616. Enhancement of Antitumor Activity of Interferons by RNAi-Mediated Silencing of SOCS Genes in Tumor Cells

Abstract

Cytokine-based cancer immunotherapy, which has already been used in clinical practice, is a promising strategy in cancer therapy. However, it has been reported that some tumor cells are not sensitive to cytokine-mediated cell death. Furthermore, tumor cells can acquire resistance to cytokines during their progression, leading to the increase in the administration dose of cytokines. However, this increase would be indispensably associated with severe side effects such as leukopenia and neuropsychiatric symptoms. Suppressor of cytokine signaling (SOCS) proteins are a family of negative regulators of cytokine signaling by inhibiting Jak activation. Of the family members, SOCS-1 and SOCS-3 are the most potent inhibitors of cytokine-induced signals. Recent studies have indicated that high expression of the SOCS proteins is found in several advanced cancer cells. Furthermore, an inverse correlation between the expressionlevel of the SOCS proteins in tumor cells and their sensitivity to cytokines was reported. These pieces of evidence suggest that the silencing of SOCS-1 or -3 in tumor cells by RNA interference can enhance the susceptibility of tumor cells to cytokines. In this study, we selected murine interferon (IFN)-|[beta]| and -|[gamma]| as antitumor cytokines, and examined their effects on the proliferation of cultured tumor cells: B16|[ndash]|BL6 melanoma cells, and CT-26 and colon 26 colon carcinoma cells. A quantitative RT-PCR analysis revealed that CT-26 cells and colon 26 cells had higher mRNA expression of SOCS-1 and -3 than B16-BL6 cells or C2C12 myoblasts, the latter used as a model of normal cells. Proliferation of the tumor cells was inhibited by the addition of IFN-|[beta]| or IFN-|[gamma]| to culture media in an IFN concentration-dependent manner. We found that B16-BL6 cells were more sensitive to IFNs than CT-26 or colon 26 cells. Then, to suppress the expression of SOCS-1 or -3, we constructed small interfering RNA (siRNA)-expressing plasmid DNA targeting one of these genes (psi-SOCS1 and psi-SOCS3). Transfection of psi-SOCS1 or psi-SOCS3 significantly reduced the mRNA expression of the corresponding target gene to 50|[ndash]|60 % of the controls. This reduction in the mRNA level of SOCS-1 or SOCS-3 significantly increased the susceptibility of CT-26 and colon 26 cells to the anti-proliferative activity of IFN-|[beta]| and IFN-|[gamma]|. On the other hand, the reduction in the mRNA levels hardly affected the susceptibility of B16-BL6 cells that showed lower mRNA expression of the SOCS genes than the others. These results indicate that the silencing of SOCS-1 and -3 expression in tumor cells can improve the anti-tumor effect of IFNs under conditions where the expression of these genes are increased.