Haein Park

Syndecan-2 Mediates Adhesion and Proliferation of Colon Carcinoma Cells

Syndecan-2 is a transmembrane heparan sulfate proteoglycan whose function at the cell surface is unclear. In this study, we examined the function of syndecan-2 in colon cancer cell lines. In several colon cancer cell lines, syndecan-2 was highly expressed compared with normal cell lines. In contrast, syndecan-1 and -4 were decreased. Cell biological studies using the extracellular domain of recombinant syndecan-2 (2E) or spreading assay with syndecan-2 antibody-coated plates showed that syndecan-2 mediated adhesion and cytoskeletal organization of colon cancer cells. This interaction was critical for the proliferation of colon carcinoma cells. Blocking with 2E or antisense syndecan-2 cDNA induced G0/G1 cell cycle arrest with concomitantly increased expression of p21, p27, and p53. Furthermore, blocking of syndecan-2 through antisense syndecan-2 cDNA significantly reduced tumorigenic activity in colon carcinoma cells. Therefore, increased syndecan-2 expression appears to be a critical for colon carcinoma cell behavior, and syndecan-2 regulates tumorigenic activity through regulation of adhesion and proliferation in colon carcinoma cells.

Transmembrane Domain-induced Oligomerization Is Crucial for the Functions of Syndecan-2 and Syndecan-4

The syndecans are known to form homologous oligomers that may be important for their functions. We have therefore determined the role of oligomerization of syndecan-2 and syndecan-4. A series of glutathione S-transferase-syndecan-2 and syndecan-4 chimeric proteins showed that all syndecan constructs containing the transmembrane domain formed SDS-resistant dimers, but not those lacking it. SDS-resistant dimer formation was hardly seen in the syndecan chimeras where each transmembrane domain was substituted with that of platelet-derived growth factor receptor (PDGFR). Increased MAPK activity was detected in HEK293T cells transfected with syndecan/PDGFR chimeras in a syndecan transmembrane domain-dependent fashion. The chimera-induced MAPK activation was independent of both ligand and extracellular domain, implying that the transmembrane domain is sufficient to induce dimerization/oligomerization in vivo. Furthermore, the syndecan chimeras were defective in syndecan-4-mediated focal adhesion formation and protein kinase Cα activation or in syndecan-2mediated cell migration. Taken together, these data suggest that the transmembrane domains are sufficient for inducing dimerization and that transmembrane domain-induced oligomerization is crucial for syndecan-2 and syndecan-4 functions.

Syndecan-2 Regulates the Migratory Potential of Melanoma Cells

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is a critical mediator in the tumorigenesis of colon carcinoma cells. We explored the function of syndecan-2 in melanoma, one of the most invasive types of cancers, and found that the expression of this protein was elevated in tissue samples from both nevus and malignant human melanomas but not in melanocytes of the normal human skin tissues. Similarly, elevated syndecan-2 expression was observed in various melanoma cell lines. Overexpression of syndecan-2 enhanced migration and invasion of melanoma cells, whereas the opposite was observed when syndecan-2 levels were knocked down using small inhibitory RNAs. Syndecan-2 expression was enhanced by fibroblast growth factor-2, which is known to stimulate melanoma cell migration; however, α-melanocyte-stimulating hormone decreased syndecan-2 expression and melanoma cell migration and invasion in a melanin synthesis-independent manner. Furthermore, syndecan-2 overexpression rescued the migration defects induced by α-melanocyte-stimulating hormone treatment. Together, these data strongly suggest that syndecan-2 plays a crucial role in the migratory potential of melanoma cells.

New Insights into Syndecan-2 Expression and Tumourigenic Activity in Colon Carcinoma Cells

Adhesion receptors play crucial roles in the neoplastic transformation of normal cells through induction of cancer-specific cellular behaviour and morphology. This implies that cancer cells likely express and utilize a distinct set of adhesion receptors during carcinogenesis. Colon cancer is an excellent model system for the study of this process, since both molecular genetic and morphological changes have been well established for this disease. We recently reported increased expression of the cell surface adhesion receptor, syndecan-2, in several colon carcinoma cell lines. Indeed, increased syndecan-2 expression was necessary for tumourigenic activity, suggesting that syndecan-2 might have value as both a new diagnostic marker and a possible therapeutic target. Here, we review recent advances in understanding the role of syndecan-2 in the carcinogenesis of colon cells, and discuss a leading role for this molecule in a new era for colon cancer treatment.

