Nitric Oxide (NO) induced sensitization of tumor cells to TRAIL-mediated apoptosis via induction of RKIP and DR5 expression and inhibition of YY1

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4868 We have recently reported that the induction of NO or the use of NO donors can reverse tumor cell resistance to death ligands via inhibition of NF-κB and the transcription repressor Yin Yang 1 (YY1) activities and upregulation of death receptors (Baritaki et al., 2007). The underlying molecular mechanisms of these NO-induced effects are not well defined. The metastatic suppressor gene Raf kinase inhibitor protein (RKIP), has been reported to inhibit NF-κB activity (Yeung et al., 1999,2000). Therefore, we hypothesized that NO may induce RKIP expression leading to inhibition of NF-κB and YY1 and upregulation of DR5, leading to sensitization to TRAIL-induced apoptosis. The involvement of RKIP and its interplay with YY1 and DR5 in the NO-induced sensitization to TRAIL was investigated using the resistant prostate tumor cells PC3 and the Non-Hodgkin’s lymphoma cell line Ramos. Treatment of tumor cells with the NO donor DETA/NONOate (1000 uM) resulted in significant sensitization to TRAIL-induced apoptosis and concomitantly induction of RKIP mRNA and protein levels. In contrast, YY1 promoter activity and YY1 expression (both in transcript and protein levels) were significantly reduced after DETA/NONOate treatment. Since we have recently shown that YY1 negatively regulates DR5 transcription and expression, tumor treatment with DETA/NONOate also resulted in upregulation of DR5. The role of YY1 in the regulation of DR5 and sensitization to TRAIL was corroborated by the use of YY1 siRNA which resulted in sensitivity to TRAIL. The direct role of RKIP in the NO-based tumor cell sensitization to TRAIL was confirmed by significant inhibition of NO-induced cell sensitization to TRAIL following treatment of tumor cells with RKIP siRNA. The above findings provide evidence that NO-induced sensitization to TRAIL is mediated, in large part, via induction of RKIP expression and subsequently, inhibition of NF-κB and YY1 activities and upregulation of DR5 expression. Preliminary findings in tissues nude mice bearing PC3 tumor xenografts and treated with DETA/NONOate showed upregulation of DR5 expression and inhibition of YY1 by IHC. These findings suggest that for successful anti-cancer immunotherapeutic approaches there is a need to use sensitizing agents that can upregulate RKIP/DR5 or inhibit NF-κB/YY1. Such sensitizing agents, like NO donors, may reverse TRAIL-resistance when used in combination with TRAIL/anti-DR4 or DR5 monoclonal antibodies.