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Departments of Hematology and Oncology,1 Pathology,2 Pharmacology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia,3 Department of Genetics, Osaka University, Suita, Osaka, Japan4
Received 1 May 2007/ Returned for modification 25 May 2007/ Accepted 9 August 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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eIF4E is phosphorylated (usually at Ser209) in many systems in response to extracellular stimuli, including growth factors, hormones, and mitogens (10, 15, 16). Phosphorylation of eIF4E increases its affinity for the cap of mRNA and may also favor its entry into initiation complexes (10, 15, 16). Although the biological significance of eIF4E phosphorylation is not completely understood, a recent genetic study in Drosophila melanogaster has demonstrated that eIF4E phosphorylation is biologically significant and is essential for normal growth and development (9). The best candidate for eIF4E phosphorylation is the mitogen-activated protein kinase (MAPK)-activated protein kinase Mnk1, which physically associates with eIF4F and directly phosphorylates eIF4E at Ser 209. In addition to Mnk1, Mnk2 also phosphorylates eIF4E, albeit to a lesser extent. Both Mnk1 and Mnk2, but particularly Mnk1, are directly phosphorylated by ERK and p38 MAPKs (10, 15).
eIF4E is required for cell cycle progression, exhibits antiapoptotic activity, and, when overexpressed, transforms cells (2, 4, 11, 16), largely due to its critical role in initiating translation of mRNAs that encode cell cycle regulators or oncogenic proteins, such as cyclin D1, ornithine decarboxylase, c-Myc, hypoxia-inducible factor 1
, fibroblast growth factor, and vascular endothelial growth factor (2, 3, 11). Therefore, it is not surprising that elevated levels of eIF4E are found in a broad spectrum of transformed cells and human cancers, including lung cancers, and are often associated with aggressive, poorly differentiated tumors (3, 4).
The mTOR inhibitor rapamycin inhibits mTOR signaling, leading to suppression of 4E-BP1 phosphorylation, which promotes binding of 4E-BP1 to eIF4E, resulting in suppression of cap-dependent protein synthesis (3). Paradoxically, our previous work showed that rapamycin actually increases eIF4E phosphorylation while inhibiting 4E-BP1 phosphorylation (20). Moreover, our results suggest that rapamycin-induced eIF4E phosphorylation is phosphatidylinositol 3-kinase (PI3K) dependent but independent of ERK or p38 MAPK (20). In the current study, we further examined the modulation of eIF4E phosphorylation by mTOR inhibition in a broader setting and determined the involvement of PI3K and Mnks in mTOR inhibitor-induced eIF4E phosphorylation. Our results clearly demonstrate that rapamycin-induced eIF4E phosphorylation is mTOR inhibition dependent and is mediated by both Mnk1 and Mnk2. In addition, the possible clinical translation significance of these findings was also demonstrated.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell lines and cell culture.
The human lung cancer cell lines and breast cancer cell lines used in this study were purchased from the American Type Culture Collection (Manassas, VA). Glioblastoma U87MG and LN229 cell lines were provided by D. Durden (Emory University). The myeloma cell lines RPMI8266, OPM-1, and KMS18 were provided by J. Chen (Emory University). These cell lines were grown in monolayer culture in RPMI 1640 medium supplemented with glutamine and 5% fetal bovine serum (FBS) at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. The colon cancer cell lines HCT116 p53+/+ and p53–/– were provided by B. Vogelstein (The Johns Hopkins Medical Institutions, Baltimore, MD). These cell lines were cultured in McCoy's 5A medium with 5% FBS. Wild-type (WT), Mnk1 knockout (1-KO), Mnk2 knockout (2-KO), and Mnk1/Mnk2 double-knockout (DKO) murine embryonic fibroblasts (MEFs) were described previously (22). WT and IRS-1–/– murine 3T3 fibroblasts (25) were obtained from C. R. Kahn (Joslin Diabetes Center and Harvard Medical School, Boston, MA). WT, p85, and p85ß DKO MEFs (p85-DKO) were provided by L. Cantley (Harvard Medical School, Boston, MA). These cell lines were cultured in Dulbecco's modified Eagle's medium with 5% FBS.
