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. Author manuscript; available in PMC: 2018 Feb 1.
Published in final edited form as: Mol Cancer Res. 2017 May 1;15(8):1117–1124. doi: 10.1158/1541-7786.MCR-16-0454

eIF2α Phosphorylation Mediates IL24-induced Apoptosis through Inhibition of Translation

Leah Persaud 1,2, Xuelin Zhong 1,2, Giselle Alvarado 1, Winchie Do 1, Jordan Dejoie 1, Anna Zybtseva 1, Bertal Huseyin Aktas 3,4, Moira Sauane 1,2
PMCID: PMC5743333  NIHMSID: NIHMS873235  PMID: 28461326

Abstract

Interleukin 24 (IL24) is an immunomodulatory cytokine that also displays broad cancer-specific suppressor effects. The tumor suppressor activities of IL-24 include inhibition of angiogenesis, sensitization to chemotherapy, and cancer-specific apoptosis. Supra-physiologic activation and/or overexpression of translation initiation factors are implicated in the initiation and progression of cancer animal models as well as a subset of human cancers. Activation and/or overexpression of translation initiation factors correlate with aggressiveness of cancer and poor prognosis. Two rate-limiting translation initiation complexes, the ternary complex and the eIF4F complex are regulated by eIF2α and 4E-BP1 phosphorylation, respectively. The work reported here provides direct evidence that IL-24 induces inhibition of translation initiation leading to apoptosis in squamous cell carcinoma. A dominant constitutively active mutant of eIF2α, which is resistant to phosphorylation, was used to determine the involvement of eIF2α in IL24-induced apoptosis. Treatment with IL-24 resulted in inhibition of protein synthesis, expression of downstream biomarkers of ternary complex depletion such as CHOP, and induction of apoptosis in cancer cells. The constitutively active non-phosphorylatable mutant of eIF2α, eIF2α-S51A, reversed both the IL24-mediated translational block and IL24-induced apoptosis. Intriguingly, IL-24 treatment also caused hypophosphorylation of 4E-BP1 which binds to eIF4E with high-affinity thus preventing its association with eIF4G and therefore preventing elF4F complex assembly.

Implications

These results demonstrate a previously unrecognized role of IL-24 in inhibition of translation, mediated through both phosphorylation of eIF2α and de-phosphorylation of 4E-BP1, and provide the first direct evidence for translation control of gene-specific expression by IL-24.

Keywords: Interleukin 24, Eukaryotic Initiation factor 2 alpha, 4E-BP1, translation regulation, apoptosis

Introduction

Cancer is caused by loss of physiologic restraints on cell proliferation and survival. Activation and/or amplification of proto-oncogenes, which cause uncontrolled cell growth, and mutations and/or deletions of tumor suppressor genes, which allow the survival of pre-cancerous cells, are prototypical examples of genetic aberrations that play a critical role in the development and progression of cancer. Recent experimental and clinical studies indicate that perturbations of some pathways associated with general cellular functions contribute to the genesis and progression of cancer (1-4). A paradigmatic example of such pathways is translation initiation, which plays a critical role in the physiological regulation of cell proliferation, differentiation and apoptosis (1,2). Unrestricted translation initiation causes malignant transformation and critically contributes to the maintenance and progression of cancers (3-6).

Phosphorylated eIF2α binds with high affinity to the guanine nucleotide exchange factor eIF2B and thereby inhibits recycling of eIF2·GDP into eIF2·GTP, depleting the ternary complex necessary to initiate a new round of translation (7-11). Depletion of the ternary complex reduces the overall rate of protein synthesis with a preferential effect on mRNAs encoding for oncogenic proteins, and up-regulating the expression of tumor-suppressor and pro-apoptotic proteins (12,13). This paradigm is consistent with the recent recognition that phosphorylation of eIF2α and the availability of the ternary complex control not only the overall rate of translation, as initially thought, but also the expression of specific gene clusters. Thus, recent cell and molecular biology work has confirmed the hypothesis that the translation initiation machinery in general and the ternary complex in particular represent very attractive targets for the development of mechanism-specific anti-cancer agents (14,15).

