High Levels Of eIF4E Research Articles

Clinical Significance of Eukaryotic Initiation Factor 4E (eIF4E) Level among Cases Suffering Basal Cell Carcinoma of Skin.

BackgroundEukaryotic initiation factor 4E (eIF4E) has been reported to act as a prognostic biomarker in various cancers, but its actual effect on basal cell cancer (BCC) of the skin is rarely reported. Our research measured eIF4E levels and discussed its consequence in BCC of the skin.Material/MethodsSemi-quantitative real-time polymerase chain reaction (RT-PCR) and western blotting analysis were used to detect relative expression level of eIF4E in specimens at both mRNA and protein levels. The relationship of eIF4E level with clinical profiles was analyzed via chi-square test. Additionally, prognostic value of eIF4E was analyzed via Kaplan-Meier and cox regression analysis.ResultsWe found that eIF4E was over-expressed in tumor tissues, in comparison to bordering cancer-free tissue samples. Besides, elevated eIF4E level exhibited a strong relation to metastasis, TNM stage, and differentiation. Kaplan-Meier analysis revealed cases harboring high eIF4E levels faced shortened overall survival compared to cases of low levels (log rank test, P=0.018). Moreover, eIF4E could act as an independent biomarker for the prognosis of BCC of the skin, according to Cox regression analysis.ConclusionsThe level of eIF4E was upregulated and significantly correlated with the development of BCC of the skin. Thus, it might be a promising prognostic biomarker and therapy target for BCC of the skin.

Abstract 4898: eIF4E reshapes the surface of migrating AML cells through regulating Hyaluronic Acid synthesis & CD44 expression

Abstract Formation of cell surface protrusions has long been associated with tumor cell migration, metastasis & bone marrow homing & retention. It was shown that increased expression of endogenous or exogenous hyaluronic acid synthesizing enzyme 3 (HAS3) induces growth of microvillus-like cell surface protrusions. Some of the biological signals triggered by HA are dependent on its recognition by the cell surface receptor CD44. In our studies we observe that overexpression of the eukaryotic translation initiation factor 4E (eIF4E) increases formation of cell surface protrusions resembling those produced by HAS3 upregulation. eIF4E regulates mRNA export & translation through binding to the m7G cap of mRNA in the nucleus & the cytoplasm, respectively. Strikingly, analysis of our two complimentary screens (immunoprecipitation of mRNAs bound to eIF4E in the nucleus, and mRNA export assay with ribavirin, an established inhibitor of eIF4E) that we carried out to identify candidate eIF4E mRNA export targets, showed that nearly all the enzymes involved in HA synthesis as well as CD44. Here, we hypothesize that eIF4E regulates HA synthesis & CD44 overexpression to reshape the surface of cancer cells producing HA-rich microvillus-like pseudopods. Following validation of the positive hits, we used immunofluorescence staining with biotinylated HA binding protein (HABP) to determine the effect of eIF4E expression on protrusion formation. Our data revealed that eIF4E overexpression increased formation of HA-rich cell surface protrusions & was correlated with increased invasiveness of eIF4E overexpressing cells in in vitro matrigel assay, compared to respective controls. Colocalization studies using confocal microscopy indicated that protrusions positively stained for CD44. Further, pharmacological inhibition with ribavirin and genetic knockdown of eIF4E (or HAS3/CD44 in the context of eIF4E overexpression) abrogated protrusion formation and decreased the invasion capacity of eIF4E overexpressing cells. Together, these data indicate that eIF4E modulates protrusion formation & tumor invasion through coordinately regulating the export of RNAs in the CD44-HA regulon. To establish the clinical relevance of our findings in AML, we examined primary AML specimens with high eIF4E levels for expression of HA synthesizing enzymes, CD44 & we directly measured HA using confocal methods. This comparison showed that these pathways are dysregulated in AML patients & that their inhibition correlates with response to eIF4E inhibition by ribavirin. While HA has long been considered as a component of the tumor microenvironment required for the migration of cancer cells though interaction with surface CD44, our results provide a novel role for eIF4E in the motility & bone marrow homing. These findings offer a rationale for potentially inhibiting tumor cell metastasis through the combinatorial inhibition of eIF4E and CD44 or HAS3. Citation Format: Hiba Zahreddine, Biljana Kraljacic-Culjkovic, Valbona Cali, Mark Lauer, Lucy Skrabanek, Nabilah Khan, Ronald Midura, Leandro Cerchietti, Vincent Hascall, Craig Jordan. eIF4E reshapes the surface of migrating AML cells through regulating Hyaluronic Acid synthesis & CD44 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4898. doi:10.1158/1538-7445.AM2017-4898

