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-three-hybrid 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]
The c-myc gene encodes a transcription factor that is a key regulator of proliferation (1). Dysregulated myc expression plays a role in Burkitt's lymphoma, murine plasmacytoma, and human multiple myeloma (MM; refs. 2–4). In myeloma, dysregulation of myc can result ftrom a gene translocation with juxtaposition of myc to the immunoglobulin enhancer (4). Another potential mechanism of dysregulated transcription is from stimulation with the myeloma growth factor interleukin (IL)-6 via a signal transducers and activators of transcription 3 (STAT-3)–dependent mechanism. However, this cytokine effect is cell type specific and IL-6 treatment of some cells actually results in decreased myc transcription (5, 6). Myc overexpression in MM cells can also be due to up-regulated translation through cap-independent mechanisms (7, 8). Cap-independent translation is the fail-safe mechanism of protein expression when cap-dependent translation is prevented or when leaders contain structural elements, which are inhibitory to scanning ribosomes (9–11). Translation initiation is then achieved via internal ribosome entry sites (IRES) in the mRNA's 5′ untranslated region (UTR). The three dimensional IRES structure directly or indirectly recruits the 40S ribosomal subunit to the mRNA for translation initiation. Several IRES-associated trans-acting factors (ITAF) are required for IRES activity. These factors bind to RNA at sites in the 5′UTR and induce conformational changes, which facilitate recruitment of the ribosome to the IRES. Several publications (7, 8) document a myeloma-specific up-regulation of myc IRES function.
In this study, we tested whether the up-regulated myc translation in MM cells could be further enhanced by the myeloma growth factor, IL-6. As IL-6 is an important growth-promoting cytokine for MM cells (12) and in MM patients (13), its ability to affect myc expression could support a pathophysiologic relevance of myc translation in this disease. In several MM cell line models as well as primary patient samples, the tumor growth factor IL-6 was shown to enhance IRES-dependent myc translation. In addition, this effect on the myc IRES was mediated by a novel myc ITAF, hnRNP A1. IL-6 induced serine phosphorylation of hnRNP A1 and significantly enhanced its binding to the myc IRES. Thus, RNP A1 is an important regulator of MM myc expression and MM tumor cell expansion.
Materials and Methods
Cell lines, constructs, reagents. The ANBL-6 wild-type (WT), K-ras–transfected ANBL-6 and U266 cell lines were gifts from Dr. Brian Van Ness (University of Minnesota, Minneapolis, MN). The pRF construct was a kind gift of Dr. A. Willis (University of Leicester, Leicester, United Kingdom). The c-myc and p27 IRESes were amplified from IMAGE clones 4667496 and 4298338, and cloned into the intercistronic region of pRF to generate pRmF and pRp27F. These have been previously described in detail (14). The pR-V-F, pSP65-R-F, and pSP65-R-vascular endothelial growth factor (VEGF)-F plasmids were gifts from Dr. Greg Goodall (University of Adelaide, Adelaide, Australia). They have also been previously described (15).
Primary myeloma samples. Primary patient myeloma cells were isolated from bone marrow by positive selection for CD38 as previously described (16). The purity was >99% plasma cells. These cells were then treated with or without IL-6 and subsequently studied for myc expression by Western blot and for myc IRES-dependent reporter expression by the dicistronic assay (see below). In addition, four primary myeloma samples that had been previously purified and cryopreserved were thawed and washed, followed by immunoprecipitation of hnRNP A1 and assessment of bound myc IRES by immunoprecipitation-reverse transcription-PCR (IP-RT-PCR) assay (see below).
Western and Northern blot. Western and Northern blot was performed as previously described (12, 14, 15).
Polysome analysis of translational state. As previously described (17), cells treated with or without IL-6 and with or without rapamycin were lysed in ice-cold lysis buffer supplemented with 100 μg/mL cycloheximide at 4°C. After removal of nuclei and mitochondria, supernatants were layered onto 15% to 50% sucrose gradients and spun at 38,000 rpm for 2 h at 4°C in a SW-40 rotor. Eleven 1-mL fractions were collected using an ISCO Density Gradient Fractionator at a flow rate of 3 mL/min. The UV absorbance at 254 nm of each fraction was measured to generate a polysome profile of the sucrose gradient that was used to differentiate between the monosome (fractions 1–3) and polysome (fractions 4–11) fractions. The RNA from individual fractions was extracted using phenol/chloroform and precipitated in ETOH. The RNA of fractions 1 to 3 (monosome) were pooled as were the RNAs from fractions 4 to 11 (polysome-associated RNA). Northern blot analysis was performed on these pooled RNA fractions for myc or actin. The percent of total mRNA present (measured by densitometry of equally exposed autoradiographs) in the polysomal fractions versus the monosomal fractions were analyzed.
