Wang Huixin, Patel Vyomesh, Miyazaki Hiroshi, Gutkind J Silvio, Yeudall W Andrew
Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298-0566, USA.
Carcinogenesis. 2009 Jan;30(1):165-74. doi: 10.1093/carcin/bgn252. Epub 2008 Nov 12.
We have investigated the role of the signaling intermediate, EPS8, in tumor progression using a model system and in vivo. HN4 primary tumor cells express low levels of EPS8, similar to normal keratinocytes, and show minimal invasion in vitro in response to epidermal growth factor, whereas HN12 cells express high levels of EPS8 and are highly motile in vitro and tumorigenic in vivo. Additional independent tumor cell lines also showed elevated EPS8 expression compared with normal keratinocytes. Using retroviral transduction, we generated HN4 cell lines expressing EPS8 (HN4/EPS8) at levels equivalent to those present in HN12 cells. HN4/EPS8 cells showed increased proliferation and migration compared with controls, together with elevated expression and activity of matrix metalloprotease (MMP)-9, which was dependent on protein kinase B (AKT) activity. Introduction of plasmids that direct synthesis of EPS8 short hairpin RNA (shRNA) into HN12 cells resulted in decreased EPS8 expression in these cells, which correlated with a decrease in their capacity to migrate and invade in vitro. In addition, shRNA-mediated knockdown of EPS8 reduced expression and activity of MMP-9 produced by these cells and reduced MMP-9 promoter activity. EPS8 knockdown cells showed decreased tumorigenicity in vivo compared with controls and lower MMP-9 expression. Conversely, overexpression of EPS8 in HN4 cells was sufficient to induce growth of these non-tumorigenic cells in orthotopic transplantation assays. Furthermore, EPS8 expression in clinical samples of squamous cell carcinoma showed variable expression levels and broadly paralleled expression of MMP-9. The data support a role for EPS8 in squamous carcinogenesis.
我们使用模型系统和体内实验研究了信号转导中间体EPS8在肿瘤进展中的作用。HN4原发性肿瘤细胞表达的EPS8水平较低，类似于正常角质形成细胞，并且在体外对表皮生长因子的反应中侵袭能力极小，而HN12细胞表达高水平的EPS8，在体外具有高度运动性且在体内具有致瘤性。与正常角质形成细胞相比，其他独立的肿瘤细胞系也显示出EPS8表达升高。通过逆转录病毒转导，我们构建了表达与HN12细胞中水平相当的EPS8（HN4/EPS8）的HN4细胞系。与对照相比，HN4/EPS8细胞显示出增殖和迁移增加，同时基质金属蛋白酶（MMP）-9的表达和活性升高，这依赖于蛋白激酶B（AKT）的活性。将指导合成EPS8短发夹RNA（shRNA）的质粒导入HN12细胞导致这些细胞中EPS8表达降低，这与它们在体外迁移和侵袭能力的降低相关。此外，shRNA介导的EPS8敲低降低了这些细胞产生的MMP-9的表达和活性，并降低了MMP-9启动子活性。与对照相比，EPS8敲低的细胞在体内致瘤性降低且MMP-9表达较低。相反，在原位移植实验中，HN4细胞中EPS8的过表达足以诱导这些非致瘤性细胞生长。此外，鳞状细胞癌临床样本中的EPS8表达显示出可变的表达水平，并且与MMP-9的表达大致平行。这些数据支持EPS8在鳞状细胞癌发生中的作用。
