- Open Access
Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells
© Lim et al.; licensee BioMed Central. 2015
Received: 22 July 2014
Accepted: 22 December 2014
Published: 27 January 2015
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.
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.
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.
Cell surface proteoglycans, notably syndecan-2, may be important regulators of breast carcinoma progression through regulation of cytoskeleton, cell adhesion and invasion.
Metastasis is a multistep process involving dissemination of cancer cells from a primary tumour to distant organs . During metastasis from solid tumours, cells change their adhesion status to facilitate migration through basement membrane and extracellular matrix, enter the bloodstream or lymphatics, extravasate into distant organs and eventually proliferate to form metastases . Understanding the molecular basis of tumour cell motility and invasion is crucial to identify attractive targets for potential therapeutic intervention.
In mammals, syndecans are a four-member family of cell surface heparan sulfate proteoglycans that are well placed to be important regulators of cell migration and tumor progression. While expression of each syndecan has some tissue- and cell type- specificity, they can bind to a wide range of proteins including growth factors, cytokines, chemokines, morphogens, extracellular matrix proteins, proteinases and proteinase inhibitors through their heparan sulfate chains [3,4]. Syndecans have an ability to regulate cell motility, cell-cell and cell-extracellular matrix adhesion through signaling to the actin cytoskeleton, with which all syndecans can interact [5,6]. Additionally, the core protein of syndecans can directly or indirectly promote integrin-mediated adhesion and integrin turnover [7-11]. Although not possessing intrinsic kinase activity, the syndecan proteoglycans can nevertheless signal through their cytoplasmic domains, mediated by specific binding partners, e.g. protein kinase Cα in the case of syndecan-4 [12-14].
In tumors, syndecan expression is frequently altered during malignant transformation and may contribute to tumor progression [15-18]. For example, syndecan-1 expression and its shedding from the cell surface appear to relate directly to myeloma progression [15,19]. Syndecan-4 may play differing roles in modulating tumor cell invasiveness depending on the cancer type. In breast carcinoma, syndecan-1 can promote cell spreading and adhesion to extracellular matrix with subsequent inhibition of cell invasion . We showed that syndecan-4 expression in human breast carcinoma tissues correlates with positive estrogen and progesterone receptor status therefore a good prognosis . Conversely, syndecan-1 expression in breast carcinoma is an indicator of poor prognosis, particularly where it is stromal [21,22]. Syndecan-2 expression is upregulated in colon cancer, pancreatic cancer, melanoma and fibrosarcoma where it enhances cell adhesion, proliferation and migration in cancer cells, suggesting that it is important in promoting tumor progression [23-26]. However, while emerging evidence suggests that syndecans have prominent regulatory roles in cancer cell behaviour, the molecular basis of these effects remains mostly obscure.
Here we show that cell surface heparan sulfate proteoglycans have multiple roles in governing invasive behavior of the MDA-MB231 breast carcinoma cell line. Specifically, a key role for a syndecan-2/caveolin-2 axis is identified and characterized. Moreover, both of these components are upregulated in breast cancers, shown by tissue microarrays.
Materials and methods
Human MDA-MB231 cells were grown in Dulbecco’s modified Eagle’s Media from Life Technologies (Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cultures were screened routinely for mycoplasma contamination.