Overexpression of eIF4E in colorectal cancer patients is associated with liver metastasis.

PurposeLiver metastasis is one of the leading causes of death in colorectal cancer (CRC) patients. The present study aimed to evaluate the value of eIF4E as a prognostic marker of colorectal liver metastasis (CLM) and identify the functional role of eIF4E in CRC metastasis.Patients and methodsThe expression level of eIF4E in CRC tissues was analyzed by immunohistochemical staining and Western blot. Expression of eIF4E in CRC cell lines was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot. Cell Counting Kit-8 (CCK-8) and Transwell assays were performed to assess the effects of eIF4E on cell proliferation, migration, and invasion. Western blot was further used to investigate the mechanism of eIF4E in tumor metastasis.ResultsThe upregulation frequency of eIF4E in the CLM group (82.5%) was higher than that in the non-CLM group (65.0%). Of the 80 patients recruited for the follow-up study, 23 were in the low eIF4E group (ratio of tumor to nontumor tissue <twofold), and 57 were in the high eIF4E group (ratio of tumor to nontumor tissue ≥twofold). In addition, the group exhibiting high eIF4E expression had a higher rate of liver metastasis (47.4%) than the group exhibiting low eIF4E expression (13.0%). In CRC cell lines, the expression of eIF4E was higher than in the normal cells. In vitro functional studies indicated that eIF4E knockdown inhibited the proliferation, migration, and invasion of Lovo and SW480 cells, and suppressed the expression of cyclin D1, VEGF, MMP-2, and MMP-9.ConclusionThe results of the present study indicated that high eIF4E levels in CRC patients predicted a high risk of liver metastasis. Knockdown of eIF4E inhibited CRC cell metastasis in part through regulating the expression of cyclin D1, VEGF, MMP-2, and MMP-9.

Identification and Characterization of an eIF4e DNA Aptamer That Inhibits Proliferation With High Throughput Sequencing.

Development of DNA aptamer screens that are both simple and informative can increase the success rate of DNA aptamer selection and induce greater adoption. High eIF4e levels contribute to malignancies, thus eIF4e presents itself as a valuable target for DNA aptamer-based inhibition screen. Here, we demonstrate a method for the rapid selection of looped DNA aptamers against eIF4e by combining negative selection and purification in a single step, followed by characterization with high throughput sequencing. The resulting aptamers show functional binding to eIF4e and inhibit translation initiation in biochemical assays. When transfected into cells, eIF4e aptamers cause a dramatic loss of cell proliferation in tumor cells as seen with eIF4e knockdown with antisense oligonucleotides, shRNAs, and siRNAs, hinting at therapeutic possibilities. With the large data set provided by high throughput sequencing, we demonstrate that selection happens in waves and that sequencing data can be used to infer aptamer structure. Lastly, we show that ligation of looped aptamers can enhance their functional effects. These results demonstrate a rapid protocol to screen and optimize aptamers against macromolecules of interest.

