Cancer Virology Program, University of Pittsburgh Medical Center, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Cancer Virology Program, University of Pittsburgh Medical Center, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
J Virol. 2018 Aug 29;92(18). doi: 10.1128/JVI.00557-18. Print 2018 Sep 15.
Nasopharyngeal carcinoma (NPC) is a metastatic Epstein-Barr virus (EBV)-associated cancer that expresses the viral oncogenic protein, latent membrane protein 1 (LMP1). During epithelial metastasis, detached cells must overcome anoikis-induced cell death and gain the ability to reattach and restore growth potential. Anoikis assays have revealed cell survival mechanisms during suspension, but few studies have tracked the fate of cells surviving anoikis-inducing conditions. In this study, a modified anoikis assay was used to examine if the expression of LMP1 confers the recovery of epithelial cells from anoikis. Cells expressing LMP1 mutants and strains were evaluated for distinguishing properties in survival during suspension, reattachment, and outgrowth potential. Expression of LMP1 promoted the outgrowth of the NPC cell line HK1 following anoikis induction that was not attributed to enhanced cell survival in suspension or reattachment. The mechanism of LMP1-induced outgrowth required Akt signaling and the conserved PXQXT motif on LMP1, which activates Akt. Deletion of any of the three LMP1 C-terminal activation regions (CTAR) abrogated anoikis recovery, suggesting that additional LMP1-regulated signaling pathways are likely involved. Of the seven LMP1 strains, only B958, China1, and Med promoted HK1 outgrowth from anoikis. This distinguishing biological property segregates LMP1 strains into two categories (anoikis recovering and nonrecovering) and suggests that LMP1 strain-specific sequences may be important in determining metastatic outgrowth potential in NPC tumors. LMP1 is one of the most divergent sequences in the EBV genome and phylogenetically segregates into seven LMP1 strains. The LMP1 strains differ in geographical distribution and NPC tumor prevalence, but the molecular basis for this potential selection is not clear. While there are signaling motifs conserved in all LMP1 sequences from circulating EBV isolates, phylogenetic studies of NPC also suggest that there may be sequence selection for tumor-associated LMP1 strains and polymorphisms. The present study describes a modified anoikis assay that can distinguish LMP1 strains into two groups by biological properties. The pleiotropic LMP1 signaling properties and sequence diversity may offer a unique opportunity to illuminate the complex mechanisms of metastasis. Although the host genomic landscape is variable between NPC tumors, the present functional-mapping studies on LMP1 support the notion that viral proteins could serve as molecular tool kits and potentially reveal sequence-associated risk factors in NPC metastatic progression.
鼻咽癌（NPC）是一种转移性的 EBV 相关癌症，表达病毒致癌蛋白潜伏膜蛋白 1（LMP1）。在上皮细胞转移过程中，分离的细胞必须克服凋亡诱导的细胞死亡，并获得重新附着和恢复生长潜力的能力。凋亡检测已经揭示了悬浮过程中细胞存活的机制，但很少有研究追踪在诱导凋亡条件下存活的细胞的命运。在这项研究中，使用改良的凋亡检测来检查 LMP1 的表达是否赋予上皮细胞从凋亡中恢复的能力。表达 LMP1 突变体和菌株的细胞在悬浮、重新附着和生长潜力方面的存活特性进行了评估。表达 LMP1 促进了 NPC 细胞系 HK1 在诱导凋亡后的生长，这归因于 LMP1 诱导的凋亡后细胞在悬浮或重新附着时的生存能力没有提高。LMP1 诱导的生长的机制需要 Akt 信号和 LMP1 上保守的 PXQXT 基序，该基序激活 Akt。任何一个 LMP1 C 端激活区（CTAR）的缺失都消除了凋亡的恢复，这表明可能涉及其他 LMP1 调节的信号通路。在七种 LMP1 株中，只有 B958、China1 和 Med 促进了 HK1 从凋亡中恢复生长。这种独特的生物学特性将 LMP1 株分为两类（能恢复凋亡和不能恢复凋亡），表明 LMP1 株特异性序列可能在决定 NPC 肿瘤的转移生长潜力方面很重要。LMP1 是 EBV 基因组中最具差异的序列之一，并且在进化上分为七种 LMP1 株。LMP1 株在地理分布和 NPC 肿瘤流行率上存在差异，但这种潜在选择的分子基础尚不清楚。虽然所有循环 EBV 分离株的 LMP1 序列都有保守的信号基序，但 NPC 的系统发育研究也表明，肿瘤相关 LMP1 株和多态性可能存在序列选择。本研究描述了一种改良的凋亡检测方法，该方法可以通过生物学特性将 LMP1 株分为两组。LMP1 的多效性信号特性和序列多样性为阐明转移的复杂机制提供了一个独特的机会。尽管 NPC 肿瘤之间的宿主基因组景观存在差异，但本研究中关于 LMP1 的功能图谱研究支持这样一种观点，即病毒蛋白可以作为分子工具包，并可能揭示 NPC 转移进展中的与序列相关的风险因素。
