Overexpression Of eIF4E Research Articles

Prognostic significance of eukaryotic initiation factor 4E in hepatocellular carcinoma.

Aberrant expression of eukaryotic initiation factor 4E (eIF4E) has been observed in human malignancies. However, its role in hepatocellular carcinoma (HCC) remains to be established. The purpose of this study was to detect eIF4E expression and to evaluate its clinical relevance. The eIF4E expression was studied in ninety HCC and randomly selected thirty-one non-tumor tissues from the same patient cohort, as well as in normal hepatic and HCC cell lines. The relation between its expression and clinicopathological parameters was also analyzed. eIF4E expression was higher in HCC samples and cell lines compared with that in non-tumor tissues (P<0.001) and hepatocyte LO2, respectively. eIF4E overexpression was significantly associated with tumor number (P=0.005) and incomplete encapsulation (P=0.001). The 5-year overall survival rate and disease-free survival rate for patients with high eIF4E expression were 32.5 and 31.2%, respectively; and for low eIF4E expression, it was 67.9 and 64.4%, respectively (P<0.001). Furthermore, subgroup analysis showed that high eIF4E level predicted poorer overall survival only for incomplete encapsulation (P=0.001) and cirrhosis (P<0.001), but not for complete encapsulation (P=0.804) and non-cirrhosis (P=0.359). Multivariate analysis revealed that eIF4E overexpression was an independent indicator for both overall survival (hazard ratio, 2.015; P=0.043) and disease-free survival (hazard ratio, 2.666; P=0.006). eIF4E protein might result in the malignant progression of HCC, and its overexpression may be a powerful prognostic biomarker and therapeutic target for HCC patients.

EIF4E promotes tumorigenesis and modulates chemosensitivity to cisplatin in esophageal squamous cell carcinoma.

Patients with esophageal squamous cell cancer are often diagnosed with advanced diseases that respond poorly to chemotherapy. Overexpression of eIF4E leads to enhance the translation of key malignancy-related proteins and enabling tumor growth and chemoresistance in a variety of human malignancies, but whether it has a role in ESCC remains obscure. We hypothesized that eIF4E promoted ESCC tumorigenesis and facilitated the development of acquired resistance to the cisplatin-based chemotherapy. In this study, we showed that eIF4E expression was increased significantly in clinical ESCC tissues and and ESCC cell lines and its expression level was correlated with lymph node metastasis, TNM stage, as well as overall and disease-free survival of ESCC. We also showed here that knockdown of eIF4E in EC9706 would dramatically reduced cell proliferation, colony formation, migration and invasion, apoptosis in vitro as well as in vivo, and vice versa. Moreover, “weak mRNAs” were demonstrated to be regulated by eIF4E in ESCC, which might interpret the above function. Overexpression of eIF4E decreased the efficacy of cisplatin-induced cell growth inhibition in ESCC cell line and xenograft model (P < 0.05). eIF4E knockdown by shRNA increased cisplatin-induced cytotoxicity in ESCC cell lines, and enhanced chemosensitivity to cisplatin in xenograft tumor models. Furthermore, we found that the PI3K/AKT pathway and Bcl-2/Bax ratio might be responsible for the eIF4E-induced cisplatin resistance in ESCC. Our data collectively show association of eIF4E expression with chemotherapeutic response in ESCC, and suggest that therapeutically targeting eIF4E may be a viable means of improving chemotherapy response in ESCC.

MTOR downstream effectors, 4EBP1 and eIF4E, are overexpressed and associated with HPV status in precancerous lesions and carcinomas of the uterine cervix.

