Inhibition Of eIF4E Phosphorylation Research Articles

Inhibition of eIF4E Phosphorylation Regulates Plaque Composition Without Affecting Atherosclerosis Development

Inhibition of eIF4E Phosphorylation Regulates Plaque Composition Without Affecting Atherosclerosis Development

The androgen receptor is a negative regulator of eIF4E phosphorylation at S209: implications for the use of mTOR inhibitors in advanced prostate cancer.

The anti-androgen bicalutamide is widely used in the treatment of advanced prostate cancer (PCa) in many countries, but its effect on castration resistant PCa (CRPC) is limited. We previously showed that resistance to bicalutamide results from activation of mechanistic target of rapamycin (mTOR). Interestingly, clinical trials testing combinations of the mTOR inhibitor RAD001 with bicalutamide were effective in bicalutamide-naïve CRPC patients, but not in bicalutamide-pre-treated ones. Here we investigate causes for their difference in response. Evaluation of CRPC cell lines identified resistant vs sensitive in-vitro models, and revealed that increased eIF4E(S209) phosphorylation is associated with resistance to the combination. We confirmed using a human-derived tumor-xenograft mouse model that bicalutamide pre-treatment is associated with an increase in eIF4E(S209) phosphorylation. Thus, AR suppressed eIF4E phosphorylation, while the use of anti-androgens relieved this suppression, thereby triggering its increase. Additional investigation in human prostatectomy samples showed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67. SiRNA-mediated knock-down of eIF4E sensitized CRPC cells to RAD001+bicalutamide, while eIF4E overexpression induced resistance. Inhibition of eIF4E phosphorylation by treatment with CGP57380 (an inhibitor of MAPK interacting serine-threonine kinases Mnk1/2, the eIF4E upstream kinase) or inhibitors of ERK1/2, the upstream kinase regulating Mnk1/2, also sensitized CRPC cells to RAD001+bicalutamide. Examination of downstream targets of eIF4E-mediated translation, including survivin, demonstrated that eIF4E(S209) phosphorylation increased cap-independent translation whereas its inhibition restored cap-dependent translation which could be inhibited by mTOR inhibitors. Thus, our results demonstrate that while combinations of AR and mTOR inhibitors were effective in suppressing tumor growth by inhibiting both AR-induced transcription and mTOR-induced cap-dependent translation, pre-treatment with AR antagonists including bicalutamide increased eIF4E phosphorylation that induced resistance to combinations of AR and mTOR inhibitors by inducing cap-independent translation. We conclude that this resistance can be overcome by inhibiting eIF4E phosphorylation with Mnk1/2 or ERK1/2 inhibitors.

Abstract B22: Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors together with AR antagonists in advanced prostate cancer

Abstract The anti-androgen bicalutamide is widely used in the treatment of advanced prostate cancer (CaP) in many countries, but its effect on castration resistant CaP (CRPC) is limited. We previously showed that resistance to bicalutamide results from increased activation of the mechanistic target of rapamycin (mTOR); indicating a role for mTOR inhibitors in CaP treatment. Interestingly, a clinical trial testing combinations of the mTOR inhibitor RAD001 with bicalutamide were initially effective (PSA decline>50%) in 62.5% CRPC patients, but many initial responders later developed resistance. Here we investigate causes for their difference in response. Evaluation of various CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E phosphorylation at S209 is associated with resistance to the combination. To recognize potential causes of resistance, we demonstrated using a human tumor xenograft mouse model, that bicalutamide use is associated with an increase in eIF4E(S209) phosphorylation. Investigations revealed that AR suppressed eIF4E phosphorylation, while the use of anti-androgens relieved this suppression, thereby triggering its increase. Additional investigation on the consequences of increased eIF4E phosphorylation in human prostatectomy samples revealed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67, indicating a role for eIF4E in tumor growth. Inhibition of eIF4E phosphorylation by siRNA suppression or treatment with CGP57380 (an inhibitor of MAPK interacting serine-threonine kinases Mnk1/2, the eIF4E upstream kinase,) sensitized CRPC cells to RAD001+bicalutamide, while eIF4E overexpression induced resistance. Our results demonstrate that bicalutamide treatment induced an increase in eIF4E phosphorylation by inhibition of AR which otherwise suppressed this phosphorylation. Increased eIF4E phosphorylation then induced resistance to the combination of anti-androgens with mTOR inhibitors by increasing proliferation. These results also indicate that resistance to RAD001+bicalutamide can be prevented by the concurrent use of a Mnk1/2 inhibitor. Citation Format: Leandro S. D'Abronzo, Michael Crapuchettes, Ryan Beggs, Ruth Vinall, Clifford Tepper, Salma Siddiqui, Maria Mudryj, Frank Melgoza, Blythe Durbin-Johnson, Ralph deVere White, Paramita Ghosh. Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors together with AR antagonists in advanced prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B22.