Dicistronic reporter assay. The reporter constructs were transfected into cell lines or primary MM cells using Lipofectamine Plus (Invitrogen) and normalized for transfection efficiency by cotransfection with pSVβGal (Promega). Transfection efficiency was generally 5% to 10%. A transfection efficiency of at least 5% was required for carrying out a dicistronic reporter assay. After 12 to 14 h, they were treated with or without IL-6 for varying durations. Cells were then harvested, and Renilla luciferase, Firefly, luciferase, and β-galactosidase activities were determined as previously described (14). To test IRES-dependent reporter expression from transfected RNA, polyadenylated dicistronic mRNAs were first generated in vitro (as previously described in ref. 15), and 500 ng RNA were transfected by electroporation (230V for 25 ms). Transfection efficiency was again controlled by assaying β-galactosidase activity.
Yeast methods and screening. All constructs used for the yeast three-hybrid assay were based on the RNA-Protein Hybrid Hunter System (Invitrogen). cDNA encoding full-length hnRNP A1 was inserted into pYesTrp3. A 233 nucleotide segment of the c-myc IRES was inserted into the hybrid RNA plasmid pRH5′. These plasmids were transformed into the strain L40uraMS2 [MATa, ura3-52, leu2-3112, his3-200, trp1-1, ade2, LYS2::(LexAop)4-HIS3, ura3:(LexAop)8-LacZ carrying pLexA/MS2/Zeo as previously described (18)].
Filter binding assay. Indicated amounts of glutathione S-transferase (GST)-hnRNP A1 were added to in vitro transcribed 32P-labeled RNAs corresponding to either the c-myc IRES or the p27 IRES in separate reactions in a volume of 10 mL in buffer containing 5 mmol/L HEPES (pH 7.6), 30 mmol/L KCL, 2 mmol/L MgCl2, 200 mmol/L DTT, 4% glycerol, and 10 ng of yeast tRNA for 10 mins at room temperature. Eight microliters of each binding reaction were applied to nitrocellulose membranes on a slot blot apparatus (Minifold II; Schleicher & Schuell). Membranes were washed, dried, and signals were quantified using a phosphorimager. Binding curves of three independent experiments were fitted by using SigmaPlot to determine the apparent dissociation constants.
Evaluation of in vivo binding of hnRNP A1 to myc IRES. The IP-RT-PCR assay was performed as previously described (19). Briefly, myeloma cell lines were treated with or without IL-6 for varying durations, treated with 1% formaldehyde, and then hnRNP A1 was immunoprecipitated and bound RNA was evaluated by RT-PCR. Real-time PCR for myc RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was performed on total extracted RNA from the immunoprecipitates.
Gene amplifications for real-time PCR were performed with an ABI PRISM 7700 sequence detection system (Applied Biosystems). Each 20-μL reaction in a 96-well plate comprised 9 μL of cDNA template, 1 μL of 20× primer mixtures for c-myc or GAPDH (containing 2 unlabeled primers for amplifying the c-myc or GAPDH sequence, respectively, and 1 6-FAM dye–labeled Taqman MGB Probe), and 10 μL 2×Taqman Universal PCR Master mixed with AmpErase UNG. Plates were sealed with adhesive optical film. After an initial 2 min at 50°C to activate ampErase and a denaturation step of 10 min at 95°C, 60 cycles of amplification were performed with denaturation for 15 s at 95°C and annealing/extension for 1 min at 60°C. All samples were run in triplicate and no template controls were included in all plates for both c-myc and GAPDH. Comparative CT method with c-myc and GAPDH in separate tubes was used according to manufacture's manual. CT corresponded to the number of cycles at which the fluorescence signal can be detected above a threshold value, which is automatically set in our case.
siRNA analysis. siRNA transfections targeting human hnRNP A1 were performed using synthetic oligonucleotides (ON-TARGETplus SMARTpool; Dharmacon) directed at sequences within the coding region and 3′UTR. An siRNA with a scrambled sequence was used as a negative-targeting control. siRNAs were introduced into cells by electroporation (230V for 25 ms).
c-Myc protein turnover assay. c-Myc protein stability was determined as previously described (20). Briefly, cells were stimulated with or without IL-6 (100 U/mL) overnight and then preincubated (15 min in DMEM without methionine and cysteine plus 5% dialyzed FCS) followed by a pulse with Tran35S-label (ICN; 300 uCi/mL) for 1 h. The cells were then chased (DMEM/10% FCS plus 400 mg/L methionine) as indicated in Fig. 1. Lysates were immunoprecipitated with anti-myc antibody (Santa Cruz).
