GSE162516
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. Overall design: 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.