Antibodies and reagents
The following antibodies were used: MMP14 (cat. no. ab-3644) and syndecan-1 (clone B-A38) from Abcam (Cambridge, UK); syndecan-2 (cat. no. H00006383-B04P) from Abnova (Taiwan); syndecan-2 (cat. no. LSB2981) and syndecan-4 (cat. no. LS-C150078) from LSBio (Seattle, WA, USA); caveolin-2 (D4A6) (cat. no. 8522), cadherin-11 (P707) (cat. no. 4442), MLC (cat. no. 3672) and phospho-MLC (Thr18/Ser19) (cat. no. 3674) from Cell Signaling (Beverly, MA, USA); caveolin-1 (clone 2297, cat. no. 610406) and flotillin (cat. no. 610820) from BD Biosciences (San Diego, CA, USA); p120-catenin (clone 6H11, cat. no. 339700) and transferrin receptor (clone H68.4, cat. no. 136800) from Life Technologies (Carlsbad, CA, USA); paxillin (clone 5H11, cat. no. 05–417) from Millipore (Billerica, MA, USA); β-tubulin (clone TUB2.1, cat. no. T4026) from Sigma-Aldrich (St Louis, MO, USA); Alexa Fluor-conjugated phalloidin and secondary antibodies used in immunofluorescence analysis were obtained from Life Technologies and peroxidase-conjugated secondary antibodies used in all western blotting analysis were purchased from Dako (Glostrup, Denmark).
The metalloproteinase inhibitor GM6001, DMSO, heparan sulfate from bovine kidney, chondroitin sulfate A from bovine trachea, and heparin from bovine intestinal mucosa were purchased from Sigma-Aldrich (St Louis, MO, USA). ROCK inhibitor (Y-27632) was obtained from Calbiochem (Darmstadt, Germany) and human antithrombin III was purchased from Alpha Diagnostic International (San Antonio, TX, USA).
siRNA and DNA transfections
Cells were transfected with siRNA targeting syndecan-1 (sc-36587, Santa Cruz Biotechnology, CA, USA), syndecan-2 (sc-41045, Santa Cruz Biotechnology, CA, USA), syndecan-4 (5′-ggccgauacuucuccggaguu-3′, Qiagen, Frederick, MD, USA), MMP14 (5′-caggcaaagcugaugcagauu-3′, Qiagen), PAR-1 (5′-aaggcuacuaugccuacuacuuu-3′, Thermo Fisher Scientific, Waltham, MA, USA), caveolin-1 (siGENOME SMARTpool, Thermo Scientific), caveolin-2 (siGENOME SMARTpool, Thermo Scientific) or non-targeting siRNA (siGENOME SMARTpool, Thermo Scientific) using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions. Cells were analyzed 48 h after transfection.
RNA isolation and quantitative reverse transcription-polymerase chain reaction
Total RNA was isolated using the RNeasy Micro Kit (Qiagen). Reverse transcription was performed with 1 μg of RNA by using TaqMan reverse transcription kit (Life Technologies) according to manufacturer’s protocol and quantitative PCR was performed using the Maxima SYBR Green qPCR Master Mix (Thermo Scientific) with the following primers:
syndecan-1 (forward: 5′-tactaatttgccccctgaagat-3′, reverse: 5′-caaggtgatatcttgcaaagca-3′), syndecan-2 (forward: 5′-actgttgactagtgctgctcca-3′, reverse: 5′-gggtccattttcctttctgagt-3′), syndecan-3 (forward: 5′-aagagtatcctggagcggaag-3′, reverse: 5′-agatgagcagtgtgaccaagaa-3′), syndecan-4 (forward: 5′-gtgtccaacaaggtgtcaatgt-3′, reverse: 5′-cggtacatgagcagtaggatca-3′), PAR-1 (forward: 5′-acttgatcctggccacagac-3′, reverse: 5′-acttgatcctggccacagac-3′), RPLPO (forward: 5′-ttcattgtgggagcagac-3′ reverse: 5′-cagcagtttctccagagc-3′).
Flow cytometry analysis
Cells were harvested with cell dissociation buffer (Life Technologies), re-suspended in ice-cold sterile filtered 1% BSA/PBS and incubated at 4°C for 30 min in the presence of syndecan-1 (1:50 dilution) syndecan-2 (1:100 dilution) or syndecan-4 (1:50 dilution) antibodies or MMP14 (2 μg/ml) antibody. Cells were washed with ice-cold sterile filtered 1% BSA/PBS and further incubated with Alexa Fluor 488-conjugated secondary antibody for 30 min on ice. Following washing with ice-cold sterile filtered 1% BSA/PBS, cells were analysed on a FACSCalibur flow cytometer and data processed by using CellQuest Pro v6.0 software (Becton Dickinson, Franklin Lakes, NJ, USA). Time course experiments were organized so that the harvesting and staining procedures were synchronised.
