IL-6–Induced Stimulation of c-Myc Translation in Multiple Myeloma Cells Is Mediated by Myc Internal Ribosome Entry Site Function and the RNA-Binding Protein, hnRNP A1

Abstract

Prior work indicates that c-myc translation is up-regulated in multiple myeloma cells. To test a role for interleukin (IL)-6 in myc translation, we studied the IL-6-responsive ANBL-6 and IL-6-autocrine U266 cell lines as well as primary patient samples. IL-6 increased c-myc translation, which was resistant to rapamycin, indicating a mechanism independent of mammalian target of rapamycin (mTOR) and cap-dependent translation. In contrast, the cytokine enhanced cap-independent translation via a stimulatory effect on the myc internal ribosome entry site (IRES). As known IRES-trans-activating factors (ITAF) were unaffected by IL-6, we used a yeast-threehybrid screen to identify novel ITAFs and identified hnRNP A1 (A1) as a mediator of the IL-6 effect. A1 specifically interacted with the myc IRES in filter binding assays as well as EMSAs. Treatment of myeloma cells with IL-6 induced serine phosphorylation of A1 and increased its binding to the myc IRES in vivo in myeloma cells. Primary patient samples also showed binding between A1 and the IRES. RNA interference to knock down hnRNP A1 prevented an IL-6 increase in myc protein expression, myc IRES activity, and cell growth. These data point to hnRNP A1 as a critical regulator of c-myc translation and a potential therapeutic target in multiple myeloma. [Cancer Res 2008;68(24):10215-22]

IRES-dependent translation of egr2 is induced under inflammatory conditions

“…Especially, PTB is considered to activate IRESs of many IRES-bearing mRNAs (Sawicka et al 2008;Yang et al 2010). Interestingly, among a number of hnRNPs, we also found hnRNP-A1, which was previously shown to be crucial for the IRES function of c-myc (Shi et al 2008) and fgf2 (Bonnal et al 2005). While no differential binding of PTB and hnRNP-A1 was seen between CM-and Ctr-treated conditions, various other RNA-interacting proteins were found to bind to egr2-59UTR as well.…”

“…Recently IL-1a was reported to activate translation of mRNAs such as IkBz and IL-6 (Dhamija et al 2010). Moreover, IL-6 was shown to enhance IRESdependent translation of prosurvival genes like c-myc (Shi et al 2011). Notably, in our experiments, depletion of IL-1b did not fully repress CM-induced egr2 translation, and treatment with recombinant IL-1b did not completely recapitulate enhanced egr2 translation as seen in response to CM (Fig.…”

Upregulated c-myc expression in multiple myeloma by internal ribosome entry results from increased interactions with and expression of PTB-1 and YB-1

“…Interestingly, the overexpression of c-myc mediated through the mutated IRES was shown to result from enhanced activity of the mutated version of the IRES Chappell et al, 2000) but other factors are clearly required as c-myc protein levels in cell lines derived from patients with MM which all harbour the mutation differ. The data suggest that this may be due to increased expression of c-myc ITAFs (Evans et al, 2003;Shi et al, 2008) and therefore we have investigated the interaction of the recently identified c-myc ITAFs PTB-1, PSF, YB-1 and p54nrb (Cobbold et al, 2008) with the mutated version of the c-myc IRES. We show that de-regulated expression of two these proteins is associated with aberrant c-myc protein expression in MM-derived cell lines.…”

IL-6-induced Enhancement of c-Myc Translation in Multiple Myeloma Cells

“…The ability of IL-6 to stimulate Myc IRES function in MM cells, up-regulate rapamycin-resistant Myc translation and expression, and stimulate MM cell proliferation were all prevented by knockdown of hnRNP A1, an RNA-binding protein (1). Because other studies (8 -10) demonstrate that hnRNP A1 (A1) can function as an IRES-trans-activating factor (ITAF), regulating Myc IRES activity, these results collectively indicate that IL-6-up-regulated c-Myc translation in MM cells was dependent on enhanced A1 ITAF function.…”

“…Primary Myeloma Specimens-Primary MM cells were purified from bone marrow of patients as described (1). The purity by microscopy and CD138 flow cytometric analysis was Ͼ99% plasma cells.…”