NCAM and attached polysialic acid affect behaviors of breast epithelial cells through differential signaling pathways

Background Neural cell adhesion molecule (NCAM), a common mammalian cell surface glycoprotein, is the major substrate of polysialic acid (PSA). Polysialylated NCAM occurs in many types of cancer, but rarely in normal adult tissues. Epithelial-mesenchymal transition (EMT) is an important process contributing to tumor metastasis. The functional role of NCAM hypersialylation in EMT is unclear. Method Expression of NCAM and PSA-NCAM in breast cancer progression were evaluated by western blot (WB), immunohistochemistry, semi-quantitative PCR and immunoprecipitation (IP). Overexpression of NCAM-140 and STX were performed to assess the functional role of NCAM hypersialylation by liposome transfection. Then, the cell proliferation ability was investigated with MTT assay. Transwell and wound closure assay were conducted to evaluate cell migratory ability. Phagokinetic gold sol assay and cell adhesion assay were performed to assess cell motility ability and cell adhesion ability, respectively. Furthermore, WB and IP were used to reveal the activated signaling pathway. Results The present study indicates that NCAM and attached polysialic acid affect behaviors of breast epithelial cells through differential signaling pathways, based on the following observations: (i) NCAM and PSA-NCAM were aberrantly regulated in breast cancer cells; (ii) NCAM and PSA-NCAM expression were upregulated in normal breast epithelial cells undergoing EMT; (iii) NCAM overexpression induced EMT in breast epithelial cells; (iv) NCAM promoted cell proliferation and migration through activation of a β-catenin/slug signaling pathway; (v) modification of PSA attached to NCAM inhibited cell adhesion and promoted cell motility through activation of an EGFR/STAT3 pathway. Conclusion These observations help clarify the molecular mechanisms whereby PSA and its major substrate NCAM modulate cell behaviors, and the significance of increased PSA expression on NCAM during EMT and tumor development. not address the possible functional role of PSA in mediating the EMT process. We found that PSA overexpression caused a significant increase of cell motility but had negligible effect on cell migration, indicating that NCAM (but not PSA) affected migration. PSA-overexpressing cells showed low adhesion to collagen IV and Matrigel, while NCAM overexpression had no effect on attachment to collagen IV, indicating that PSA-NCAM and NCAM have differing effects on adhesion to various ECM components.


Aberrant regulation of NCAM and PSA-NCAM during EMT
EMT is a basic and highly conserved process that plays crucial roles in embryogenesis, cancer invasion and metastasis [27] . Expression of NCAM and PSA-NCAM was studied in an in vitro EMT model established by TGF-β1 induction in MCF10A and NMuMG cells. Typical morphology shift from epithelial to mesenchymal was observed in the two cell lines (Suppl. Fig. 1a). Changes of protein levels associated with EMT were observed, including increase of N-cadherin (N-cad) ("cadherin switch"), and decreases of E-cadherin (E-cad) and the tumor markers vimentin (VM) and fibronectin (FN) (Suppl. Fig. 1b). Expression at the mRNA level of various NCAM isotypes was elevated in NMuMG cells undergoing EMT (Fig. 2a). NCAM-140 expression at the protein level was maximal in MCF10A and NMuMG cells undergoing EMT (Fig. 2b). It is known that NCAM can be posttranslationally polysialylated by the polysialyltransferases ST8Sia II (STX) and ST8Sia IV (PST), resulting in PSA-NCAM glycoforms. We therefore analyzed STX and PST expression in the two model cell lines. Increased STX expression and reduced PST expression were observed at the mRNA level (Fig. 2c). PSA-NCAM expression was much higher in cells undergoing EMT (Fig. 2d), which was validated by immunoprecipitation assay (Fig. 2e), indicating that upregulation of NCAM and PSA-NCAM are involved in EMT process.