Cells were plated on glass coverslips in complete medium (10% FBS) and after 24 h were then treated for 24 h with 20 μg/ml heparan sulfate, heparin or chondroitin sulfate, either in serum-free or serum-containing medium. Control cultures were not treated with glycosaminoglycans. Cultures were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. After washing with PBS, free aldehydes were quenched with 0.1 M ammonium chloride, followed by blocking in 5% heat-denatured BSA. Cells were incubated with Alexa Fluor-conjugated phalloidin (1:1000) and/or primary antibody recognising cadherin-11 (1:100), p120-catenin (1:100) or paxillin (1:100) overnight at 4°C. After primary incubation, cells were washed with PBS and incubated with an appropriate fluorescent conjugated secondary antibody (1:2000) for 1 h at room temperature. Coverslips were mounted with ProLong antifade mounting medium (Life Technologies) and analysed on a Zeiss Axioplan-2 microscope (Carl Zeiss, Oberkochen, Germany). Images were processed using Metamorph (version 6.2r6) and Adobe Photoshop (version 11.0.2). ImageJ (version 1.44p) was used to quantify cell areas. For co-localization studies of syndecan-2 and caveolin-2, cells were double-stained with antibodies against syndecan-2 (1:100) and caveolin-2 (1:100), followed by Alexa-conjugated secondary antibodies. Images were acquired using a Zeiss LSM-510 confocal microscope (Carl Zeiss) equipped with a diode laser (405 nm), an argon laser (488 nm) and two helium-neon lasers (543 nm and 633 nm) and the Zen 2009 software. A 63X numerical aperture (NA) 1.4 oil-immersion Plan-Apochromat objective (Carl Zeiss) was used.
In vitro invasion assay
Invasion assay were performed as previously described . The membrane on the top chamber (12-well insert; pore size 8 μm, Millipore, Billerica, MA, USA) was coated with a mixture of 3 mg/ml acid-soluble type I collagen (Cellmatrix type 1-A, Nitta Gelatin, Osaka, Japan) and 10× RPMI medium (Sigma-Aldrich, St Louis, MO, USA) in a 9:1 ratio. The pH of the collagen mixture was adjusted to pH 8 with 1 M NaOH on ice. The collagen mixture was further diluted with DMEM medium to a final concentration of 2 mg/ml and incubated for 30 min at 37°C. Cells were plated on the top chamber in medium without serum and medium with serum was placed in lower chamber as a chemoattractant. The cells were incubated for 24 h and non-invasive cells were removed by cotton swab. The invasive cells were fixed, stained for DAPI and analysed on a Zeiss Axioplan-2 microscope (Carl Zeiss). Numbers of invaded cells on each whole membrane were quantified. In further control experiments, uncoated filters were used in place of collagen-coated filters.
Collagen degradation assays
Collagen degradation assays were performed according to . 12-well cell culture plates were coated with a thin layer of approx. 2.7 mg/ml PureCol™ collagen (Nutacon, Leimuiden, The Netherlands) containing 10× RPMI medium (pH 8). Plates were incubated for 1 h at 37°C to form fibrillar collagen. Cells were cultured on the fibrillar collagen for 48 h then removed by trypsin-EDTA (Life Technologies). The collagen films were fixed with 4% paraformaldehyde for 30 min, stained with Coomassie Brilliant Blue R250 and analysed on an Axiovert 135 microscope (Carl Zeiss). The clear unstained zones indicated areas of degraded collagen. Images were quantitated using Volocity 6.0.1 software.
