NEU4L INDUCES ALTERATIONS ON CELL PROLIFERATION AND DIFFERENTIATION IN NEUROBLASTOMA CELL LINE, SK-N-BE

Sialidases are glycohydrolytic enzymes that remove sialic acid residues from gangliosides, sialoglycoproteins and oligosaccharides. The most recently identified member of the human sialidase family is Neu4 and is found in two forms, NEU4 long (Neu4L) and NEU4 short (Neu4S), differing in the presence of a 12-amino-acid sequence at the N-terminus (1-2). Overall, Neu4 is scarcely expressed in adult tissues, in contrast to fetal tissues, where its biological role is currently unclear. We evidenced that the more aggressive neuroblastoma cell lines, a tumor derived from the neural crest, characteristically show a high expression of Neu4L. Here, we demonstrated that this feature is connected to the distorted control of proliferation and differentiation in neuroblastoma. In order to understand Neu4L role in neuroblastoma, we stably over-expressed Neu4L in human neuroblastoma cells, SK-N-BE. The first important consequence was a marked acceleration of proliferation rate assessed by increase of 3[H]thymidine incorporation (+ 36%), by cell count for up to 4 days (+36%), by MTT test for up 4 days (+26) and by soft agar assay. Moreover, Neu4L over-expressing cells were impaired to differentiate under FBS depletion culture, as we assessed following the formation and the length of neurites, MTT assay and the content of src, as neuronal marker. These phenotypical modifications were associated to a marker activation of Wnt/-Catenin pathway, a well-known regulator of proliferation and self-renewal in many cell types. We assessed an increased content of intracellular, not phosphorylated -Catenin (+30%) in Neu4L over-expressing SK-N-BE cells. In parallel, the main inhibitor of Wnt pathway, GSK3, decreased its expression as mRNA (-30%), while c-myc, cyclin D2 and axin2, which have been identified as target genes of -Catenin (3), underwent a significant increase, as mRNA content, in Neu4L over-expressing SK-N-BE cells. In order to further confirm the activation of the Wnt canonical pathway, we transiently transfected the TOP-Flash luciferase vector into mock and Neu4L over-expressing cells, as gene reporter assay responsive to -Catenin. We detected an increased activation of luciferase in Neu4L over-expressing cell (+52%), confirming our previous results. In order to confirm the greater ability of control cells to differentiate, we studied the neuronal marker , v-src. During the process of differentiation, we observed that Neu4L over-expressing cells had a lower content of this protein in contrast with control cells. Moreover, we identified the precise localization of Neu4L in SK-N-BE cells. We observed that Neu4L mainly localized in mitochondria (40%) and in endoplasmatic reticulum (60%). The direct substrates of Neu4L, in SK-N-BE cells, seem to be soluble glycoproteins in the range of 60 kDa; on the base of literature data, it could be hypothesized that one of these glycoproteins could belong to the Wnt family or to inhibitor-glycoproteins interacting with Wnt signaling. Therefore, we provided the evidence that Neu4L promotes the activation of Wnt/-Catenin signaling in neuroblastoma, inducing proliferation and inhibiting differentiation at the same time, enhancing self-renewal of malignant neuroblasts. 1 Monti E et al. (2004) Genomics 83: 445–53 2 Yamaguchi K et al. (2005) Biochem J 390: 85–93 3 Clevers H et al. (2006) Cell 127; 469-480.