NCAM-140 overexpression induces EMT in NMuMG cells
To evaluate the effects of two isoforms of NCAM on cell behaviors, we cloned the genes encoding NCAM-120 and NCAM-140, and transfected them separately into NMuMG cells. In contrast to a previous finding that ectopic expression of NCAM caused cell death of NMuMG[28], we obtained stable transfectants of the two isoforms, termed NG/120 and NCAM-140 cells (Suppl. Fig. 1c). NG/140 had single, motile mesenchymal cell morphology, whereas NG/120 retained epithelial morphology similar to that of non-transfected cells ("NG") (Suppl. Fig. 1d). N-cad and VM were upregulated in NG/120 and NG/140, but not in NG or NG/3.1. Compared to NG/120, NG/140 showed nearly complete loss of E-cad expression, significantly enhanced FN expression (Fig. 3a,b), increased proliferation and migration (Fig. 3c,d,e). However, motility of NG/140 was similar to that of the other NG cell lines (Fig. 3f). These findings suggest that NCAM-140 overexpression switches cells to an EMT-like process, with consequent alteration of proliferation and migration.

Differential effects of PSA-NCAM and NCAM on cell behaviors
We observed previously that PSA, catalyzed by STX, facilitated NCAM-mediated cell migration in a polysialyltransferase-specific manner [25]. To assess the role of PSA in modulating NCAM-mediated cell behaviors, we cloned the STX gene into NG, and the resulting cell lines were termed NG/STX (Suppl. Fig. 2a,b). In comparison with NG and NG/140, PSA-NCAM level was elevated in NG/STX (Fig.   4a). Proliferation of NG/140 was significantly higher after 36 h and 48 h culture, and after 60 h NG/140 cells were almost completely confluent (Fig. 4b). These findings suggest that cell proliferation was increased by NCAM overexpression, and this effect was further enhanced by PSA.
The effects of PSA-NCAM and NCAM on cell motility and migration were examined. Cell motility was slightly increased in NG/140 and significantly increased in PSA-overexpressing NG/STX (Fig. 4c), consistent with results shown in Fig. 3f. Cell migration was increased in NG/140 but not in NG/STX, indicating that this cell behavior was affected by NCAM, but not by PSA (Fig. 4d,e). Because NCAM is a type of adhesion molecule, we examined the effects of PSA-NCAM and NCAM on cell adhesion, based on attachment of our experimental cell lines to extracellular matrix (ECM) components (FN, laminin, collagen IV, Matrigel) (Suppl. Fig. 3). PSA-overexpressing NG/STX showed reduced attachment to collagen IV and Matrigel, whereas attachment of NCAM-overexpressing NG/140 to collagen IV was the same as that of NG (Fig. 4f). Thus, PSA-NCAM and NCAM had differing effects on cell adhesion to ECM components.

PSA-NCAM-mediated EGFR/STAT3 signaling pathway
The EGFR/STAT3 signaling pathway plays an important role in human BC [29,30]. We examined the possible effects of PSA-NCAM and NCAM on this pathway. EGFR expression differed significantly between NG/STX and the other NG cell lines (Fig. 5a). TGF-β1-induced EMT increased total EGFR (tEGFR) in NG/STX but had no effect on β-catenin expression (Fig. 5a). EGFR phosphorylation in NG/STX differed before vs. after EMT (Fig. 5b). EGFR phosphorylation was reduced in NG/140 undergoing EMT, suggesting that the EGFR/STAT3 signaling pathway was activated by PSA but not by NCAM. When PSA was knocked down by silencing STX (STXi) (Suppl. Figs. 2c), phosphorylated EGFR and STAT3 were downregulated in each of the cell lines (Fig. 5c). Expression of β-catenin and FN, which were upregulated by NCAM, were notably reduced by STX knockdown (Fig. 5c). These findings indicate that PSA is involved in activation of the EGFR/STAT3 signaling pathway.

