Unique Pattern of Raf-1 Kinase Inhibitory Protein (RKIP) Expression in Multiple Myeloma (MM) Compared to Other Tumor Types: Overexpression of RKIP and a Phosphorylated RKIP Is Common in MM Tumor Cells.

Objective and Rationale Raf-1 kinase inhibitory protein (RKIP) is a modulator of cell signaling and survival that functions as an endogenous inhibitor of multiple kinases, including kinases involved in the Raf/MEK/ERK and NF-κB pathways. RKIP has been identified as a metastasis suppressor gene and an immune surveillance cancer gene, since loss of RKIP protein expression has been associated with tumor progression, metastasis and escape from immune surveillance. Further, RKIP expression has been associated with prognostic significance in many cancers. Recently, we have demonstrated that induction of RKIP expression in tumors with low RKIP levels results in increased tumor cell sensitivity to immuno- or chemo-therapy via inhibition of the above pathways. However, multiple myeloma (MM) cells have been shown to express high RKIP levels compared to other tumors and still remain highly resistant to conventional cytotoxic therapies. These findings were unexpected and thus, it was plausible that the high level of RKIP expression was not functionally active. It has been reported that phosphorylation of RKIP at Ser-153 renders the cells inactive (Rosner et al., 2003, J Biol Chem 278:13061–8). Thus, we examined the expression and the phosphorylation status of the RKIP protein in several multiple myeloma cell lines and tissues and compared them with other cell lines with low RKIP expression. Hypothesis We hypothesized that MM tumor cells express high levels of the inactive phoshorylated RKIP protein which antagonizes the active non-phoshorylated RKIP form in the inhibition of the survival signaling pathways.

Experimental Designs and Methods Multiple myeloma (IM-9, RPMI 8226, MM1S, U266 cell lines and fresh bone marrow samples from MM patients), PC-3 prostatic carcinoma and Ramos B-NHL cell lines were examined for total and phosphorylated RKIP expression by IHC and Western Blot analyses. The total RKIP protein was significantly elevated in multiple myeloma cell lines compared to the prostate and B-NHL lines. The predominant RKIP form in multiple myeloma tumors was the phosphorylated RKIP protein with high nuclear localization, as assessed by IHC, while the phosphorylated RKIP levels in the non-myeloma tumors were relatively low. It has been reported that the phosphorylation of RKIP is mediated by protein kinase C (Rosner et al., 2003, J Biol Chem 278:13061–8). Additional studies in multiple myeloma cell lines also revealed high expression of the zeta isoform of PKC (PKCζ), known to phosphorylate and inactivate RKIP.

Conclusions and Implications The present findings demonstrate that the aberrant RKIP phosphorylation in multiple myeloma tumors may result in the inhibition of the suppressive effect of RKIP on tumor survival signaling pathways. We postulate that the high expression of RKIP may be due to inhibition of proteasome degradation. The present findings also suggest that screening of RKIP levels and RKIP phosphorylation status in MM may be useful as prognostic factors of tumor cell response to anti-tumor therapies.