Gene knockdown by siRNA. mTOR small interfering RNA (siRNA) (catalog no. 6381) was purchased from Cell Signaling Technology, Inc. p70S6K (catalog no. 1027217) and control (nonsilencing) (catalog no. 1022076) siRNAs were ordered from QIAGEN (Valencia, CA). Raptor siRNA that targets 5'-AAGGCTAGTCTGTTTCGAAAT-3' was described previously (18) and was synthesized by QIAGEN. Stealth MNK1 siRNA that targets the sequence 5'-CCTTGCCAGGAAAGTTTGAAGATAT-3' and stealth control siRNA that targets the sequence 5'-CCTACCAGGGAATTTAAGAGTGTAT-3' were synthesized by Invitrogen (Carlsbad, CA). The IRS-1 siRNA/siAb assay kit, which contains IRS-1 and nonspecific control SMARTpool siRNAs, was purchased from Upstate. The transfection of siRNA was conducted in a 24- or 12-well plate using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Forty-eight hours after the transfection, the cells were treated with dimethyl sulfoxide, rapamycin, or RAD001 for the indicated times and then subjected to detection of the indicated proteins by Western blot analysis.
We also used lentiviral control (scramble), mTOR, and raptor and rictor short hairpin RNAs (shRNAs), which were described previously (18) and ordered from Addgene Inc. (Cambridge, MA). The viral package and production and cell infection were the same as described previously (18).
Western blot analysis. The procedures for preparation of whole-cell protein lysates and for Western blotting were described previously (21). Whole-cell protein lysates (50 µg) were electrophoresed through 7.5%, 10%, or 12% denaturing polyacrylamide slab gels and transferred to an Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) by electroblotting. The blots were probed or reprobed with the primary antibodies, and then antibody binding was detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL) according to the manufacturer's protocol.
PI3K activity assay. Whole-cell protein lysates were prepared as described for Western blot analysis and then used for detection of PI3K activity as described previously (1).
Growth inhibition assays. Growth inhibition was evaluated by determining the cell numbers in a 3-day monolayer culture using the sulforhodamine B assay and by counting colony formations on culture plates after a 10-day treatment, as described previously (20).
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RESULTS |
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Rapamycin increases eIF4E phosphorylation independently of the IGF-1/IGF-1R/IRS-1 axis. To determine whether the increase of eIF4E phosphorylation by rapamycin is dependent on the presence of growth factors, we examined the effect of rapamycin on eIF4E phosphorylation in the absence of serum. Specifically, the cells were serum starved for approximately 24 h and then exposed to rapamycin. As presented in Fig. 1A, rapamycin induced a quick and sustained increase in p-eIF4E levels, which was detected between 1 h and 24 h post-rapamycin treatment. Since the IGF-1/IGF-1R/IRS-1 axis is involved in mTOR-mediated feedback inhibition of Akt (14, 24), we determined whether this axis also plays a role in mTOR inhibition-mediated eIF4E phosphorylation. Therefore, we examined the effect of rapamycin on eIF4E phosphorylation in the presence of IGF-1. As shown in Fig. 1B, IGF-1 substantially increased Akt phosphorylation, but not eIF4E phosphorylation. The presence of IGF-1 did not alter the modulation of eIF4E phosphorylation by rapamycin. Collectively, these results suggest that rapamycin increases eIF4E phosphorylation independently of activation of a growth factor/receptor, particularly IGF-1 signaling. Moreover, we examined the effect of rapamycin in the presence of an IGF-1R inhibitor and found that rapamycin still increased eIF4E phosphorylation (Fig. 1C). This result further indicates that rapamycin induces eIF4E phosphorylation independently of IGF-1/IGF-1R signaling. We also analyzed the effects of rapamycin on eIF4E phosphorylation in cells where IRS-1 expression was knocked down or in IRS-1-deficient cells. As presented in Fig. 1D and E, rapamycin still increased eIF4E phosphorylation in cells where IRS-1 was silenced (Fig. 1D) or in IRS-1 KO cells (Fig. 1E). These findings indicate that mTOR inhibitors induce an IRS-1-independent eIF4E phosphorylation. Taking these data together, we conclude that mTOR inhibitors increase eIF4E phosphorylation independently of the IGF-1/IGF-1R/IRS-1 axis.