Abstract A28: Eukaryotic initiation factor 4E phosphorylation in human malignant melanoma

Abstract Malignant melanoma continues to be a challenge both in terms of understanding the biologic mechanisms behind its behavior, as well as applying that information toward developing new therapeutic interventions. From 1992 through 2006, the incidence of malignant melanoma increased annually in the United States among non-Hispanic whites in all age groups, with death rates increased in patients 65 years and older. Approximately 70,000 new cases of invasive melanoma are anticipated in 2014. The need for better therapeutic strategies to combat these alarming statistics is paramount. While melanoma is a disease that primarily arises in the skin, it is a derivative of neural crest. During embryogenesis, an epithelial to mesenchymal transition occurs in order to form these cells. Once formed, these neural crest cells migrate to various locations in the body and differentiate into cells of the central and peripheral nervous system, medullary cells of the adrenal glands, various craniofacial connective tissues, and melanocytes. The histopathologic progression in melanocytic transformation is described by Chin, et al., (1998) as follows: (1) Normal skin has an even distribution of dendritic melanocytes throughout the basal layer; (2) Benign proliferation of melanocytes - in a nevus, nevoid melanocytes are organized into uniform nests of cells; (3) Melanocyte dysplasia - a dysplastic nevus has irregular and bridging nests of cells consisting of large atypical melanocytes; (4) In-situ melanoma, radial growth phase (RGP) - single dysplastic cells are present in the epidermis (Pagetoid spread); (5) Malignant melanoma, the vertical growth phase (VGP). Breslow thickness, the thickness of the melanoma from the most superficial nucleated layer, the granular layer, to the deepest level of invasion, has a strong predictive value in the prognosis of patients with non-metastatic melanomas. In our present study of 75 FFPE archived surgical specimens from 52 melanoma patients, we examined immunohistochemically detected cytoplasmic and nuclear expression of eukaryotic translation initiation factor 4E (eIF4E) and phosphorylated eIF4E in the four major subtypes of cutaneous melanoma (superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma and acral lentiginous melanoma), as well as in benign nevi, in relation to clinical outcome. Although the importance of activation of eIF4E through phosphorylation for cell proliferation and transformation in cultured cells and animal models of epithelial derived carcinomas has been documented, the role of eIF4E phosphorylation in transformation and histopathologic progression of melanocytes to melanomas has not previously been reported. We find that both eIF4E and phosphorylated eIF4E are increased in all major subtypes of human cutaneous malignant melanomas relative to benign dermal nevi. Importantly, in a subset of these specimens, nuclear phosphorylated eIF4E is significantly increased in nodular and metastatic melanomas compared to superficial spreading melanomas (P = 0.02). Survival analyses indicate that increased nuclear phosphorylated eIF4E is significantly associated with reduced survival (P = 0.0019, 3.994 Hazard Ratio). Since it has recently been reported (Konicek, et al., 2011) that inhibition of eIF4E phosphorylation with an orally bioavailable inhibitor of MAPK-interacting kinases (MNK) reduces lung metastases in the B16 mouse melanoma model, the present data may point to a novel cancer therapeutic strategy for treatment of human malignant melanoma, namely inhibition of eIF4E phosphorylation. Citation Format: Julia H. Carter, Reed Spaulding, James A. Deddens, Bruce M. Colligan, Grant Lewis, Jackson O. Pemberton, Jeremy R. Graff, Larry E. Douglass. Eukaryotic initiation factor 4E phosphorylation in human malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A28.

Abstract 3385: Increased eIF4E phosphorylation prevents response of prostate cancer cells to mTOR inhibitors.

