-Rad apparatus (Bio-Rad, Hercules, CA, USA) and washed once with PBS. The membrane was then blocked in 0.05% Tween-20 in Tris-buffered saline (TBS: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) with 5% skim milk, washed, and probed with appropriate primary antibodies for 24 hr at 4°C, followed by species-specific horseradish peroxidaseconjugated secondary antibodies from AbClon. The signals were detected by enhanced chemiluminescence (ECL; AbClon).

Migration assay
For Transwell migration assay, the lower surface of Transwell inserts (Corning) was coated with gelatin (10 μg/ml), and the membranes were allowed to dry for 1 hr at room temperature. The Transwell chambers were assembled on a 24-well plate, and the lower chambers were filled with fibroblast growth factor-2 (100 ng/ ml) in fresh media. Cells were added to each upper chamber, and the plates were incubated at 37°C in 5% CO 2 for 16 hr (HCT116 cells), 30 hr (HT29 cells), and 6 hr (B16F10 cells). The cells that had migrated to the lower surface of the filters were stained with 0.6% hematoxylin and 0.5% eosin, and counted. Four visual fields were randomly chosen, and the migrated cells were counted under a light microscope. The data are presented as the average cell number per field. For the studies of cell migration in real time, the xCELLigence system (Roche Diagnostics GmbH, Switzerland) was used. To examine cell migration, the lower chambers of a CIM-plate 16 (8-μm pore size) were filled with fresh medium containing 10% FBS. The upper chambers were filled with serum-free medium (30 μl/well), and the plate was incubated at 37°C in 5% CO 2 for 1 hr. The background was measured using the RTCA DP Analyzer. The HCT116 (2.5 × 10 4 cells/well) or HT29 cells (3 × 10 4 cells/well) were added to each well, and the plate was incubated at 25°C. After 30 min, the CIMplate was assembled onto the RTCA DP Analyzer, and cell migration was assessed at 5 min intervals at 37°C in 5% CO 2 . The obtained data were analyzed using the provided RTCA software.

Purification of the recombinant 6-His-tagged syndecan-2 extracellular domain
The expression vector pET32a-syndecan-2 extracellular domain (His-SDC2E, amino acids 29-154) was established and transformed into E. coli BL21 (DE3). Cells were allowed to grow until they reached the optical density of around 0.6-0.8 at 600 nm and induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM for 16 hr at 25°C. The expression of Tobacco Etch Virus (TEV) protease was induced by adding IPTG to a final concentration of 1 mM for 6 hr at 37°C. After induction with IPTG, the cells were harvested by centrifugation at 6,000 xg for 30 min and resuspended in lysis buffer (20 mM sodium phosphate buffer pH 7.5 containing 500 mM NaCl, 5 mM β-mercaptoehanol, 1% TX-100), and then lysed by sonication on ice. The lysates was centrifuged at 14,000 xg for 30 min and supernatant was loaded onto Ni-NTA affinity column (Qiagen) according to the protocol specified by the manufacturer. The loaded column was washed with lysis buffer with 30 mM imidazole and His-SDC2E was eluted with the elution buffer (20 mM sodium phosphate buffer pH 7.5 containing 300 mM NaCl, 5 mM β-mercaptoehanol, 500 mM imidazole, 1% TX-100, pH 7.5). The elution fraction was dialyzed and incubated with TEV protease for 6 hr at 25°C. The digested products were purified through Ni-NTA affinity column and analyzed by SDS-PAGE.

Cell proliferation assay
Proliferation of HCT116 cells was measured by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) as described [29] in the presence or absence of purified syndecan-2 fragments. This assay was tested on three separate occasions with triplicate samples, and the absorbance values were averaged.

Anchorage-independent growth in soft agarose
Each well of a 6-well culture plate was coated with 3 ml of bottom agar mixture (McCoy's 5A, 10% FBS, 0.6% agar). After the bottom layer had solidified, 1 ml of top agar mixture (McCoy's 5A, 10% FBS, 0.3% agar) containing either HCT116 or HT29 cells (1 × 10 5 cells/well) was added to each well with various concentrations of shed syndecan-2 fragments, and the cultures were incubated at 37°C in a 5% CO 2 atmosphere. Colony formation was monitored daily with a light microscope. Colonies in soft agar were photographed after incubation for 14 days with a digital camera. Colonies were fixed with methanol, stained with 0.1% crystal violet, and colonies with a diameter greater than 20 μm were counted in each well under a microscope. melanoma cells were transfected with VEC, Flag-WT-or -NC-syndecan-2. Syndecan-2 mRNA expression was evaluated by RT-PCR using the indicated primer using β-actin as a control (top). Conditioned medium was subjected to slot blotting with the anti-Flag antibody. Ponceau S staining was done to determine loading for protein blots (bottom). (B) Transfected cells were allowed to migrate on gelatincoated (10 μg/ml) Transwell plates, and migrated cells were stained with hematoxylin and eosin. Data are shown as mean ± s.d., n = 3; **p < 0.01. (A) His-tagged thioredoxin (Trx)-syndecan-2 extracellular domain (His-Trx-SDC2E) fusion proteins were affinity purified over Ni-NTA columns and incubated with TEV protease. Each fractions and cleavage products were separated by 15% SDS-PAGE and CBB R-250 stained. Arrowhead indicates His-removed SDC2E, arrows indicate His-Trx-SDC2E or His-Trx. (B) HT29 and HCT116 cells were treated with 0.2 μg/ml of purified recombinant SDC2E, and allowed to migrate on RTCA CIM-Plate wells. (C) HCT116 cells were treated with 0.2 μg/ml of purified recombinant SDC2E, and allowed to migrate on Transwell apparatus. Data are shown as mean ± s.d., n = 3; **P < 0.01. (D) HCT116 cells were treated with 0.2 μg/ml of purified recombinant SDC2E, and cell numbers were evaluated with MTT assay for the indicated times. The experiments were performed in duplicate, with triplicate samples. (E) Mixture of indicated cells (1 × 10 5 cells/well) with 0.2 μg/ml of purified recombinant SDC2E was added to bottom agar and colonies were counted after 2 weeks. Data are shown as mean ± s.d., n = 3; **P < 0.01.