Abstract 3830: Novel small molecule translation inhibitors with safety and efficacy in triple negative breast cancer (TNBC) animal models

Abstract Although the overall survival rate for early stage breast cancer is high, triple negative breast cancers are particularly aggressive and are more likely to recur than other breast cancer subtypes, resulting in a significantly increased risk of death. Eukaryotic mRNAs possess a capped 5′-end that is required for initiation of protein synthesis known as cap-dependent translation, which in normal cells is tightly controlled. Deregulation of translation initiation can lead to uncontrolled growth, a hallmark of cancer cells. Eukaryotic initiation factor 4E (eIF4E) is a critical component of the machinery that carries out cap-dependent translation in the cell and a strong determinant for the rate of translational initiation. Greater than 60% of triple negative breast tumors have high levels of eIF4E, and elevated levels of eIF4E have been shown to correlate with higher rates of recurrence and increased risk of death. We have recently developed two new classes of translation initiation inhibitors using in vitro and cellular assays of protein translation and cancer cell proliferation. These inhibitors are novel drug-like molecules with molecular weights &amp;lt; 500, micromolar IC50s in TNBC cells lines, desirable PK properties, and no significant toxicity. A xenograft study (MDA-MB-231 s.c. in nude mice) was performed on lead compounds NM043 and NM891 and compared to vehicle control and paclitaxel. Daily i.p. injections were initiated when mean tumor volumes reached 100 mm3 (Day 0). Mean tumor volumes at Day 31 were as follows; vehicle: 898 ±260 mm3, paclitaxel: 493± 123 mm3, NM043: 394 ± 143 mm3, and NM891: 276± 119 mm3. Analysis of AUC indicated that there were significant differences between the vehicle control group and groups treated with NM043 (p=0.003) and NM891 (p=0.001), but not paclitaxel. The groups treated with NM043 and NM891 were subdivided into 2 groups on Day 32. For the next 10 days, one group continued to receive treatment while the other did not. Statistically significant differences were noted between the vehicle control group and all treatment groups (including the drug withdrawal groups) on Day 42. Although tumor growth resumed quickly after NM043 treatment was withdrawn, tumor growth did not resume within 10 days of the withdrawal of NM891. On day 45, mice treated with NM891 were sacrificed and detectable levels of NM891 were found in both plasma and tumor samples from the mice that discontinued treatment on Day 31, indicating that less frequent dosing of NM891 will likely be an option. NM043 and NM891 are promising clinical candidates and represent potential first in class small molecule therapeutics aimed at reducing recurrence and increasing survival rates for TNBC. Future efforts will be focused on GLP toxicology and studies to determine dosing range, route, and schedule in support of an IND to advance these drugs into Phase I/II clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3830. doi:1538-7445.AM2012-3830

Abstract 4984: Phosporylation of eIF4E confer resistance to cellular stress

Abstract Eukaryotic initiation factor (eIF) 4E mediates eIF4F association with the mRNA 5′ cap structure and is an essential factor for canonical protein translation. eIF4E can act as an oncogene when overexpressed in cell lines and high levels of eIF4E and phosporylated peIF4E have been described in a large number of human tumors. eIF4E has also been described as a component of cytoplasmic stress granules and processing bodies. Importantly, non-phosphorylated 4E-BP1 (often in response to stress) binds eIF4E to suppress protein translation. We demonstrated previously that high levels of peIF4E were associated with resistance to this suppressor effect in carcinoma cell lines. Because the role of peIF4E is not yet well established, we evaluated the role of eIF4E phosphorylation on in vitro stress resistance. Breast carcinoma cell lines (MDA-MB 231, MBA- MB 468 and HeLa cells) and an immortalized human keratinocyte cell line (HaCaT) were infected with retrovirus expressing wild type, hyperphosporylated (S209D) or hypophosporylated (S209D) mutants of eIF4E. Cells were subjected to different types of stress including oxidative (Arsenite), nutrient starving and cisplatin treatment. Immediate and long term response to stress was evaluated using colony formation, MTT cell proliferation assays, co-inmunoprecipitation and immunofluorescence microscopy. A significantly greater recovery after all forms of stress was observed in all the cell lines expressing hyperphosporylated eIF4E-S209D. After treatment with arsenite there was an increase of peIF4E, 4E-T and Hur and importantly higher increase of levels of Hur, CyclinD1, Mcl-1 and eIF4G in eIF4E-S209D expressing cells. Under normal condition we observed a unique colocalitzation of 4E-T and eIF4E-S209D in cytoplasmic bodies. This complex colocalize with Ago2 but not with markers of the stress granules (TIA-1) and processing bodies (DCP1A). By co-immunopreciptation, we observed that peIF4E binds to its carrier protein 4E-T, Ago2 and Hur. Conclusions: Phosphorylated eIF4E confer resistance to several stress conditions, including oxidative stress, DNA damage and nutrient deprivation. The fact that there was a relevant and significant increase of proteins such as Cyclin D1, Mcl-1 and Hur suggests that phosporylated eIF4E has an important role in selectively stimulating RNA translation under stress conditions, possibly via the formation of a unique type of 4E-T/peIF4E cytoplasmic bodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4984. doi:1538-7445.AM2012-4984