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It is known that mTOR inhibitors suppress the phosphorylation of p70S6K due to inhibition of mTOR/raptor activity. To determine whether eIF4E phosphorylation induced by mTOR inhibitors is secondary to p70S6K inhibition, we also determined the impact of silencing of p70S6K on rapamycin-induced eIF4E phosphorylation. As shown in Fig. 2E, transfection of p70S6K substantially reduced p70S6K levels at either 24 h or 48 h posttransfection. Rapamycin increased p-eIF4E in both control siRNA- and p70S6K-transfected cells with comparable potencies, indicating that rapamycin increases eIF4E phosphorylation regardless of p70SK expression. Collectively, these results demonstrate that rapamycin increases eIF4E independently of p70S6K inhibition.
Rapamycin increases eIF4E phosphorylation independently of Akt activation. Our previous work showed that rapamycin increases the phosphorylation of both Akt and eIF4E (20). To determine whether eIF4E phosphorylation induced by rapamycin is associated with the increase in Akt phosphorylation, we examined the effect of rapamycin on eIF4E phosphorylation when Akt expression was silenced using Akt siRNA. As shown in Fig. 3A, inhibition of Akt (phosphorylation) by silencing its expression using Akt siRNA actually increased eIF4E phosphorylation while minimally decreasing p-p70S6K levels. In Akt siRNA-transfected cells, where Akt phosphorylation is inhibited, the increase in eIF4E phosphorylation by rapamycin was further enhanced (Fig. 3B). Taken together, these results indicate that Akt actually exerts a negative effect on eIF4E phosphorylation. Therefore, it is unlikely that rapamycin increases eIF4E phosphorylation secondary to Akt activation.
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and p85ß DKO)-deficient MEFs (p85-DKO). We found that rapamycin increased p-eIF4E levels at both 4 h and 8 h posttreatment in WT MEFs but not in p85-DKO MEFs (Fig. 4C), indicating that intact p85 or PI3K is essential for rapamycin to increase eIF4E phosphorylation. To further demonstrate the relationship between PI3K and eIF4E phosphorylation, we activated PI3K by expressing the constitutively active p110 catalytic subunit of PI3K through Ad infection and then examined its effect on eIF4E phosphorylation. As presented in Fig. 4D, infection of both H157 and A549 cells with Ad-p110* resulted in increased levels of p-eIF4E, in addition to increased levels of p-Akt, an indicator of PI3K activation. Thus, it is conceivable that PI3K indeed regulates eIF4E phosphorylation.
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We noted that LY294002 (4 and 6 µM) or wortmannin (0.5 and 1 µM) alone at relatively high concentrations that inhibited p70S6K phosphorylation increased p-eIF4E levels as well in H157 cells (Fig. 4A and B). However, we did not find that wortmannin alone increased p-eIF4E levels in A549 cells (Fig. 4B). Thus, whether the increase in eIF4E phosphorylation by a PI3K inhibitor in certain cancer cell lines is due to inhibition of mTOR signaling needs to be investigated further.