Syndecan-2 Functions as a Docking Receptor for Pro-matrix Metalloproteinase-7 in Human Colon Cancer Cells

Although elevated syndecan-2 expression is known to be crucial for the tumorigenic activity in colon carcinoma cells, how syndecan-2 regulates colon cancer is unclear. In human colon adenocarcinoma tissue samples, we found that both mRNA and protein expression of syndecan-2 were increased, compared with the neighboring normal epithelium, suggesting that syndecan-2 plays functional roles in human colon cancer cells. Consistent with this notion, syndecan-2-overexpressing HT-29 colon adenocarcinoma cells showed enhanced migration/invasion, anchorage-independent growth, and primary tumor formation in nude mice, paralleling their morphological changes into highly tumorigenic cells. In addition, our experiments revealed that syndecan-2 enhanced both expression and secretion of matrix metalloproteinase-7 (MMP-7), directly interacted with pro-MMP-7, and potentiated the enzymatic activity of pro-MMP-7 by activating its processing into the active MMP-7. Collectively, these data strongly suggest that syndecan-2 functions as a docking receptor for pro-MMP-7 in colon cancer cells.

Focal adhesion kinase regulates syndecan-2-mediated tumorigenic activity of HT1080 fibrosarcoma cells.

Expression of syndecan-2, a transmembrane heparan sulfate proteoglycan, is crucial for the tumorigenic activity in colon carcinoma cells. However, despite the high-level expression of syndecan-2 in mesenchymal cells, few studies have addressed the function of syndecan-2 in sarcoma cells. In HT1080 fibrosarcoma cells, we found that syndecan-2 regulated migration, invasion into Matrigel, and anchorage-independent growth but not cell-extracellular matrix adhesion or proliferation, suggesting that syndecan-2 plays different functional roles in fibrosarcoma and colon carcinoma cells. Consistent with the increased cell migration/invasion of syndecan-2-overexpressing HT1080 cells, syndecan-2 overexpression increased phosphorylation and interaction of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), membrane localization of T-lymphoma invasion and metastasis gene-1 (Tiam-1), and activation of Rac. Syndecan-2-mediated cell migration/invasion of HT1080 cells was diminished when (a) cells were cotransfected with nonphosphorylatable mutant FAK Y397F or with other FAK mutants lacking PI3K interactions, (b) cells were treated with a specific PI3K inhibitor, or (c) levels of Tiam-1 were knocked down with small interfering RNAs. Furthermore, expression of several FAK mutants inhibited syndecan-2-mediated enhancement of anchorage-independent growth in HT1080 cells. Taken together, these data suggest that syndecan-2 regulates the tumorigenic activities of HT1080 fibrosarcoma cells and that FAK is a key regulator of syndecan-2-mediated tumorigenic activities.

Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin α2

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is known to serve as an adhesion receptor, but details of the regulatory mechanism governing syndecan-2 cell adhesion and migration remain unclear. Here, we examined this regulatory mechanism, showing that overexpression of syndecan-2 enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells (RIE1), and increased integrin alpha2 expression levels. Interestingly, RIE1 cells transfected with either syndecan-2 or integrin alpha2 showed similar adhesion and migration patterns, and a function-blocking anti-integrin alpha2 antibody abolished syndecan-2-mediated adhesion and migration. Consistent with these findings, transfection of integrin alpha2 siRNA diminished syndecan-2-induced cell migration in HCT116 human colon cancer cells. Taken together, these results demonstrate a novel cooperation between syndecan-2 and integrin alpha2beta1 in adhesion-mediated cell migration and invasion. This interactive dynamic might be a possible mechanism underlying the tumorigenic activities of colon cancer cells.

Decreased syndecan-2 expression correlates with trichostatin-A induced-morphological changes and reduced tumorigenic activity in colon carcinoma cells

The inhibition of histone deacetylase activity is known to induce morphological changes of transformed cells. In this study, we investigated the effect of the specific HDAC inhibitor, trichostatin A (TSA), on colon carcinoma cell lines. Treatment of human colorectal carcinoma cells, KM1214 and KM12SM, with TSA induced distinct morphological changes. Both cell lines, which normally piled up in layers without clear boundary, became more flattened, and formed monolayers with evident boundaries between cells, with concomitant increased actin filament organization. Cell–cell interaction was not affected much, based on expression level, membrane localization, and interaction of E-cadherin with β-catenin. In contrast, syndecan-2 expression was dramatically reduced and it was correlated with the morphological changes of colon carcinoma cells. Consistently, downregulation of syndecan-2 expression by antisense cDNA clearly mimicked the morphological changes in KM12SM and reduced anchorage-independent growth of colon cancer cells. All these results indicate that reduced syndecan-2 expression correlates with TSA-induced morphological changes and reduced tumorigenic activity in colon carcinoma cells.

Focal Adhesion Kinase Is Negatively Regulated by Phosphorylation at Tyrosine 407

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr407 was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr407 increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr397 phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr397 and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr407 phosphorylation negatively regulates the enzymatic and biological activities of FAK.