The present study aims to investigate the expression levels of two critical mammalian target of rapamycin (mTOR) downstream effectors, 4E binding protein 1 (4EBP1) and eukaryotic initiation factor 4E (eIF4E) proteins, in precancerous squamous intraepithelial lesions and cancer of the uterine cervix, and their association with human papilloma virus (HPV) infection status. Uterine cervical biopsies from 73 patients were obtained, including 40 fresh-frozen samples and 42 archival formalin-fixed, paraffin-embedded tissue specimens. Whole protein extracts were analyzed for the expression of 4EBP1 and eIF4E proteins using western blotting. In addition, distribution of 4EBP1 and eIF4E protein expression and 4EBP1 phosphorylation (P-4EBP1) were analyzed by immunohistochemistry in archival tissues and correlated with the degree of dysplasia. The presence of high-risk HPV (HR-HPV) types was assessed by polymerase chain reaction. Using western blot analysis, high expression levels of 4EBP1 and eIF4E were observed in all uterine cervical carcinomas, which significantly correlated with the degree of dysplasia. By immunohistochemistry, overexpression of 4EBP1 and eIF4E was detected in 20 of 21 (95%) and 17 of 21 (81%) samples, respectively, in patients with high-grade dysplasia and carcinomas, compared with 1 of 20 (5%) and 2 of 20 (10%) samples, respectively, in patients with low-grade lesions or normal histology. All 4EBP1-positive cases tested were also positive for P-4EBP1. Furthermore, overexpression of 4EBP1 and eIF4E significantly correlated with the presence of HR-HPV oncogenic types. The present study demonstrated that critical effectors of mTOR signaling, which control protein synthesis initiation, are overexpressed in cervical high-grade dysplasia and cancer, and their levels correlate with oncogenic HPV types. These findings may provide novel targets for investigational therapeutic approaches in patients with cancer of the uterine cervix.

Abstract 4761: Translational regulation by eIF4E and its contribution to tamoxifen resistance in breast cancer

Abstract Breast cancer is one of the prevalent causes of cancer in women and its occurrence has been rising in the last few decades. Two thirds of breast cancer patients are ER-positive and can receive tamoxifen (TAM) treatment. Unfortunately, many of the patients eventually develop resistance to tamoxifen. Identifying a molecular marker associated with tamoxifen resistance would help in designing better therapeutic strategies to overcome this. In eukaryotes, translation initiation is the most regulated step. Abnormal translation machinery underlies a variety of human diseases including cancers. Eukaryotic translation initiation factor 4E (eIF4E) is the least abundant initiation factor and is believed to be the rate limiting step for translation initiation. Overexpression of eIF4E in cells selectively promotes translation of mRNAs involved in cell proliferation, apoptosis and tumor progression. Recently, eIF4E overexpression has been implicated in drug resistance in melanoma1. We hypothesized that eIF4E may also be involved in tamoxifen resistance. The aim of this study was to characterize the role of eIF4E in tamoxifen resistance and identify novel molecular targets that may be translationally regulated. Immunohistochemistry (IHC) staining of eIF4E showed up-regulation in human breast cancer samples compared with non-tumor counterparts. In silico analysis predicted that ERá and FOXM1 have a long and highly structured 5’-UTR, which are sensitive to the expression level of eIF4E. Polysomal fractionation confirmed that ERá and FOXM1 mRNA were more actively translated in MCF7 than in MCF10A. The correlation between eIF4E and ERá/FOXM1 were further confirmed in tissue microarray (TMA). The role of eIF4E in conferring TAM resistance was studied by MTT assays. Overexpression of eIF4E induced TAM resistance while knockdown eIF4E sensitized cells to TAM. Additionally, overexpression of eIF4E upregulated ERá/FOXM1 expression at protein level while knockdown eIF4E downregulated their expression at protein level. Our results show that eIF4E induced TAM resistance possibly through translational regulation of ERá/FOXM1. eIF4E is a promising biomarker for anti-estrogen drug responsiveness and a future therapeutic target for treating drug resistant breast cancer patients. Reference: 1. Zhan Y, et al. The role of eIF4E in response and acquired resistance to vemurafenib in melanoma J Invest Dermatol. 2015 May;135(5):1368-76. (2178 characters without spaces) Citation Format: Chun Gong, Ka Chun Mok, Yuen-Nei CHEUNG, Pui-Sum Man, EWF Lam, Ui-Soon Khoo. Translational regulation by eIF4E and its contribution to tamoxifen resistance in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4761.

Abstract 2134: Rational design of selective MNK 1 and 2 kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia patients

