Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Summer 8-15-2016 The p38MAPK-MK2-HSP27 axis regulates the mRNA stability of the pro-tumorigenic senescence-associated secretory phenotype Hayley Reynolds Moore Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Recommended Citation Moore, Hayley Reynolds, "The p38MAPK-MK2-HSP27 axis regulates the mRNA stability of the pro-tumorigenic senescence- associated secretory phenotype" (2016). Arts & Sciences Electronic Theses and Dissertations. 873. https://openscholarship.wustl.edu/art_sci_etds/873 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Molecular Cell Biology Dissertation Examination Committee: Sheila A. Stewart, Chairperson Peter Burgers Nicholas Davidson Sergej Djuranovic Joshua Rubin Zhongsheng You The p38MAPK-MK2-HSP27 axis regulates the mRNA stability of the pro-tumorigenic senescence-associated secretory phenotype by Hayley R. Moore A dissertation presented to the Graduate School of Arts & Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2016 St. Louis, Missouri Table of Contents List of figures ................................................................................................................................. v Acknowledgements ...................................................................................................................... vii Abstract ......................................................................................................................................... ix Chapter 1: Background and Significance, Senescence .............................. 1 1.1 Overview and significance ..................................................................................... 2 1.2 Mechanisms governing senescence ....................................................................... 4 1.3 The senescence-associated secretory phenotype ................................................... 5 1.4 Transcriptional regulation of the SASP ................................................................. 7 1.5 Post-transcriptional regulation of SASP mRNAs.................................................. 9 1.5.1 AUF1 and AU-rich-element-mediated mRNA degradation ............................ 10 1.5.2 The p38MAPK-MK2 mRNA degradation pathway ........................................ 11 1.6 Summary .............................................................................................................. 13 References ........................................................................................................................ 17 Chapter 2: The p38MAPK-MK2-HSP27 axis regulates the mRNA stability of the senescence-associated secretory phenotype .......................... 23 Abstract ............................................................................................................................ 24 Introduction ...................................................................................................................... 24 Materials and Methods ..................................................................................................... 28 Results .............................................................................................................................. 33 HSP27 destabilizes SASP factor mRNA in pre-senescent cells ........................... 33 p38MAPK and MK2 regulate mRNA stability in senescent cells ........................ 35 The p38MAPK-MK2-AUF1 axis regulates mRNA stability in senescent cells through HSP27 phosphorylation ...................................................................... 37 The p38MAPK-MK2-HSP27 axis promotes stromal-supported tumor cell growth .............................................................................................. 40 Discussion ........................................................................................................................ 41 Acknowledgements .......................................................................................................... 46 References ........................................................................................................................ 54 Chapter 3: Conclusions and Future Directions, Senescence ................ 58 3.1 Summary .............................................................................................................. 59 3.2 The p38MAPK-MK2-HSP27-AUF1 axis regulates post-transcriptional stabilization of SASP factor mRNAs in senescence ........................................ 60 ii 3.3 Inhibiting the p38MAPK-MK2-HSP27 pathway abrogates the growth promotion of epithelial cells by senescent fibroblasts ................... 63 3.4 Conclusions .......................................................................................................... 65 References ........................................................................................................................ 67 Chapter 4: Background and Significance, DNA metabolism ............... 69 4.1 Overview and significance .................................................................................. 70 4.2 Genomic instability as a driver of disease ........................................................... 70 4.2.1 DNA replication fidelity .................................................................................. 71 4.2.2 DNA repair and replication fork progression .................................................. 72 4.2.3 Telomere maintenance ..................................................................................... 73 4.3 Dna2 functions to ensure genomic stability ......................................................... 75 4.3.1 Dna2’s function in lagging-strand DNA replication ........................................ 77 4.3.2 Dna2 participates in homologous recombination ............................................ 78 4.3.3 Dna2 protects telomere integrity ...................................................................... 78 4.4 Summary .............................................................................................................. 80 References ........................................................................................................................ 84 Chapter 5: An Okazaki fragment processing-independent role for human Dna2 during DNA replication ............................... 90 Abstract ............................................................................................................................ 91 Introduction ...................................................................................................................... 92 Materials and Methods ..................................................................................................... 94 Results ............................................................................................................................ 101 hDna2 contributes to genomic stability ............................................................. 101 hDna2’s nuclease and helicase activities are essential ..................................... 102 hDna2 interacts with the replisome protein And-1 in a replication dependent manner ................................................................. 104 hDna2 depletion leads to replication checkpoint activation ............................. 105 Defects in Okazaki fragment processing do not alter replication fork progression ........................................................................... 107 hDna2 depletion does not lead to detectable defects in maturation of newly synthesized DNA ........................................................ 108 Discussion ...................................................................................................................... 110 Acknowledgements ........................................................................................................ 114 References ...................................................................................................................... 124 iii Chapter 6: Conclusions and Future Directions, DNA metabolism ..130 6.1 Summary ............................................................................................................ 131 6.2 Dna2 has non-Okazaki fragment processing roles in DNA replication ............ 131 6.3 Dna2 is required for replication fork restart ...................................................... 133 6.4 Dna2 knockout induces a stringent cell cycle arrest and abnormal metaphase chromosomes ......................................................... 135 6.5 Conclusions ....................................................................................................... 137 Materials and methods ................................................................................................... 138 References .....................................................................................................................