IL-24 is a tumor suppressing protein that is currently in phase II clinical trials. We and others have shown that IL-24 releases Ca++ from ER-stores (16); induces ROS and ceramide production (17-21); and induces inhibitory phosphorylation of eIF2α (22). We also have demonstrated that Sigma 1 Receptor (Sig1R) interacts with IL-24 and that IL-24:Sig1R is a critical upstream signal for IL24-induced ER-stress, calcium mobilization, and notably, phosphorylation of eIF2α and apoptosis in cancer cells (16-22).

The present studies show that IL-24 causes phosphorylation of eIF2α in a wide variety of cancer cells. Furthermore, our results indicate that IL-24 induces apoptosis through phosphorylation of eIF2α, restricting the amount of the ternary complex, and thereby inhibiting translation initiation. These results demonstrate that eIF2α phosphorylation plays a major role in IL-24-mediated apoptosis in cancer cells. Interestingly, we show that, in cells treated with IL-24 not only eIF2α is phosphorylated, but also eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) is dephosphorylated. Ternary complex and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the 4E-BP pathway, respectively. We found that activation of 4E-BP1 by IL24-dependent de-phosphorylation inhibits eIF4F complex assembly, whereas IL-24-mediated phosphorylation of eIF2α inhibits Ternary complex.

Materials and Methods

Virus infection

The IL-24 expressing replication defective (Ad.IL-24) and corresponding empty adenovirus vector lacking exogenous gene, used as a control (Ad.vector) were custom engineered by Vector Biolabs, Inc. (Philadelphia, PA).

Reagents

The PERK inhibitor I, GSK2606414 (EMD Millipore, Billerica, MA), was dissolved in dimethyl sulfoxide (DMSO; Sigma–Aldrich), and stored at 100 μM. The final concentration to be used was 0.5 μM GSK2606414. The PKR inhibitor C16, an imidizole-oxindole compound was obtained from Sigma–Aldrich, dissolved in DMSO. The final concentration to be used was 2.5μM.

Cells and culture conditions

Squamous cell carcinoma (KLN cells) expressing either wild-type (eIF2α-WT) or S51A mutant eIF2α (eIF2α-S51A) or bidirectional promoter/enhancer complex (KLN-tTA/pBISA-DL(ATF4)) stable transfected cells were described previously (23,24). Briefly for the dual luciferase TC assay we engineered cancer cell lines that express firefly (F) and renilla (R) luciferase open reading frame (ORF) under the control of a bi-directional promoter/enhancer complex. This assay was developed to identify compounds that reduce the availability of the eIF2•GTP•tRNAiMet TC. Reduced availability of the TC inhibits translation of most mRNAs but paradoxically increases translation of some mRNAs that contain multiple tandem upstream ORF in their 5′-untranslated regions (5′UTRs). In this assay, firefly luciferase mRNA is fused to 5′UTR of activating transcription factor 4 (ATF-4) mRNA that has multiple tandem uORFs while renilla luciferase mRNA is fused to a 5′UTR lacking any uORFs as previously described (23,24). To determine if phosphorylation of eIF2α is necessary for the activity of IL-24 in the ternary complex assay, KLN cancer cell lines endogenous eIF2α was replaced by either a non-phosphorylatable eIF2α mutant (eIF2α-S51A) or a recombinant wild type eIF2α (eIF2α-WT) as previously described (23,24). All other cell lines were purchased from American Type Culture Collection (ATCC), maintained per ATCC protocols and utilized within 6 months of thawing each vial. All cell lines were cultured in humidified atmosphere at 37°C with 5% CO2 and media was replaced every alternate day.