Abstract 3165: Anti-tumor activity of the eIF4E-targeted drug ribavirin in breast cancer cells

Abstract In this study, we explored the potential of targeting the eukaryotic translation initiation factor (eIF4E) with ribavirin as a novel anti-tumor agent in breast cancer. eIF4E is an oncogene that facilitates nuclear export and translation of specific, growth-stimulatory mRNAs, including cyclins, c-myc, survivin, VEGF and others, thereby promoting cell survival. Overexpression of eIF4E also leads indirectly to activation of Akt, providing a positive feed-back loop for eIF4E activation and Akt signaling effects. Ribavirin is an antiviral drug that has been shown to inhibit oncogenic transformation mediated by eIF4E and reduce the clonogenic potential of cancer cells with high eIF4E levels. Ribavirin specifically inhibits translation and/or nuclear export of eIF4E targets in cells both in vitro and in patients, as shown in a recent phase I/II proof-of-principle trial in patients with AML. In this trial, dramatic clinical improvements were observed and reductions in eIF4E levels and activity correlated with clinical response. Importantly, ribavirin is largely non-toxic even at high doses, possibly due to an eIF4E oncogene addiction specific to tumor cells. eIF4E is overexpressed in more than 50% of breast cancers, and high levels are associated with increased angiogenesis, clinical progression and poor prognosis. Targeting eIF4E with ribavirin may therefore be an attractive therapeutic strategy for this malignancy. We studied the effects of ribavirin in a panel of breast cancer cells, representing luminal and basal-type tumors with various ER, PR and Her2 status. Western blot analysis showed that eIF4E was overexpressed compared to normal breast tissue and predominantly cytoplasmic in all of the cell lines. In addition, we examined eIF4E levels in metastatic skin lesions of three breast cancer patients and found highly elevated levels compared to normal skin. Ribavirin anti-proliferative activity was assessed using a cell viability assay and clonogenic assays were performed to examine changes in both anchorage dependent and -independent growth. At clinically relevant concentrations, the majority of the cell lines responded to ribavirin, with varying sensitivity. Inhibition of cell growth was associated with decreased protein levels of eIF4E targets such as cyclin D1 and survivin, and a reduction in phosphorylation of Akt as well as eIF4E binding protein 1 (4E-BP1) were observed. Cell cycle analysis showed that ribavirin caused a significant S-phase arrest in sensitive cells, while apoptosis was only observed at elevated concentrations of the drug. This data encourages further study of ribavirin as a breast cancer therapeutic and identification of potential combination regimens. A clinical trial of single agent ribavirin in patients with advanced metastatic breast cancer is planned in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3165.

Regulation of eIF4F complex assembly by Mnk1/2 kinase

The rate of translation initiation in most eukaryotic cells depends on both the features of the mRNA and the level and activity of the eIF4F complex, which consists of eIF4G, eIF4E and eIF4A. mRNAs with highly structured 5′UTR encode a disproportionate share of proteins involved in cell growth and cell cycle progression, growth factors, growth factor receptors, and cyclins. Translation of these mRNAs requires high levels of eIF4E that presumably acts through the eIF4F complex. One current model suggests that the eIF4F integrity is regulated by the PI3K/Akt/mTor cascade, while the MAPK pathway targets the eIF4F cap‐binding activity. The activation of mTor kinase leads to increase of eIF4E·eIF4G complex formation via phosphorylation of eIF4E‐binding protein 4E‐BP that releases eIF4E. MAPK signaling targets the Mnk kinase via Erk1/2 and p38 pathways. Mnk is the only known physiological kinase that phosphorylates eIF4E at single site (S209, human eIF4E). Mnk binds eIF4G and phosphorylates eIF4E primarily in a complex with eIF4G. Earlier we showed that inhibition of Mnk activity in cell‐free translation system affects translation of mRNA that contains both a cap‐ and a structured 5′UTR and also affects binding of eIF4E with eIF4G. We suggest the involvement of Mnk kinase in the dynamics of eIF4F assembly. Supported by grant R01GM20818 from the NIGMS.