EMSA. The c-myc IRES, Apaf-1 IRES, or GAPDH RNA were in vitro transcribed (mMessage T7 transcription kit; Ambion) as previously described (14) and 5′ end labeled with (32P) γ-ATP (GE Healthcare) using T4 polynucleotide kinase. Radiolabeled RNA was incubated with or without purified GST-hnRNP A1 in binding buffer containing 10 mmol/L HEPES (pH 7.5), 90 mmol/L potassium acetate, 1.5 mmol/L magnesium acetate, 2.5 mmol/L DTT, and 40U SUPERase-IN (Ambion) for 30 mins at 30°C. Subsequent EMSA procedures were performed as described previously (21).
Statistics. The t test was used to determine significance of differences between groups.
Effects of IL-6 on myc expression and myc IRES activity in MM cells. We initially studied the ANBL-6 MM cell line, which expresses functional IL-6 receptors. ANBL-6 WT cells are IL-6 dependent in that their growth ceases after ∼1 to 2 weeks of IL-6 depletion. In contrast, “K-RAS” cells are ANBL-6 MM cells stably transfected with an activated K-Ras allele (22). These cells become IL-6 independent in that they proliferate in the absence of IL-6, although they still respond to exogenous IL-6 with enhanced growth. Activating mutations of K-RAS are frequently found in primary MM specimens and correlate with aggressive disease (23). The ANBL-6 cell line does not contain a myc-Ig translocation.
Both cell lines [WT empty vector–transfected and K-RAS–transfected (K-RAS) ANBL-6 cells] were initially exposed to 100 U/mL of recombinant IL-6. Each cell line showed a significant increase in growth as previously shown (22, 24). This growth was accompanied by up-regulation of c-myc protein expression in both lines (Fig. 1A,, top). In contrast, Fig. 1A (bottom) shows that induction of c-myc RNA only occurred in WT ANBL-6 cells. Two additional experiments confirmed these results and also showed no significant RNA induction in K-RAS mutated cells at earlier time points (30–120 minutes). Northern data from all three experiments are summarized as bar graphs in Fig. 1A. The latter K-RAS–mutated cells may express heightened constitutive myc RNA expression, which results from the RAS mutation with little room for further stimulation when exposed to IL-6. Nevertheless, these data show that the IL-6–induced up-regulation of c-myc protein expression in mutant K-RAS–containing MM cells is not accompanied by increased RNA expression.
Additional studies, using the pulse-chase technique, showed that the increased myc protein expression in IL-6–treated K-RAS cells was not due to increased protein stability. As shown in Fig. 1B, the opposite result was detected, namely a slight decrease in protein stability due to IL-6 exposure. A second possible explanation for enhanced protein expression could be an IL-6–induced stimulation of cap-dependent translation. We had previously shown that IL-6 could activate mammalian target of rapamycin (mTOR) in MM cells (24, 25), which could achieve this effect. We, thus, addressed effects of IL-6 on myc translation using the methodology of Zong and colleagues (26), which is based on the observation that well translated transcripts are associated with polysomes, whereas poorly translated mRNAs are monosomal. ANBL-6 cells were treated with or without IL-6, polysomes were separated from monosomes on a sucrose gradient, and the associated RNA from these two groups was assessed by Northern analysis. Densitometric analysis of the signals obtained from monosomal versus polysomal fractions confirmed an enhancement in c-myc translation induced by IL-6. IL-6 induced an increase in polysome c-myc RNA from 28% to 44% in WT ANBL-6 cells and from 25% to 52% in K-RAS mutant cells (Fig. 1C). Interestingly, however, the mTOR inhibitor rapamycin (at 1 and 10 nmol/L) had no effect on enhanced translation although both concentrations inhibited mTOR mediated phosphorylation of p70S6kinase with complete ablation at 10 nmol/L (Fig. 1D). This indicated that the increase in c-myc translation induced by IL-6 was not mediated by mTOR and cap-dependent translation, and suggested a role for cap-independent translation.