mTOR inhibitors increase eIF4E phosphorylation through an Mnk-mediated mechanism. It is well known that Mnks, particularly Mnk1, regulate eIF4E activation through its phosphorylation at Ser209 (10, 22). Rapamycin increases eIF4E phosphorylation at Ser209 (20), as did rapamycin and RAD001, as has been demonstrated (see Fig. S1 in the supplemental material). Therefore, we determined whether rapamycin increased eIF4E phosphorylation through a Mnk-dependent mechanism. To this end, we knocked down Mnk1 expression and then analyzed its impact on rapamycin-induced eIF4E phosphorylation. As shown in Fig. 5A, knockdown of Mnk1 expression decreased basal levels of p-eIF4E but could not prevent its phosphorylation being increased by rapamycin treatment. Modulation of p-Akt and p-p70S6K were not affected by Mnk1 silencing. This result clearly suggests that Mnk1 silencing is not sufficient to prevent eIF4E phosphorylation by rapamycin. It is possible that both Mnk1 and Mnk2 are responsible for rapamycin-induced eIF4E phosphorylation. Loss of Mnk1 function can be compensated for by Mnk2. As a result, knockdown of either gene's expression may not be sufficient to abrogate rapamycin-induced increase in eIF4E phosphorylation. To test this possibility, we examined the effects of mTOR inhibitors on eIF4E phosphorylation in WT, 1-KO, 2-KO, and Mnk1/Mnk2 DKO MEFs. Both rapamycin and RAD001 increased eIF4E phosphorylation in WT, 1-KO, and 2-KO MEFs but not in Mnk1/Mnk2 DKO MEFs. In fact, we did not detect basal levels of p-eIF4E in Mnk1/Mnk2 DKO MEFs. In contrast, p-p70S6K levels were inhibited and p-Akt levels were increased in all of the cell lines (Fig. 5B), indicating that Mnk deficiency does not affect mTOR inhibitor-induced inhibition of p70S6K phosphorylation and increase in Akt phosphorylation. Collectively, these results indicate that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism that involves both Mnk1 and Mnk2. We noted that the basal levels of p-p70S6K and p-Akt in Mnk1/Mnk2 DKO MEFs were lower than those in WT, 1-KO, and 2-KO MEFs (Fig. 5B). Whether Mnks are linked to regulation of the basal levels of p-Akt and p-p70S6K needs to be investigated in the future.
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Cotargeting mTOR and Mnk/eIF4E signaling augments inhibition of the growth of cancer cells. Given that the increase in eIF4E phosphorylation might counteract rapamycin's inhibitory effect on cap-dependent translation initiation, we hypothesized that cotargeting mTOR and Mnk/eIF4E signaling would result in augmented growth-inhibitory effects in cancer cells. Thus, we examined the effects of rapamycin combined with the Mnk1 inhibitor CGP57380 on the growth of several lung cancer cell lines. Although CGP57380 was originally identified as a Mnk1 inhibitor, this agent in fact also inhibits Mnk2-mediated eIF4E phosphorylation (8). As presented in Fig. 6A, in a 3-day assay, the combination of rapamycin and CGP57380 was more potent than each single agent alone in inhibiting the growth of four human lung cancer cell lines, which was either additive or synergistic. Moreover, we confirmed the effect of this combination in a long-term colony formation assay. Compared with the effects of rapamycin alone, the addition of CGP57380 enhanced rapamycin's ability to inhibit colony formation and growth (Fig. 6B). To demonstrate whether CGP57380 indeed blocked rapamycin-induced eIF4E phosphorylation, we further treated cells with rapamycin in the absence and presence of CGP57380 and analyzed eIF4E phosphorylation in these cells by Western blotting. As shown in Fig. 6C, the presence of CGP57380 inhibited rapamycin-induced eIF4E phosphorylation, particularly at an early time (e.g., 1 h), whereas inhibition of p70S6K phosphorylation by rapamycin was not affected. Thus, these data clearly show that CGP57380 abrogates rapamycin-induced eIF4E phosphorylation without interfering with rapamycin's inhibitory effect on p70S6K phosphorylation. We noted that rapamycin alone did not apparently reduce the levels of cyclin D1, a cap-dependent protein known to be regulated by mTOR signaling. However, the cotreatment with rapamycin and CGP57380 did reduce cyclin D1 levels at early times (e.g., 1 h), at which rapamycin-induced eIF4E phosphorylation was substantially inhibited by CGP57380 (Fig. 6C).