Western blot analysis

Protein extracts were prepared with RIPA buffer containing a mixture of protease inhibitors as described (19). Briefly, fifty micrograms of protein were applied to a 12% SDS/PAGE and transferred to nitrocellulose membranes. Membranes were incubated with Odyssey blocking buffer (Li-Cor) prior to incubation with polyclonal or monoclonal antibodies to phospho-eIF2α, total eIF2α, CHOP, cyclin D1, cyclin E, survivin, Bcl2, α-tubulin, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Hsp27, c-jun, and β-actin overnight at 4°C. Goat anti-rabbit IgG (H+L) 800 CW, goat anti-rabbit (680 RD) and/or goat anti-mouse (H+L) was applied for 60 minutes at room temperature (1:25000, LI-COR) prior to washing with 1× Phosphate Buffered Saline Tween-20 (PBS-T). Visualization and quantification was carried out with the LI-COR OdysseyH scanner and software (LI-COR Biosciences).

Cap-binding affinity assay

Cells were treated with Ad.IL-24 for the indicated time and lysed in lysis buffer [50 mM HEPES-KOH (pH 7.5), 150 mM KCl, 1 mM EDTA, 2 mM DTT and 0.2 % Tween] containing protease inhibitors. Cell lysates were incubated with m7GTP-agarose for 2 hours at 4°C (Sigma–Aldrich). Cap-binding proteins were washed four times using the lysis buffer, and then m7GTP-bound proteins were assessed by western blotting as described (16,22).

MTT assays

Cells were plated in 96-well dishes (1×103) DMEM containing 10% FBS and allowed to attach for 12 h prior to treatment(s). Cell growth and viable cell numbers were monitored by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining as previously described (20).

Annexin V binding assays

Cells were trypsinized, washed once with complete medium and PBS, resuspended in 0.5ml of binding buffer containing 2.5 mmol/L CaCl2, and stained with allophycocyanin-labeled Annexin V (Becton Dickinson Biosciences, Palo Alto, CA) and propidium iodide (PI) for 15 min at room temperature. Flow cytometry assays were performed as previously described (22).

RT-PCR

Total RNA was isolated by using the RNAeasy kit (Qiagen). Reverse transcription (RT) was performed on 5 ng of total RNA with an oligo(dT) primer. cDNA corresponding to 5 ng of total RNA was amplified for 35 cycles by PCR with specific primers as previously described (21). Sequence-validated QuantiTec probes for bcl2, survivin, mTOR, cyclin D1, cyclin E, c-Myc, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin purchased from Qiagen Bio-technology were used for these mRNAs.

Dual luciferase assay

Translation of the reporter mRNAs harboring 5′UTR of human ATF-4 was monitored by dual-luciferase assay as previously described (25). Stably transfected KLN cells with a bi-directional plasmid in which a common promoter/enhancer complex drives the transcription of Firefly luciferase ORF fused to the 5′UTR of ATF-4, and of the Renilla luciferase ORF fused to a simple 90-nucleotide 5′UTR derived from the plasmid, were seeded in a six-well plate in triplicate and the luciferase assay was performed at 48 h post infection with cells at 80% confluency. Cells were collected in Passive Lysis Buffer, and Firefly and Renilla luciferase activities were measured using the Dual-luciferase Reporter Assay kit (Promega, Inc., Madison, WI) according to the manufacturer's instructions. Experiments were repeated three times independently, whereby each biological replicate consisted of a technical quadruplicate.

Protein Synthesis

Total protein synthesis was measured as previously described (25). Briefly, wild-type (eIF2α-WT) or S51A mutant eIF2α (eIF2α-S51A) stable transfected cells were seeded in six-well plates, serum starved for 16 h and then deprived of methionine and cysteine for an additional 2 h using DMEM without the above amino acids (Gibco). Cells were then treated with 10% serum containing 10 μCi ml-1 of 35S-Met/Cys (Perkin Elmer). Cells were lysed in RIPA buffer and protein concentration was determined by bicinchoninic acid assay (Pierce). Equal amounts of protein were separated by SDS/PAGE or an aliquot of lysate was trichloroacetic acid-precipitated and counted in a scintillation counter (Beckman Coulter).