To test whether IL-6 enhanced IRES-dependent cap-independent translation, we used the myc discistronic vector in which the myc 5′UTR, containing its IRES, was subcloned into the intracistronic space between the Renilla and Firefly luciferase open reading frames in the parental pRF vector (ref. 14; Fig. 2A). This generated the pRmF vector whose firefly luciferase translation would be driven by the 5′UTR. As a control, we also subcloned the p27 IRES into pRF, generating the pRp27F vector. After transfection of WT or K-RAS cells with the vectors, the mRNAs were tested for their ability to direct cap-independent translation (Firefly luciferase). Results (Fig. 2B) are normalized for transfection efficiency by cotransfection with a β-galactosidase construct. The presence of the myc 5′UTR IRES in pRmF markedly enhanced Firefly luciferase expression (versus pRF) in both WT and K-RAS lines while having no effect on Renilla luciferase expression, a monitor of cap-dependent translation. When exposed to 100 U/mL IL-6, a time-dependent increase in Firefly luciferase was shown in both cell lines. As a negative control, Fig. 2B shows that similar IL-6 treatment of K-RAS cells did not increase firefly luciferase driven by the p27 IRES (right). It is difficult to see in Fig. 2 because of the range of luciferase activity assayed (Y axis), but there was a very modest but significant increase in Renilla luciferase expression also after IL-6 exposure [to 150–175% of control values (no IL-6)]. This is consistent with the positive effect of IL-6 on global cap-dependent translation secondary to activation of mTOR. A representative experiment with raw data are shown in Supplementary Fig. S1.
To further support an effect of IL-6 on myc IRES activity in MM cells, we studied a third cell line, the U266 MM line, in which an autocrine IL-6 growth pathway exists. This cell line secretes IL-6, which, subsequent to its binding to IL-6 receptors, results in activation of many pathways and cell growth. As shown in Fig. 2C, U266 cells exhibit a huge amount of myc IRES activity, shown by the marked increase in Firefly luciferase expression in pRmF compared with pRF. When the cells are exposed to neutralizing anti–IL-6 and blocking anti-IL-6R antibodies (+anti–IL-6 AB), Firefly luciferase is greatly inhibited, whereas identical concentrations of isotype-specific control antibodies have no effect. To the right of Fig. 2C is shown an immunoblot assay for STAT-3 phosphorylation (STAT-3-P) used as a monitor for IL-6 signal transduction. It shows that the anti–IL-6/anti-IL6R antibodies interrupted the IL-6 autocrine loop, whereas the control antibodies had no effect. Thus, these results in three IL-6–responsive MM cell lines show that this growth factor enhanced RAP-resistant myc translation and myc IRES activity. This up-regulation of IRES activity completely explains the enhanced myc protein expression in IL-6–treated mutant RAS-containing cells and works together with enhanced RNA expression in WT cells.
To rule out the possibility that IL-6 increased myc firefly luciferase expression because of effects on a cryptic myc promoter located in the 5′UTR, we exploited RNA transfection using dicistronic RNAs to avoid cryptic promoter activity. To achieve this, the control pRF dicistronic reporter construct or the corresponding construct containing the myc 5′UTR in the dicistronic space was inserted into the pSP65-20B vector, which contains the SP6 RNA polymerase promoter and a 38 nucleotide polyadenylic acid tail (gift of Dr. Greg Goodall). Polyadenylated dicistronic RNAs were generated (Fig. 3A) and transfected into ANBL-6 WT cells. As an additional control, we also used the dicistronic RNA, which contains the VEGF 5′UTR in the intracistronic space (pR-V-F, generated as previously described in ref. 15). The Renilla and Firefly luciferase activity was then measured, and the relative luciferase activity of pRmF RNA was compared with the pRF control RNA in MM cells treated with or without IL-6. As shown in Fig. 3B, IL-6 significantly enhanced firefly luciferase expression in pRmF RNA-transfected cells by 2.5-fold while having no effect on expression in cells transfected with control pRF RNA or in the additional control cells transfected with the VEGF 5′UTR-containing RNA (pR-V-F). This ruled out the possibility of a cryptic promoter in the myc IRES DNA that could have responded to IL-6 stimulation.
