Poster Presentation 27th Lorne Cancer Conference 2015

Using genome-wide analysis of translational activity to investigate the response of cancer cells to therapies targeting the ribosome (#193)

Eric P Kusnadi 1 , Katherine M Hannan 1 2 , Ross D Hannan 1 2 3 4 5 , Richard B Pearson 1 2 3 4
  1. Division of Cancer Research, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
  2. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia
  3. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia
  4. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
  5. School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia

Protein synthesis is rate limiting for cell growth and division, and therefore requires coordinate regulation to maintain cellular homeostasis [1]. Protein synthesis rates are dependent on how fast the ribosomes can translate the mRNAs (i.e. “efficiency”) and the number of functional ribosomes (i.e. “capacity”). However, elevated translation efficiency may eventually be limited by capacity. Ribosome biogenesis is a complex multistep process that includes the synthesis of ribosomal (r) RNAs and rproteins, rRNA processing and ribosomal subunits assembly [2, 3]. Importantly, elevated ribosome biogenesis and protein synthesis are characteristics of rapidly proliferating cancer cells [1, 3]. Targeting signaling pathways to alter ribosome function (e.g. mTORC1 inhibitor Everolimus) [4] or targeting ribosome synthesis itself (RNA Polymerase I inhibitor CX-5461) [5] can selectively kill lymphoma cells in vivo while sparing normal cells, thus are promising approaches to treat cancer. We hypothesise that therapies combining targeting ribosome synthesis and function will act synergistically via specific changes in the translatome (mRNAs being actively translated). Characterisation of these translatome will provide new understanding of the mechanisms of cellular response to these agents and identify potential targets for improving the efficacy of treatments targeting the ribosome. This project utilises polysome profiling as a high-throughput translational landscape analysis. Extracts from HeLa and Eμ-Myc model of B-cell lymphoma cells were subjected to sucrose density gradient ultracentrifugation and fractionation, which separates the mRNAs based on the number of ribosomes bound to them. Quantitative real-time polymerase chain reaction (qRT-PCR) and RNA-seq analyses were used to monitor changes in the translation efficiency of specific mRNAs in response to modulated amino acids levels or Everolimus and CX-5461 treatment. In conclusion, we have optimised a technique to analyse genome-wide mRNA translation as a tool for understanding the mechanism by which treatments targeting ribosome synthesis and function mediate cellular response(s) in malignant cells.

  1. Ruggero, D., Translational control in cancer etiology. Cold Spring Harbor perspectives in biology, 2013. 5(2): p. a012336
  2. Hein, N., et al., The nucleolus: an emerging target for cancer therapy. Trends in molecular medicine, 2013. 19(11): p. 643-654
  3. Bywater, M.J., et al., Dysregulation of the basal RNA polymerase transcription apparatus in cancer. Nature Reviews Cancer, 2013. 13(5): p. 299-314
  4. Devlin, J.R., et al., AKT signalling is required for ribosomal RNA synthesis and progression of Eμ‐Myc B‐cell lymphoma in vivo. FEBS Journal, 2013. 280(21): p. 5307-5316
  5. Bywater, M.J., et al., Inhibition of RNA polymerase I as a therapeutic strategy to promote cancer-specific activation of p53. Cancer cell, 2012. 22(1): p. 51-65