Results

IL24-dependent Phosphorylation of eIF2α is necessary and sufficient to mediate Apoptosis

Translation in eukaryotic cells starts with the assembly of the eIF2.GTP-Met-tRNA ternary complex, recruitment of the 40S ribosomal subunit, followed by binding to mRNA at its 5′ UTR end (14,15,26). The ribosomal complex then migrates along the 5′ UTR of the mRNA in a process facilitated by an array of initiation factors including eIF2 and eIF4 (14,15,26). At the end of each initiation event, GTP is hydrolyzed, and the eIF2.GDP complex is released (26). Regeneration of the eIF2.GTP complex by a GDP-GTP exchange reaction catalyzed by eIF2B is required to start a new round of translation initiation (26). Phosphorylation of serine 51 in the α subunit of eIF2 inhibits GDP-GTP exchange, suppressing the initiation of translation (26). We have previously demonstrated that IL-24 induces phosphorylation of eIF2α on serine 51 in prostate cancer (22). Now we demonstrate that IL-24 also induces phosphorylation of eIF2α on serine 51 (Fig. 1A) in melanoma (HO-1, WM35, MeWo), breast (MCF-7, MDA-MB-231) and cervical cancer cells (HeLa). Similarly, IL-24 exposure also reduced viability of cancer cells (Fig. 1B). The requirement for eIF2α phosphorylation for IL-24-induced apoptosis was analyzed by IL-24 treatment of squamous cell carcinoma (KLN cells) expressing either wild-type (eIF2α-WT) or S51A mutant eIF2α (eIF2α-S51A), the dominant negative mutant of eIF2α. Compared with KLN cells expressing wild-type eIF2α, S51A mutant eIF2α (eIF2α-S51A) cells were resistant to the inhibitory action of IL-24 on both cell growth (Fig. 2A, B and C) and protein synthesis (Fig. 2D). These results show that phosphorylation of eIF2α is responsible for the inhibitory effect of IL-24 on protein synthesis and cell growth.

Figure 1. Effect of IL-24 on Phosphorylation of eIF2α and proliferation in cancer cells.

Figure 1

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A. Melanoma (HO-1, WM35, MeWo), breast (MCF-7, MDA-MB-231) and cervical cancer cells (HeLa) were treated for 48 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Cells were collected, protein purified, and subjected to Western Blot analysis to detect phospho-eIF2α protein. B. Cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell), and cell viability was determined by the MTT proliferation assay 5 days after treatment. Numbers represent the ratio of specific treatments to values in control cells (Ad.vector). C. Cells were treated as described in B, and then assayed for cell death using Annexin V staining a measure of apoptosis, was determined 48 h later by FACS analysis using the CellQuest software (Becton Dickinson). An average of three independent experiments is shown ± SD.

Figure 2. IL-24-dependent Phosphorylation of eIF2α is necessary to mediate apoptosis.

Figure 2

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A. Growth-inhibitory effects of IL-24 in wild type eIF2α (left panel) or S51A mutant eIF2α (right panel) expressing cells. Cells were treated with Ad.IL-24 (25, 50, or 100 pfu per cell) or Ad.vector (25, 50, or 100 pfu per cell), and cell viability was determined by the MTT proliferation assay. Numbers represent the ratio of specific treatments to values in control cells (Ad.vector). An average of three independent experiments is shown ± SD. B. Cells were treated as described in A, and then assayed for cell death using Annexin V staining, a measure of apoptosis, was determined by FACS. C. Induction of eIF2α phosphorylation protein after treatment with different concentrations of Ad.IL-24 was determined by Western blot analysis. D. Wild type eIF2α or S51A mutant eIF2α expressing cells were serum starved (16 h) and followed by 10% serum stimulation (FBS) with increasing concentrations of Ad.IL-24. Global protein synthesis was monitored by 35S-Met/Cys incorporation.

