Inhibition of the RKIP and PTEN-Transcription Repressor Snail in B-NHL by the Proteasome Inhibitor NPI-0052: Roles of RKIP and PTEN Induction in the Reversal of Resistance to TRAIL Apoptosis.

Abstract 3938

Poster Board III-874

Several strategies have been examined to reverse tumor cell resistance to immunotherapy. For instance, proteasome inhibitors such as Bortezomib, MG-132 and NPI-0052 have been reported to sensitize hematopoietic tumor cells to TRAIL-mediated apoptosis. NPI-0052 mediates its cytotoxic and sensitizing effects in lymphoma cells through the inhibition of the NF-κB and PI3K/Akt survival pathways. The molecular mechanisms and associated gene products involved in NPI-0052-induced inhibition of these pathways are not known. We have reported that NPI-0052 induced the expression of Raf-1 kinase inhibitor protein (RKIP) via inhibition of NF-κB activity and downstream the RKIP transcription repressor Snail (Baritaki et al., Oncogene. 2009, PMID: 19633685). Also, Snail has been reported to repress phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and allowing the Akt pathway to remain activated. Based on these findings, we hypothesized that treatment of B-NHL cells with NPI-0052 will result in the induction of both RKIP and PTEN through inhibition of Snail and, consequently, will result in the inhibition of the NF-κB and Akt pathways, respectively. Treatment of Ramos cells with NPI-0052 resulted in the inhibition of NF-κB activity and Snail expression along with the induction of both RKIP and PTEN expression, as assessed by western. The direct roles of RKIP and PTEN in B-NHL cell sensitization to TRAIL were demonstrated by the use of cells transfected with siRNA RKIP and siRNA PTEN, whereby reversal of sensitivity occurred. In contrast, Snail knockdown by siRNA Snail resensitized the cells to TRAIL apoptosis. The inhibition by NPI-0052 of both the NF-κB and PI3K/Akt pathways resulted downstream in the inhibition of transcription and/or activity of anti-apoptotic gene products such as Bcl-2 family members. The direct role of Bcl-2 family anti-apoptotic gene products in the reversal of resistance by NPI-0052 was demonstrated by the use of the Bcl-2 family chemical inhibitor 2MAM3. Altogether, these findings demonstrate that NPI-0052 modifies a tightly regulated loop in the resistance of B-NHL cells, namely, the constitutive activation of NF-κB and Akt survival pathways that crosstalk and induce Snail. Snail in turn, represses RKIP and PTEN and, thus, the cells continue to survive and proliferate. However, upon treatment with NPI-0052, the inhibition of PI3K/Akt and NF-κB activities results downstream in the inhibition of Snail and the inhibition of Snail, in turn, derepresses the transcription of both RKIP and PTEN. In a feedback loop, the induction of RKIP represses the NF-κB pathway and the induction of PTEN represses the PI3K/Akt pathway. Hence, the inhibition by NPI-0052 of the NF-κB/Akt/Snail/RKIP/PTEN regulatory resistance loop in B-NHL cells results in the reversal of resistance. Further, these findings identify several new targets for potential therapeutic intervention in the reversal of resistance.