Inhibition of Multiple Myeloma Cell Proliferation and Increase of Apoptosis through Regulation of the NF-κB and JNK Pathways by Silencing TRAF6 C-Domain mRNA.

Several members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF5, and TRAF6 have been implicated in regulating signal transduction from various TRAF family members. However, the unique biological function of TRAF6 is largely determined by its TRAF-C domain, which does not interact with peptide motifs that are recognized by TRAF1, -2, -3 or -5.

Based on TRAF6, CD40, and RANKL sequences and crystal structures, we targeted the TRAF6 C-domain residues from 420 to 440 because the TRAF6 interaction domain with CD40 or RANKL resides in residues 333 to 508. We found that silencing TRAF6 mRNA with the C-terminal domain siRNA significantly inhibited MM cell proliferation maximally at 72 hours whereas effects on inducing MM cell apoptosis were most prominent at 48 hours. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration (of siRNA)-dependent fashion. Furthermore, NF-κB mRNA expression and protein levels were also reduced using siRNA of the TRAF6 C-domain. We also examined the effect of TRAF6 siRNA on the JNK pathway since this signaling pathway is also associated with cell cycle effects in myeloma. We measured JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. The results showed that the phosphorylation of JNKK is clearly reduced after knockdown of TRAF6 gene expression by siRNA. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The transcription activity of c-Jun is regulated by SAPK/JNK binding to c-Jun and phosphorylation of c-Jun at Ser63/73. We found that total endogenous c-Jun is reduced after silencing TRAF6 mRNA in the RPMI8226 MM cell line. In contrast, our data demonstrated that TRAF6 C-domain does not affect TRAF5 or TRAF2 gene expression. In addition, introduction of siRNA derived from the Zn-finger sequence into RPMI8226 MM cells failed to inhibit TRAF6 production. These studies suggest that the TRAF6 C-Domain may be an excellent target to block myeloma cell signaling important for the survival and proliferation of MM cells.