Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells.
Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells.
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DOI：
10.1371/journal.pone.0148075
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Shiloach J
中科院分区：
文献类型：
--
作者：
Chu C;Bottaro DP;Betenbaugh MJ;Shiloach J
上皮-间充质转化（EMT）是一种复杂的癌症进展，其可以通过诱导迁移、细胞粘附丧失和促进在锚定非依赖性条件下的增殖来增强转化细胞的转移潜力。进行DNA微阵列分析，比较亲本锚定依赖性MDCK细胞和经工程改造以表达人siat 7 e（ST 6 GALNAC 5）的锚定非依赖性MDCK细胞。比较确定了几个基因参与EMT过程中的差异表达之间的锚定依赖性和锚定非依赖性MDCK细胞系。发现肝细胞生长因子基因（hgf）在工程改造的MDCK-siat 7 e细胞中在转录和蛋白表达水平上过度表达。MET受体酪氨酸激酶的磷酸化分析证实了MDCK-siat 7 e细胞中HGF/ MET信号通路的自分泌环的激活。当使用小分子抑制剂药物PF-02341066（克唑替尼）抑制MET活性时，锚定非依赖性MDCK-siat 7 e细胞恢复到亲本锚定依赖性MDCK细胞的细胞形态。这些观察结果表明，MET受体在MDCK细胞的生长特性中起着核心作用，其磷酸化状态可能依赖于唾液酸化。对MET网络中下游信号靶标的进一步研究表明，MDCK细胞粘附的程度与基质金属蛋白酶MMP 1的分泌水平相关，表明金属蛋白酶在EMT过程中的作用。这些结果表明，除了其在生物技术过程中的应用，MDCK-siat 7 e可以作为转移研究的模型细胞，以破译导致EMT激活的事件序列。
The epithelial-mesenchymal transition (EMT) is a complex cancer progression that can boost the metastatic potential of transformed cells by inducing migration, loss of cell adhesion, and promoting proliferation under anchorage-independent conditions. A DNA microarray analysis was performed comparing parental anchorage-dependent MDCK cells and anchorage-independent MDCK cells that were engineered to express human siat7e (ST6GALNAC5). The comparison identified several genes involved in the EMT process that were differentially expressed between the anchorage-dependent and the anchorage-independent MDCK cell lines. The hepatocyte growth factor gene (hgf) was found to be over-expressed in the engineered MDCK-siat7e cells at both transcription and protein expression levels. Phosphorylation analysis of the MET receptor tyrosine kinase confirmed the activation of an autocrine loop of the HGF/ MET signaling pathway in the MDCK-siat7e cells. When MET activities were suppressed by using the small-molecular inhibitor drug PF-02341066 (Crizotinib), the anchorage-independent MDCK-siat7e cells reverted to the cellular morphology of the parental anchorage-dependent MDCK cells. These observations indicate that the MET receptor plays a central role in the growth properties of the MDCK cells and its phosphorylation status is likely dependent on sialylation. Further investigation of the downstream signaling targets in the MET network showed that the degree of MDCK cell adhesion correlated with secretion levels of a matrix metalloproteinase, MMP1, suggesting a role of metalloproteinases in the EMT process. These results demonstrate that in addition to its application in biotechnology processes, MDCK-siat7e may serve as a model cell for metastasis studies to decipher the sequence of events leading up to the activation of EMT.