NCAM-mediated β-catenin/slug signaling pathway
NCAM-140 overexpression in NG caused reduction of E-cad level (Fig. 3a,b). Dissociation of E-cad/βcatenin complex is a key step in EMT, and alterations in localization and expression levels of β-catenin have been observed in various types of cancers. The well-known EMT regulator/ transcription factor slug has been shown to inhibit E-cad expression and promote cell metastasis [31,32]. The Wnt signaling pathway associated with β-catenin, a key downstream effector, is involved in BC development [33]. We examined the possible role of NCAM-140 in regulation of the β-catenin signaling pathway. β-catenin expression was increased in NG/140 nuclei, but reduced in NG/STX nuclei (Fig.   6a). These findings suggest that NCAM induces translocation of β-catenin from cytoplasm into the nucleus, and that such translocation is inhibited by PSA overexpression. Slug was accumulated in nuclei of NCAM-overexpressing cells. β-catenin transcription was enhanced in NG/140 but reduced in NG/STX (Fig. 6b). Expression of genes targeted by β-catenin (axin 2, c-myc, CCND1) was significantly upregulated (Fig. 6c). It thus appears that upregulation of the β-catenin/slug signaling pathway leads to increased expression of axin 2, slug, c-myc, and CCND1 genes in NG/140. In contrast, expression of receptor genes (fzd 7, wnt 3a) of the Wnt signaling pathway was not notably altered in association with the NCAM-mediated β-catenin/slug signaling pathway (Fig. 6c).
In the absence of Wnt ligands, β-catenin is usually phosphorylated by GSK-3 at Ser33 and Ser37, leading to its ubiquitin-dependent degradation through incorporation of APC and CK1α[34]. We measured APC, GSK-3β, and CK1α mRNA levels in our NG cell lines. CK1α level was notably reduced in NG/140 whereas GSK-3β was increased in PSA-overexpressing NG/STX, suggesting that CK1α is downregulated in NG/140 to block β-catenin degradation, whereas GSK-3β is upregulated in NG/STX to promote β-catenin degradation (Fig. 6c,d).

Discussion
Modulation of tumor cell adhesion molecules is crucial in control of the metastatic cascade. NCAM, the major substrate of PSA, has been associated with tumor invasion and formation of metastatic deposits in many types of cancer [35,36]. PSA molecules are able to affect NCAM function, and are associated with malignant and metastatic phenotype [37,38]. Because of the large negative charge of PSA, its presence inhibits the adhesive properties of NCAM. We previously examined the role of PSA in NCAM function using cell line ldlD-14, an essential model for functional study of glycoproteins and glycolipids. NCAM-140 strongly enhanced cell adhesion to FN and reduced adhesion to Matrigel, and loss of E-cad expression, NCAM function, focal adhesion assembly, and cell migration and invasion, it did not address the possible functional role of PSA in mediating the EMT process. We found that PSA overexpression caused a significant increase of cell motility but had negligible effect on cell migration, indicating that NCAM (but not PSA) affected migration. PSA-overexpressing cells showed low adhesion to collagen IV and Matrigel, while NCAM overexpression had no effect on attachment to collagen IV, indicating that PSA-NCAM and NCAM have differing effects on adhesion to various ECM components.
PSA and NCAM are closely associated with cell adhesion, migration, and invasion, and help mediate tumor development and progression [18]. Our findings suggest that PSA overexpression stimulates the EGFR/STAT3 signaling pathway. NCAM induced translocation of β-catenin from cytoplasm into the nucleus, and promoted the β-catenin/slug signaling pathway. Such translocation was inhibited by PSA overexpression, indicating that PSA and NCAM affect cell adhesive properties through different signaling pathways. PSA decoration of NCAM appears to play crucial roles in its altered expression during EMT, and its modulating effects on cell behaviors.

Conclusion
Collectively, we propose that NCAM-140 overexpression in NMuMG cells inhibits CK1α expression and β-catenin phosphorylation, and promotes translocation of released β-catenin into nuclei, with consequent downregulation of E-cad expression and upregulation of FN, VM, and slug expression.
Consistent with this hypothesis, β-catenin downstream genes axin 2, c-myc, and CCND1 were activated in the Wnt-independent β-catenin/slug signaling pathway ( Fig. 6c and Fig. 7). As PSA accumulated on NCAM, it stimulated of EGFR/STAT3 signaling pathway, reduction of cell adhesion, and increased cell motility (Fig. 7). The degree of PSA attachment on NCAM molecules is a crucial factor in mediation of various pathways by NCAM. NG/STX cells contain relatively few NCAM molecules, each with a high amount of attached PSA. As a result, PSA dominates the pathway that mediates cell adhesion and motility.         Transition between NCAM-mediated β-catenin/slug signaling pathway and PSA-NCAMmediated EGFR/STAT3 signaling pathway.

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