Gopal Shashi K, Greening David W, Hanssen Eric G, Zhu Hong-Jian, Simpson Richard J, Mathias Rommel A
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
Oncotarget. 2016 Apr 12;7(15):19709-22. doi: 10.18632/oncotarget.7573.
The metastatic cascade describes the escape of primary tumour cells to distant secondary sites. Cells at the leading tumour edge are thought to undergo epithelial-mesenchymal transition (EMT), to enhance their motility and invasion for spreading. Whether EMT cells directly promote tumour angiogenesis, and the role of exosomes (30-150 nm extracellular vesicles) remains largely unknown. We examined the functional effects of exosomes from MDCK cells, MDCK cells stably expressing YBX1 (MDCKYBX1, intermediate EMT), and Ras-transformed MDCK cells (21D1 cells, complete EMT). 2F-2B cell motility and tube formation (length and branching) was significantly increased following supplementation with MDCKYBX1 or 21D1 exosomes, but not MDCK exosomes. Next, Matrigel™ plugs containing exosome-supplemented 2F-2B cells were subcutaneously injected into mice. Systemic perfusion was only observed for plugs supplemented with MDCKYBX1 or 21D1 exosomes. Comparative proteomics revealed that 21D1 exosomes contained VEGF-associated proteins, while MDCKYBX1 exosomes were enriched with activated Rac1 and PAK2. To validate, 2F-2B cells and HUVECs were pre-treated with PAK inhibitors prior to exosome supplementation. PAK inhibition nullified the effects of MDCKYBX1 exosomes by reducing the tube length and branching to baseline levels. By contrast, the effects of 21D1 exosomes were not significantly decreased. Our results demonstrate for the first time that oncogenic cells undergoing EMT can communicate with endothelial cells via exosomes, and establish exosomal Rac1/PAK2 as angiogenic promoters that may function from early stages of the metastatic cascade.
转移级联反应描述了原发性肿瘤细胞向远处继发性位点的转移过程。肿瘤前沿的细胞被认为会经历上皮-间质转化（EMT），以增强其迁移和侵袭能力从而实现扩散。EMT细胞是否直接促进肿瘤血管生成，以及外泌体（30 - 150纳米的细胞外囊泡）的作用在很大程度上仍不清楚。我们研究了来自MDCK细胞、稳定表达YBX1的MDCK细胞（MDCKYBX1，处于中间EMT阶段）和Ras转化的MDCK细胞（21D1细胞，处于完全EMT阶段）的外泌体的功能效应。用MDCKYBX1或21D1外泌体处理后，2F - 2B细胞的迁移能力以及管腔形成（长度和分支）显著增加，但MDCK外泌体处理后无此现象。接下来，将含有经外泌体处理的2F - 2B细胞的基质胶栓皮下注射到小鼠体内。仅在补充了MDCKYBX1或21D1外泌体的栓子中观察到全身灌注。比较蛋白质组学显示，21D1外泌体含有与血管内皮生长因子（VEGF）相关的蛋白质，而MDCKYBX1外泌体富含活化的Rac1和PAK2。为进行验证，在补充外泌体之前，先用PAK抑制剂预处理2F - 2B细胞和人脐静脉内皮细胞（HUVECs）。PAK抑制通过将管腔长度和分支减少到基线水平，消除了MDCKYBX1外泌体的作用。相比之下，21D1外泌体的作用没有显著降低。我们的结果首次证明，经历EMT的致癌细胞可通过外泌体与内皮细胞进行通讯，并确定外泌体Rac1/PAK2为血管生成促进因子，其可能在转移级联反应的早期阶段发挥作用。
