Wayne State University DigitalCommons@WayneState Wayne State University Dissertations 1-1-2012 Identification of transcriptional mechanisms downstream of nf1 gene defeciency in malignant peripheral nerve sheath tumors Daochun Sun Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Recommended Citation Sun, Daochun, "Identification of transcriptional mechanisms downstream of nf1 gene defeciency in malignant peripheral nerve sheath tumors" (2012). Wayne State University Dissertations. Paper 558. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. IDENTIFICATION OF TRANSCRIPTIONAL MECHANISMS DOWNSTREAM OF NF1 GENE DEFECIENCY IN MALIGNANT PERIPHERAL NERVE SHEATH TUMORS by DAOCHUN SUN DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2012 MAJOR: MOLECULAR BIOLOGY AND GENETICS Approved by: _______________________________________ Advisor Date _______________________________________ _______________________________________ _______________________________________ © COPYRIGHT BY DAOCHUN SUN 2012 All Rights Reserved DEDICATION This work is dedicated to my parents and my wife Ze Zheng for their continuous support and understanding during the years of my education. I could not achieve my goal without them. ii ACKNOWLEDGMENTS I would like to express tremendous appreciation to my mentor, Dr. Michael Tainsky. His guidance and encouragement throughout this project made this dissertation come true. I would also like to thank my committee members, Dr. Raymond Mattingly and Dr. John Reiners Jr. for their sustained attention to this project during the monthly NF1 group meetings and committee meetings, Dr. Gregory Kapatos for his understanding and support at the critical time, and Dr. James Garbern for his suggestions for this project at the very beginning. I gratefully acknowledge Dr. Janice Kraniak for her generous help during these years, her assistance with my experiments, and for especially teaching me the importance of precision in science. I also want to thank Mrs. Suzanne Shaw and Mrs. Mary Ann from CMMG, and Mrs. J’nice Stork from Karmanos Cancer Institute for their consideration and assistance throughout my study, and making my overseas education feel like home. iii TABLE OF CONTENTS Dedication ...................................................................................................................... ii Acknowledgements ....................................................................................................... iii List of Tables ................................................................................................................. vi List of Figures .............................................................................................................. vii List of Supplementary Tables ........................................................................................ ix List of Abbreviations ...................................................................................................... x Chapter I: Neurofibromin, a RAS-GAP and more ......................................................... 1 Introduction ............................................................................................................. 1 Chapter II: Identification of downstream gene expression regulation of neurofibromin ...................................................................................................................................... 21 Summary ............................................................................................................... 21 Materials and Methods .......................................................................................... 23 Results ................................................................................................................... 28 Discussion ............................................................................................................. 48 Chapter III: The role of BMP2 in MPNST cells .......................................................... 59 Summary ............................................................................................................... 59 Materials and Methods .......................................................................................... 61 Results ................................................................................................................... 64 Discussion ............................................................................................................. 72 iv Chapter IV: Conclusions and significance ................................................................... 80 References .................................................................................................................. 185 Abstract ...................................................................................................................... 207 Autobiographical Statement ....................................................................................... 209 v LIST OF TABLES Table 1: Mutations assayed for each of the 19 oncogenes in the OncoCarta v1.0 Mutation Panel. ..................................................................................... 53 Table 2: Definition of data set and pathway association ............................................ 54 Table 3: EDN1 related gene expression changes in DS-6 with literature supports. .. 55 Table 4: Canonical pathways associated to the down-regulated genes in DS-9 ........ 55 Table 5: Literature based pathways associated to the downregulated genes in DS-9 .............................................................................................................. 56 Table 6: Canonical pathways associated to the upregulated genes in DS-9 .............. 56 Table 7: Literature based pathways associated to the upregulated genes in DS-9 ..... 57 Table 8: Canonical pathways associated to the downregulated genes in DS-10 ....... 57 Table 9: Literature based pathways associated to the downregulated genes in DS-10 ............................................................................................................ 58 Table 10: Canonical pathways associated to the upregulated genes in DS-10 .......... 58 vi LIST OF FIGURES Fig. 1 Domain scheme of neurofibromin with reported protein-ligand interactions. .. 6 Fig. 2 The signaling pathways downstream of RAS .................................................. 14 Fig. 3 Neurofibromin and Phos-ERK1/2 in MPNST cell lines ................................. 32 Fig. 4 Scheme of intersection analysis of gene profiles ............................................. 32 Fig. 5 Verification of interference conditions in MPNST cell lines ........................... 33 Fig. 6 Quantitative RT-PCfR verification of Edn1 expression in microarrays .......... 38 Fig. 7 EDN1 related molecules network built by the IPA ......................................... 39 Fig. 8 Quantitative RT-PCR analysis of Edn1 mRNA expression in MPNST cell lines ................................................................................................................. 40 Fig. 9 Bmp2 mRNA expression in NF1-associated tissue samples ........................... 44 Fig. 10 Quantitative RT-PCR verification of Bmp2 mRNA change in different comparisons .................................................................................................... 45 Fig. 11 Neurofibromin status influences the Bmp2 mRNA expression in STS26T (Nf1+/+) ........................................................................................................... 46 Fig. 12 Inducible RAS expression system in STS26T(Nf1+/+) .................................. 46 Fig. 13 Inducible oncogenic RAS failed to change the expression of Bmp2 mRNA 47 Fig. 14 Status of phosphorylated SMAD1/5/8 in MPNST cell lines ......................... 66 Fig. 15 siBmp2 inhibited Bmp2 mRNA accumulation and the phosphorylation of SMAD1/5/8 ................................................................................................... 67 Fig. 16 NRAS and BMP2 influence the activity of SMAD1/5/8 independently ....... 67 vii Fig. 17 Efficacy of LDN-193189 concentration series to inhibit SMAD1/5/8 in T265(Nf1-/-) ............................................................................................... 69 Fig. 18 LDN-193189 time dependent inhibition of phosphorylation of SMAD1/5/8 in T265(Nf-/-). ................................................................................................. 69 Fig. 19 Migration of MPNST cell lines with LDN-193189 at 24 h in scratch assay with different FBS concentration .................................................................. 76 Fig. 20 The growth curve of T265NonKD ................................................................ 76 Fig. 21 Bmp2 knockdown by lentivirus in ST88-14(Nf1-/-) impaires cell migration ........................................................................................................ 77 Fig. 22 BMP2 and LDN-193189 influenced the invasion ability of ST88-14(Nf1-/-) and T265(Nf1-/-) ................................................................... 78 Fig. 23 Bmp2 stable knockdown lines indicated decreased invasion