Division of Cancer Glycosylation Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai.
Division of Cancer Molecular Biology, Graduate School of Medicine, Tohoku University, Sendai, Japan.
Int J Cancer. 2015 Oct 1;137(7):1560-73. doi: 10.1002/ijc.29527. Epub 2015 Apr 1.
The plasma membrane-associated sialidase NEU3 is a key enzyme for ganglioside degradation. We previously demonstrated remarkable up-regulation of NEU3 in various human cancers, with augmented malignant properties. Here, we provide evidence of a close link between NEU3 expression and Wnt/β-catenin signaling in colon cancer cells by analyzing tumorigenic potential and cancer stem-like characteristics. NEU3 silencing in HT-29 and HCT116 colon cancer cells resulted in significant decrease in clonogenicity on soft agar and in vivo tumor growth, along with down-regulation of stemness and Wnt-related genes. Analyses further revealed that NEU3 enhanced phosphorylation of the Wnt receptor LRP6 and consequently β-catenin activation by accelerating complex formation with LRP6 and recruitment of GSK3β and Axin, whereas its silencing exerted the opposite effects. NEU3 activity-null mutants failed to demonstrate the activation, indicating the requirement of ganglioside modulation by the sialidase for the effects. Under sphere-forming conditions, when stemness genes are up-regulated, endogenous NEU3 expression was found to be significantly increased, whereas NEU3 silencing suppressed sphere-formation and in vivo tumor incidence in NOD-SCID mice. Increased ability of clonogenicity on soft agar and sphere formation by Wnt stimulation was abrogated by NEU3 silencing. Furthermore, NEU3 was found to regulate phosphorylation of ERK and Akt via EGF receptor and Ras cascades, thought to be additionally required for tumor progression. The results indicate an essential contribution of NEU3 to tumorigenic potential through maintenance of stem-like characteristics of colon cancer cells by regulating Wnt signaling at the receptor level, in addition to tumor progression via Ras/MAPK signaling.
质膜相关唾液酸酶 NEU3 是神经节苷脂降解的关键酶。我们之前的研究表明，NEU3 在各种人类癌症中显著上调，具有增强的恶性特性。在这里，我们通过分析致瘤潜能和癌症干细胞样特征，提供了 NEU3 表达与结肠癌细胞中 Wnt/β-catenin 信号之间密切联系的证据。HT-29 和 HCT116 结肠癌细胞中 NEU3 的沉默导致软琼脂克隆形成和体内肿瘤生长显著减少，同时下调了干性和 Wnt 相关基因。分析进一步表明，NEU3 通过与 LRP6 加速复合物形成并募集 GSK3β 和 Axin，增强 Wnt 受体 LRP6 的磷酸化，从而增强 β-catenin 的激活，而其沉默则产生相反的效果。NEU3 活性缺失突变体未能显示出激活作用，表明该酶对神经节苷脂的调节对于这种作用是必需的。在形成球体的条件下，当干性基因上调时，内源性 NEU3 表达被发现显著增加，而 NEU3 沉默则抑制了 NOD-SCID 小鼠中的球体形成和体内肿瘤发生率。Wnt 刺激增加的软琼脂克隆形成能力和球体形成能力被 NEU3 沉默所阻断。此外，发现 NEU3 通过 EGF 受体和 Ras 级联调节 ERK 和 Akt 的磷酸化，这被认为是肿瘤进展所必需的。结果表明，NEU3 通过调节 Wnt 信号在受体水平上维持结肠癌细胞的干细胞样特征，除了通过 Ras/MAPK 信号促进肿瘤进展外，对肿瘤发生潜能也有重要贡献。