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous work clearly demonstrated that rapamycin increases eIF4E phosphorylation while inhibiting 4E-BP1 phosphorylation (20). eIF4E function is known to be negatively regulated by its reversible association with the 4E-BPs, including 4E-BP1. Hyperphosphorylated 4E-BPs dissociate from eIF4E, leading to eIF4E activation, whereas hypophosphorylated 4E-BPs associate strongly with eIF4E and inhibit its phosphorylation and activity (15). Because eIF4E, when bound to 4E-BP1, is no longer a substrate for Mnks (15), it is unlikely that eIF4E phosphorylation induced by an mTOR inhibitor is secondary to inhibition of 4E-BP1 phosphorylation. Moreover, we found that knockdown of p70S6K expression using p70S6K siRNA did not affect rapamycin's effect on the increase of eIF4E phosphorylation, suggesting that rapamycin-induced eIF4E phosphorylation is not secondary to inhibition of p70S6K phosphorylation. Taken together, we suggest that eIF4E phosphorylation is likely a parallel event to inhibition of phosphorylation of p70S6K and 4E-BP1 during mTOR inhibition (Fig. 7).
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We previously suggested that rapamycin increases eIF4E phosphorylation through a PI3K-mediated mechanism using a PI3K inhibitor (20). In the current study, we further showed that the PI3K inhibitor LY294002 or wortmannin blocked eIF4E phosphorylation by RAD001, a derivative of rapamycin. Moreover, in PI3K-deficient MEFs, rapamycin lost its activity in increasing eIF4E phosphorylation. Thus, these results clearly indicate that mTOR inhibitors indeed increase eIF4E phosphorylation in a PI3K-dependent fashion. This notion is further supported by our finding that activation of PI3K by enforcing expression of constitutively active p110 elevated p-eIF4E levels (Fig. 4).
It has been suggested that mTOR activation (e.g., by insulin) initiates a feedback inhibition of PI3K/Akt through p70S6K activation and its subsequent phosphorylation and degradation of IRS-1 (Fig. 7). Rapamycin suppresses p70S6K and thus may relieve this negative-feedback inhibition of Akt (6, 12). In our current study, we have demonstrated that rapamycin-induced eIF4E phosphorylation is independent of p70S6K and IRS-1 (Fig. 1), although rapamycin does activate PI3K (Fig. 4E) and induce PI3K-mediated eIF4E phosphorylation (Fig. 4A to D). Thus, these findings argue that mTOR inhibition may activate PI3K signaling through another unknown mechanism(s), which is currently under investigation in our laboratory.
It is known that Mnks, particularly Mnk1, phosphorylate eIF4E at Ser209 (15, 16). In our study, we found that knockdown or KO of Mnk1 was not sufficient to abrogate mTOR inhibitors’ effects on eIF4E phosphorylation. Also, KO of Mnk2 was not able to abrogate mTOR inhibitors’ effects on eIF4E phosphorylation. eIF4E phosphorylation by mTOR inhibitors was abolished only when both Mnk1 and Mnk2 were knocked out. Thus, we conclude that both Mnk1 and Mnk2 are responsible for mTOR inhibitor-induced eIF4E phosphorylation. The question remained how mTOR inhibition increases Mnk activity. Mnks are known to be activated by ERK and p38 MAPKs (10, 15). However, our preliminary results suggest that rapamycin increases eIF4E phosphorylation independently of ERK and p38 because a MEK inhibitor or a p38 inhibitor failed to inhibit rapamycin-induced eIF4E phosphorylation (20). Interestingly, our previous work (20) and current results demonstrate that rapamycin, as well as RAD001, induced a PI3K-dependent phosphorylation of eIF4E because these mTOR inhibitors failed to increase eIF4E phosphorylation in the presence of the PI3K inhibitor LY294002 or wortmannin or in PI3K (p85)-deficient cells. Moreover, PI3K (p85) deficiency attenuated rapamycin's ability to increase Mnk1 phosphorylation, an event that activates Mnk1. Thus, we suggest that mTOR inhibition by mTOR inhibitors induced PI3K-dependent activation of Mnks, leading to an increase in eIF4E phosphorylation (Fig. 7). To the best of our knowledge, this is the first demonstration that links PI3K to modulation of Mnks.
