Mozzi Alessandra, Forcella Matilde, Riva Alice, Difrancesco Carlotta, Molinari Francesca, Martin Vittoria, Papini Nadia, Bernasconi Barbara, Nonnis Simona, Tedeschi Gabriella, Mazzucchelli Luca, Monti Eugenio, Fusi Paola, Frattini Milo
Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Milano 20126, Italy.
Laboratory of Molecular Pathology, Institute of Pathology, Locarno CH-6600, Switzerland.
Glycobiology. 2015 Aug;25(8):855-68. doi: 10.1093/glycob/cwv026. Epub 2015 Apr 28.
Several studies performed over the last decade have focused on the role of sialylation in the progression of cancer and, in particular, on the association between deregulation of sialidases and tumorigenic transformation. The plasma membrane-associated sialidase NEU3 is often deregulated in colorectal cancer (CRC), and it was shown that this enzyme co-immunoprecipitates in HeLa cells with epidermal growth factor receptor (EGFR), the molecular target of most recent monoclonal antibody-based therapies against CRC. To investigate the role of NEU3 sialidase on EGFR deregulation in CRC, we first collected data on NEU3 gene expression levels from a library of commercial colon cell lines, demonstrating that NEU3 transcription is upregulated in these cell lines. We also found EGFR to be hyperphosphorylated in all cell lines, with the exception of SW620 cells and the CCD841 normal intestinal cell line. By comparing the effects induced by overexpression of either the wild-type or the inactive mutant form of NEU3 on EGFR, we demonstrated that the active form of NEU3 enhanced receptor activation without affecting EGFR mRNA or protein expression. Moreover, through western blots and mass spectrometry analysis, we found that EGFR immunoprecipitated from cells overexpressing active NEU3, unlike the receptor from mock cells and cells overexpressing inactive NEU3, is desialylated. On the whole, our data demonstrate that, besides the already reported indirect EGFR activation through GM3, sialidase NEU3 could also play a role on EGFR activation through its desialylation.
过去十年进行的多项研究聚焦于唾液酸化在癌症进展中的作用，特别是唾液酸酶失调与肿瘤发生转化之间的关联。质膜相关唾液酸酶NEU3在结直肠癌（CRC）中常出现失调，并且已表明该酶在HeLa细胞中与表皮生长因子受体（EGFR）共免疫沉淀，EGFR是最近基于单克隆抗体的CRC治疗的分子靶点。为了研究NEU3唾液酸酶在CRC中EGFR失调方面的作用，我们首先从商业结肠细胞系文库中收集了NEU3基因表达水平的数据，证明这些细胞系中NEU3转录上调。我们还发现除SW620细胞和CCD841正常肠细胞系外，所有细胞系中的EGFR均发生过度磷酸化。通过比较野生型或无活性突变型NEU3过表达对EGFR的影响，我们证明NEU3的活性形式增强了受体激活，而不影响EGFR mRNA或蛋白质表达。此外，通过蛋白质印迹和质谱分析，我们发现从过表达活性NEU3的细胞中免疫沉淀的EGFR，与来自模拟细胞和过表达无活性NEU3的细胞中的受体不同，是去唾液酸化的。总体而言，我们的数据表明，除了已经报道的通过GM3间接激活EGFR外，唾液酸酶NEU3还可能通过其去唾液酸化作用在EGFR激活中发挥作用。
