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Thyroid Hormone Receptor SS University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2017 Thyroid Hormone Receptor SS (trß) Regulation Of Runt-Related Transcription Factor 2 (runx2) In Thyroid Tumorigenesis: Determination Of The Trß Nuclear Protein Complexes That Associate With The Runx2 Gene. Thomas Howland Taber University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Cell Biology Commons, and the Oncology Commons Recommended Citation Taber, Thomas Howland, "Thyroid Hormone Receptor SS (trß) Regulation Of Runt-Related Transcription Factor 2 (runx2) In Thyroid Tumorigenesis: Determination Of The rT ß Nuclear Protein Complexes That Associate With The Runx2 Gene." (2017). Graduate College Dissertations and Theses. 820. https://scholarworks.uvm.edu/graddis/820 This Thesis is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. THYROID HORMONE RECEPTOR ß (TRß) REGULATION OF RUNT-RELATED TRANSCRIPTION FACTOR 2 (RUNX2) IN THYROID TUMORIGENESIS: DETERMINATION OF THE TRß NUCLEAR PROTEIN COMPLEXES THAT ASSOCIATE WITH THE RUNX2 GENE. A Thesis Presented by Thomas Howland Taber to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Master of Science Specializing in Pharmacology October, 2017 Defense Date: August 15th, 2017 Thesis Examination Committee: Frances E. Carr, Ph.D., Advisor Jeanne Harris, Ph.D., Chairperson George Wellman, Ph.D. Karen M. Lounsbury, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Thyroid Tumorigenesis is typically a well understood process, with well delineated oncogenic factors. Follicular and papillary thyroid cancers are typically survivable, with 5- year survival rates being >95% for Stage I-III of both cancer types. Anaplastic thyroid cancer, in contrast, lacks this prognosis, and is the most lethal of all endocrine-related cancers. The median survival time after a diagnosis is generally between 6-8 months, with a 5-year survival rate of <10%. Current treatment for anaplastic thyroid cancers routinely meet roadblocks, as resistance is quickly developed. Even non-discriminatory kinase inactivators, such as sorafenib, which are generally considered a drug of last resort, are unable to effect survival rates. As such, there is a clear need for further investigation of the causes of anaplastic thyroid cancer mechanisms. Previous work in the Carr lab revealed a novel regulatory pathway of an oncogene that is associated with several other endocrine-related cancers, as well as other non- endocrine-related cancers. Specifically, the Runt-related transcription factor 2 (Runx2) was found to be suppressed via direct binding of the thyroid hormone receptor beta 1 isoform (TRß1) to its proximal promotor. Runx2 was previously shown to be associated with increasing malignancy, with Runx2 occurring at low-levels in indolent cell lines, whilst occurring at high-levels in more malignant cell lines. TRß1, conversely, exhibited the opposite relationship. Endogenous levels of TRß1 were found to be high in indolent cell lines and were depleted in malignant cell lines. These findings were further confirmed via tissue microarrays. Restoration of TRß1 in malignant cell lines diminished Runx2 mRNA and protein levels, which was corroborated by evidence from electrophoretic mobility-shift assays, and chromatin immunoprecipitations that TRß1 was able to directly bind Runx2 promotor 1. Current studies have investigated the nuclear protein profile that associates with TRß1 to alter Runx2 transcription. Through EMSA-to-Mass Spectrometry methodologies, as well as novel DNA pulldown techniques, binding partners have been elucidated. Findings have also been confirmed via classical immunoprecipitations. Specifically, our findings show that TRß1 complexes with the brahma-related gene 1 (BRG1) protein, the nuclear co-repressor (NCOR), and BRG1-associated protein 60 (BAF60). BRG1 functions by preferentially recruiting histone deacetylases (HDAC), with BRG1 and the HDAC’s acting to alter chromatin, and thus transcription. Future studies aim at examining whether other proteins complex with TRß1 to alter Runx2 transcription, and whether these complexes are altered in aggressive cell lines. ACKNOWLEDGEMENTS I would like to first acknowledge my mentor of the past three years, Dr. Frances E. Carr. As challenging of a student as I may have been, Dr. Frances E. Carr has been an incredibly effective and thoughtful mentor to me throughout my studies at the University of Vermont. Not only has she shown a willingness to accommodate whatever issues I may be going through in life, she has always treated me with the highest respect and dignity. Dr. Frances E. Carr truly serves as an archetype of the definition of what a mentor is. Dr. Frances E. Carr truly embodies the selflessness and dedication that academia represents. I would also like to acknowledge all the members of the Carr lab during my tenure as both an undergraduate and graduate student at the University of Vermont. Jeffery White, Jennifer Tomczak, Michael Barnum, Noelle Gillis, Eric Bolf, Katherine Amidon and Caitlin Beaudet have all been excellent colleagues and all strive to be as helpful as possible, whenever possible. Even though I have been the definition of a train wreck, they have all provided excellent conversation, advice and are truly skilled at denigrating me in the most humorous of manners. I should also acknowledge my family, as I wouldn’t exist without them - literally. Thanks mom and dad. Humor aside, I don’t think I could have made it through the past two years without your love, dedication and support. You guys are truly the real MVP’s. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ ii CHAPTER 1: COMPREHENSIVE LITERATURE REVIEW ..........................................1 1.1 Introduction ................................................................................................................1 1.2 Thyroid Cancer ..........................................................................................................1 1.2.1 Thyroid Cancer Classifications and Statistics ....................................................1 1.2.2 Pathways Associated with Thyroid Tumorigenesis: RAS/PTEN ......................3 1.2.3 Pathways Associated with Thyroid Tumorigenesis: EGFR ...............................5 1.3 Runt-related transcription factor 2 (Runx2)’s role in thyroid tumorigenesis ............8 1.3.1 Runx2’s role in skeletal development and maintenance ....................................8 1.3.2 Runx2’s role in tumorigenesis .........................................................................10 1.3.3 Runx2’s emerging impact in thyroid tumorigenesis ........................................11 1.4 Thyroid Hormone Receptor ß Isoform 1’s role as a tumor suppressor ....................14 1.4.1 Structure and function .......................................................................................14 1.4.2 Isoforms and generalized actions ......................................................................16 1.4.3 Actions of cognate ligand: T3 and T4 ...............................................................18 1.4.4 TRß1 as a tumor suppressor ..............................................................................20 1.4 Ancillary Effectors ...................................................................................................21 1.4.1 Nuclear co-repressors ........................................................................................21 1.4.2 Nuclear co-activators ........................................................................................23 1.4.3 Brahma-related gene 1 ......................................................................................24 1.4.4 Histone deacetylases .........................................................................................25 CHAPTER 2: MATERIALS AND METHODS ...............................................................28 2.1 Research Goals .........................................................................................................28 2.2 Experimental Protocols ............................................................................................29 2.2.1 Cell Culture and Nuclear Protein Extraction ....................................................29 2.2.2 Oligonucleotide Biotinylation ..........................................................................29 2.2.3 Electrophoretic Mobility Shift Assays (EMSA’s) ...........................................31 2.2.4 Immunoblot ......................................................................................................32 2.2.5 Immunoprecipitation ........................................................................................33 2.2.6 DNA Pulldown Assay ......................................................................................33 2.2.7 DNAse I Hypersensitivity Assay .....................................................................34 2.2.8 Sample Preparation for Mass Spectrometry Analysis .....................................35 2.2.9 Analysis of Mass Spectrometry Results ..........................................................36
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