P133: DNA inhibits dsRNA-activated NF-kB-based inflammation in tumour cells: The role of Ku protein

The strong connection between cancer-related inflammation and tumour development with pattern – recognizing receptors (PRRs) activation results in identification of new target molecules that could lead to improved cancer diagnosis and treatment. TLR3, RIG1 and MDA5 synthetic ligand poly(I:C) was shown to trigger apoptosis in cancer cells. However, TLR3 signaling also includes NF-kB transcription factor which has emerged as endogenous tumour promoter via stimulation of pro-inflammatory tumour microenvironment, enhancement of angiogenesis, tumour cell proliferation and metastasis. Thus down regulation of NF-kB-mediated effects after TLR3 activation is needed for implementation of TLR3 ligand-based therapy into clinical trials. We have earlier demonstrated that DNA and sequence specific ODNs inhibit poly(I:C)-induced production of pro-inflammatory cytokines in human primary fibroblasts and endothelial cells (Cherepanova et al., Immunobiology, 2013). Using these specific ODNs and affinity modification/isolation approach combined with subsequent MALDI-TOF the main cellular targets for these ODNs were identified as Ku protein – heterodimer of KU70 and KU80 (Cherepanova et al., Exp. Opin. Biol. Ther., 2012). The goals of this study are to reveal whether the ODNs target poly(I:C)-induced activation in tumour cells: cervical carcinoma (Hela) and epidermoid carcinoma (A431) and to confirm Ku protein involvement in the observed effect. Poly(I:C) was shown to activate IL-6 production in Hela and A431 cells, and specific ODNs inhibit this activation with comparable efficacy as in primary cells. ODNs decrease both IL-6 concentration and mRNA expression rate in poly(I:C)-activated cells, indicating that NF-kB activation is also impaired. To reveal whether Ku protein is involved in the inhibiting effect of ODNs, Ku70 level in Hela cells was reduced by siRNA, which were shown to inhibit the expression of Ku70 by up to 70%. Inhibiting efficacy of ODN is almost twice lower in Ku70 knock-down cells as compared to control siRNA, indicating that ODN binding to Ku protein is important for the inhibition. Whether Ku is directly involved in nucleic acid pattern recognition or have auxiliary functions, mediating ODN localization or poly(I:C)-proteins interactions, remains to be investigated. Nevertheless, efficient NF-kB pathway targeting by specific ODNs may improve dsRNA-based cancer therapy.