EIF4E Expression Predicts Apoptosis in Response to Epidermal Growth Factor Receptor Inhibition and Mammalian Target of Rapamycin Inhibition in Triple Negative Breast Cancers.

Abstract Background: Triple negative (TN) breast cancers account for 15% of all breast cancers, are two-fold higher in African American women, have high histological grade, a poor prognosis and until recently no viable therapeutic targets. Epidermal growth factor receptor (EGFR) is over-expressed in more than 50% of triple negative breast cancers, but the use of agents blocking EGFR has produced disappointing results in metastatic breast cancer. Inhibitors of Mammalian Target of Rapamycin (mTOR) have demonstrated moderate activity as single agents in metastatic breast cancer. mTOR inhibitors have been demonstrated to activate the Akt pathway by a possible feedback mechanism, which could potentially sensitize TN breast cancer cells to upstream inhibitors. We have previously demonstrated synergistic antitumor effects with combined mTOR and EGFR inhibition in TN breast cancers. Eukaryotic translation initiation factor (eIF4E) is a protein downstream of mTOR and a target of PI3K/Akt and Erk signaling, and is important in mRNA translation, cell proliferation and apoptotic resistance. We evaluated the effects of EGFR and mTOR inhibition on apoptotic markers, and correlated these effects with eIF4E protein expression.Methods: Apoptotic assay and colony formation analysis were performed following mTOR inhibition with rapamycin and EGFR inhibition with lapatinib or erlotinib in TN breast cancer cells (MDA-MB-231, MDA-MB-468, HCC1806). Effects of EGFR and mTOR inhibition on downstream proteins in TN breast cancer in vitro and in vivo were examined through western blotting analysis with p-EGFR, pAkt, p-Erk, p-S6, and p-eIF4E.Results: We observed differential apoptotic effects of EGFR and mTOR inhibition in TN breast cancers cells. The combination resulted in a significant increase in apoptosis in MDA-MB-468 cells in vitro, but no increase in apoptosis was seen in MDA-MB-231 or HCC1806 cells. As expected, correlating with the apoptotic effects, expression of p-eIF4E was decreased in MDA-MB-468 cells treated with the combination of mTOR and EGFR inhibition in vitro. In contrast, both MDA-MB-231 and HCC1806 cells had high baseline levels of eIF4E, which increased in response to treatment with EGFR and mTOR inhibitors. Using a colony forming assay, we demonstrated that the combination of EGFR and mTOR inhibition resulted in cytostatic effects in HCC1806 cells. The combination of EGFR and mTOR inhibition did not result in apoptosis in MDA-MB-231 cancers in vivo, and these xenograft tumors had high level of eIF4E at baseline.We are currently evaluating the effects of lapatinib and rapamycin on apoptotic pathways in MDA-MB-468 cancers in vivo.Conclusion: Based on our previous findings, we are developing a clinical trial in which patients with metastatic TN breast cancers are treated with lapatinib and everolimus. These results suggest that this combination may be more effective in TN breast cancers with low levels of eIF4E, and this will be explored using metastatic research biopsies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5078.

