Browsing by Subject "eIF4E"
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Item Evaluation of Eukaryotic Initiation Factor 4E (eIF4E) antagonist 4Ei-1 in mammalian breast cancer and lung cancer cells : chemosensitizaiton with low cytotoxicity.(2012-08) Li, ShuiThe development of cancer and fibrotic diseases has been shown to be highly dependent on disregulation of cap-dependent translation. Binding protein eIF4E to N7-methylated guanosine capped mRNA has been found to be the rate-limiting step governing translation initiation; and therefore represents an attractive target for drug discovery. Our group has found that 7-benzyl guanosine monophosphate (7Bn-GMP) is a potent antagonist of eIF4E cap binding (Kd = 0.8 uM). Recent X-ray crystallographic studies have revealed that the cap-dependent pocket undergoes a unique structural change in order to accommodate the benzyl group. Unfortunately, 7Bn-GMP is not cell permeable. Recently, we have prepared a tryptamine phosphoramidate prodrug of 7Bn-GMP, 4ei1, and shown that it is a substrate for human histidine triad nucleotide binding protein (hHINT1) and is inhibit eIF4E initiated epithelial-mesenchymal transition (EMT) by Zebra fish embryo cells. To assess the intracellular uptake of 4ei1 and conversion to 7Bn-GMP by cancer cells, we developed a sensitive assay using LC-ESI-MS/MS for the intracellular quantitation of 4ei1 and 7Bn-GMP. When incubated with the breast cancer cell line MDA-231; or lung cancer cell lines H460, H383 and H2009, 4ei1 was found to be rapidly internalized and converted to 7Bn-GMP. Since oncogenic mRNAs are predicted to have the highest eIF4E requirement for translation, we carried out chemosensitization studies with 4ei1. The prodrug was found to chemosensitize both breast and lung cancer cells to non-toxic levels of gemcitabine. Further mechanistic studies revealed that the expressed levels of eIF4E were substantially reduced in cells treated with 4ei1 in a dose dependent manner. The levels of eI4E could be restored by treatment with the proteasome inhibitor MG-132. Taken together, our results demonstrate that 4ei1 is likely to inhibit translation initiation by eIF4E cap binding by both antagonizing eIF4E cap binding and initiating eIF4E proteasomal degradation.Item Genome-wide pharmacological modulation of cap-dependent translational control(2013-12) Braziunas, Jeffrey JosephThe first step of cap-dependent translation is mediated by the mRNA cap-binding protein eukaryotic initiation factor 4E (eIF4E). Although involved in translating nearly all cellular transcripts, mRNAs vary widely in their translational response to eIF4E activity changes. Prior studies of mRNA structure revealed several features governing eIF4E responsiveness; however, most of this knowledge is based on comparison of two levels of eIF4E activity with unclear physiological relevance. To identify mRNA structural features that govern genome-wide ribosome recruitment across a full range of physiological eIF4E activities, we precisely modulated eIF4E activity using an eIF4E-inducible system together with 4Ei-1, an inhibitor of the eIF4E-5'mRNA cap association. We identified genes that were more (4E hypersensitive) or less (4E hyposensitive) responsive to eIF4E activity changes than average. Distinct characteristics associated with each class: 4E hypersensitive genes had longer 5'UTRs with higher GC content, longer 3'UTRs with lower GC content; more AU-rich elements and a higher density of unique microRNA targets sites than typical genes. Importantly, these structural characteristics predicted the translational response across the dose range of 4Ei-1. Gene ontology analysis showed an association between 4E hypersensitive genes and proliferation; and cell cycle experiments with 4Ei-1 validated this result. A search for the outcome and mechanism of this proliferative gene activation in a physiological setting revealed that abrupt gain of eIF4E function in quiescent cells first triggers G0 exit and then cell cycle transit at least partially by increasing ribosome recruitment to cyclins C and D1. Whereas cyclin C is not necessary for this effect; cyclin D1 is indispensable, although not sufficient. Our findings provide important insights into mRNA properties of eIF4E-modulated translational control.Item Translational control of cancer: an exploration of eIF4E and its role in cellular oncogenic transformation.(2009-04) Underwood II, Jon MichaelCancer is currently the 2nd leading cause of death in the U.S., responsible for approximately one quarter of the annual death rate. While cancer is genetically diverse, we suspect there may be common components of the cellular machinery where oncogenic signals converge, and that these critical regulatory nodes may represent molecular targets for new therapies. My approach derives from recent work on human breast and lung carcinoma, and murine models of lymphoid malignancies; which identify the cap-dependent protein synthesis machinery as a critical point of convergence and amplification of oncogenic signals emanating from the Ras/PI3K/Akt cascade. The rate limiting step in protein synthesis is initiation, mediated by the trimeric protein eukaryotic initiation factor 4F (eIF4F). The limiting component of eIF4F is the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E). Over expression of eIF4E transforms immortalized rodent fibroblasts and confers primary human cells with several cancer-related functions including decreased growth factor requirement