Regulation of Bim Expression by IGF-1 in the 5T33MM Murine Model for Multiple Myeloma.

Insulin-like growth factor-1 (IGF-1) is an important factor in proliferation, cell survival and migration of multiple myeloma (MM) cells. Bcl-2 like 11 (Bim) is a pro-apoptotic protein belonging to the BH3-only group of Bcl-2 family members. Three major forms of Bim are known: BimEL, BimL and BimS. In leukemia, beta1 integrin mediated adhesion has been shown to reduce Bim levels by enhanced proteasome activation. We used the 5T33MM syngeneic, immunocompetent murine model for MM. Both the exclusively in vivo growing 5T33MMvv cells and the stroma independent in vitro growing 5T33MMvt cells were used. The present study showed that Bim levels were reduced in MM cells after IGF-1 treatment. IGF1-mediated regulation of Bim expression in 5T33MMvv cells was demonstrated to be a result of reduced gene expression on the one hand and increased proteasomal-mediated degradation of Bim protein levels on the other hand. Reduced expression was related to activation of the Akt pathway and inactivation of the transcription factor FoxO3a. Increased degradation of Bim was related to activation of the mitogen-activated protein kinase pathway, as IGF-1 treatment resulted in phosphorylation of ERK1/2. Reducing Bim levels by transducing 5T33MMvt cells with a lentiviral vector with a short hairpin RNA cassette inhibited serum starved and the clinical relevant deacetylase inhibitor panobinostat (LBH589, Novartis) induced cell death. Together our data indicate that disrupting IGF-1-mediated regulation of Bim degradation may increase the efficacy of drugs. In the past few years, methylation of DNA and acetylation of histones have been proven to be important regulators of gene expression. Moreover, acetylation of non-histone proteins has emerged as a dynamic posttranslational modification regulating numerous cellular processes. Co-treatment of 5T33MMvv and 5T33MMvt cells for 24h with the demethylation agent 5-Aza-2′deoxycytidine and the deacetylase inhibitor panobinostat resulted in a strong induction of Bim expression at protein level. In the future, we will investigate the contribution of IGF-1 to the epigenetic silencing of Bim by i) comparing the Bim promoter methylation pattern of both human IGF-1 responsive cell lines and murine 5T33MMvv cells treated or not with IGF-1 using bisulfite PCR sequencing and ii) using the epigenetic modulating agents 5-Aza-2′deoxycytidine and panobinostat.