Background: The tumor microenvironment plays a pivotal role in the pathogenesis of classical HL (cHL) because of the multiple and complex interactions of Hodgkin and Reed-Sternberg cells with inflammatory cells through numerous cytokines and chemokines. The innate immune responses can be regulated by the cGAS-STING pathway, which may be activated by cytosolic DNA in neoplastic cells. The cGAS-STING signaling, in turn, activates transcription factors IRF3 and NF-κB via kinases TBK1 and IKK, respectively. IRF3 and NF-κB can induce the expression of interferons (IFNs), cytokines and chemokines. We investigated for the first time the effects of the natural compound sulforaphane (SFN) on the cell growth and anti-tumor immune responses in cHL.
Methods: The in vitro system included 6 cHL cell lines (MDAV, L1236, L428, L540, KMH2, HDLM2) as well as HUT78 control cells. The cHL cells were treated with increasing concentrations of SFN or a STING agonist. Silencing of STING, IRF3, RelA, and RelB genes was performed using transient transfection (Nucleofector) with siRNA constructs. Expression of proteins was analyzed by Western blot, and gene expression (mRNA) of type 1 IFNs, including IFN-β, CXCL10 and IFN-γ, by RT-qPCR. 51Cr-based NK cell killing, cytokine arrays and flow cytometry methods were also utilized to assess the anti-tumor immune responses.
Results: Treatment with SFN resulted in decreased cell growth and induction of IFN-β and CXCL10 gene expression, and substantially modified the cytokine profile in vitro (Fig. 1). SFN treatment also led to a dramatic increase in the protein level of NK ligand MIC A/B and to a lesser degree altered expression of other NK ligand, which were associated with significant increase in functional NK cell-mediated killing of co-cultured cHL cells. MIC A/B expression is upregulated by cGAS-STING signaling, which is functional in cHL cells since stimulation with STING agonist resulted in increased gene expression of IFN-β and/or CXCL10. SFN treatment resulted in activation of the cGAS-STING pathway as shown by phosphorylation/ activation of TBK1 kinase and its downstream target IRF3. Inversely, STING gene silencing using specific siRNA constructs resulting in decreased IFN-β and CXCL10 gene expression, and altered the chemokine and cytokine profile of cHL cells in vitro.
Conclusion: SFN is a strong immunomodulatory agent that induces NK cell-mediated anti-tumor immune responses in cHL, in part through STING-dependent mechanisms.
Ioanna Xagoraris, Ying Yang, Erofili Bougka, Dora Trogrlic, Persa Xyderou, Konstantina Stathopoulou, Christina An Bihn Nordentoft, Nikolas Herold, Andreas Lundqvist, Georgios Z. Rassidakis