Stability of Eukaryotic Translation Initiation Factor 4E mRNA Is Regulated by HuR, and This Activity Is Dysregulated in Cancer

Eukaryotic translation initiation factor 4E (eIF4E) is encoded by a potent oncogene which is highly elevated in many human cancers. Few studies have investigated how the level, and thus activity, of eIF4E is regulated in healthy (noncancerous) cells and how they become elevated in malignant cells. Here, our studies reveal a novel mechanism by which eIF4E levels are regulated at the level of mRNA stability. Two factors known to modulate transcript stability, HuR and the p42 isoform of AUF1, compete for binding to the 3' untranslated regions (3'UTRs) of eIF4E mRNAs. We identified a distinct AU-rich element in the 3'UTR of eIF4E which is responsible for HuR-mediated binding and stabilization. Our studies show that HuR is upregulated in malignant cancer specimens characterized by high eIF4E levels and that its depletion leads to reduction in eIF4E levels. Further, HuR and eIF4E regulate a common set of transcripts involved in cellular proliferation (cyclin D1 and c-myc) and neoangiogenesis (vascular endothelial growth factor), which suggests a functional connection between HuR and eIF4E in the regulation of these important processes. In summary, we present a novel model for the regulation of eIF4E expression and show that this model is relevant to elevation of eIF4E levels in malignant cells.

EIF4E-Targeted Suicide Gene Therapy in a Minimal Residual Mouse Model for Metastatic Soft-Tissue Head and Neck Squamous Cell Carcinoma Improves Disease-Free Survival

Translation initiation factor eIF4E unwinds long 5'-untranslated regions of certain tightly regulated mRNAs and, thereby, facilitates their translation into proteins. eIF4E has been shown to be overexpressed in a majority of solid tumors, including head and neck cancers. To exploit this dysregulation, a long 5'-untranslated region was spliced upstream of a thymidine kinase (Tk) gene to enhance translation of this "suicide" gene within cells overexpressing eIF4E. We investigated the efficacy of therapy with an adenovirus incorporating this novel suicide gene (Ad-HSV-UTk) following cytoreductive tumor surgery in improving disease-free and overall survival in a mouse soft-tissue metastasis model for head and neck squamous cell carcinoma. SCC-7 (orally-derived mouse SCCa) cells were treated with Ad-HSV-Tk, Ad-HSV-UTk, Ad-null, or saline and characterized for eIF4E and Tk levels by Western blot analysis. Cytotoxicities for cells treated with Ad-HSV-Tk, Ad-HSV-UTk, or Ad-null were quantified by MTS assay. Mice bearing SCC-7-induced tumors received cytoreduction followed by Ad-HSV-UTk + ganciclovir (GCV) or control treatment and were followed for disease-free and overall survival. SCC-7 cells showed uniformly high levels of eIF4E but elevated Tk for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells over Ad-null-treated cells. Cytotoxicities for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells were, correspondingly, observed to be 100-fold more sensitive than Ad-null-treated cells to GCV treatment. Cytoreduced mice receiving Ad-HSV-UTk + GCV treatment showed significantly longer disease-free survival (P = 0.0045) than control arm mice. Ad-HSV-UTk suicide gene therapy prolonged disease-free survival in a mouse minimal residual soft-tissue head and neck squamous cell carcinoma metastasis model.

Troglitazone Exerts a Cell-Dependent Effect on the Extracellular Secretion of VEGF165 Independently from the Rapamycin Sensitivity or the Level of eIF4E Expression in AML-Derived Cells.