Abstract The marketed BCR-ABL tyrosine kinase inhibitor (TKI), imatinib (Gleevec™) is a very successful targeted anti-cancer therapy. It has revolutionized the treatment of early stage or chronic phase (CP) chronic myeloid leukemia (CML). Unfortunately, a proportion of CP patients experience suboptimal responses to BCR-ABL TKIs, and progress to blast crisis (BC) stage of CML with poor survival rate. A potential cause of the resistance to TKI is the elevated level of phosphorylated eukaryotic initiation factor 4E (eIF4E), which has been found to be a consistent feature in patient-derived BC-CML samples. Importantly, both in vivo and in vitro studies have demonstrated that the MAP kinase-interacting serine/threonine-protein kinases 1 and 2 (MNK1/2) phosphorylate eIF4E on Ser209, and that the overexpression of eIF4E drives oncogenesis in a variety of cancers including BC-CML. Furthermore, several reports have indicated that eIF4E phosphorylation at Ser209, as well as eIF4E overexpression, is critical to tumor progression. We found that a BC-CML cell line, K562, that expresses a serine to alanine phospho-mutant at position 209 of eIF4E, shows reduced ability to form tumors in mice compared to wildtype eIF4E. In addition, our recent work has demonstrated the importance of the MNK-eIF4E axis in activating BC leukemia stem cell (LSC) function (Lim et al., PNAS18; 110(25):E2298-307, 2013). These data highlight the critical importance of MNK1/2-dependent eIF4E phosphorylation in cancer progression and maintenance, and suggests that inhibition of MNK1/2 is an attractive therapeutic approach to treat BC-CML. Consequently, we set out to identify selective inhibitors of the MNK1/2 kinases to treat BC-CML patients. Here, we report our hit finding strategy, as well as our hit to lead optimization process. Results describing structure activity relationships, pharmacokinetics properties, and biochemical characteristics of a highly specific MNK1/2 inhibitor, are presented. Our data demonstrate that drug-like molecules can be developed to potently and specifically inhibit the MNK kinases. We also show that simultaneous inhibition of MNK and BCR-ABL is effective at inhibiting BCR-ABL-driven growth and proliferation, as well as inhibiting the MNK-eIF4E-dependent self-renewal function of BC-LSCs. A combination of selective MNK and BCR-ABL inhibitors may provide clinical benefit to BC-CML patients. Citation Format: Kassoum Nacro, Haiyan Yang, Melvyn Wai Tuck Ho, Yoon Sheng Yeap, Lohitha Rao Chennamaneni, Shi Hua Ang, Eldwin Sum Wai Tan, Athisayamani Jeyaraj Duraiswamy, Sharon Lim, Boping Liu, Esther Hongqian Ong, Meng Ling Choong, Shi Jing Tai, Vithya Manoharan, Vishal Pendharkar, Lijun Ding, Yun Shan Chew, Joma Kanikadu Joy, John LW Kuan, Perlyn Z. Kwek, Anders Poulsen, May Ann Lee, Kanda Sangthongpitag, Charles Chuah, Tiong S. Ong, Jeffrey Hill, Thomas H. Keller, Alex Matter. Rational design of selective MNK 1 and 2 kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2134.

Expression of phosphorylated eIF4E-binding protein 1, but not of eIF4E itself, predicts survival in male breast cancer

Background:Male breast cancer is rare and treatment is based on data from females. High expression/activity of eukaryotic initiation factor 4E (eIF4E) denotes a poor prognosis in female breast cancer, and the eIF4E pathway has been targeted therapeutically. Eukaryotic initiation factor 4E activity in female breast cancer is deregulated by eIF4E overexpression and by phosphorylation of its binding protein, 4E-BP1, which relieves inhibitory association between eIF4E and 4E-BP1. The relevance of the eIF4E pathway in male breast cancer is unknown.Methods:We have assessed expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 (p4E-BP1) using immunohistochemistry in a large cohort of male breast cancers (n=337) and have examined correlations with prognostic factors and survival.Results:Neither eIF4E expression nor estimated eIF4E activity were associated with prognosis. However, a highly significant correlation was found between p4E-BP1 expression and disease-free survival (DFS), linking any detectable p4E-BP1 with poor survival (univariate log rank P=0.001; multivariate HR 8.8, P=0.0001).Conclusions:Our data provide no support for direct therapeutic targeting of eIF4E in male breast cancer, unlike in females. However, as p4E-BP1 gives powerful prognostic insights that are unrelated to eIF4E function, p4E-BP1 may identify male breast cancers potentially suitable for therapies directed at the upstream kinase, mTOR.

The Gene Expression Status of the PI3K/AKT/mTOR Pathway in Gastric Cancer Tissues and Cell Lines.

