Critical role of galectin-9 in EBV-driven transformation of human B-lymphocytes

Background: In several types of malignancies, especially EBV-associated nasopharyngeal carcinomas, high Galectin-9 (Gal-9) expression is indicative of an aggressive tumor phenotype. The contribution of Gal-9 to the oncogenesis of B-cell lymphomas (BCLs) has not yet been investigated. Methods: The expression of Gal-9, STING, and EBNA1 was measured by immunohistochemical (IHC) staining on tumor sections from 66 BCL patients. Artificial EBV infection of normal primary B cells in vitro was used as an experimental model. The dynamic changes of the transcriptome of EBV-infected B cells undergoing transformation were investigated by bulk RNA-sequencing and bioinformatics analysis. The oncogenic role of Gal-9 was investigated in vitro by addition of recombinant Gal-9 to EBV-infected primary B-cells, and growth assays. The underlying molecular mechanisms were investigated by immunoblotting and immunofluorescent (IF) staining, CHIP and luciferase reporter assays. Results: In clinical specimens of BCLs, Gal-9 expression was significantly associated with tumor stage, latent EBV infection and abundance of the viral latent protein EBNA1. Looking at the transcriptome changes occurring in vitro during EBV-driven B-cell transformation, we could identify a series of genes undergoing long term activation and remaining highly expressed in mature LCLs. The Gal-9 gene is one of them. Its expression is enhanced during the transformation process at the mRNA level and even more at the protein level. This up-regulation results at least in part from its transactivation by EBNA1 which can bind its promoter. Reciprocally, we find that addition of extra-cellular Gal-9 promotes B-cell transformation and establishment of the latent phase of EBV-infection. Concomitantly, extra-cellular Gal-9 blocks STING signaling and enhances STAT3 phosphorylation. Inhibition of Sting signaling or STAT3 phosphorylation blocks B-cell transformation, even in the presence of Gal-9. Conclusion: our data unveil a role of amplification and acceleration for Gal-9 in the process of EBV-driven B-cell transformation. This process might be relevant to the pathogenesis of EBV-associated BCLs. The primary B cells were selected by human anti-CD3 beads (eBioscience) from the peripheral blood of healthy donors. Then the selected cells infected in vitro by EBV collected at multiple time points including D0, 3, 7, 14 ,21 and mature LCL were subjected to serial bulk RNA-seq assessment.

背景：在多种恶性肿瘤中，尤其是EB病毒（Epstein-Barr Virus, EBV）相关的鼻咽癌，高表达半乳糖凝集素-9（Galectin-9, Gal-9）提示肿瘤表型具有侵袭性。目前尚未明确Gal-9在B细胞淋巴瘤（B-cell lymphomas, BCLs）发生发展中的作用。方法：采用免疫组织化学（immunohistochemical, IHC）染色法，检测66例BCLs患者肿瘤组织切片中Gal-9、STING（干扰素基因刺激因子，Stimulator of Interferon Genes）以及EB病毒核抗原1（Epstein-Barr Nuclear Antigen 1, EBNA1）的表达水平。本研究以体外人工感染EBV的正常原代B细胞作为实验模型。通过批量RNA测序（bulk RNA-sequencing）与生物信息学分析，探究转化过程中EBV感染B细胞的转录组动态变化。通过向EBV感染的原代B细胞中添加重组Gal-9并开展生长实验，在体外探究Gal-9的致癌作用。采用免疫印迹、免疫荧光（immunofluorescent, IF）染色、染色质免疫沉淀（chromatin immunoprecipitation, CHIP）以及荧光素酶报告基因实验，解析其潜在分子机制。结果：在BCLs临床标本中，Gal-9的表达水平与肿瘤分期、潜伏性EBV感染以及病毒潜伏蛋白EBNA1的丰度显著相关。针对体外EBV诱导的B细胞转化过程中的转录组变化分析显示，一系列基因被长期激活，并在成熟淋巴母细胞样细胞系（lymphoblastoid cell lines, LCLs）中持续高表达，Gal-9基因即为其中之一。在转化过程中，Gal-9的mRNA表达水平上调，其蛋白表达上调更为显著。这种上调至少部分源于EBNA1的反式激活作用——EBNA1可结合其启动子区域。反之，本研究发现胞外添加Gal-9可促进B细胞转化以及EBV感染潜伏相的建立。同时，胞外Gal-9可阻断STING信号通路并增强信号转导与转录激活因子3（Signal Transducer and Activator of Transcription 3, STAT3）磷酸化。抑制STING信号通路或STAT3磷酸化，即便存在Gal-9，也可阻断B细胞转化。结论：本研究数据揭示了Gal-9在EBV诱导的B细胞转化过程中发挥扩增与加速作用。该过程可能与EBV相关BCLs的发病机制密切相关。本研究通过抗CD3磁珠（eBioscience）从健康志愿者外周血中分离原代B细胞，随后在体外以EBV感染这些分选得到的细胞，并在多个时间点（D0、3、7、14、21天）以及成熟LCLs中开展连续批量RNA测序评估。