Epstein-Barr Virus-Encoded LMP1 Interacts with FGD4 to Activate Cdc42 and Thereby Promote Migration of Nasopharyngeal Carcinoma Cells

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a human malignancy notorious for its highly metastatic nature. Among EBV-encoded genes, latent membrane protein 1 (LMP1) is expressed in most NPC tissues and exerts oncogenicity by engaging multiple signaling pathways in a ligand-independent manner. LMP1 expression also results in actin cytoskeleton reorganization, which modulates cell morphology and cell motility— cellular process regulated by RhoGTPases, such as Cdc42. Despite the prominent association of Cdc42 activation with tumorigenesis, the molecular basis of Cdc42 activation by LMP1 in NPC cells remains to be elucidated. Here using GST-CBD (active Cdc42-binding domain) as bait in GST pull-down assays to precipitate active Cdc42 from cell lysates, we demonstrated that LMP1 acts through its transmembrane domains to preferentially induce Cdc42 activation in various types of epithelial cells, including NPC cells. Using RNA interference combined with re-introduction experiments, we identified FGD4 (FYVE, RhoGEF and PH domain containing 4) as the GEF (guanine nucleotide exchange factor) responsible for the activation of Cdc42 by LMP1. Serial deletion experiments and co-immunoprecipitation assays further revealed that ectopically expressed FGD4 modulated LMP1-mediated Cdc42 activation by interacting with LMP1. Moreover, LMP1, through its transmembrane domains, directly bound FGD4 and enhanced FGD4 activity toward Cdc42, leading to actin cytoskeleton rearrangement and increased motility of NPC cells. Depletion of FGD4 or Cdc42 significantly reduced (∼50%) the LMP1-stimulated cell motility, an effect that was partially reversed by expression of a constitutively active mutant of Cdc42. Finally, quantitative RT-PCR and immunohistochemistry analyses showed that FGD4 and LMP1 were expressed in NPC tissues, supporting the potential physiologically relevance of this mechanism in NPC. Collectively, our results not only uncover a novel mechanism underlying LMP1-mediated Cdc42 activation, namely LMP1 interaction with FGD4, but also functionally link FGD4 to NPC tumorigenesis.

EB病毒（Epstein-Barr virus, EBV）与鼻咽癌（nasopharyngeal carcinoma, NPC）密切相关，而鼻咽癌是一种以高转移性著称的人类恶性肿瘤。在EB病毒编码的基因中，潜伏膜蛋白1（latent membrane protein 1, LMP1）在多数鼻咽癌组织中均有表达，且通过不依赖配体的方式激活多条信号通路，从而发挥致癌作用。LMP1的表达还会引发肌动蛋白细胞骨架（actin cytoskeleton）重排，进而调控细胞形态与细胞迁移——这一细胞过程由Rho小GTP酶（RhoGTPases）家族成员调控，例如细胞分裂周期蛋白42（Cdc42）。尽管Cdc42的激活与肿瘤发生存在显著关联，但LMP1在鼻咽癌细胞中激活Cdc42的分子机制仍有待阐明。本研究以谷胱甘肽S-转移酶-Cdc42结合结构域（GST-CBD, active Cdc42-binding domain）为诱饵，通过GST下拉实验（GST pull-down assays）从细胞裂解液（cell lysates）中沉淀活性形式的Cdc42，结果证实LMP1可通过其跨膜结构域（transmembrane domains），在包括鼻咽癌细胞在内的多种上皮细胞（epithelial cells）中优先诱导Cdc42激活。本研究结合RNA干扰（RNA interference）与回补实验（re-introduction experiments），鉴定出含FYVE、RhoGEF和PH结构域蛋白4（FGD4, FYVE, RhoGEF and PH domain containing 4）是介导LMP1激活Cdc42的鸟苷酸交换因子（GEF, guanine nucleotide exchange factor）。系列缺失实验（serial deletion experiments）与免疫共沉淀实验（co-immunoprecipitation assays）进一步证实，异位表达的FGD4可通过与LMP1相互作用，调控LMP1介导的Cdc42激活。此外，LMP1可通过其跨膜结构域直接结合FGD4，并增强FGD4对Cdc42的活性，进而引发肌动蛋白细胞骨架重排，提升鼻咽癌细胞的迁移能力。敲低FGD4或Cdc42可显著降低（约50%）LMP1诱导的细胞迁移能力，而过表达组成型激活的Cdc42突变体（constitutively active mutant）可部分逆转这一效应。最后，定量逆转录聚合酶链反应（quantitative RT-PCR）与免疫组织化学（immunohistochemistry）分析结果显示，FGD4与LMP1在鼻咽癌组织中均有表达，证实了该机制在鼻咽癌中潜在的生理相关性。综上，本研究不仅揭示了LMP1介导Cdc42激活的全新机制——即LMP1与FGD4相互作用，还从功能上将FGD4与鼻咽癌的肿瘤发生过程关联起来。