The PI3K/AKT/mTOR pathway plays a crucial role in the regulation of multiple cellular functions including cell growth, proliferation, metabolism and angiogenesis. Emerging evidence has shown that deregulation of this pathway has a role promoting gastric cancer (GC). The aim was to assess the expression of genes involved in this pathway by qPCR in 23 tumor and 23 non-tumor gastric mucosa samples from advanced GC patients, and in AGS, MKN28 and MKN45 gastric cancer cell lines. Results showed a slight overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1, EIF4EBP1 and EIF4E genes, and a slightly decreased PTEN and TSC1 expression. In AGS, MKN28 and MKN45 cells a significant gene overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1 and EIF4E, and a significant repression of PTEN gene expression were observed. Immunoblotting showed that PI3K-β, AKT, p-AKT, PTEN, mTOR, p-mTOR, P70S6K1, p-P70S6K1, 4E-BP1, p-4E-BP1, eIF4E and p-eIF4E proteins were present in cell lines at different levels, confirming activation of this pathway in vitro. This is the first time this extensive panel of 9 genes within PI3K/AKT/mTOR pathway has been studied in GC to clarify the biological role of this pathway in GC and develop new strategies for this malignancy.

Elevated Translation Initiation Factor eIF4E Is an Attractive Therapeutic Target in Multiple Myeloma.

eIF4E is the key regulator of protein translation and critical for translation. The oncogenic potential of tumorigenesis, which is highly contingent on cap-dependent eIF4E, also arises from the critical role in the nuclear export and cytosolic translation of oncogenic transcripts. Inhibition of Exportin1 (XPO1), which is the major nuclear export protein for eIF4E-bound oncoprotein mRNAs, results in decreased tumor cell growth in vitro and in vivo, suggesting that eIF4E is critical in multiple myeloma. Indeed, we found that eIF4E is overexpressed in myeloma cell lines and primary myeloma cells compared with normal plasma cells. Although stable overexpression of eIF4E in multiple myeloma cells significantly increases tumorigenesis, knockdown of eIF4E impairs multiple myeloma tumor progression in a human xenograft mouse model. Using a tet-on-inducible eIF4E-knockdown system, eIF4E downregulation blocks multiple myeloma tumor growth in vivo, correlating with decreased eIF4E expression. Further overexpression and knockdown of eIF4E revealed that eIF4E regulates translation of mRNAs with highly complex 5'-untranslated regions, such as c-MYC and C/EBPβ, and subsequently proliferation in multiple myeloma cells, but not in nonmalignant bone marrow stromal cells. Because many transcription factors that are critical for multiple myeloma proliferation exhibit a higher dependency on protein translation, eIF4E is an ideal and selective tool to target multiple myeloma cell growth. Mol Cancer Ther; 15(4); 711-9. ©2016 AACR.

Antibiotic drug rifabutin is effective against lung cancer cells by targeting the eIF4E-β-catenin axis

The essential roles of overexpression of eukaryotic translation initiation factor 4E (eIF4E) and aberrant activation of β-catenin in lung cancer development have been recently identified. However, whether there is a direct connection between eIF4E overexpression and β-catenin activation in lung cancer cells is unknown. In this study, we show that antibiotic drug rifabutin targets human lung cancer cells via inhibition of eIF4E-β-catenin axis. Rifabutin is effectively against lung cancer cells in in vitro cultured cells and in vivo xenograft mouse model through inhibiting proliferation and inducing apoptosis. Mechanistically, eIF4E regulates β-catenin activity in lung cancer cells as shown by the increased β-catenin phosphorylation and activity in cells overexpressing eIF4E, and furthermore that the regulation is dependent on phosphorylation at S209. Rifabutin suppresses eIF4E phosphorylation, leads to decreased β-catenin phosphorylation and its subsequent transcriptional activities. Depletion of eIF4E abolishes the inhibitory effects of rifabutin on β-catenin activities and overexpression of β-catenin reverses the inhibitory effects of rifabutin on cell growth and survival, further confirming that rifabutin acts on lung cancer cells via targeting eIF4E- β-catenin axis. Our findings identify the eIF4E- β-catenin axis as a critical regulator of lung cancer cell growth and survival, and suggest that its pharmacological inhibition may be therapeutically useful in lung cancer.

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.