Western blotting and co-immunoprecipitation
Cells were lysed in sample buffer containing 62.5 mM Tris–HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% β-mercaptoethanol, and 0.001% bromophenol blue. For phosphorylated protein detection, cells were lysed with cold lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 25 mM NaF, 2 mM NaVO4 and protease inhibitor cocktail (Roche, Mannheim, Germany). Cell lysates were resolved on 10% SDS-PAGE, proteins were transferred electrophoretically to PVDF membranes (Bio-Rad, USA) and blotted with the indicated antibodies. Blots were quantified using TotalLab TL100 software (Biosystematica, Devon, UK). For co-immunoprecipitation experiments, cells were lysed in ice cold buffer containing 20 mM HEPES pH7.5, 150 mM NaCl, 1% Triton-X100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail. The cell lysates were sheared with 25G needles and left mixing for 1 h at 4°C. The lysates were centrifuged at 13,000 rpm for 5 min at 4°C and the supernatants were pre-cleared with protein A agarose beads (Sigma-Aldrich, St Louis, MO, USA) for 1 h at 4°C. The pre-cleared lysates were incubated with caveolin-2 antibody and rabbit IgG as a control overnight at 4°C and further incubated with protein A-agarose beads for 1 h at 4°C. The beads were washed and eluted followed by electrophoresis and immunoblot analysis.
Isolation of detergent-resistant membranes
Two confluent 15 cm dishes of MDA-MB231 cells were each scraped in 2 ml PBS on ice after 3 washes in ice cold PBS (divalent cation free). Cells were pelleted at 2°C at 900 rpm for 5 min. Some dishes were untreated, while others had been heparan sulfate (20 μg/ml) treated for the final 24 h. The two pellets were each resuspended in 0.6 ml ice-cold MNE (25 mM MES pH6.5, 150 mM NaCl, 2 mM EDTA) containing 1% Triton X-100, protease inhibitors (Roche protease inhibitor cocktail, pepstatin and 1 mM PMSF). The lysates were incubated on ice for 15 min and sheared by passage through a 25G needle 15 times, all on ice. The lysates were mixed with 0.6 ml ice-cold 80% (w/v) sucrose/MNE and divided into 2 × 1.4 ml centrifuge tubes. Each was overlaid with 0.5 ml 30% and 0.25 ml 5% sucrose/MNE in a precooled Beckman benchtop MAX-XP ultracentrifuge TLS-55 swingout rotor (Beckman Coulter, Fullerton, CA, USA). Centrifugation was carried out at 200,000 × g for 20 h at 2°C. The Triton-soluble fraction was defined as the bottom 0.2 ml of the 40% sucrose layer. The detergent-resistant membrane (DRM) fraction was clearly visible as an opaque ring at the 30-40% boundary. Each tube was fractionated into 9 × 150 μl fractions. One set from control and HS treated samples were heated with 5× sample buffer. Gels were run (10% SDS-PAGE) at 125 V. Electrotransfer to PVDF membrane was at 75 V for 90 min. The membranes were blocked in 5% milk/1% heat treated BSA in TBS for 1 h at RT, then the membranes were incubated with three antibodies simultaneously. Antibodies against flotillin were combined with those against caveolin-2 and transferrin receptor, all at 1 μg/ml in 1% milk/1% BSA/TBST at 4°C on a rocking platform. In other experiments, both antibodies against syndecan-2 or syndecan-4 were used (1:500 dilution). Membranes were washed at least 4 × over 30 min with TBST, then incubated at RT for 1 h in 1:2500 each of GAR and GAM-HRP (combined). Washing 3 × TBST over 30 min, then TBS with no Triton. Membranes were incubated with EZ-ECL (Biological Industries, Beit Haemek, Israel). Blots were visualized and photographed in the LAS4010 instrument (GE Healthcare, Freiburg, Germany).
