Targeting alternative translational initiation of oncogenes in cancer cells Prof. Dr. Matthias Selbach & PD Dr. med. Patrick Hundsdörfer We aim to evaluate mechanisms of internal ribosomal entry site (IRES)-­‐mediated translation as an innovative avenue towards new cancer treatment strategies. IRES-­‐
mediated translation represents an alternative protein synthesis method used by tumor cells to maintain oncogene expression even during cellular stress, when global (cap-­‐dependent) translation is impaired.1-­‐7 This complex posttranscriptional regulation mechanism is controlled by IRES trans-­‐acting factors (ITAFs) including EIF4G2 and MDM2 (Fig. 1).8-­‐11 Interestingly, among the limited number of eukaryotic mRNAs harboring IRESes, several encode important oncogenes (e.g. MYC, MYCN, VEGFA, HIF1A, XIAP).12-­‐16 Based on data obtained in previous projects, we hypothesize that (i) IRES-­‐regulated translation represents a general survival mechanism of malignant cells and (ii) targeting IRES-­‐mediated translation might be an efficient and promising new treatment strategy simultaneously inhibiting several major oncogenes in clinically relevant cancers including neuroblastoma. 8
Fig. 1: Schematic representation of Cap-­‐ (A) vs. IRES-­‐mediated translation initiation The main objectives of this project are to (i) evaluate the impact of IRES-­‐mediated translation for regulating oncogene expression in neuroblastoma as a representative model system and (ii) validate EIF4G2 and MDM2 as candidate targets for specific inhibition of IRES-­‐mediated translation. Firstly, we will analyze the effect of silencing EIF4G2 or MDM2 on de novo protein synthesis in neuroblastoma cells (EIF4G2: SHEP, MDM2: p53-­‐mutant NMB7) using pSILAC (pulsed stable labeling of amino acids in cell culture).17,18 Suppressing global translation by applying cell stress (γ-­‐irradiation) will allow the proteome-­‐wide mass spectrometry-­‐based quantification of cap-­‐independent protein synthesis. Simultaneous pulsed labeling of newly synthesized mRNA with 4-­‐thiouridine (4sU) followed by next-­‐generation sequencing (NGS) will enable us to determine the transcriptional effects of EIF4G2/MDM2 silencing. 17 These analyses will both determine the impact of EIF4G2/MDM2 on the expression of known IRES-­‐regulated oncogenes and identify further target genes translationally regulated by EIF4G2 and MDM2. In a second step, we will identify EIF4G2/MDM2 target mRNAs using PAR-­‐CLIP (photoactivatable ribonucleoside-­‐enhanced crosslinking and immunoprecipitation). Incorporation of 4sU into nascent mRNA allows efficient UV-­‐crosslinking of proteins to target mRNAs. Following EIF4G2/MDM2 immunoprecipitation, NSG will be used to identify crosslinked mRNAs and exactly localize the EIF4G2/MDM2 binding sites.19 1
These data will help validate EIF4G2/MDM2-­‐regulated candidates identified by pSILAC as putative IRES-­‐regulated genes. Finally, we will directly determine the impact of EIF4G2/MDM2 on IRES activity in a unique neuroblastoma-­‐based cell-­‐free translation system recapitulating cellular IRES-­‐
mediated translation in vitro (Fig. 2).9,20-­‐22 Fig. 2: Determination of IRES activity using in vitro transcribed reporter mRNAs (A) in neuroblastoma cell-­‐derived cytoplasmic extracts (B) By silencing EIF4G2/MDM2 prior to cytoplasmic extract preparation or addition of the (recombinant) proteins, this system allows direct analysis of EIF4G2/MDM2 effects on IRES translation. Established IRESes (e.g. MYC, XIAP) and newly identified candidates (pSILAC, PAR-­‐CLIP) can easily be studied in this stable biochemical system. In parallel, the IRES reporter plasmids can be transfected into neuroblastoma cells, and IRES activity can be evaluated in living cells under stress conditions. This Ph.D. project will contribute to testing the hypothesis whether IRES-­‐