IL-24 restricts formation of the ternary complex

The availability of the ternary complex not only determines the overall rate of translation initiation but also differentially affects the translation of specific mRNAs. When the ternary complex is scarce, mRNA translation is generally down-regulated. Paradoxically, translation of some mRNAs such as ATF-4 mRNA is significantly more efficient under conditions that limit the availability of the ternary complex because their 5′ UTRs contain multiple uORF (27-29). To confirm that IL-24 restricts the abundance of the eIF2.GTP.Met-tRNAi ternary complex, we used a bi-directional plasmid in which a common promoter/enhancer complex drives the transcription of firefly luciferase (F-luc) ORF fused to the 5′UTR of ATF-4, and of the renilla luciferase (R-luc) ORF fused to a simple 90-nucleotide 5′UTR derived from the plasmid (Fig. 3A). The relative expression of each luciferase was established by the F-luc to R-luc ratio determined in a dual luciferase assay (3-6). In stably transfected KLN cells, IL-24 increased the F-luc to R-luc ratio in a dose-dependent manner (Fig. 3B), indicating that IL-24 increases translation modulated by a multiple-uORF 5′UTR relative to translation modulated by a simple 90-nucleotide 5′UTR. To validate IL-24 as a bona fide inhibitor of the ternary complex formation, we took advantage of the fact that reducing the abundance of the ternary complex up-regulates CHOP mRNA and protein expression, a direct transcriptional target of ATF-4. We measured the effect of IL-24 on KLN cells on the expression of CHOP mRNA by real time PCR (Fig. 3C) and CHOP protein by Western blot (Fig. 3D). Results of these secondary assays showed that IL-24 restricts formation of the ternary complex and also induces expression of both CHOP protein, and mRNA, without any effect on the expression of the housekeeping β-actin gene.

Figure 3. Identification and validation of IL-24 as modifier of the ternary complex abundance.

Figure 3

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A. The Firefly luciferase (F) ORFs and the Renilla luciferase (R) ORFs were cloned into pBISA plasmid to transcribe two reporter mRNAs. The 5′UTR of ATF4 mRNA including first two codons of bona-fide ORF was cloned in frame with respect to the start codon of F-luc ORF (pBISA-DL(ATF4)). The mRNA products of pBISA-DL(ATF4) plasmid are shown. B. KLN-tTA/pBISA-DL(ATF4) cells were incubated with the indicated concentrations of Ad.IL-24 (25, 50, or 100 pfu per cell) and the normalized F/R ratio was determined by Dual-Luciferase Reporter Assay (Promega). C. KLN-tTA/pBISA-DL(ATF4) cells were incubated with indicated concentrations of Ad.IL-24 and expression of endogenous CHOP mRNA was determined by real-time PCR. D. IL-24 induces CHOP expression in KLN cells. KLN-tTA/pBISA-DL(ATF4) cells were incubated with the indicated concentrations of Ad.IL-24 (25, 50, or 100 pfu per cell) and expression of endogenous CHOP protein was determined by Western blot analysis.

IL-24 preferentially inhibits expression of oncogenic proteins

Expression of most proteins involved in cell proliferation and malignant transformation is translationally controlled and is highly dependent on the activity of translation initiation factors (5,30). To determine if IL-24 translationally down-regulates expression of oncogenic proteins, we performed Western blot and quantitative real time PCR analyses of lysates from squamous-cell carcinoma cells treated with IL-24 or control (Ad.vector). Figure 4 shows that IL-24 significantly reduced the expression of Bcl2, c-Myc, Survivin, Cyclin D1, and Cyclin E while the expression of housekeeping proteins such as β-actin, GAPDH, α-tubulin, as well as Hsp27 and c-jun was not affected. Down-regulation of most oncogenic proteins was likely translational because IL-24 has minimal effects on the levels of the respective mRNAs (Fig. 4B). In eIF2α-S51A cells that are resistant to its growth-inhibitory effect, Ad.IL-24 did not affect Bcl2, c-Myc, Survivin, Cyclin D1, or Cyclin E expression (data not shown). These findings are consistent with the view that inhibitors of translation initiation preferentially affect the expression of oncogenic proteins.

