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

Abstract

Neural cell adhesion molecule (NCAM), a common mammalian cell surface glycoprotein, is the major substrate of polysialic acid (polySia). Polysialylated NCAM occurs in many types of cancer, but rarely in normal adult tissues. The functional role of NCAM hypersialylation in the epithelial-mesenchymal transition (EMT) process remains unclear. The present study indicates that NCAM and attached polysialic acid affect behaviors of breast epithelial cells through differential signaling pathways. NCAM and polysialylated NCAM are aberrantly regulated in breast cancer cells. They are both upregulated in normal breast epithelial cells undergoing EMT. Western blot analysis demonstrates that NCAM-140 overexpression induces EMT in breast epithelial cells and promotes cell proliferation and migration through activation of the β-catenin/slug signaling pathway. Modification of polySia attached to NCAM modulates cell adhesion and promotes cell motility through activation of the EGFR/STAT3 pathway. These observations contribute to clarifying the molecular mechanisms by which polysialic acid and its major substrate, NCAM, modulate cell behaviors, and highlight the significance of increased polysialylated expression on NCAM during EMT and tumor development.

1. Introduction

Sialic acid is a vital monosaccharide often found at the terminal position of cell-surface glycan chains [1]. Elevated expression of sialylation is correlated with tumor aggressiveness, invasion, drug resistance and poor prognosis in cancer patients [2,3]. Aberrant sialylation is primarily due to abnormal regulation of sialyltransferases, the enzymes that catalyze the linkage of sialic acid to other carbohydrates [4,5]. For example, overexpression of ST3GalI in breast cancer cells promotes tumorigenesis in a murine model [6], and upregulation of ST6GalI is essential for the maintenance of cancer cells stemness [7].

Polysialyltransferases II (ST8SiaII) and IV (ST8SiaIV) transfer sialic acid from CMP-sialic acid to sialic acid residues of other sialoglycans to yield 2,8-linked polymerized structures (polySia) [8]. PolySia is associated with tumor development, including glioblastoma [9], lung cancer [10], and many other cancers [11,12]. It has been reported that polySia is expressed in breast cancer MCF7 cells [13], but few studies have focused on the biological role of polySia in breast cancer progression. Neural cell adhesion molecule (NCAM) is the major polySia substrate in cancer cells [14]. Polysialylated NCAM inhibits cell-cell/cell-ECM interactions due to the steric effects of polySia. Therefore, polySia-NCAM is used as diagnostic marker because of the highly invasive and proliferative characteristics in polySia-expressing cancers [15].

NCAM is a member of the immunoglobulin superfamily cell adhesion molecules and has three isoforms (NCAM-120, NCAM-140, NCAM-180) based on alternative splicing [16]. NCAM-140 and -180 are transmembrane proteins; NCAM-120 is a glycosylphosphatidylinositol (GPI)-anchored protein. The extracellular region of NCAM comprises five immunoglobulin-like (Ig1-5) domains with six N-glycosylation sites and two fibronectin type-III-like (FN1-2) domains [15]. Elevated NCAM expression has been reported in pancreatic cancer [17], neuroblastoma [18], small cell lung cancer [19], and other cancers. Polysialylated NCAM levels are also correlated with tumor metastasis [20]. NCAM plays critical roles as a signal transducer in regulating cell migration proliferation, apoptosis and EMT in multiple cancer cells [21], but polySia has not been specifically evaluated in that context. The effects of hypersialylation or hyposialylation of NCAM on various cell behaviors remain unclear.