mediated translation is a general mechanism enabling tumor cells to maintain expression of oncogenes under stress conditions, thereby, contributing as a survival mechanism to their oncogene addiction. It will also deliver information on EIF4G2/MDM2 as candidates for novel, IRES-­‐targeted treatment strategies in neuroblastoma. References 1. Gu L, Zhu N, Zhang H, Durden DL, Feng Y, Zhou M. Regulation of XIAP translation and induction by MDM2 following irradiation. Cancer Cell 2009;15:363-­‐75. 2. Holcik M, Lefebvre C, Yeh C, Chow T, Korneluk RG. A new internal-­‐ribosome-­‐entry-­‐site motif potentiates XIAP-­‐mediated cytoprotection. Nat Cell Biol 1999;1:190-­‐2. 3. Warnakulasuriyarachchi D, Cerquozzi S, Cheung HH, Holcik M. Translational induction of the inhibitor of apoptosis protein HIAP2 during endoplasmic reticulum stress attenuates cell death and is mediated via an inducible internal ribosome entry site element. J Biol Chem 2004;279:17148-­‐57. 4. Ungureanu NH, Cloutier M, Lewis SM, et al. Internal ribosome entry site-­‐mediated translation of Apaf-­‐1, but not XIAP, is regulated during UV-­‐induced cell death. J Biol Chem 2006;281:15155-­‐63. 5. Morfoisse F, Kuchnio A, Frainay C, et al. Hypoxia induces VEGF-­‐C expression in metastatic tumor cells via a HIF-­‐1alpha-­‐independent translation-­‐mediated mechanism. Cell Rep 2014;6:155-­‐67. 6. Subkhankulova T, Mitchell SA, Willis AE. Internal ribosome entry segment-­‐mediated initiation of c-­‐Myc protein synthesis following genotoxic stress. Biochem J 2001;359:183-­‐92. 7. Lang KJ, Kappel A, Goodall GJ. Hypoxia-­‐inducible factor-­‐1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol Biol Cell 2002;13:1792-­‐801. 8. Komar AA, Hatzoglou M. Cellular IRES-­‐mediated translation: the war of ITAFs in pathophysiological states. Cell cycle 2011;10:229-­‐40. 9. Hundsdoerfer P, Thoma C, Hentze MW. Eukaryotic translation initiation factor 4GI and p97 promote cellular internal ribosome entry sequence-­‐driven translation. PNAS 2005;102:13421-­‐6. 10. Marash L, Liberman N, Henis-­‐Korenblit S, et al. DAP5 promotes cap-­‐independent translation of Bcl-­‐2 and CDK1 to facilitate cell survival during mitosis. Mol Cell 2008;30:447-­‐59. 2
11. Lewis SM, Cerquozzi S, Graber TE, Ungureanu NH, Andrews M, Holcik M. The eIF4G homolog DAP5/p97 supports the translation of select mRNAs during endoplasmic reticulum stress. Nucleic Acids Res 2007. 12. Nanbru C, Lafon I, Audigier S, et al. Alternative translation of the proto-­‐oncogene c-­‐myc by an internal ribosome entry site. J Biol Chem 1997;272:32061-­‐6. 13. Stoneley M, Paulin FE, Le Quesne JP, Chappell SA, Willis AE. C-­‐Myc 5' untranslated region contains an internal ribosome entry segment. Oncogene 1998;16:423-­‐8. 14. Jopling CL, Willis AE. N-­‐myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene 2001;20:2664-­‐70. 15. Miller DL, Dibbens JA, Damert A, Risau W, Vadas MA, Goodall GJ. The vascular endothelial growth factor mRNA contains an internal ribosome entry site. FEBS Lett 1998;434:417-­‐20. 16. Lang KJ, Kappel A, Goodall GJ. Hypoxia-­‐inducible factor-­‐1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol Biol Cell 2002;13:1792-­‐801. 17. Schwanhausser B, Busse D, Li N, et al. Global quantification of mammalian gene expression control. Nature 2011;473:337-­‐42. 18. Schwanhausser B, Gossen M, Dittmar G, Selbach M. Global analysis of cellular protein translation by pulsed SILAC. Proteomics 2009;9:205-­‐9. 19. Hafner M, Landthaler M, Burger L, et al. Transcriptome-­‐wide identification of RNA-­‐binding protein and microRNA target sites by PAR-­‐CLIP. Cell 2010;141:129-­‐41. 20. Bergamini G, Preiss T, Hentze MW. Picornavirus IRESes and the poly(A) tail jointly promote cap-­‐independent translation in a mammalian cell-­‐free system. RNA 2000;6:1781-­‐90. 21. Rakotondrafara AM, Hentze MW. An efficient factor-­‐depleted mammalian in vitro translation system. Nature protocols 2011;6:563-­‐71. 22. Thoma C, Bergamini G, Galy B, Hundsdoerfer P, Hentze MW. Enhancement of IRES-­‐mediated translation of the c-­‐myc and BiP mRNAs by the poly(A) tail is independent of intact eIF4G and PABP. Mol Cell 2004;15:925-­‐
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