Abstract Inhibitors of the mammalian target of rapamycin (mTOR) had been used in various clinical trials, but with limited efficacy. The overall goal of the present studies was to identify predictive markers of response to mTOR inhibitors in prostate cancer (PCa) cells. We previously showed that the mTOR pathway is activated in castration resistant PCa (CRPC), while its inhibition upregulates androgen receptor (AR) signaling, resulting in resistance to mTOR inhibitors (Wang et al, Oncogene, 2008). mTOR forms two complexes, mTORC1 and mTORC2: in this study we compared the effects of RAD001, an mTORC1 inhibitor, BEZ235, a dual mTOR/PI3K inhibitor, and Torin1, which inhibits both mTORC1 and mTORC2. Comparison of the effects of RAD001, alone, or in combination with the AR inhibitor bicalutamide in various CRPC cell lines showed that although C4-2 cells responded well to the combination, other cell lines, such as LNCaP-AI, PC-346C and CWR-R1, did not. The resistance of LNCaP-AI and CWR-R1 was partially overcome by treatment with BEZ-235 or Torin1, alone or in combination with bicalutamide; however, PC-346C cells were resistant to all three drugs. In an effort to identify the mechanism of resistance to mTOR inhibitors, therefore, the molecular effects of the drugs in downstream targets p70S6 kinase, 4E-BP1, eIF4G, Akt and eIF4E were compared in C4-2 (least resistant) and PC-346C (most resistant) cell lines. Western blots revealed that p70S6 kinase was downregulated by all the drugs whereas 4E-BP1 phosphorylation was effectively downregulated by BEZ235, but not Torin1, or RAD001. Phosphorylation of both p70S6K and 4E-BP1 results in activation of the translation initiation complex: a complex of eIF4E and eIF4G, therefore, inactivation of the translation initiation complex requires inhibition of eIF4E phosphorylation, which is independent of mTOR activation. In C4-2 cells, eIF4E phosphorylation was increased by RAD001 but suppressed by the other two drugs. However, in PC-346C cells, despite inhibition of 4E-BP1 phosphorylation, eIF4E phosphorylation was increased by all 3 drugs. Our data indicate eIF4E phosphorylation as a likely cause of resistance to mTOR inhibitors in PCa. Citation Format: Leandro S. D'Abronzo, Ryan Beggs, Swagata Bose, Paramita Ghosh. Increased eIF4E phosphorylation prevents response of prostate cancer cells to mTOR inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3385. doi:10.1158/1538-7445.AM2013-3385

Increased eIF4E Expression and Phosphorylation in Late Phase Chronic Myelogenous Leukemia Occurs in a Bcr-Abl-Dependent Manner, and Can Be Targeted by a Novel Mnk Kinase Inhibitor, CGP57380, To Overcome Imatinib-Resistance.