Immunostaining was performed on formalin-fixed paraffin-embedded breast tissue arrays obtained from USBiomax (Rockville, MD, USA, cat. no. BRC961, BRC962, BR486 and BRM961). The material included 24 cases of normal, reactive or benign breast tissue, 168 cases of breast cancer, from which 48 were triple negative cases and another 48 had matching lymph node metastasis or adjacent normal breast tissue. The sections were deparaffinised with xylene and rehydrated through graded alcohols into distilled water. Heat induced antigen retrieval in 0.01 M citrate buffer, pH 6.0 was performed . The EnVision + System-HRP Labelled Polymer anti-Rabbit and Liquid DAB + Substrate Chromogen System (DAKO) were used for the detection. Polyclonal or monoclonal rabbit anti-human antibodies were used to detect syndecan-2 (diluted 1:50; LSBio) and caveolin-2 (diluted 1:50). Slides were counterstained with haematoxylin, dehydrated and mounted in permanent mounting medium (Eukitt quick-hardening mounting medium; Sigma-Aldrich). Slides were scanned at a 40× magnification using the NanoZoomer 2.0-HT digital slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). The immunohistochemical staining was quantified and analysed. TMA cores which were either lost or fragmented were excluded from quantification analysis. Intensity and area of syndecan-2 and caveolin-2 staining were measured using BioPix iQ, version 2.1.8 (Gothenburg, Sweden) after excluding blood vessels, fat tissues and necrotic areas. Intensity per area in ln scale was plotted.
Data are presented as standard error of mean. All western blots were quantified using TotalLAB software (Biosystematica, Devon, UK) and values are shown as ratio to controls. Two-tailed paired t-test was used to compare between groups and one-way ANOVA with Tukey’s post-hoc test was used for comparison of more than two groups. p < 0.05 was considered significant. All statistical analysis and graphs were plotted using GraphPad Prism 5 (La Jolla, CA, USA).
Cell invasion and matrix degradation is blocked by exogenous heparan sulfate or heparin
Treatment of MDA-MB231 cells with Y-27632, which inhibits the Rho kinases ROCK I and II, led to total loss of microfilament bundles in cells treated with either heparin or heparan sulfate, consistent with a normal actomyosin constitution and function (Figure 1D). Consistent with this, heparan sulfate and heparin-treated cells had higher levels of Thr18/Ser19 phosphorylated myosin light chain than control or chondroitin sulfate treated cells (Figure 1E). However, further experiments showed that the cell spreading seen with heparan sulfate or heparin was more complex than simply elevated myosin light chain phosphorylation. Transfection of control cells with a cDNA encoding a mutated MLC where Thr18 and Ser19 were changed to aspartate (as a phosphomimetic ) led to fine microfilament bundles but no pronounced spreading (Figure 1F).
Heparin and heparan sulfate treatments markedly reduced collagen gel invasion and degradation compared to untreated controls or equivalent levels of chondroitin sulfate addition (Figure 1G, H). Since heparin is known to interact with type I collagen , it was important to establish whether the effects seen resulted from interactions with the matrix, or the cells themselves. In control experiments, therefore, collagen matrices were pretreated with glycosaminoglycans, and then washed before addition of mammary carcinoma cells. Under these circumstances, matrix pretreatment did not retard migration or degradation (not shown), indicating that the effects of exogenous heparin and heparan sulfate were cell-mediated.
Inhibition of the thrombin/PAR-1 receptor signalling pathway
Hierarchical roles for Syndecans-2 and −4 in carcinoma cell behaviour
No effects on cell adhesion or junction formation were noted where syndecan-1 or syndecan-4 were depleted on their own. Moreover, no additional effects were noted where syndecan-1 was depleted in combination with syndecan-2 knockdown (Figure 4H). However, double depletion of syndecan-2 and −4 had one clearly observable effect, a decrease in the size and number of focal adhesions, compared with syndecan-2 knockdown alone (Figure 4H). These data are consistent with previously published roles for syndecan-4 in focal adhesion assembly . As a whole the data suggest that syndecan-2 in breast carcinoma plays a major regulatory role in maintaining the invasive phenotype and appears to suppress the focal adhesion promoting role of syndecan-4.
