Radiotherapy induced expression of DNA sensors and cytokines in tumor cells - Prof. Maja Cemazar
Radiotherapy is one of the commonly used treatments with high antitumor effectiveness. It is based on random interactions of ionizing radiation with targeted tumor, immune and other stromal cells within tumors. Ionizing radiation causes breaks of chemical bonds and oxidation of molecules in the irradiated cells. The most important consequences are single or double-stranded DNA breaks further causing mutations, cell death or carcinogenesis. DNA, which is released in to the cytosol, is detected by pattern recognition receptors (PRRs) called DNA sensors. Binding of DNA fragments to DNA sensors causes a signaling cascade leading to cytokine release, which activate the immune response. Our research groups have already demonstrated reduced cell survival and tumor regression due to increased expression of DNA sensors and interferon β (IFN β), after gene electrotransfer of control plasmid DNA in B16F.10 melanoma cells and tumors. We also conducted preliminary experiments of DNA sensor expression after irradiation of B16F10 melanoma cells in vitro. In both cases, the expression of DAI (DNA-dependent activator of IFN-regulatory factors) / ZBP1, IFI16 and DDX60 cytosolic DNA sensors was increased. Binding of DAI sensor with poly (dA: dT) results in production of IFNβ, whereas STING (stimulating gene for IFN) -signaling pathways additionally results in the production of tumor necrosis factor alpha (TNFα). Similarly, after DNA binding to the IFI16 sensor, the signaling pathway leading to production IFNβ is triggered. DDX60 binds both RNA as well as DNA and is involved in increased expression of type I interferons. Type I interferons produced as a result of DNA sensor activation promote antitumor immunity by activation of the innate and acquired immune systems (dendritic cells, T lymphocytes, NK cells) and has negative impact on tumors suppressor cells (MDSC, Treg). Type I interferons also have intrinsic effects on tumor cells, resulting in inhibition of proliferation, regulation of different types of cell death, differentiation, migration and antigen expression on the cell surface. The production of IFN type I can occur in tumor or immune cells and affect the activation of immune cells, regulating antigen and cytokine expression and cell death signals.
The main goal of collaboration among research groups is to validate and elucidate our hypothesis that after irradiation, DNA released to the cytosol to activate one or more DNA sensors. Therefore, we will conduct experiments, using shRNA technology, where each of the mentioned DNA sensors will be silenced, first individually and later a combination of two or three DNA sensors. We are also planning to silence STING and determine if there is any other existing signaling pathway through which DNA sensors could trigger cytokine production.