Activation Of Cap-dependent Translation Research Articles

The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1

The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.

Activation of EIF4E by Aurora Kinase A Depicts a Novel Druggable Axis in Everolimus-Resistant Cancer Cells.

Purpose: Aurora kinase A (AURKA) is overexpressed in several cancer types, making it an attractive druggable target in clinical trials. In this study, we investigated the role of AURKA in regulating EIF4E, cap-dependent translation, and resistance to mTOR inhibitor, RAD001 (everolimus).Experimental Design: Tumor xenografts and in vitro cell models of upper gastrointestinal adenocarcinomas (UGC) were used to determine the role of AURKA in the activation of EIF4E and cap-dependent translation. Overexpression, knockdown, and pharmacologic inhibition of AURKA were used in vitro and in vivoResults: Using in vitro cell models, we found that high protein levels of AURKA mediate phosphorylation of EIF4E and upregulation of c-MYC. Notably, we detected overexpression of endogenous AURKA in everolimus-resistant UGC cell models. AURKA mediated phosphorylation of EIF4E, activation of cap-dependent translation, and an increase in c-MYC protein levels. Targeting AURKA using genetic knockdown or a small-molecule inhibitor, alisertib, reversed these molecular events, leading to a decrease in cancer cell survival in acquired and intrinsic resistant cell models. Mechanistic studies demonstrated that AURKA binds to and inactivates protein phosphatase 2A, a negative regulator of EIF4E, leading to phosphorylation and activation of EIF4E in an AKT-, ERK1/2-, and mTOR-independent manner. Data from tumor xenograft mouse models confirmed that everolimus-resistant cancer cells are sensitive to alisertib.Conclusions: Our results indicate that AURKA plays an important role in the activation of EIF4E and cap-dependent translation. Targeting the AURKA-EIF4E-c-MYC axis using alisertib is a novel therapeutic strategy that can be applicable for everolimus-resistant tumors and/or subgroups of cancers that show overexpression of AURKA and activation of EIF4E and c-MYC. Clin Cancer Res; 23(14); 3756-68. ©2017 AACR.

Downregulation of p68 RNA Helicase (DDX5) Activates a Survival Pathway Involving mTOR and MDM2 Signals.

The DEAD box p68 RNA helicase (DDX5) is required to manipulate RNA structures implicated in mRNA/rRNA processing and transcript export, and acts as a co-activator for a range of transcription factors. Previous research has indicated that p68 RNA helicase may also be important in tumour development. Wild-type HeLa and stable HeLa (clone 13) cell cultures containing RNAi-mediated depletion of p68 RNA helicase induced by doxycycline (DOX) were used to study how the p68 RNA helicase affects the mTOR cell signalling pathway. Relevant results were repeated using transient transfection with pSuper/pSuper-p68 RNA helicase, containing RNAi-mediated depletion of p68 RNA helicase, to avoid DOX interference. Here we provide strong evidence for the participation of p68 RNA helicase in mTOR regulation. In detail, depletion of this helicase decreases cell growth and activates the mTOR/MDM2 cell survival mechanism, which ultimately leads to inhibition of the pro-apoptotic activity. p68 RNA helicase downregulation strongly stimulates 4E-BP1 phosphorylation, thereby provoking activation of cap-dependent translation. In contrast, the IRES-dependent translation of c-myc is reduced when p68 RNA helicase is depleted, thus indicating that at least this specific translation requires p68 RNA helicase activity to manipulate the complex 5' end of this mRNA. Interestingly, p68 RNA helicase depletion decreases cell growth while activating the mTOR/MDM2 cell survival mechanism. As MDM2 is a known negative regulator of p53, we infer that the activation of the cell survival mechanism may result in inhibition of the pro-apoptotic factor p53. Finally, p68 RNA helicase depletion activates capdependent translation and inhibits c-MYC IRES-mediated translation.

HPIP promotes gastric cancer cell proliferation through activation of cap-dependent translation

Cap-dependent translation has an essential role in the control of cell proliferation by initiating the translation of oncogenes involved in the regulation of cell cycle progression, such as cyclinD1, and its deregulation contributes to the development and progression of various types of cancers. Hematopoietic pre-B-cell leukemia transcription factor interacting protein(HPIP) was found to be overexpressed in gastric cancer(GC) tissues compared to normal tissues and to promote GC growth invitro and invivo. However, the mechanism by which HPIP promotes GCcell proliferation remains unknown. In the present study, we found that HPIP activated cap-dependent translation in an AKT/mTORC1 pathway-dependent manner. Blocking cap‑dependent translation with 4EGI-1, a specific eIF4E/eIF4G interaction inhibitor, profoundly abrogated the ability of HPIP to promote G1/Sphase transition and GC cell proliferation, while activation of cap-dependent translation by silencing 4E-BP1 expression significantly reversed the inhibitory effect of HPIP knockdown on GC cell proliferation. Furthermore, targeting translation initiation with 4EGI-1 effectively suppre-ssed the ability of HPIP to promote gastric tumor growth in a xenograft mouse model invivo. All these data indicate that HPIP promotes GC cell proliferation through positive regulation of cap-dependent translation and mproves our understanding of the underlying mechanisms involved in the regulation of GC cell proliferation by HPIP.

