A proteomic approach to identify endosomal cargoes controlling cancer invasiveness.
A proteomic approach to identify endosomal cargoes controlling cancer invasiveness.
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DOI：
10.1242/jcs.190835
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发表时间：
2017-02-15
影响因子：
4
通讯作者：
Norman JC
中科院分区：
文献类型：
--
作者：
Diaz-Vera J;Palmer S;Hernandez-Fernaud JR;Dornier E;Mitchell LE;Macpherson I;Edwards J;Zanivan S;Norman JC
我们以前已经证明，Rab17是一个与上皮极性相关的小GTP酶，它被ERK2(也称为MAPK1)信号特异性抑制，从而促进侵袭性表型。然而，Rab17丢失允许侵袭性的机制，以及负责调节这一过程的内体货物，尚不清楚。利用基于定量质谱学的蛋白质组学，我们发现Rab17的敲除导致v-SNARE(Vamp8)细胞水平的高度选择性降低。此外，蛋白质组学和免疫荧光显示Vamp8与Rab17在内体晚期相关。Vamp8水平降低促进导管原位癌(DCIS)向更具侵袭性的表型转化。我们开发了一种无偏倚的蛋白质组学方法来阐明在内体和质膜之间重新分布的受体的补体，并将针尖神经毛细蛋白-2(NRP2)作为Rab17和Vamp8调节的运输的关键亲侵袭货物。事实上，Rab17或Vamp8水平的降低会导致含有NRP2的晚期内小体的动员增加，并上调细胞表面NRP2的表达。最后，我们证明了NRP2是伴随DCIS向更具侵袭性表型转变的基底膜破坏所必需的。本文描述了一种新的蛋白质组学方法来识别前侵袭性货物，我们已经使用该方法来确定Rab17控制Vamp8水平以驱动神经毛细蛋白-2运输，促进侵袭性。
We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype. This paper describes a novel proteomic approach to identify pro-invasive cargoes, which we have used to determine that Rab17 controls Vamp8 levels to drive neuropilin-2 trafficking, promoting invasion.