Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes -: Inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase
Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes -: Inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase
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DOI：
10.1074/jbc.m611518200
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发表时间：
2007-07-27
影响因子：
4.8
通讯作者：
Pierce, Michael
中科院分区：
文献类型：
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作者：
Guo, Hua-Bei;Randolph, Matthew;Pierce, Michael
N 连接聚糖分支的糖基转移酶表达的变化可以改变几种类型的细胞表面受体和葡萄糖转运蛋白的功能。为了详细研究 N-乙酰氨基葡萄糖转移酶 V（GnT-V、GnT-Va 和 Mgat5a）表达改变引起的异常 N-糖基化调节某些癌症中发现的侵袭性相关表型的机制，我们利用特定的小干扰 RNA (siRNA) 选择性敲低高度转移和侵袭性人乳腺癌细胞系 MDA-MB231 中的 GnT-V 表达。正如预期的那样，通过 siRNA 表达敲低 GnT-V 对表皮生长因子受体表达水平没有影响，但降低了表皮生长因子受体上 N 连接 β(1,6) 分支的表达。与对照细胞相比，GnT-V 的敲低导致形态变化和细胞从基质脱离的显着抑制，这通常在表皮生长因子 (EGF) 刺激后出现。 GnT-V 表达的减少导致 EGF 诱导的粘着斑激酶 (FAK) 去磷酸化受到显着抑制，这与表达 GnT-V siRNA 的细胞中缺乏细胞形态变化一致。在 GnT-V 敲低细胞中，显着观察到 EGF 介导的磷酸化和酪氨酸磷酸酶 SHP-2 激活的减弱，并且可以通过将 GnT-V 重新引入这些细胞来挽救这些效应，表明 EGF 介导的 SHP-2 激活的减少与 GnT-V 相关。与此同时，GnT-V 的敲低导致 EGF 介导的 ERK 信号传导和肿瘤细胞侵袭相关表型减少，包括对肌动蛋白重排和细胞运动的影响。然而，敲除 GnT-V 后，没有观察到 EGF 结合的变化。我们的结果表明，人乳腺癌细胞中 siRNA 表达导致 GnT-V 活性降低，导致 EGF 刺激的 SHP-2 激活受到抑制，从而导致 EGF 诱导的 FAK 去磷酸化减弱。这些作用抑制了 EGF 介导的下游信号传导和侵袭相关表型，表明 GnT-V 作为潜在的治疗靶点。
Changes in the expression of glycosyltransferases that branch N-linked glycans can alter the function of several types of cell surface receptors and a glucose transporter. To study in detail the mechanisms by which aberrant N-glycosylation caused by altered N-acetylglucosaminyltransferase V(GnT-V, GnT-Va, and Mgat5a) expression can regulate the invasiveness-related phenotypes found in some carcinomas, we utilized specific small interfering RNA (siRNA) to selectively knock down GnT-V expression in the highly metastatic and invasive human breast carcinoma cell line, MDA-MB231. Knockdown of GnT-V by siRNA expression had no effect on epidermal growth factor receptor expression levels but lowered expression of N-linked beta(1,6)-branching on epidermal growth factor receptor, as expected. Compared with control cells, knockdown of GnT-V caused significant inhibition of the morphological changes and cell detachment from matrix that is normally seen after stimulation with epidermal growth factor (EGF). Decreased expression of GnT-V caused a marked inhibition of EGF-induced dephosphorylation of focal adhesion kinase (FAK), consistent with the lack of cell morphology changes in the cells expressing GnT-V siRNA. The attenuation of EGF-mediated phosphorylation and activation of the tyrosine phosphatase SHP-2 was dramatically observed in GnT-V knockdown cells, and these effects could be rescued by reintroduction of GnT-V into these cells, indicating that reduced EGF-mediated activation of SHP-2 was GnT-V related. Concomitantly, knockdown of GnT-V caused reduced EGF-mediated ERK signaling and tumor cell invasiveness-related phenotypes, including effects on actin rearrangement and cell motility. No changes in EGF binding were observed, however, after knockdown of GnT-V. Our results demonstrate that decreased GnT-V activity due to siRNA expression in human breast carcinoma cells resulted in an inhibition of EGF-stimulated SHP-2 activation and, consequently, caused attenuation of the dephosphorylation of FAK induced by EGF. These effects suppressed EGF-mediated downstream signaling and invasiveness-related phenotypes and suggest GnT-V as a potential therapeutic target.