Figure 4. IL-24 preferentially inhibits expression of oncogenic proteins.

Figure 4

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A. The wild type eIF2α or S51A mutant eIF2α expressing cells were treated with Ad.vector (100 pfu per cell) or Ad.IL-24 (100 pfu per cell) for 72 h, lysates were prepared and probed with antibodies specific to Bcl2, Survivin, c-Myc, Cyclin D1, Cyclin E, α-tubulin, β-actin, GAPDH, Hsp27, and c-jun. B. The wild type eIF2α or S51A mutant eIF2α expressing cells were incubated with Ad.vector (100 pfu per cell) or Ad.IL-24 (100 pfu per cell) and expression of Bcl2, Survivin, c-Myc, Cyclin D1, Cyclin E, α-tubulin and β-actin mRNA was determined by real-time PCR.

PKR and PERK in eIF2α phosphorylation induced by IL-24

Pataer et al. demonstrated that IL-24 activates the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) in lung cancer cells (31). Yacoub, A. et. al. demonstrated that PKR-like endoplasmic reticulum-resident protein kinase (PERK) is activated by IL-24 treatment in glioma cancer cells (32). Here, we explored whether PERK and/or PKR are involved in IL-24-induced eIF2α phosphorylation. Figure 5A shows that IL-24 was partially unable to trigger eIF2α phosphorylation in the presence of PKR inhibitor 2-aminopurine or PERK inhibitor, GSK2606414. These results indicate that PKR and PERK are necessary for eIF2α phosphorylation.

Figure 5. IL24-mediated activation of 4E-BP1 appears to inhibit eIF4F complex assembly.

Figure 5

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A. KLN cells were incubated with the PKR or PERK inhibitors with or without Ad.IL-24 (100 pfu per cell) and expression of phoshpho-eIF2α protein was determined by Western blot analysis. B. Western blots of extracts from KLN cells treated for 48 h with Ad.IL-24. Blots were developed as indicated with antibodies specific for total 4E-BP1 and its phosphorylated forms (Thr37/46 and 70), and its phosphorylated form (Ser65), and β-actin (as a loading control). C. Cells treated for 48 hours with increasing doses Ad.IL-24 (25, 50, or 100 pfu per cell) or Ad.vector (25, 50, or 100 pfu per cell) were subjected to m7GTP pull-down. Amounts of the indicated proteins in the input or pull-down were determined by Western blotting. β-actin served as a loading control (input) and to exclude contamination (m7GTP pull-down) (left panel). Cells were pretreated with or without Ad.IL-24 prior to Insulin treatment (right panel). Amounts of the indicated proteins in the input or pull-down were determined by Western blotting. β-actin served as a loading control (input) and to exclude contamination (m7GTP pull-down). D. Model illustrating the possible molecular mechanism of cancer cell-selective apoptosis induction by IL-24 through inhibition of translation in squamous cell carcinoma.

