Contribution of sialidase NEU1 to suppression of metastasis of human colon cancer cells through desialylation of integrin β4

Abstract

We previously found an inverse relationship between sialidase Neu1 expression and metastatic potential of murine cancer cells. To elucidate the mechanism underlying the cellular events, the human sialidase gene NEU1 was overexpressed or silenced in colon cancer HT-29 cells. When NEU1-overexpressing cells were injected transsplenically into mice, in vivo liver metastasis was significantly reduced. NEU1 suppressed cell migration, invasion and adhesion in vitro, whereas the silencing resulted in the opposite. One of the major molecular changes by NEU1 was decreased sialylation of integrin b4, assessed by PNAand MAL-II-lectin blotting of immunoprecipitates with anti-integrin b4 antibody. The desialylation was accompanied by decreased phosphorylation of the integrin followed by attenuation of focal adhesion kinase and Erk1/2 pathway. Moreover, NEU1 caused downregulation of matrix metalloproteinase-7, overexpression of which is associated with cancer metastasis. Treatment of the cells with GalNAc-a-O-benzyl, an inhibitor of O-glycosylation, showed increased PNA-positive integrin b4 with its decreased phosphorylation, indicating that sialic acid removal from the integrin O-glycans results in the decreased phosphorylation. Biotinylation and immunofluorescence staining exhibited some NEU1 molecules to be at the cell surface accessible to the integrin. These results suggest that NEU1 is important in regulation of integrin b4-mediated signaling, leading to suppression of metastasis.

NEU1 Sialidase Expressed in Human Airway Epithelia Regulates Epidermal Growth Factor Receptor (EGFR) and MUC1 Protein Signaling

“…4) implicates NEU1 in airway epithelial repair and wound healing, tumorigenesis, and metastatic potential (1,74). In fact, NEU1 overexpression has been reported to suppress anchorage-independent growth and metastasis of murine B16 melanoma cells (75) and human colon cancer cells (76). Because NEU1 desialylates MUC1 (Fig.…”

Regulation of Sialyl Lewis Antigen Expression in Colon Cancer Cells by Sialidase NEU4

“…Cell Surface Biotinylation-Cell surface biotinylation was carried out as described previously (16). Cell surface proteins were labeled with sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer's instructions.…”

“…Human sialidases are classified into four groups according to their enzymatic properties and subcellular localization (abbreviated to NEU1, NEU2, NEU3, and NEU4), and their expression levels indeed change in response to various cellular phenomena and especially in relation to cancer phenotype (14,15). We previously demonstrated that NEU1 shows down-regulation in cancers, promoting anchorage-independent growth and contributing to metastatic ability (16), whereas NEU3 exhibits marked up-regulation, playing as an essential factor for cancer cell survival (17,18). Among them, NEU4 is a unique sialidase efficiently removing sialic acid residues from mucins.…”

“…The ORF of human NEU4S tagged with HA at the C-terminal was subcloned into pcDNA 3.1 (Invitrogen), and a pCAGGS expression plasmid (a generous gift from Dr. Miyazaki, Osaka University School of Medicine), and then transfected with Lipofectamine 2000 reagent (Invitrogen). Expression vectors for NEU1, NEU2, and NEU3 were prepared as previously reported (24), and for NEU1 expression, the plasmid for the protective protein (carboxypeptidase A) pp11 gene was co-transfected (16). To obtain stable transfectants, G418 (Sigma) was used with HT29 cells at 600 g/ml and with DLD-1 at 300 g/ml.…”

Novel monoclonal antibodies against pancreatic juice from pancreatic cancer patients and their possible application in differential diagnosis

“…cell surface proteins in oncogenically transformed cells (14)(15)(16)(17). Thus, the >250 kDa PC-specific antigen in 6P-3-2-4 might be a cancer-related antigen with aberrant glycosylation; further investigation is required to confirm this.…”