Department of Medical and Molecular Genetics, Institute of Biomedical Research, University of Birmingham, Birmingham, UK.
Oncogene. 2010 Jul 29;29(30):4307-16. doi: 10.1038/onc.2010.192. Epub 2010 May 31.
The Ras-assocation domain family (RASSF) of tumor suppressor proteins until recently contained six proteins named RASSF1-6. Recently, four novel family members, RASSF7-10, have been identified by homology searches for RA-domain-containing proteins. These additional RASSF members are divergent and structurally distinct from RASSF1-6, containing an N-terminal RA domain and lacking the Sav/RASSF/Hpo (SARAH) domain. Here, we show that RASSF8 is ubiquitously expressed throughout the murine embryo and in normal human adult tissues. Functionally, RNAi-mediated knockdown of RASSF8 in non-small-cell lung cancer (NSCLC) cell lines, increased anchorage-independent growth in soft agar and enhanced tumor growth in severe combined immunodeficiency (SCID) mice. Furthermore, EdU staining of RASSF8-depleted cells showed growth suppression in a manner dependent on contact inhibition. We show that endogenous RASSF8 is not only found in the nucleus, but is also membrane associated at sites of cell-cell adhesion, co-localizing with the adherens junction (AJ) component beta-catenin and binding to E-cadherin. Following RASSF8 depletion in two different lung cancer cell lines using alternative small interfering RNA (siRNA) sequences, we show that AJs are destabilized and E-cadherin is lost from the cell membrane. The AJ components beta-catenin and p65 are also lost from sites of cell-cell contact and are relocalized to the nucleus with a concomitant increase in beta-catenin-dependent and nuclear factor-kappaB (NF-kappaB)-dependent signaling following RASSF8 depletion. RASSF8 may also be required to maintain actin -cytoskeletal organization since immunofluorescence analysis shows a striking disorganization of the actin- cytoskeleton following RASSF8 depletion. Accordingly, scratch wound healing studies show increased cellular migration in RASSF8-deficient cells. These results implicate RASSF8 as a tumor suppressor gene that is essential for maintaining AJs function in epithelial cells and have a role in epithelial cell migration.
Ras-association 结构域家族（RASSF）的抑癌蛋白，直到最近才包含六个蛋白，分别命名为 RASSF1-6。最近，通过同源性搜索 RA 结构域蛋白，发现了四个新的家族成员，RASSF7-10。这些额外的 RASSF 成员是不同的，与 RASSF1-6 在结构上有明显区别，含有一个 N 端 RA 结构域，缺乏 Sav/RASSF/Hpo（SARAH）结构域。在这里，我们表明 RASSF8 在整个小鼠胚胎和正常成人组织中广泛表达。功能上，非小细胞肺癌（NSCLC）细胞系中 RNAi 介导的 RASSF8 敲低，增加了软琼脂中的无锚定生长，并增强了严重联合免疫缺陷（SCID）小鼠中的肿瘤生长。此外，RASSF8 耗竭细胞的 EdU 染色显示出依赖于接触抑制的生长抑制。我们表明，内源性 RASSF8 不仅存在于核内，而且还与细胞-细胞黏附部位的膜相关，与黏着连接（AJ）成分β-连环蛋白共定位，并与 E-钙黏蛋白结合。在使用两种不同的肺癌细胞系中的替代小干扰 RNA（siRNA）序列耗尽 RASSF8 后，我们表明 AJ 不稳定，E-钙黏蛋白从细胞膜丢失。AJ 成分β-连环蛋白和 p65 也从细胞-细胞接触部位丢失，并重新定位到核内，同时伴随着 RASSF8 耗尽后β-连环蛋白依赖性和核因子-κB（NF-κB）依赖性信号的增加。RASSF8 可能还需要维持肌动蛋白细胞骨架组织，因为免疫荧光分析显示 RASSF8 耗尽后肌动蛋白细胞骨架发生明显紊乱。相应地，划痕愈合研究表明，RASSF8 缺陷细胞的细胞迁移增加。这些结果表明 RASSF8 是一种肿瘤抑制基因，对于维持上皮细胞 AJ 的功能是必不可少的，并且在上皮细胞迁移中起作用。