Abstract 2889: Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail

Abstract The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular mechanisms underlying the translational activation for cancer metastatic progression remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer snail followed by repression of E-cadherin expression and promotion of cell migration and invasion as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates snail expression and inhibits cell migratory/invasive capacities and metastasis. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the eIF4F translation initiation also profoundly attenuates snail translation/expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings reveal a novel role of 4E-BP1 in the regulation of EMT and metastasis through translational control of snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking snail-mediated metastatic potential of cancer. Citation Format: Qing Ye, Weijia Cai, Qing-Bai She. Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail. [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 2889.

Abstract B23: The 5 UTR of many oncogenes and transcription factors encodes a targetable dependence on the eIF4A RNA helicase

Abstract We report a mechanism of translational control that is determined by a requirement for eIF4A RNA helicase activity and underlies the anticancer effects of Silvestrol and related compounds. Briefly, activation of cap-dependent translation contributes to T-cell leukemia (T-ALL) development and maintenance. Accordingly, inhibition of translation initiation factor eIF4A with Silvestrol produces powerful therapeutic effects against T-ALL in vivo. We used transcriptome-scale ribosome footprinting on Silvestrol-treated T-ALL cells to identify Silvestrol-sensitive transcripts and the hallmark features of eIF4A-dependent translation. These include a long 5 UTR and a 12-mer sequence motif that encodes a guanine quartet (CGG)4. RNA folding algorithms as well as experimental evidences pinpoint the (CGG)4 motif as a common site of RNA G-quadruplex structures within the 5 UTR. In T-ALL these structures mark approximately eighty highly Silvestrol-sensitive transcripts that include key oncogenes and transcription factors and contribute to the drug's anti-leukemic action. Hence, the eIF4A-dependent translation of G-quadruplex containing transcripts emerges as a gene-specific and therapeutically targetable mechanism of translational control. Citation Format: Kamini Singh, Andrew L. Wolfe, Yi Zhong, Gunnar Rätsch, Hans-Guido Wendel. The 5 UTR of many oncogenes and transcription factors encodes a targetable dependence on the eIF4A RNA helicase. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B23.

Loss of 4E-BP1 function induces EMT and promotes cancer cell migration and invasion via cap-dependent translational activation of snail.

The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular basis of the translation activation for metastatic progression of cancer remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer Snail followed by repression of E-cadherin expression and promotion of cell migratory and invasive capabilities as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates Snail expression and suppresses cell migration and invasion. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the translation initiation also profoundly attenuates Snail expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of Snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated Snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings indicate a novel role of 4E-BP1 in the regulation of EMT and cell motility through translational control of Snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking Snail-mediated metastatic potential of cancer.

ERK and AKT signaling cooperate to translationally regulate survivin expression for metastatic progression of colorectal cancer

The MEK/ERK and PI3K/AKT pathways are often concurrently activated by separate genetic alterations in colorectal cancer (CRC), which is associated with CRC progression and poor survival. However, how activating both pathways is required for CRC metastatic progression remains unclear. Our recent study showed that both ERK and AKT signaling are required to activate eIF4E-initiated cap-dependent translation via convergent regulation of the translational repressor 4E-BP1 for maintaining CRC transformation. Here, we identified that the activation of cap-dependent translation by cooperative ERK and AKT signaling is critical for promotion of CRC motility and metastasis. In CRC cells with coexistent mutational activation of ERK and AKT pathways, inhibition of either MEK or AKT alone showed limited activity in inhibiting cell migration and invasion, but combined inhibition resulted in profound effects. Genetic blockade of the translation initiation complex by eIF4E knockdown or expression of a dominant active 4E-BP1 mutant effectively inhibited migration, invasion and metastasis of CRC cells, whereas overexpression of eIF4E or knockdown of 4E-BP1 had the opposite effect and markedly reduced their dependence on ERK and AKT signaling for cell motility. Mechanistically, we found that these effects were largely dependent on the increase in mTORC1-mediated survivin translation by ERK and AKT signaling. Despite the modest effect of survivin knockdown on tumor growth, reduction of the translationally-regulated survivin profoundly inhibited motility and metastasis of CRC. These findings reveal a critical mechanism underlying the translational regulation of CRC metastatic progression, and suggest that targeting cap-dependent translation may provide a promising treatment strategy for advanced CRC.