A Tailor-Made Protein Synthesis Program Drives Erythroid Development and Disease

Erythropoiesis constitutes the largest demand on the hematopoietic system due to its extraordinary production on a daily basis. The erythroid proteome requires an integration of multiple external cues to coordinate programs of differentiation as well as maintenance of erythroid precursors. The biomedical relevance of this critical process is underscored by recent findings showing impaired ribosome function in an entire class of clinical disorders with severe impairments in erythroid differentiation, known as ribosomopathies, which remain poorly understood. One of the main signaling pathways controlling post-transcriptional gene expression during erythropoiesis is the mTOR pathway. mTOR activation downstream of SCF/Epo in erythroid progenitors controls the activity of the major cap-binding protein eIF4E. However, the functional role of eIF4E during erythropoiesis and protein synthesis control in this cell type remains unexplored. Here we show that eIF4E activity, through mTOR-dependent phosphorylation of its inhibitory protein 4EBP1, unexpectedly undergoes a dynamic switch between early erythroid precursor populations and during terminal erythrocyte maturation, where eIF4E becomes progressively silenced. Employing a unique eIF4E transgenic mouse model, we strikingly show that overexpression of eIF4E in the bone marrow compartment results in an early accumulation of erythrocyte precursors and a block in erythrocyte differentiation. Surprisingly, this new role of eIF4E in erythropoiesis is independent from control of global protein synthesis but instead may promote a specialized program of translation control that is customized for erythroid cell function. Employing state of the art unbiased proteomics, our work is uncovering distinct networks of proteins, whose expression levels are controlled by eIF4E dosage during specific phases of erythrocyte maturation. Together, our research highlights a novel molecular program linking exquisite regulation of eIF4E activity to specialized translational control underlying erythroid development, providing unprecedented insight into the etiology of erythroid dysfunction in ribosomopathies. DisclosuresNo relevant conflicts of interest to declare.

Immunohistochemical expression levels of p53 and eIF4E markers in histologically negative surgical margins, and their association with the clinical outcome of patients with head and neck squamous cell carcinoma.

Molecular markers can be used to identify residual cancer at the surgical margins of head and neck squamous cell carcinoma (HNSCC) and assist in evaluating the complete resection of the tumour. The purpose of the present study was to investigate the expression levels of prognostic molecular markers at the histological tumour free surgical margins. In the present clinical retrospective study, 24/48 patients were selected with negative surgical margins for further analysis with immunohistochemical staining. Contingency tables and Fisher's exact tests were used to investigate the association between the expression levels of p53 and eukaryotic translation imitation factor 4E (eIF4E) with the clinical outcomes for patients with HNSCC. The expression levels of p53 and eIF4E were 54.2 and 87.5%, respectively, in the surgical margins of patients with HNSCC. A total of 3/7 patients with recurrent cancer (42.8%) were identified with p53-positive margins, and 6 (85.7%) patients exhibited recurrence with eIF4e-positive margins. No statistically significant differences were identified for the recurrence risk between the overexpression of p53 and eIF4E in the surgical margins (P=0.88 and P=0.99, respectively). The eIF4E marker appears to be a more marked prognosticator compared with p53, as overexpression of eIF4E was identified in the margins of 6/7 patients with local recurrence.

MiR-34c-3p functions as a tumour suppressor by inhibiting eIF4E expression in non-small cell lung cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression and mediate diverse physiological processes. In this study, we investigated functions of miRNA miR-34c-3p in non-small cell lung cancer (NSCLC). miR-34c-3p expression was evaluated by qPCR. Cell viability was examined by MTT and proliferation by cell cycle analysis. Cell migration and invasion were tested using Transwells with/without Matrigel coating. Western blot analysis was performed for eIF4E, c-Myc, Cyclin D1, survivin and Mcl-1 protein expression. miR-34c-3p expression was significantly reduced in tissues and serum samples from NSCLC patients and in NSCLC cell lines A549, H460, H23, H157 and H1299. Overexpression of miR-34c-3p in A549 and H157 cells reduced cell proliferation, migration and invasion, whereas transfection with miR-34c-3p inhibitor (miR-34c-3p-in) produced opposite effects. Target analysis using algorithms miRanda, TargetScan and DIANA identified eIF4E as a potential target of miR-34c-3p. Luciferase assay using the eIF4E 3'-UTR reporter carrying a putative miR-34c-3p target sequence revealed eIF4E to be a specific target of miR-34c-3p. Overexpression of miR-34c-3p in NSCLS cell lines led to significant reduction in mRNA and protein levels of eIF4E, whereas inhibition of miR-34c-3p resulted in significant increase in eIf4e protein levels, confirming eIF4E to be a direct target of miR-34c-3p in NSCLS. Overexpression of eIF4E in A549 cells promoted cell proliferation, migration and invasion, which were partially reversed by miR-34c-3p. miR-34c-3p directly targeted eIF4E and reduced miR-34c-3p expression in NSCLC, promoting cell cycle progression, proliferation, migration and invasion.

Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity.

Abstract 1649: Potent anticancer activity of an orally bioavailable small molecule, ON 013100, and its water soluble derivative, briciclib, a clinical-stage eIF4E-targeted agent

Abstract Introduction: Eukaryotic translation initiation factor 4E (eIF4E) is a master regulator that controls translation of mRNA in mammalian cells. eIF4E is a proto-oncogene that promotes translation of several genes essential for cellular proliferation (cyclin D1, c-Myc, mTOR), survival (Akt, survivin), angiogenesis (VEGF), and metastasis (MMP9). Overexpression of eIF4E has been observed in almost all major groups of cancers and has been shown to induce increased expression of cyclin D1 and c-Myc. Briciclib is a small molecule, water soluble derivative of ON 013100 that binds to eIF4E. An intravenous formulation of briciclib is currently being investigated in a Phase 1 clinical trial. Recent advancements in formulation technology have made feasible a stable, orally bioavailable version of ON 013100, which may allow for more convenient administration. In this study we investigated and compared the anticancer activity of briciclib to ON 013100. We determined the susceptibility of various breast, mantle cell leukemia (MCL), gastric, and esophageal cancer cell lines to treatment with briciclib or ON 013100. In addition, we investigated the effect of briciclib and ON 013100 on expression of markers associated with eIF4E activity (cyclin D1 and c-Myc) and apoptosis (P53 and Cleaved Caspase 3). Methods: MTT cell viability assays, Western blot analysis, and ELISA assays were used to evaluate cellular viability, survival, and protein expression levels. Results: Briciclib and ON 013100 inhibited the proliferation of MCL (JEKO-1 and MINO), breast (MCF7 and MDA-MB-231), gastric (AGS), and esophageal (OE19, OE33, and FLO-1) cancer cell lines at nanomolar concentrations (Briciclib: GI50 = 9.8 - 12.2 nM; ON 013100 GI50 = 6.7 - 11.2 nM). By comparison, briciclib and ON 013100 were relatively non-toxic to normal endothelial cells. Western blot analysis indicated that treatment with briciclib or ON 013100 significantly reduced the expression of cyclin D1 and c-Myc in breast and MCL cancer cell lines within 8 hours and in a dose-dependent manner. These observations were supported by ELISA analysis of cyclin D1 and c-Myc protein levels. Furthermore, treatment with these agents enhanced the expression of P53 and Cleaved Caspase 3 pro-apoptotic proteins in breast and MCL cancer cell lines. Our ongoing tumor xenograft experiments are in agreement with the aforementioned in vitro observations. Conclusions: Our findings suggest that both an orally bioavailable ON 013100, and its water soluble derivative, briciclib, have the same novel mechanism of action involving translation. Our in vitro and in vivo data demonstrate the potential of briciclib in targeting eIF4E for hematopoietic and solid cancers and the possibility for developing an oral version of this promising clinical agent. Citation Format: Neel Jasani, Bina Desai, Justine M. Betzu, Tanmay Dichwalkar, Samhita Bapat, V. J. Rajadhayksha, Benjamin S. Hoffman, Manoj Maniar, Vikas Sehdev. Potent anticancer activity of an orally bioavailable small molecule, ON 013100, and its water soluble derivative, briciclib, a clinical-stage eIF4E-targeted agent. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1649. doi:10.1158/1538-7445.AM2015-1649

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T37/T46, followed by T70 and S65. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, (54)YXXXXLΦ(60) (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m(7)GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP150-84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S65 and T70). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.

Dehydrocostus lactone suppressed the proliferation, migration, and invasion of colorectal carcinoma through the downregulation of eIF4E expression.