Caveolin-2 regulates invasive phenotype of MDA cells
To investigate whether the formation of microfilament bundles was Rho kinase dependent, cells after syndecan-2 or caveolin-2 depletion were treated with the ROCK inhibitor, Y-27632. Similar to heparan sulfate or heparin treatment, Y-27632 treatment inhibited microfilament bundle formation triggered by syndecan-2 or caveolin-2 depletion (Figure 6H). In addition, synedecan-2 or caveolin-2 depleted cells, but not caveolin-1 depleted cells, had higher levels of Thr18/Ser19 phosphorylated myosin light chain compared to controls (Figure 6I). The data suggest that microfilament bundle formation observed in the absence of syndecan-2 or caveolin-2 was Rho kinase-dependent.
Syndecan-2 expression is elevated in metastatic breast tumour
Heparin and heparan sulfate are frequently used as exogenous competitors of cell surface proteoglycan-mediated events. These glycosaminoglycans potently influenced the behavior and cytoskeletal organization of the invasive MDA-MB231 cell line, which is triple negative (i.e. lacking estrogen, progesterone and Her2Neu/ErbB2 receptors). Their effects appeared to be due to inhibition of two major signaling pathways, thrombin interactions with PAR-1 receptor, and that mediated by a syndecan-2/caveolin-2 pathway.
Thrombin is a serine protease with key roles in the coagulation cascade and can be inhibited by heparin . Thrombin signaling is mediated through a family of small family of G-protein-coupled protease activated receptors (PARs). Of the four PAR family members, PAR-1, is the prototypical member and has been identified as a potent oncogene based on its ability to stimulate focus formation and induce NIH3T3 cell transformation . In addition to their normal physiological roles in vascular biology, there is substantial evidence for aberrant expression of thrombin and PAR-1 in several cancers, including breast cancer [32,41-43], melanoma [44,45] and prostate cancer [46,47]. In breast cancer, increased PAR-1 expression was closely associated with the invasive capacity of primary breast tissue specimens and breast carcinoma cell lines [32,41,43,48,49], suggesting PAR-1 has a critical role in tumor progression. We have demonstrated that heparan sulfate and heparin may inhibit PAR-1 activation by inhibiting thrombin, leading to cell spreading and attenuated invasiveness of MDA-MB231 cells. However, PAR-1 depletion did not alter the cell surface expression levels of the major metalloproteinase, MMP14, which was confirmed here to be essential for collagen invasion and degradation.
We also show that a heparan sulfate proteoglycan, syndecan-2 is a regulator of breast carcinoma invasiveness. Cell invasion and degradation of type I collagen were remarkably inhibited after syndecan-2 depletion. Furthermore, reduction in cell invasiveness was accompanied by changes in actin cytoskeletal organization and cell-cell adhesion, where cell spreading, microfilament bundles, focal adhesions, and cadherin-11 containing adherens junctions were all enhanced. Thus depletion of syndecan-2 reveals a mechanism by which it controls the invasiveness of MDA-MB231 cells. Consistent with this heparan sulfate treatment was shown by FACS analysis to lead to specific syndecan-2 loss from the cell surface, while levels of syndecan-4 increased and syndecan-1 was unchanged. Focal adhesion and microfilament bundle formation in response to loss of syndecan-2 was shown to depend on the presence of syndecan-4. Very recent work suggests that this mechanism involves p190ARhoGAP, which associates with syndecan-4 and active β1 integrin at the cell margins in the absence of syndecan-2 . RhoGAP promotes conversion of GTP-Rho to the inactive GDP-bound form but is inactivated by Src-mediated tyrosine phosphorylation. Previous work has demonstrated the importance of syndecan-4 regulation of p190RhoGAP distribution  and our data correspondingly show that Rho kinases (ROCKs) and their downstream target, myosin light chain, are required for focal adhesion and microfilament bundle formation in MDA-MB231 cells upon heparan sulfate addition or syndecan-2 depletion.