Abstract 3787: ERK and AKT signaling cooperate to translationally regulate survivin expression for promotion of cell motility and metastasis in colorectal cancer.

Abstract The MEK/ERK and PI3K/AKT pathways are often concurrently activated by separate genetic alterations in colorectal cancer (CRC), which is associated with CRC progression and poor survival. However, how activating both pathways is required for CRC metastatic progression remains unclear. Our recent study showed that both ERK and AKT signaling are required to activate eIF4E-initiated cap-dependent translation via convergent regulation of the translational repressor 4E-BP1 for maintaining CRC transformation. Here, we identified that the activation of cap-dependent translation by cooperative ERK and AKT signaling is critical for promotion of CRC motility and metastasis. In CRC cells with coexistent mutational activation of ERK and AKT pathways, inhibition of either MEK or AKT alone showed limited activity in inhibiting cell migration and invasion, but combined inhibition resulted in profound effects. Genetic blockade of the translation initiation complex by eIF4E knockdown or expression of a dominant active 4E-BP1 mutant effectively inhibited migration, invasion and metastasis of CRC cells, whereas overexpression of eIF4E or knockdown of 4E-BP1 had the opposite effect and markedly reduced their dependence on ERK and AKT signaling for cell motility. Mechanistically, we found that these effects were largely dependent on the increase in mTORC1-mediated survivin translation by ERK and AKT signaling. Despite the modest effect of survivin knockdown on tumor growth, reduction of the translationally-regulated survivin profoundly inhibited motility and metastasis of CRC. These findings reveal a critical mechanism underlying the translational regulation of CRC metastatic progression, and suggest that targeting cap-dependent translation may provide a promising treatment strategy for advanced CRC. Citation Format: Qing Ye, Weijia Cai, Yongbin Zheng, B. Mark Evers, Qing-Bai She. ERK and AKT signaling cooperate to translationally regulate survivin expression for promotion of cell motility and metastasis in colorectal cancer. [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 3787. doi:10.1158/1538-7445.AM2013-3787

Activation of Cap-Dependent Translation by Mucosal Human Papillomavirus E6 Proteins Is Dependent on the Integrity of the LXXLL Binding Motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.

Targeting Translation Bypasses Pim Kinase Activity, a Common and Adverse Prognostic Marker In Lymphoma

AbstractAbstract 119The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, but rapamycin-analog (rapalog) mTOR inhibitors have shown only modest benefits in clinical trials on lymphoma patients. To better understand the resistance of lymphomas to rapalogs, we have undertaken a study of the Pim family kinases, which signal in parallel to PI3K/AKT/mTOR and whose expression has been detected in multiple subtypes of non-Hodgkin lymphoma (NHL). The two pathways converge on activation of cap-dependent translation, suggesting new treatment possibilities by targeting this common downstream output. To assess the clinical relevance of Pim activity, we have quantified Pim1 and Pim2 expression in multiple NHL subtypes using tissue microarray (TMA) technology. We find common expression of Pim1, Pim2, or both proteins in diffuse large B-cell lymphoma (DLBCL, 65.5%), follicular lymphoma (58%), small lymphocytic lymphoma (76.5%) and mantel cell lymphoma (89.7%). Importantly, Kaplan-Meier survival analysis of clinical data linked to our DLBCL TMAs show a strong trend toward a worse overall survival when Pim expression is present in diagnostic tumor samples compared to Pim-negative tumors (p=0.0965). Studies in vivo demonstrate Pim's ability to accelerate oncogenesis in a manner similar to AKT in model systems specific to Burkitt's lymphoma (Eμ -Myc) and follicular lymphoma (VavP-Bcl2). Treatment studies in secondary recipient animals show that Pim promotes resistance to anthracycline chemotherapy like AKT. However, Pim tumors completely resist rapamycin in stark contrast to AKT tumors. To elaborate on these findings, we have employed a genetically defined system of rapamycin sensitivity lacking mTOR's upstream repressor TSC2. These studies show that Pim's ability to mediate rapamycin resistance is dependent on its ability to maintain inhibitory phosphorylation of the translation repressor 4EBP1. Specifically, a phosphorylation-site deficient mutant of 4EBP1 completely abrogates Pim's ability to maintain the viability of the TSC2 −/− cells. We have expanded on this finding using the drug silvestrol, which inhibits cap-dependent translation by targeting eIF4A. Silvestrol shows high potency against Pim-expressing TSC2 −/− cells (IC50 < 1 nM) and also against a panel of Pim-expressing lymphoma cell lines (IC50 1–10 nM). Indeed, targeting cap-dependent translation appeared more effective than the Pim kinase inhibitor SGI-1776 (IC50 1–10 μ M against lymphoma cell lines), which has significantly higher potency against Pim1 than Pim2. In conclusion, we have more clearly defined Pim kinase activity as a major mediator of oncogenesis in multiple NHL subtypes and as a likely negative prognostic marker in DLBCL. Our mechanistic and treatment studies provide a strong rational basis for targeting cap-dependent translation as a treatment strategy to bypass Pim activity and improve lymphoma patients' responses to both cytotoxic and rapalog therapies. Disclosures:No relevant conflicts of interest to declare.