Coexpression of vascular endothelial growth factor-165 (VEGF165) and its receptors as components of an autocrine loop has been well documented in hematopoietic malignancies. Therapeutic strategies that aim at blocking VEGF-induced signaling are currently being investigated for the treatment of malignancies. Furthermore, eukaryotic initiation factor 4E (eIF4E) allows a fast constitutive translation of mRNAs with short unstructured 5′-UTRs. Longer highly structured 5′-UTR mRNAs such as VEGF, which are less preferentially translated in normal conditions, become highly translated via an increase of eIF4E during malignant transformation. The thiazoladinedione troglitazone (TRO) has recently been reported to either increase or decrease the secretion of VEGF165 in several non hematopoietic cell lines tested. In the current study, we evaluated the action of TRO in HL-60 and U937 cells, derived from acute myelogenous leukemia (AML) known to be VEGF165 secretors, and to express VEGF receptors. The level of VEGF165 measured by ELISA as pg/ml/106 cells, and expressed as percentage of increase/decrease over control (no treatment), was reduced to 40% ± 15, and 14% ± 4.5 as compared to controls in HL-60 cells treated with 10m and 50m TRO for 24 h, respectively (P = 0.04 and 0.01). The number of HL-60 viable cells (Trypan blue) concurrently decreased to 55% ± 3.4 and 45% ± 2.3 as compared to controls after treatment with 10m and 50m TRO for 24 h, respectively (P = 0.01 and 0.006). On the other hand, the same doses of TRO did not significantly reduce the level of secreted VEGF165 and the number of viable cells in U937 cells treated for 24 h. Surprisingly, the level of VEGF165 was increased compared to controls (126% ± 2.5) when U937 cells were incubated with 50m TRO. This observation was in agreement with previous findings in the same cell line (Arterioscler Thromb Vasc Biol , 2001; 21:560). To investigate whether the action of TRO on the level of VEGF165 secretion and cell viability was mediated through mTOR/eIF4E pathway in HL-60 cells, we measured the level of total eIF4E by image analysis software of Western blots and expressed as percentage of increase/decrease over controls (treated vs not treated). HL-60 cells present high levels of eIF4E (41% ± 7.2) compared to normal controls (circulating mononucleated cells from healthy donors). There was a slight decrease in level of eIF4E in cells treated with TRO and this was not statistically significant. Furthermore, the level of secreted VEGF165 was unchanged and the number of viable cells was not significantly reduced (80% ± 3 vs control, P = 0.06), when HL-60 cells were treated with 500 nM Rapamycin (mTOR/eIF4E pathway inhibitor) for 24 h. Our data show that TRO may exert an action on the VEGF-proliferative-autocrine loop that is cell dependent and independent from the mTOR/eIF4E pathway in HL-60 cells. These results deserve further investigation to elucidate the mechanism in an attempt to define a new therapeutic modality.

Proto oncogene/eukaryotic translation initiation factor (eIF) 4E attenuates mevalonate-mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase synthesis.

The rate-limiting enzyme for mevalonate synthesis in mammalian cells is 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Products of mevalonate synthesis are required for cell cycle progression as well as cell growth and survival. In tumor cells, HMG-CoA reductase is generally elevated because of attenuated sterol-mediated regulation of transcription. However, tumor cell HMG-CoA reductase remains sensitive to post-transcriptional regulation by mevalonate-derived isoprenoid intermediates of cholesterol synthesis. Isoprenoids suppress HMG-CoA reductase synthesis through a mechanism that reduces initiation of translation on HMG-CoA reductase mRNA. Because HMG-CoA reductase mRNA transcripts have 5'-untranslated regions (UTR) that are GC rich and contain stable secondary structure, we tested the hypothesis that overexpression of eIF4E would attenuate isoprenoid-mediated regulation of HMG-CoA reductase. eIF4E is elevated in many tumor cells and behaves as a proto-oncogene by aberrantly translating mRNAs whose translation is normally suppressed by 5-UTRs that are GC rich. A CHO cell line expressing high levels of eIF4E (rb4E) was developed by infecting cells with retroviruses containing a full-length mouse cDNA for eIF4E. Levels of reductase synthesis were elevated fivefold in rb4E cells compared to noninfected CHO cells; HMG-CoA reductase mRNA levels were not increased in rb4E cells compared to normal CHO cells. Total cellular protein synthesis was only increased by approximately 15% in rb4E cells compared to CHO cells. The mTOR inhibitor rapamycin lowered HMG-CoA reductase synthesis by 50 and 60% in rb4E and CHO cells, respectively; no equivalent effect was observed for HMG-CoA reductase mRNA levels with rapamycin treatment. These results indicate that HMG-CoA reductase mRNA is in a class of mRNAs with highly structured 5'-UTRs whose m(7)GpppX cap-dependent translation is closely linked to the rapamycin-sensitive mitogen activated pathway for protein synthesis.

Expression of constitutively active 4EBP-1 enhances p27Kip1 expression and inhibits proliferation of MCF7 breast cancer cells

BackgroundEukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells.ResultsA constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1.ConclusionsExpression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.