IL24-mediated activation of 4E-BP1 appears to inhibit eIF4F complex assembly

Two major rate-limiting steps in translation initiation regulation are the ternary complex (TC) and eIF4F complex assembly, primarily via the phosphorylation of eIF2α and the de-phosphorylation of 4E-BP1, respectively (33). We therefore investigated whether eIF4F complex assembly, via 4E-BP1 phosphorylation, plays a role in IL-24. As it can be seen in Figure 5B, a decrease in 4E-BP1 phosphorylation was detected after IL-24-treatment. IL-24 strongly decreased the phosphorylation of 4E-BP1 (on Thr37/46. Thr70 and Ser65). To determine whether IL-24 affects eIF4F complex formation in KLN cells, we performed a cap-column assay to measure the association of either 4E-BP1 or eIF4G with eIF4E. As shown in Figure 5C, Ad.IL-24 treatment resulted in decreased eIF4G and increased 4E-BP1 association with eIF4E. An increase in 4E-BP1 binding to eIF4E, and a corresponding decrease in eIF4G association is indicative of translational repression. Together, these results demonstrate that IL-24-induced apoptosis and translational repression in KLN cells is mediated by 4E-BP1 activation. These findings indicate that the regulation of the availability of eIF4E, via its sequestration by dephosphorylated 4E-BP1, also contribute to the translational repression mediated by IL-24. In agreement with the phosphorylation of eIF2α, IL-24 exhibited inhibition of 4E-BP1 phosphorylation, supporting the global translation inhibition in these cells.

Discussion

Involvement of translation initiation factors in human cancers has been recognized (14,26,34). Increased activation and/or overexpression of translation initiation factors have been associated with transformation to and maintenance of cancer cell phenotype (35-37). Inhibition of translation initiation could therefore be a powerful mechanism harnessed for cancer therapy. The work reported here provides direct evidence that inhibition of translation initiation with IL-24 induces apoptosis in cancer cells. Specifically, IL-24 induces eIF2α phosphorylation, which reduces the abundance of the ternary complex (TC). Importantly, we show that depletion of the TC by IL-24 results in the up-regulation of pro-apoptotic proteins such as CHOP. Thus, we provide the first direct evidence for translational control of gene-specific expression by IL-24. This paradigm is consistent with the evolving notion in the field of translational regulation of gene expression, that phosphorylation of eIF2α and the availability of the ternary complex control not only the overall rate of translation, as initially thought, but also have differential effects on the translation and expression of specific genes (5). We show that through this mechanism IL-24 targets the expression of specific oncogenic proteins such as Bcl2, Survivin, c-Myc, Cyclin D1, and Cyclin E on which cancer cells depend to maintain their transformed phenotype. This approach has a clear advantage over the more generally genotoxic conventional chemotherapy. On the other hand, though selective, IL-24 mediated inhibition of translation, is not reduced to targeting one single oncogene as other approaches do but multiple oncogenic proteins, which has the potential to circumvent the problem of resistance to treatments with targeted therapy (38). It is plausible that the effect of IL-24 on cell cycle regulation also related to a broader effect of IL-24 on cell cycle regulation, possibly involving molecular players other than eIF2α. IL-24-mediated inhibition of translation initiation could be exploited for the combination therapy with existing therapies that relay on induction of eIF2α phosphorylation as primary or secondary targets or with mechanism specific translation initiation inhibitors at various stages of development.

IL-24 is a pleiotropic cytokine that displays a broad range of activities including antibacterial and antiviral responses, tissue remodeling, wound healing, and anti-tumor effects. The effects of IL-24 seem to be quite complex because its role can vary depending on the cellular source, target, and phase of the immune response. It remains unclear whether the antitumor effects of IL-24 reflect its other biological functions, specifically, it is plausible to speculate that translation regulation has an important role in IL-24's other functions besides its anti-tumor activity, such as its antibacterial- and antiviral- effects.

In summary, the identification of translation initiation inhibition as a key mediator of IL-24-cancer-specific apoptosis significantly broadens its therapeutic potential against tumors.

Acknowledgments

We are very grateful to Dr. J. A. Halperin for his invaluable support and expert advice. We are also grateful to Dr. J. A. Halperin, and Dr. N. Sonenberg for kindly providing constructs and stable transfected cells, and for their invaluable expert advice. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award number SC1 CA2005 (M. Sauane) and 1RO1CA152312 (B.H. Aktas).

Footnotes

Conflicts of Interest: The authors declare no potential conflicts of interest.

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