X-shaped DNA potentiates therapeutic efficacy in colitis-associated colon cancer through dual activation of TLR9 and inflammasomes

Background: Immunotherapy has been extensively pursed as a promising strategy for the treatment of cancer. Pattern-recognition receptors (PRRs) play important roles in triggering activation of innate and adaptive immunity. Therefore, agents that stimulate PRRs could be useful for cancer immunotherapy. We developed two kinds of X-shaped double-stranded oligodeoxynucleotides (X-DNA), a single unit of X-DNA (X-S-DNA) composed of four strands of DNA and a ligated X-DNA complex (X-L-DNA) formed by crosslinking each X-S-DNA to the other, and investigated if they had immunostimulatory activity and could be applied to anti-cancer immunotherapy. Methods: Activation of MAPKs and NF-kappa B was determined by immunoblotting in bone marrow-derived primary dendritic cells (BMDCs). Immune cytokines and co-stimulatory molecules were measured by ELISA and flow cytometry analysis. Anti-cancer efficacy was examined in an azoxymethane/dextran sulfate sodium-induced colitis-associated colon cancer mouse model. Association of X-DNA and TLR9 was determined by co-immunoprecipitation followed by immunoblotting. The involvement of TLR9 and inflammasomes was determined using TLR9- or caspase-1-deficient BMDCs. Inflammasome activation was examined by degradation of pro-caspase-1 to caspase-1 and cleavage of pro-IL-1 beta to IL-1 beta in BMDCs. Results: X-L-DNA and X-S-DNA induced activation of MAPKs and NF-kappa B and production of immune cytokines and co-stimulatory molecules in BMDCs. BMDCs stimulated by X-L-DNA induced differentiation of naive CD4(+) T cells to T(H)1 cells. Intravenous injection of X-L-DNA into mice resulted in increased serum IFN-gamma and IL-12 levels, showing in vivo efficacy of X-L-DNA to activate T(H)1 cells and dendritic cells. X-L-DNA greatly enhanced the therapeutic efficacy of doxorubicin, an anti-cancer drug, in colitis-associated colon cancer. X-L-DNA directly associated with TLR9. In addition, immunostimulatory activities of X-DNA were abolished in TLR9-deficient dendritic cells. Furthermore, X-DNA induced caspase-1 degradation and IL-1 beta secretion in BMDCs, which were abolished in caspase-1-deficient cells. Conclusions: X-DNA induced the activation of dendritic cells as shown by the expression of immune-cytokines and co-stimulatory molecules, resulting in the differentiation of T(H)1 cells, mediated through dual activation of TLR9 and inflammasomes. X-DNA represents a promising immune adjuvant that can enhance the therapeutic efficacy of anti-cancer drugs by activating PRRs.