A very recent study illustrated that syndecan-2 depletion in MDA-MB231 derived 1833 cell line with enhanced bone tropism led to inhibition of breast tumor growth and metastasis to bone in a mouse xenograft model, in part through enhanced apoptosis . No effects on junctions or actin cytoskeleton were reported in this study. However, we did not observe changes in growth or cleaved caspase-3 staining after syndecan-2 depletion (data not shown), indicating that the parental MDA-MB231 cells and MDA-MB231 derived 1833 cells might signal through different pathways to regulate invasive behaviour. The current data strongly suggest that syndecan-2 is a tumor promoter by regulating cytoskeletal organization and tumorigenic activity in breast cancer cells.
In MDA-MB231 cells, syndecan-2 combines with caveolin-2 to promote an aggressive phenotype. Caveolin is the major structural component of plasma membrane microdomains that play numerous pivotal roles in intracellular trafficking and signal transduction [53,54]. The MDA-MB231 cell line is known to carry a KRas G13D mutation . Upon oncogenic Ras transformation in BalbC/3 T3 cells, syndecan-2 has been shown to form a complex with caveolin-2 , which was also shown here. The interaction of caveolin-2 with syndecan-2 is specific, since caveolin-2 did not form a complex with syndecan-4. This interaction and its consequences deserve further attention. Strikingly, caveolin-2 depletion yielded the same cell behavior and cytoskeletal organization as syndecan-2 depletion, with increased cell spreading, microfilament bundles, larger and more numerous focal adhesions and diminished type I collagen invasion and degradation. Unlike caveolin-2, caveolin-1 depletion did not give rise to an altered phenotype, though cell invasion was reduced in these cells. Caveolin-1, but not caveolin-2 has been identified as being important for the formation and activity of invadopodia . Therefore, reduced cell invasion in caveolin-1 depleted cells may result from impaired invadopodia function. Caveolin-2, on the other hand, might stimulate cell invasiveness through different mechanisms than caveolin-1, which awaits further elucidation.
In breast carcinoma, caveolin-2 expression is frequently upregulated and correlates with poor prognostic status [57,58]. However, there is no previous evidence for involvement of a syndecan-2/caveolin-2 axis in breast tumor cell metastasis. Our patient tissue microarray data showed that syndecan-2 expression and caveolin-2 were significantly increased in breast cancer patients regardless of tumor grade. More importantly, syndecan-2 expression was upregulated in lymph node samples compared directly with the primary tumours and in triple negative cases, expression of syndecan-2 and caveolin-2 were correlated.
Syndecan-2 has remained very elusive with regard to its signaling ability. The syndecan-2/caveolin-2 relationship may provide an important insight into regulation of a specific membrane microdomain in tumour, and perhaps untransformed cells. Previous work has shown that syndecan-1 upregulation in breast carcinoma is associated with poor prognosis, particularly when present in the stroma [21,59]. Recent data also suggests that syndecan-2, normally considered a mesenchymal proteoglycan, is upregulated and a potential target in colon carcinoma . It was also noted as upregulated by gene expression profiling in breast cancer  but insight into its function has been lacking. Given the importance of syndecans in breast, colon and other cancers such as myeloma  their role on the cell surface is increasingly important to understand. The fact that exogenous heparan sulfate and heparin inhibit two pathways that support an invasive phenotype suggests that cell surface proteoglycans are a promising area for development of reagents and understanding of breast carcinoma invasion and metastasis.
This work was supported by The Danish National Research Foundation and Novo Nordisk Fonden.
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