Identification of hnRNP A1 as a potential IL-6–responsive c-myc ITAF. We initially hypothesized that an IL-6–induced increase in enhancing ITAFs or a decrease in inhibitory ITAFs could explain an increased IRES activity. However, no significant effects of IL-6 were found on protein levels of known myc ITAFs (including PTB, hnRNP K, hnRNP E1/E2, hnRNP C1/C2, and unr) or their electrophoretic mobility (data not shown). We, thus, used a yeast three-hybrid screen (Supplementary Fig. S2) to identify novel proteins, which specifically bound to the smallest construct containing sequences from the myc 5′UTR, which retained full IRES activity, a nucleotide segment spanning −396 to −165 upstream of the initiation codon. From this screen, we identified several clones encoding hnRNP A1, which specifically bound to the c-myc IRES sequence and was dependent on both the IRES sequence and the hnRNP A1 activation domain fusion for reporter expression.
Filter binding assays (Fig. 4A) and EMSA analysis (Fig. 4B) showed specific binding of hnRNP A1 to the myc IRES. The kDa for A1 binding to the myc IRES was ∼200 nmol/L, whereas binding to the p27 IRES (Fig. 4A,, darkened circles) was undetectable. In EMSAs, addition of recombinant hnRNP A1 retarded the migration of radiolabeled c-myc IRES but had no effect on migration of the Apaf-1 IRES or GAPDH RNA (Fig. 4B). In addition, when added as recombinant protein to extracts prepared from U87 cells in which endogenous hnRNP A1 had been silenced, hnRNP A1 markedly enhanced myc IRES-dependent reporter expression (27). These data indicate hnRNP A1 is an ITAF for the myc IRES.
Although IL-6 had no effect on the expression level of hnRNP A1 in MM cells (Fig. 4C), it increased the binding of A1 to the myc IRES in vivo as determined in an IP-RT-PCR assay (Fig. 4D). In this experiment, we treated both ANBL-6 WT and “K-RAS” cell lines with IL-6 and, after varying incubation periods, immunoprecipitated hnRNP A1, and performed PCR analysis on the precipitate for detection of myc IRES sequences. As shown in Fig. 4D, myc IRES RNA binding to hnRNP A1 was undetected in resting WT ANBL-6 MM cells, whereas a modest amount was bound to A1 in K-RAS cells. As a control, immunoprecipitation with nonspecific IgG could not bring down any myc IRES sequences. Exposure to IL-6 induced an increase in binding of hnRNP A1 to the myc IRES in both cell lines in a time-dependent fashion with optimal binding at 0.5 to 3 hours. To further confirm these data, we performed real-time PCR for quantification of myc IRES sequences bound to hnRNP A1. As shown in Fig. 4E, the real-time PCR data are consistent with a maximal induced binding after 3 hours of IL-6 exposure up to 7-fold over control (no IL-6). Thus, IL-6 increased the amount of hnRNP A1 bound to the myc IRES inside MM cells, and this was not due to any increase in A1 expression.
Although IL-6 did not alter total expression of hnRNP A1, A1 became serine phosphorylated as shown in Fig. 4F. In this experiment, ANBL-6 MM cells were treated with or without IL-6 or, as a positive control for A1 phosphorylation, sorbitol (28). HN-RNP A1 was then immunoprecipitated and immunoblotted for phospho-serine or total A1. As shown, A1 serine phosphorylation due to IL-6 exposure was comparable with that of sorbitol treatment. In a time course experiment, serine phosphorylation of A1 in MM cells was identified by 2 hours of exposure to IL-6 (Fig. 4F).
Effect of hnRNP A1 knockdown. To directly test a role for hnRNP A1 in the IL-6–induced stimulation of myc IRES function, A1 was silenced by siRNA, using a pool of three siRNA sequences. As shown in Fig. 5A (top gel), effective knockdown of A1 was accomplished in K-RAS cells with a pool of scrambled sequences having no effect. These cell lines were then treated with or without IL-6, and the IRES-dependent discistronic reporter assay was performed. As shown in Fig. 5B, A1 knockdown prevented the ability of IL-6 to stimulate myc IRES activity. In addition, there was a corresponding absence of myc protein up-regulated expression in these IL-6–treated cells (Fig. 5A,, bottom gel). Finally, the A1 siRNA silencing also prevented the ability of IL-6 to stimulate cell growth in vitro (Fig. 5C).
Studies in primary MM specimens. To test whether IL-6 could enhance myc IRES function in primary MM specimens, three patient bone marrow samples were processed (see Materials & Methods) to isolate pure myeloma tumor cell populations (>99% pure). The cells were treated with or without IL-6 (100 u/mL for 6 hours) and then transiently transfected with the dicistronic reporters to test IRES activity. As shown in Fig. 6A and B, the patient samples showed a significant enhancement of firefly luciferase expression (IRES activity) and myc protein expression after exposure to IL-6.