mTOR inhibitors suppress 4E-BP1 phosphorylation, which presumably inhibits eIF4E phosphorylation or activity, leading to inhibition of cap-dependent translation (3). Paradoxically, our results clearly show that mTOR inhibitors also increase eIF4E phosphorylation while still inhibiting 4E-BP1 phosphorylation. Since eIF4E phosphorylation by mTOR inhibitors occurs very rapidly, which almost parallels inhibition of 4E-BP1 phosphorylation (20), we assume that an increase in eIF4E phosphorylation by mTOR inhibitors will counteract their effects on inhibition of cap-dependent translation. Thus, the net effect of an mTOR inhibitor on cap-dependent translation depends on whether the mTOR inhibitor-induced suppression of 4E-BP1 phosphorylation overrides its effect on eIF4E phosphorylation. Indeed, we found that rapamycin did reduce cyclin D levels in human prostate cancer cells while having minimal effects on downregulation of cyclin D1 in several lung cancer cell lines (our unpublished preliminary data). It needs to be pointed out that several other studies have also shown that eIF4E phosphorylation may not necessarily be involved in the assembly of the eIF4F complex or general protein synthesis (7, 8, 13, 22). Nonetheless, the impact of mTOR inhibition-induced eIF4E phosphorylation on cap-dependent translation, particularly in cancer cells, needs further investigation.
eIF4E phosphorylation or activation is associated with cell proliferation, transformation, tumor progression, and apoptosis resistance. Therefore, an increase in eIF4E phosphorylation during mTOR inhibition may counteract mTOR inhibitors’ growth-inhibitory effects on cancer cells. Since mTOR inhibitor-induced eIF4E phosphorylation is PI3K and Mnk dependent, one strategy to improve an mTOR inhibitor's efficacy against cancer, particularly those with high levels of eIF4E, is to prevent eIF4E phosphorylation by combining an mTOR inhibitor with a PI3K inhibitor or a Mnk inhibitor. Our previous work indeed showed that the combination of rapamycin and LY294002 exhibited synergistic growth-inhibitory effects in human lung cancer cells (20). In this study, we have further demonstrated that the combination of rapamycin and the Mnk1 inhibitor CGP57380 (which inhibits Mnk2, as well) also exerts augmented growth-inhibitory effects on a panel of human lung cancer cells (Fig. 7). Thus, our findings in this study are of translational significance from a clinical cancer treatment point of view using mTOR inhibitors.
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ACKNOWLEDGMENTS |
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This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award (to S.-Y.S.), National Institute of Health grant RO1 CA118450-01 (to S.-Y.S.), and Department of Defense grant IMPACT W81XWH-05-0027 (Project 5 to F.R.K. and S.-Y.S.). S.-Y. S. and F.R.K. are Georgia Cancer Coalition Distinguished Cancer Scholars.
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FOOTNOTES |
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Published ahead of print on 27 August 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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250 cells per well were seeded in 12-well plates. On the second day, the cells were treated with the indicated concentrations of rapamycin (Rap) alone, CGP57380 (CGP) alone, and their combination. The same treatments were repeated every 3 days. After 10 days, the plates were stained for the formation of cell colonies with crystal violet. The colonies in each well were counted. The bars are means of three replicate determinations plus standard deviations. (C) H157 cells were pretreated with the indicated concentrations of CGP57380 (CGP) for 30 min and then cotreated with 10 nM rapamycin (Rap) for 1 h or 24 h. The cells were then subjected to preparation of whole-cell protein lysates for detection of the indicated proteins using Western blotting.