for proliferation and survival, colony formation and anchorage independence. However, until very recently all experiments examining the oncogenic potential of eIF4E have involved constitutive over expression in stably transfected cell lines; leaving uncertain precisely which oncogenic functions could be ascribed directly to eIF4E, and which relate to its ability to suppress apoptosis - thus creating a permissive environment for subsequent oncogenic mutations. I have developed a high fidelity eIF4E inducible system which allows me to abruptly activate eIF4E over expression. With this system I am able to establish a direct connection between eIF4E induction and autonomous cell proliferation. I have also been able to specify the cell cycle kinetics which occurs after eIF4E induction, including the ability of eIF4E to bypass growth factor initiation on the canonical proliferative pathway. The growth factor bypass properties of eIF4E are due to its ability to translationally activate cyclin D1, an occurrence I report for the first time. I also demonstrate the potential of pharmacological intervention targeting hyperactive translational activity. With the use of small molecular compounds which antagonize translation initiation, I demonstrate the ability to reverse eIF4E triggered proliferation in the eIF4E inducible system and the selective elimination of cancer cells with minimal toxicity in normal cells. This study has lead to a better understanding of how cap-dependent translation regulates cell proliferation and advanced the novel concept for cancer biology and therapeutics focused on translational control.Item Two barriers to Ras mediated oncogenesis: translational control checkpoint and proliferative block by autophagy.(2009-06) Kim, Yong YeanAccording to the American Cancer Society, cancer is the second leading cause of mortality in the United States, accounting for more than half a million deaths per year. Cancer is a complex disease with multiple factors influencing its genesis, maintenance, growth, and invasion which makes the treatment and prevention of the disease challenging. Despite the high mortality and morbidity associated with cancer, it is a disease of the old, with the median age for cancer incidence being 66 years old according to the National Cancer Institute. Therefore, the human body is remarkably adept at protecting itself from malignant transformation. In my thesis, I explore the oncogene Ras which is represented in approximately 30% of all human cancers, making Ras one of the most commonly activated oncogenes. I analyzed the innate barriers to oncogenic Ras (RasV12) induced transformation in the cell in an attempt to better understand tumor defense systems which can be mimicked for novel cancer therapy. My results confirmed one putative barrier which had good experimental grounding and identified a completely new barrier. One putative barrier to RasV12 oncogenesis was the existence of a translational control check point in tumor defense. Activation of translation initiation has been shown to be on the causal pathway to cancer and is activated by Ras via two different pathways. The rate limiting translation initiation factor 4E (eIF4E) is up regulated in many cancers and over expression of eIF4E confers cells with transformed phenotypes. Therefore, if activation of translation is oncogenic, then it is reasonable to posit the existence of a translational control checkpoint in tumor defense. The logical guardians of this checkpoint would be the primary negative regulators of translational initiation, the eIF4E binding protein (4E-BP) family of proteins. I show that translational control checkpoint does indeed exist in tumor defense and that the 4E-BPs are the guardians of this checkpoint. Mice lacking two of the three 4E-BPs (4ebp1-/-/4ebp2-/-) were more sensitive to tobacco carcinogen NNK induced lung tumors and showed tumors with increased vascularity. Also, 4ebp1-/-/4ebp2-/- genotype was associated with a skewing of the genome wide translational profile towards growth and proliferation even before NNK treatment indicating a cancer primed state. Lastly, I showed that the cytochrome P450 2A5, the protein that metabolizes NNK to its carcinogenic product, was translationally up regulated increasing the carcinogenic potency of NNK. The second barrier to RasV12 oncogenesis was an unexpected discovery. In an effort to determine the mechanism of RasV12 oncogenesis and its defense, I discovered that RasV12 triggered proliferative block even in cells which have bypassed the senescence barriers. This was unexpected since previous reports had shown that in this setting, RasV12 actually caused anchorage independent growth and invasion in vitro. The nature of the proliferative block was not senescence although it has many of the characters of senescence. Due to a striking phenotypical change where large vacuoles accumulate, I explored the possibility that autophagy was playing a role in the proliferative block. I show that RasV12 expressing cells displayed hallmarks of autophagy such as double membraned vacuoles with pieces of organelles, acidic nature of the vacuoles, and positivity for early and late markers of autophagy. My study validates the current efforts to develop targeted therapy against the translation initiation complex and provides autophagy as a new potential target for caner therapy. By learning from the cell's innate cancer barriers, I hope that we will be able to develop more effect therapies for cancer.