We were also able to investigate if hnRNP A1 binds the myc IRES in primary MM cells. Cryopreserved patient-isolated myeloma cell specimens (>99% pure tumor cells, n = 4) were thawed, RNA-protein binding was secured by treatment with 1% formaldehyde, and A1 was immunoprecipitated with anti-A1 antibody or control IgG. The immunoprecipitates were then analyzed for bound myc IRES RNA by RT-PCR. As shown in Fig. 6C, the myc IRES was present in the anti-A1 immunoprecipitates obtained from all the myeloma patient samples. Thus, in vivo binding between hnRNP A1 and the myc IRES is present in primary MM cells.
The results of this study indicate that part of the up-regulation of myc protein expression in MM cells stimulated with IL-6 is due to enhanced IRES-dependent translation. This was shown in the ANBL-6 MM cell model, the U266 cell line and in primary MM specimens. Furthermore, hnRNP A1 is an important protein that mediates IL-6–induced enhancement of myc translation. Importantly, siRNA knockdown of A1 expression in RAS-mutated MM cells prevented the stimulation of myc IRES activity, curtailed up-regulation of myc protein expression, and abrogated IL-6–enhanced cell growth.
HnRNP A1 has recently been identified as an ITAF for the fibroblast growth factor-2 and XIAP IRESes (29, 30), and our data (27) support its role as a myc ITAF. The ability of IL-6 to increase the binding between A1, and the myc IRES suggests a possible mechanism that could explain how A1 mediates the IL-6 effect on IRES activity. This increased binding may facilitate the ability of A1 to induce the proper conformational change in the IRES required for recruitment to the 40S ribosomal subunit. Whether or not the detected IL-6–induced serine phosphorylation of hnRNP A1 is critical for the enhanced A1-IRES binding remains to be examined.
An additional potential mechanism that must be considered is that IL-6 enhances A1 cytoplasmic localization. Initial binding between A1 and the myc IRES presumably occurs in the nucleus where hnRNP A1 is primarily localized. However, A1 is a nuclear-cytoplasmic shuttling protein and is likely involved in the nuclear export of mRNAs such as myc to the cytoplasm, the translationally competent compartment. Cytoplasmic shuttling is regulated by phosphorylation of A1 on 4 serine residues in the F peptide, an 18 amino acid segment in the COOH terminus of the protein (31, 32). Such phosphorylation is mediated by MNK kinases (33) that are activated downstream of p38 mitogen-activated protein kinase (MAPK; ref. 34). Although IL-6 does not activate the p38MAPK cascade, it is well-accepted that it can activate the RAS-RAF-MAP/ERK kinase-extracellular signal-regulated kinase (ERK) MAPK cascade (12). Interestingly, MNKs can also be activated by ERK (35). Thus, an additional potential mechanism of IL-6 stimulation of A1-IRES function is via stimulation of ERK with subsequent activation of MNKs, MNK-dependent phosphorylation of A1 at the F peptide and enhanced shuttling of A1/Myc RNA to the cytoplasmic translational compartment. Experiments are under way to determine which residues of A1 become serine phosphorylated by IL-6 and whether there is an IL-6–dependent increase in A1 cytoplasmic localization.
Recent work (36) supports the hypothesis that dysregulation of c-myc expression is key to the progression from the premalignant MGUS syndrome to overt myeloma. Gene set enrichment analysis identified a myc activation gene set signature that was significantly associated with MM compared with MGUS. Because myc translocations to the immunoglobulin enhancer element only occur in ∼15% of newly diagnosed patients (37), it is possible that a heightened myc translation is also important in progression and this may be facilitated by hnRNP A1 after IL-6 stimulation.
In summary, our data underscore the potential importance of a myc RNA-binding protein, hnRNP A1, in the enhanced myc translation that occurs in myeloma cells whose growth is stimulated by IL-6. A further understanding of the regulation of A1 and myc IRES function may allow future manipulation of myeloma tumor growth by targeting A1 therapeutically.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Grant support: Veteran's Administration, the Multiple Myeloma Research Foundation, and NIH grants RO1CA111448 (A.K. Lichtenstein) and RO1CA109312 (J. Gera).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Carolyne Bardeleben for assistance.