探查EB病毒感染中RON 酪胺酸激酶的表現及其對EB病毒相關疾病的致病機轉之影響

Abstract

EB病毒是一具致癌性的疱疹病毒，與許多惡性疾病有高度相關，包含Burkitt 氏淋巴瘤、Hodgkin氏症、T細胞淋巴瘤、NK細胞淋巴瘤、移植後淋巴增生疾病及鼻咽癌。EB病毒可以透過多種調控機制不朽化(immortalize)人類初代B細胞轉形為具有不斷分裂能力的淋巴母細胞株(LCL)。然而，對於酪胺酸激酶在EB病毒所造成的B細胞不斷增生的關係尚不清楚。在此研究中，我們利用kinase display方法發現RON酪胺酸激酶會表現在淋巴母細胞株中，但不表現在正常的B淋巴球中。進一步，我們發現在B細胞株及淋巴母細胞株中均可觀察到EB病毒所表現的潛伏膜蛋白1 (LMP1)可以直接透過其上的CTAR1 domain活化NF-kB結合到RON之啟動子上，而增加RON的表現及活化其酪胺酸激酶的活性。剔除RON的表現就可成功地抑制淋巴母細胞株的生長，而在LMP1剔除的細胞中，表現RON則可以回復剔除LMP1所造成的生長抑制。免疫組織化學分析發現移植後淋巴增生性疾病病人的切片檢體可以觀察到LMP1和RON的表現具有相關性，証實LMP1所誘導之RON不僅為影響淋巴母細胞株生長所必需而且RON在移植後淋巴增生疾病的致病機轉中扮演重要角色。 另一方面，鼻咽癌不同於其他頭頸部的癌症是因為具有高度的轉移性且和EB病毒有高度相關性。我們發現LMP1可以誘導RON表現並活化其酪胺酸激酶活性而刺激上皮間質細胞轉化epithelial- mesenchymal transition (EMT)和促進細胞移動及入侵。此外，ERK為LMP1和RON下游之訊息傳遞鏈的重要分子。剔除RON可以回復LMP1所誘導的上皮間質細胞轉化和抑制LMP1所引起的細胞移動及入侵和抑制ERK的活化。在分子機制方面，可以觀察到LMP1透過其CTAR1 domain活化NF-kB結合到RON之啟動子而促進RON表現。免疫組織化學染色顯示RON大量表現在原位鼻咽癌和轉移鼻咽癌的病人檢體中而不表現在控制組組織，而且LMP1和RON的表現有顯著的相關性。 這些結果提供RON在移植後淋巴增生性疾病及鼻咽癌的致病機轉中的新觀點，推測RON可能可以成為在EB病毒相關疾病中新穎的治療標靶分子。

Epstein-Barr virus (EBV), an oncogenic human herpesvirus, is associated with several malignancies, including Burkitt’s lymphoma, Hodgkin’s lymphoma, T cell lymphoma, NK cell lymphoma, post-transplantation lymphoproliferative disorder (PTLD), and nasopharyngeal carcinoma (NPC). EBV can transform primary B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) through multiple regulatory mechanisms. However, the involvement of protein tyrosine kinases in the infinite proliferation of B cells is not clear. In this study, we performed kinase display assays to investigate this subject and identified a specific cellular target, Recepteur d’Origine Nantais (RON) tyrosine kinase, expressed in LCLs but not in primary B cells. Furthermore, we found that latent membrane protein 1 (LMP1), an important EBV oncogenic protein, enhanced RON expression and activation through its carboxyl-terminal activation region-1 (CTAR1) by promoting nuclear factor (NF)-kB binding to the RON promoter in B cells and LCLs. RON knockdown decreased the proliferation of LCLs and transfection with RON compensated for the growth inhibition caused by knockdown of LMP1. Immunohistochemical analysis revealed a correlation between LMP1 and RON expression in biopsies from PTLD, suggesting that LMP1-induced RON expression not only is essential for the growth of LCLs but also may contribute to the pathogenesis of EBV-associated PTLD. In addition, NPC is distinct from other human head and neck cancers because of its highly metastatic character and strong association with EBV. Here we show that the LMP1 induces and activates RON to stimulate epithelial-mesenchymal transition (EMT) and promotes cell migration and invasion. Furthermore, ERK is known to be involved in the downstream signaling of LMP1-RON pathways. Knockdown of RON in cells expressing LMP1 significantly reverses LMP1-induced EMT, suppresses LMP1-induced cell migration and invasion, and inhibits ERK activation. At the molecular level, LMP1 stimulates NF-kB binding to the RON promoter through its CTAR1 domain to induce expression of RON. Immunohistochemical staining showed expression of RON in NPC biopsies but not in control tissues and revealed a significant correlation of LMP1 and RON expression in biopsies from primary and metastatic NPC. These results may provide a new insight into the pathogenesis of RON receptor tyrosine kinase in PTLD and NPC, suggesting that RON may be a novel therapeutic target for EBV-associated diseases.