The molecular mechanisms which mediate progression of chronic phase (CP) CML to accelerated and blast phase (BP) disease remain unclear, although one feature that correlates with progression is increased expression of the Bcr-Abl protein itself (Barnes et al., Can. Res. 2005). Increased Bcr-Abl expression is likely to contribute to the more aggressive behavior of BP disease, but the downstream factors that are dysregulated by the increased amounts of Bcr-Abl protein remain to be determined. In these studies we turned our attention to eIF4E since forced expression of eIF4E is transforming, and because increased levels of eIF4E have been found in BP but not CP CML (Topisirovic et al., Mol. Cell. Bio. 2003). eIF4E plays a critical role in cap-dependent translation and allows recruitment of the translation machinery to mRNA. eIF4E is phosphorylated at Ser209, and phosphorylation correlates with exposure to growth factors and increased cap-dependent translation. Using a panel of primary CML cells representing patients at various stages of disease, we confirmed that both Bcr-Abl and eIF4E protein levels were elevated in BP samples compared to those in CP, and furthermore that phosphorylation at Ser209 was dependent on Bcr-Abl kinase activity in BP but not CP samples. We next went on to explore the role of eIF4E phosphorylation in BP CML. Because eIF4E is exclusively phosphorylated at Ser209 by the MAPK signal-integrating kinases (Mnk1/2), we used a small molecule inhibitor of Mnk1/2, CGP57380, to inhibit eIF4E phosphorylation (kind gift of Dr. H. Gram, Novartis). Using MTS assays, we found that CGP57380 exhibited synergistic activity with imatinib mesyalte (IM) against Ba/F3-Bcr-Abl and K562 cells, and that this was associated with increased caspase-3 activation. Consistent with a role for eIF4E phosphorylation in cap-dependent translation, we found that CGP57380 augmented the IM-mediated inhibition of cap-binding complex (eIF4F) formation, as well as loading of mRNA onto polysomes. Interestingly, we also uncovered the existence of a novel negative-feedback loop regulating Mnk kinase. Here, treatment with CGP57380 resulted in increased phosphorylation of Mnk1 as well as its upstream activator, ERK, in a time- and dose-dependent manner. Because activation of the MEK/ERK pathway is essential to Bcr-Abl-mediated transformation, this finding suggested that the full activity of CGP57380 might be obscured by this feedback loop. In support of this, the addition of the MEK inhibitor, U0126, to the IM/CGP57380combination resulted in increased activity against CML cells. The triple combination was also effective against Ba/F3-Bcr-Abl cells harboring the E255K and T315I mutations, but not parental Ba/F3 cells (reduced by 50, 23, and 15% respectively of DMSO-treated controls by MTS assay). Colony forming assays also demonstrated the activity of the IM/CGP57380 combination against CML progenitor cells. In conclusion, our data demonstrate that:eIF4E protein expression and phosphorylation are upregulated in a Bcr-Abl-dependent manner in BP CML;Inhibition of eIF4E phosphorylation by the novel Mnk kinase inhibitor, CGP57380, synergizes with IM in killing CML cells, as well as overcomes certain forms of IM-resistance;The addition of CGP57380 to IM results in inhibition of key steps in cap-dependent mRNA translation, and may provide a mechanistic explanation for the activity of this agent in CML.

The proteasome inhibitor, MG132, promotes the reprogramming of translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complex.

The eukaryotic translation initiation factor (eIF) 4E, is regulated by modulating both its phosphorylation and its availability to interact with the scaffold protein, eIF4G, to form the mature eIF4F complex. Here we show that treatment of C2C12 myoblasts with the proteasomal inhibitor, MG132 (N-carbobenzoxyl-Leu-Leu-leucinal), resulted in an early decrease in protein synthesis rates followed by a partial recovery, reflecting the reprogramming of translation. The early inhibition of protein synthesis was preceded by a transient increase in eIF2alpha phosphorylation, followed by a sustained increase in eIF4E phosphorylation. Inhibition of eIF4E phosphorylation with CGP57380 failed to prevent translational reprogramming or the moderate decrease in eIF4F complexes at later times. Prolonged incubation with MG132 resulted in the increased expression of heat shock protein (hsp)25, alphaB-crystallin and hsp70, with a population of hsp25 associating with the eIF4F complex in a p38 mitogen-activated protein kinase-dependent manner. Under these conditions, eIF4GI, and to a lesser extent eIF4E, re-localized from a predominantly cytoplasmic distribution to a more perinuclear and granular staining. Although MG132 had little effect on the colocalization of eIF4E and eIF4GI, it promoted the SB203580-sensitive association of eIF4GI and hsp25, an effect not observed with alphaB-crystallin. Addition of recombinant hsp25 to an in vitro translation assay resulted in stimulation of on-going translation and a moderate decrease in de novo translation, indicating that this modified eIF4F complex containing hsp25 has a role to play in recovery of mRNA translation following cellular stress.

Regulation of eukaryotic initiation factor 4E phosphorylation in the nervous system of Aplysia californica.

