Transfer RNA-mediated enhancement of RSK/MSK signaling and its connection to tumorigenesis

다국어 초록 (Multilingual Abstract)

Transfer RNA (tRNA) is essential RNA for protein synthesis, transporting amino acids to the ribosome. tRNA has been known for house-keeping RNA for a long time, however, recent studies suggest that tRNA can be a multi-functional molecule that controls various cellular events including translation initiation and cell apoptosis.
In various cancer cell lines and tissues, although increased expression of tRNA was observed, the function of the overexpressed tRNA was unclear. We hypothesized that tRNA would play a pro-oncogenic role during tumorigenesis, therefore we overexpressed tRNA in 293T cells and investigated the effect of tRNA overexpression on cell proliferation and survival. Interestingly, 293T cells with high expression level of tRNA, especially tRNALeu, showed increased resistance to cell death and enhanced phosphorylation of 90-kDa S6 kinase under amino acid starvation condition. Phosphorylation of 90-kDa S6 kinase was reduced by anti-tRNALeu RNA, p38MAPK inhibitor and ERK inhibitor, but not by treatment of mTOR inhibitor, rapamycin. RSK1 (Ribosomal S6 kinase 1) and MSK2 (Mitogen-and stress-activated protein kinase 2) were identified as the tRNALeu-responsive effectors among the tested 7 candidate proteins categorized as 90-kDa S6 kinase. Considering that RSK1 and MSK2 are downstream regulators of p38MAPK and ERK and play an important role in hormonal cancer such as estrogen-responsive breast cancer, we analyzed the expression level of tRNALeu in breast cancer cell lines and its responsiveness to ß-estradiol. Estrogen receptor (ER) positive MCF7 cell line showed the highest expression levels of tRNALeu isotypes compared with ER negative cell lines and its tRNALeu expression was induced by ß-estradiol treatment. We also investigated the protein binding partner of tRNALeu under amino acid starvation condition using SILAC (Stable Isotope Labeling by/with amino acids in cell culture) mass spectrometry and identified EBP1 (ErbB3-binding protein), which is the upstream regulator of RSK/MSK signal pathway.
These results indicate that tRNALeu can be induced by estrogen and enhance EGFR signaling via binding with EBP1, subsequently activating RSK/MSK to increase survival of breast cancer cells under amino acid starvation condition. This study also suggests a possibility that tRNA can work as a new cellular regulator beyond translation and its novel function in tumorigenesis.

목차 (Table of Contents)

• ABSTRACT 01
• CONTENTS 04
• LIST OF FIGURES 06
• Ⅰ. INTRODUCTION 07
• Ⅱ. MATERIALS AND METHODS 10
• 1.Materials 10
• 2.Cell culture 10
• 3.Establishment of Tet-On stable cell line 11
• 4.Inhibitor treatment 12
• 5.Thymidine incorporation assay 12
• 6.Methionine incorporation assay 13
• 7.1-dimensional electrophoresis and Western blot 13
• 8.Semi-quantitative RT-PCR 15
• 9.Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) 15
• 10. RNA interference 16
• 11.SILAC Mass spectrometry 17
• 12.Anchorage independent transformation assay 19
• 13. Incucyte (Live Cell Kinetic Imaging System) 19
• Ⅲ. RESULTS 21
• 1. tRNALeu increases cell survival 21
• 2. tRNALeu increases phosphorylation of 90-kDa S6 kinase in amino acid starvation condition 22
• 3. Phosphorylation of 90-kDa S6 kinase by tRNALeu is independent to mTOR pathway 23
• 4. tRNALeu is overexpressed in breast cancer cell and induced by β-estradiol treatment 24
• 5. tRNALeu mediates RSK/MSK signaling by binding EBP1 25
• 6. tRNALeu promotes cell transformation 26
• Ⅳ. DISCUSSION 40
• Ⅵ. REFERENCES 44
• Ⅶ. 국문 초록 46