IL-6 Enhances C-myc Translation in Myeloma Cells Via Activation of the Myc Internal Ribosomal Entry Site (IRES) and an IRES-Binding Trans Factor, hnRNP A1.

Dysregulated c-myc expression in multiple myeloma (MM) can be due to a translocation with juxtaposition of the myc gene to the Ig enhancer resulting in constitutive transcription. However, myc protein levels can also be increased in MM cells when it is due to heightened translation. Activated translation can be mediated via an mTOR-mediated, cap-dependent mechanism, thus named because the translation initiation complex utilizes the cap structure on mRNA to recruit the ribosomal subunit. However, an alternative mechanism is cap-independent where the ribosomal subunit binds a transcript via an IRES in the RNA’s 5′UTR. IRES function is regulated by IRES trans activating factors (ITAFs) which bind to the 5′UTR. We studied myc expression in ANBL-6 wild type and activated K-RAS-transfected MM cells which proliferatively respond to IL-6 but do not contain myc translocations. IL-6 increased myc protein expression in both cell lines in a concentration and time-dependent fashion but RNA expression was only enhanced in the wild type cells. The increased protein expression in K-RAS mutant cells was unaffected by rapamycin, indicating independence from mTOR and cap-dependent mechanisms. Using an IRES-dependent reporter vector, reporter expression was shown to be increased by IL-6, confirming IL-6 activates myc IRES activity. This was also demonstrated in the IL-6 autocrine U266 MM cell line where myc IRES-dependent reporter expression was ablated following exposure to anti-IL-6 antibodies. In addition, primary MM cells treated with IL-6 also demonstrated an increase in myc IRES activity. Since a yeast three-hybrid screen for proteins specifically binding to the myc IRES identified several clones encoding hnRNP A1, we evaluated hnRNP A1 as a mediator of myc IRES activity. IL-6 increased the cytoplasmic localization of hnRNP A1 in ANBL-6 MM cells. Furthermore, in MM cells where hnRNP A1 expression was knocked down, myc IRES activity was greatly diminished and there was no enhancement with addition of IL-6. These results indicate that IL-6 can enhance c-myc translation in MM cells via activation of the myc IRES and that hnRNP A1 is a critical myc ITAF involved in the IL-6 response. hnRNP A1 may serve as a new therapeutic target in myeloma.