NUCLEAR RECEPTOR REGULATION OF A LUMINAL BREAST CANCER STEM CELL POPULATION by LYNSEY MAE FETTIG B.A., University of North Dakota, 2009 B.S., University of Minnesota, 2011 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Cancer Biology Program 2019 This thesis for the Doctor of Philosophy degree by Lynsey Mae Fettig has been approved for the Cancer Biology Program by Rebecca Schweppe, Chair David Bentley Peter Kabos Maranke Koster Diana Cittelly Jennifer Richer Carol A. Sartorius, Advisor Date: May 17, 2019 ii Fettig, Lynsey Mae (Ph.D., Cancer Biology) Nuclear Receptor Regulation of a Luminal Breast Cancer Stem Cell Population Thesis directed by Professor Carol A. Sartorius ABSTRACT Crosstalk between nuclear receptors is emerging as important in regulating breast cancer (BC) treatment response and progression. Estrogen receptor (ER), progesterone receptor (PR), and retinoic acid receptor (RARα) are all co-expressed in luminal subtype BCs that are generally associated with good prognosis. This is mainly due to the success of endocrine therapies targeting ER. However, eventual resistance to endocrine therapy is a long-standing clinical problem that affects ~30% of patients. About half of all luminal tumors contain subpopulations of ER negative cells that express cytokeratin 5 (CK5). CK5+ cells exhibit cancer stem cell (CSC) properties such as resistance to chemo- and endocrine therapies, and enhanced tumor initiating potential. In previous work we identified that progesterone (P4) expands the population of CK5+ BC cells and that retinoids block such expansion. Thus, we decided to investigate the mechanisms by which PR, RARα, and their cognate ligands regulate breast CSC activity, expression of the CK5 gene, and tumor growth. P4-expanded CK5+ cells were more tumorigenic than CK5− cells in vivo, and P4 treated BC cells formed larger mammospheres in vitro. Either silencing of CK5 via shRNA or co-treatment with retinoic acid (RA) abolished this P4 effect in mammospheres. Using promoter deletion constructs, we identified a region 1.1 kb upstream of the CK5 transcriptional start site (TSS) that is necessary for P4 activation. We next performed ChIP and found that P4 induces association of both PR and RARα with the CK5 promoter at two distinct sites: a distal site (~1.1 kb upstream of the TSS) containing a putative progesterone response element and a proximal site (~130 bp upstream of the TSS) near a known retinoic acid iii response element. RA blocked recruitment of RARα and other essential co-factors to the CK5 promoter. In ongoing studies, we are assessing genome-wide crosstalk between PR and RARα in BC cells using RNA-seq and ChIP-seq. Treatment of BC xenografts in vivo with a retinoid reduced the accumulation of CK5+ cells during estrogen depletion. This reduction, together with the inhibition of CK5+ cell expansion through RAR/PR crosstalk, may explain the efficacy of retinoids in prevention of some BC recurrences. The form and content of this abstract are approved. I recommend its publication. Approved: Carol A. Sartorius iv DEDICATION I wish to thank the incredible people I have met who have encouraged me and helped me get to this point in my life. In particular, my teachers Phil Johnson and Brad Bachmeier who fostered my love of learning and showed me that, even though I am not an artist, I can still think creatively. Also, Dr. Kirsten Nielsen, Dr. Annabell Oh, Dr. Douglas Yee, and Kelly LaPara, who all saw something in me that I did not know was there and stepped in to help guide me through the process of becoming a scientist. I would especially like to thank my mentor, Dr. Carol Sartorius, for taking a chance on me. Even though I came to you under unusual circumstances, you welcomed me into the lab. You have not only been supportive of my career and allowed me to explore science in my own way, you have created an environment where I have been allowed to grow as a person and find my confidence. I cannot thank you enough. I am immensely grateful for my lab mates and graduate school friends. If it were not for you, I would not have survived. I cannot imagine having a more supportive group of people by my side as we tried to find our paths through all of this. You helped me see the good sides and funny sides, and helped pull me through the hard parts. I will forever be thankful for each of our friendships. I would also like to thank my parents, Kevin and Colleen, and my sister, Annie. Never did you tell me there were things in this life that were beyond my reach; instead you helped me figure out ways to make them happen, regardless of how asinine they initially seemed. The times you have come to my rescue – literally and figuratively – are innumerable. You have been there every time I have asked, and many where I did not have to. Without you wonderful people I would crumble. v To my babies, Elliot and Wesley – you make life so much better on the hard days. I hope I can make you proud. Lastly, I wish to thank my husband, Jayden. You have an incredible talent for knowing how I need to be supported, whether it is letting me work by myself at home while you enjoy your time on the ski hill, celebrating with me when it has been a good day for science, or bringing home ice cream when it has in fact not been a good day for science. Our lives have changed so much since we started this adventure, but six years and two kids later I am so glad that you have been my partner through it all. Here’s to the next chapter in our life together. And I promise, no more school. Really. I’m done this time. vi ACKNOWLEDGEMENTS I wish to thank the University of Colorado Cancer Center Flow Cytometry Core, Biorepository Core, Genomics and Microarray Core, and Tissue Culture Core supported by P30CA046934 for their technical assistance and services. This work was supported by UL1 TR002535 via the Colorado Clinical and Translational Sciences Institute (LMF) and National Institutes of Health grants NIH F31 CA210519 (LMF) and NIH 2R01 CA140985 (CAS). For animal work, our IACUC approval number is #000160. vii TABLE OF CONTENTS CHAPTER I. INTRODUCTION ..................................................................................................................... 1 Breast Cancer Incidence, Subtypes, and Treatment ............................................................ 1 Cancer Stem Cells ............................................................................................................... 4 Cytokeratin Structure and Function .................................................................................... 6 Cytokeratins in Breast Cancer ............................................................................................ 9 Nuclear Receptors ............................................................................................................. 12 Estrogen Receptors ............................................................................................... 14 Estrogen in Cancer ................................................................................................ 14 Progesterone Receptors ......................................................................................... 15 Progesterone Receptor in Breast Cancer ............................................................... 16 Retinoic Acid Receptors ....................................................................................... 18 Retinoic Acid Receptors in Cancer ....................................................................... 19 Crosstalk Among Nuclear Receptors .................................................................... 20 Thesis Direction and Summary ......................................................................................... 21 II. MATERIALS AND METHODS .......................................................................................... 23 Cell Culture and shRNA ................................................................................................... 23 CK5 Promoter Deletion Constructs .................................................................................. 23 Luciferase Reporter Assay and siRNA ............................................................................. 23 Chromatin Immunoprecipitation Assay ............................................................................ 24 Mammosphere Formation Assay and Proliferation Assays .............................................. 24 Immunoblotting................................................................................................................. 25 viii Immunohistochemistry, Immunocytochemistry, and Proximity Ligation Assays ............ 25 Quantitative Reverse Transcription PCR and RNA-sequencing ...................................... 26 Limiting Dilution Analysis and Tumor Growth ............................................................... 26 Statistical Methods ............................................................................................................ 27 III. CYTOKERATIN 5 IS DIRECTLY REGULATED BY PROGESTERONE AND IS IMPORTANT IN MEDIATING PROGESTERONE-INDUCED CANCER STEM CELL PROPERTIES ............................................................................................................................. 29 Introduction ....................................................................................................................... 29 Results ..............................................................................................................................