Abstract 5038: AZD8055 is an effective inhibitor of mTOR kinase signaling and breast cancer growth while relieving feedback inhibition of HER-kinase signaling

Abstract The PI3K signaling pathway is often dysregulated in human cancer. mTOR kinase is a component of this pathway that mediates activation of cap-dependent translation, other AGC-kinases and feedback inhibition of upstream components of the pathway. The natural product rapamycin binds to and inhibits the mTOR-containing TORC1, but not the TORC2 complex. Rapamycin-like TORC1 inhibitors have modest clinical activity in renal cell and other cancers, but only marginal activity in glioblastoma, prostate and breast cancers in which PI3K/AKT signaling is often activated. The limited antitumor activity of rapamycin may be due to inefficient inhibition of TORC1, induction of TORC1-dependent feedback, or lack of inhibition of TORC2. Potent mTOR kinase inhibitors could obviate these issues by inhibiting both TORC1 and TORC2 and by preventing induction of phosphorylation of AKT at Serine 473. We have now compared the effects of the specific mTOR kinase inhibitor AZD8055 and rapamycin in breast cancer models with amplified HER2 or mutant p110alpha PI3K. As expected, AZD8055 and rapamycin both inhibit S6K and S6 phosphorylation, but only the former inhibits AKT phosphorylation at Serine 473, which is, instead, induced by rapamycin. Moreover, AZD8055 is a more potent inhibitor of 4EBP-1 phosphorylation, CAP-dependent translation, and D-cyclin expression. Thus, the mTOR kinase inhibitor inhibits mTOR functions more effectively than rapamycin. Whereas both drugs cause tumor cells to undergo autophagy and G1 arrest, only AZD8055 induces significant apoptotic cell death. The more effective inhibition of 4EBP1 phosphorylation, cap-dependent translation and enhancement of apoptosis compared to that caused by rapamycin may be due to more complete inhibition of TORC1 function or to abolition of feedback activation of AKT by AZD8055. As previously established with rapamycin, we now show that AZD8055 causes relief of feedback inhibition of HER kinase signaling in these tumors, resulting in marked induction of both ERK and PI3K signaling. These results suggest that mTOR kinase inhibition with drugs such as AZD8055 can more effectively inhibit TOR activity than rapamycin. They therefore have the potential for increased antitumor activity, but relief of TOR kinase-dependent negative feedback could create a rationale for combination therapy. 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 5038.

Translational Control of the Antiapoptotic Function of Ras

Activated Ras has been shown to provide powerful antiapoptotic signals to cells through well defined transcriptional and post- translational pathways, whereas translational control as a mechanism of Ras survival signaling remains unexplored. Here we show a direct relationship between assembly of the cap-dependent translation initiation apparatus and suppression of apoptosis by oncogenic Ras in vitro and in vivo. Decreasing protein synthesis with rapamycin, which is known to inhibit cap-dependent translation, increases the susceptibility of Ras-transformed fibroblasts to cytostatic drug-induced apoptosis. In contrast, suppressing global protein synthesis with equipotent concentrations of cycloheximide actually prevents apoptosis. Enforced expression of the cap-dependent translational repressor, the eukaryotic translation initiation factor (eIF) 4E-binding protein (4E-BPI), sensitizes fibroblasts to apoptosis in a manner strictly dependent on its ability to sequester eIF4E from a translationally active complex with eIF4GI and the co-expression of oncogenic Ras. Ectopic expression of 4E-BP1 also promotes apoptosis of Ras-transformed cells injected into immunodeficient mice and markedly diminishes their tumorigenicity. These results establish that eIF4E-dependent protein synthesis is essential for survival of fibroblasts bearing oncogenic Ras and support the concept that activation of cap-dependent translation by extracellular ligands or intrinsic survival signaling molecules suppresses apoptosis, whereas synthesis of proteins mediating apoptosis can occur independently of the cap.