We have used an antibody that specifically recognizes eukaryotic initiation factor 4E (eIF4E) when it is phosphorylated at Ser(207) to characterize eIF4E phosphorylation in the nervous system of APLYSIA: The level of phosphorylated eIF4E, but not the level of total eIF4E, was significantly correlated with the basal rate of translation measured from different animals. Serotonin (5-HT), a transmitter that regulates the rate of translation in APLYSIA: neurons, had mixed effects on eIF4E phosphorylation. 5-HT decreased eIF4E phosphorylation in sensory cell clusters through activation of protein kinase C. 5-HT increased eIF4E phosphorylation in the whole pleural ganglia. In the APLYSIA: nervous system, eIF4E phosphorylation correlated with phosphorylation of the p38 MAP kinase, but not the p42 MAP kinase (ERK). Furthermore, an inhibitor of the p38 MAP kinase significantly decreased basal eIF4E phosphorylation, but an inhibitor of the MAP or ERK kinase (MEK) did not. Despite the correlation of eIF4E phosphorylation with the basal rate of translation, inhibition of eIF4E phosphorylation by an inhibitor of the p38 MAP kinase did not significantly decrease the rate of translation.

Adenovirus-specific translation by displacement of kinase Mnk1 from cap-initiation complex eIF4F.

Translation of cellular mRNAs involves formation of a cap-binding translation initiation complex known as eIF4F, containing phosphorylated cap-binding protein eIF4E, eIF4E kinase Mnk1, eIF4A, poly(A)-binding protein and eIF4G. Adenovirus is shown to prevent cellular translation by displacing Mnk1 from eIF4F, thereby blocking phosphorylation of eIF4E. Over expression of an eIF4E mutant that cannot be phosphorylated by Mnk1 impairs translation of cellular but not viral late mRNAs. Adenovirus 100k protein is shown to bind the C-terminus of eIF4G in vivo and in vitro, the same region bound by Mnk1. In vivo, 100k protein displaces Mnk1 from eIF4G during adenovirus infection, or in transfected cells. Purified 100k protein also evicts Mnk1 from isolated eIF4F complexes in vitro. A mutant adenovirus with a temperature-sensitive 100k protein that cannot inhibit cellular protein synthesis at restrictive temperature no longer blocks Mnk1 binding to eIF4G, or phosphorylation of eIF4E. We describe a mechanism whereby adenovirus selectively inhibits the translation of cellular but not viral mRNAs by displacement of Mnk1 from eIF4G and inhibition of eIF4E phosphorylation.

Distinct Signalling Pathways Mediate Insulin and Phorbol Ester-stimulated Eukaryotic Initiation Factor 4F Assembly and Protein Synthesis in HEK 293 Cells

Stimulation of serum-starved human embryonic kidney (HEK) 293 cells with either the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), or insulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kinase, eIF4F assembly, and protein synthesis. All these effects were blocked by rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase and protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p70 S6 kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (PD098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, and TPA-stimulated protein synthesis. Transient transfection of constitutively active mitogen-activated protein kinase interacting kinase 1 (the eIF4E kinase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other signals emanating from MEK. This report also provides evidence that increased eIF4E phosphorylation alone does not affect the assembly of the eIF4F complex or general protein synthesis.

A late adenovirus factor induces eIF-4E dephosphorylation and inhibition of cell protein synthesis

Adenovirus prevents host cell protein synthesis during its late phase of replication in large part by causing the underphosphorylation of translation initiation factor eIF-4E, a component of initiation factor eIF-4F (cap-binding protein complex). Late adenovirus mRNAs are preferentially translated because they possess a reduced requirement for eIF-4F. This study continues the characterization of the mechanism by which adenovirus inhibits cellular protein synthesis. First it is shown that adenovirus blocks the addition of phosphate to eIF-4E rather than enhancing its removal, establishing that the virus impairs a signalling pathway or protein kinase activity involved in eIF-4E phosphorylation. It is then shown that shutoff of cell protein synthesis and translation of late viral mRNAs are uncoupled, in that shutoff actually occurs a short time (1 to 3 h) after late adenovirus mRNAs are already undergoing translation. Finally, by using a variety of genetic mutants stalled at different stages in the viral life cycle, it was found that dephosphorylation of eIF-4E and inhibition of cell translation are not caused by early adenovirus gene products acting at late times or by events related to viral DNA replication. Instead, it is shown that inhibition of eIF-4E phosphorylation and cell translation are mediated upon activation of the viral major late transcription unit. These and other results presented indicate that the adenovirus signal which induces eIF-4E dephosphorylation and shutoff of cell protein synthesis is linked either to an activity of one or more late viral polypeptides, to double-stranded RNA produced by opposition of the early and late viral transcription units, or to both.