Dehydrocostus lactone (DHC) is the main active ingredient extracted from a traditional Chinese medicine called Radix Aucklandiael. A few studies recently showed that DHC has anticancer potential. However, no reports exist as yet on the effects of DHC on colorectal carcinoma (CRC). This study aimed to determine whether and how DHC functions in CRC cells. After treatment with DHC, both Lovo and SW480 cells were significantly inhibited in their proliferation, cell cycle progression, migration, and invasion abilities in a dose-dependent and/or treatment time-dependent manner. Also, DHC significantly increased the apoptosis rate of SW480 cells, but not Lovo cells. The expression of eukaryotic translation initiation factor 4E (eIF4E), which was originally highly expressed in both cells, was significantly decreased by DHC. The inhibition of proliferation, migration, and invasion was significantly attenuated by the ectopic transfection of eIF4E, and was promoted by the knockdown of eIF4E in Lovo cells. To the best of our knowledge, this is the first time it has been shown that DHC suppressed the proliferation, cell cycle progression, antiapoptosis, and migration and invasion capabilities of CRC cells by the downregulation of eIF4E expression. In terms of the overexpression of eIF4E in many cancers, it was speculated that DHC might also play an anticancerous role by suppressing eIF4E expression. This discovery could lay the foundations for advancing our understanding of the anticancerous mechanism of DHC and developing DHC into a novel and effective natural anticancer therapeutic.

Ligustrazine Suppresses the Growth of HRPC Cells through the Inhibition of Cap- Dependent Translation Via Both the mTOR and the MEK/ERK Pathways.

Ligustrazine (TMP) has recently been used for the treatment of various cancers. However, its exact mechanisms of action, particularly the functions and the mechanisms of Ligustrazine in human hormone-refractory prostate cancer (HRPC), have not yet been extensively studied. Recently, our findings suggest that Ligustrazine dose- and time-dependently inhibits the growth of HRPC cells by reducing their proliferation and promoting apoptosis. Interestingly, the treatment of hormone-refractory prostate cancer (PC-3) cells with Ligustrazine results in a significant inhibition of the activation of mTOR and related downstream targets, which are critical for cell growth. Furthermore, pull-down assays with 7-methyl- GTP Sepharose 4B beads indicate that Ligustrazine reduces the available eIF4E for translation initiation. Accordingly, the results from the translation assay using a luciferase reporter system further demonstrate that Ligustrazine indeed inhibits cap-dependent translation. In addition, the transient overexpression of eIF4E or MNK1 prevents the Ligustrazine-induced inhibition of proliferation and confers significant protection against Ligustrazine-induced apoptosis. Therefore, the present study provides evidences that Ligustrazine may be a candidate for therapeutic reagent for the treatment of HRPC and certifies that Ligustrazine modulates the availability of eIF4E mainly through the mTOR and MEK/ERK signaling pathways to inhibit cap-dependent translation. Taken together, our results indicate that the inhibition of cap-dependent translation is likely an essential mechanism in Ligustrazine-induced apoptosis.

Growth-factor dependent expression of the translationally controlled tumour protein TCTP is regulated through the PI3-K/Akt/mTORC1 signalling pathway

Translationally controlled tumour protein TCTP (gene symbol: TPT1) is a highly-conserved, cyto-protective protein implicated in many physiological and disease processes, in particular cancer, where it is associated with poor patient outcomes. To understand the mechanisms underlying the accumulation of high TCTP levels in cancer cells, we studied the signalling pathways that control translation of TCTP mRNA, which contains a 5′-terminal oligopyrimidine tract (5′-TOP). In HT29 colon cancer cells and in HeLa cells, serum increases the expression of TCTP two- and four-fold, respectively, and this is inhibited by rapamycin or mTOR kinase inhibitors. Polysome profiling and mRNA quantification indicate that these effects occur at the level of mRNA translation. Blocking this pathway upstream of mTOR complex 1 (mTORC1) by inhibiting Akt also prevented increases in TCTP levels in both HeLa and HT29 colon cancer cells, whereas knockout of TSC2, a negative regulator of mTORC1, led to derepression of TCTP synthesis under serum starvation. Overexpression of eIF4E enhanced the polysomal association of the TCTP mRNA, although it did not protect its translation from inhibition by rapamycin. Conversely, expression of a constitutively-active mutant of the eIF4E inhibitor 4E-BP1, which is normally inactivated by mTORC1, inhibited TCTP mRNA translation in HEK293 cells. Our results demonstrate that TCTP mRNA translation is regulated by signalling through the PI3-K/Akt/mTORC1 pathway. This explains why TCTP levels are frequently increased in cancers, since mTORC1 signalling is hyperactive in ~80% of tumours.

Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma.

Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.