Understanding the Biology of Triple Negative Breast Cancer and Breast Cancer Stem Cells in Patients of Diverse Ethnicities. by Evelyn Mawunyo Jiagge A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cancer Biology) In The University of Michigan 2017 Doctoral Committee: Professor Sofia D Merajver Co-Chair Professor Max S Wicha, Co-Chair Dr. Gary D Hammer Dr. Elizabeth R. Lawlor Dr. Jacques Eduardo Nor Acknowledgement My profound gratitude to my advisor, Dr. Sofia D Merajver for her guidance, time, ever-present support and mentorship. My sincere gratitude to my thesis co-chair, Dr. Max S Wicha for his support and guidance. I am very thankful to the members of my dissertation committee: Dr. Gary Hammer, Dr. Beth Lawlor and Dr. Jacques E Nor for their great support, time and for directing my work. Thank you very much, Dr. Lisa A Newman and ICSBCS team for the enormous amount of resource and time you invested into this work. Thank you to the many wonderful people in the Merajver and Wicha labs who contributed in diverse ways to my work. Special thanks to Dr. Sean McDermott for teaching me to be a scientist and Dr. Rabia Gilani for contributing significantly to my work. Many thanks to our collaborators at the Komfo Anokye teaching hospital, Ghana who sacrificed their time and energy to prepare patients for this work. Thank you to our collaborators at TGEN, especially Shukmei Wong for the many hours they invested into preparing and sequencing our sample. Thank you to Dr. Jun Li and the Li lab for analyzing my data. ii My sincere thanks to the University of Michigan flow Cytometry core for their time and patience in analyzing my many samples. I am grateful to the ULAM and Cancer center pathology labs for the work you performed on my samples. A special thanks to all the patients in Ghana and in the US who consented to their samples being used for this work My sincere gratitude and love to my dear husband and the love of my life, Dr. Sylvester Antwi for sacrificing everything to make this work possible. Thank you Nyameye and Judah for allowing me to do this work. This work was supported in part by grants: Komen promise grant, the Breast Cancer Research Foundation (MW, SDM), the Metavivor Foundation (SDM), the UM CCC core grant and Rackham Barbour Scholarship. iii Preface A version of Chapter II, Breast Cancer and African Ancestry: Lessons Learned at the 10-Year Anniversary of the Ghana-Michigan Research Partnership and International Breast Registry was published is accepted in Journal of Global Oncology and online February 5, 2016. The list of authors is as follows: Evelyn Jiagge, Joseph Kwaku Oppong, Jessica Bensenhaver, Francis Aitpillah, Kofi Gyan, Ishmael Kyei, Ernest Osei-Bonsu, Ernest Adjei, Michael Ohene-Yeboah, Kathy Toy, Karen Eubanks Jackson, Marian Akpaloo, Dorcas Acheampong, Beatrice Antwi, Faustina Obeng Agyeman, Zainab Alhassan, Linda Ahenkorah Fondjo, Osei Owusu-Afriyie, Robert Newman Brewer, Amma Gyamfuah, Barbara Salem, Timothy Johnson, Max Wicha, Sofia Merajver, Celina Kleer, Judy Pang, Emmanuel Amankwaa-Frempong, Azadeh Stark, Francis Abantanga, Lisa Newman, Baffour Awuah A portion of chapter III, Comparative Analysis of Breast Cancer Phenotypes in African American, White American, and West Versus East African patients: Correlation Between African Ancestry and Triple-Negative Breast Cancer, is accepted in Annals of Surgical Oncology and was published online November 23, 2016. The list of authors are as follows Evelyn Jiagge, Aisha Souleiman Jibril, Dhananjay Chitale, Jessica M. Bensenhaver, Baffour Awuah, iv Mark Hoenerhoff , Ernest Adjei, Mahteme Bekele, Engida Abebe, S. David Nathanson, Kofi Gyan, Barbara Salem, Joseph Oppong, Francis Aitpillah, Ishmael Kyei, Ernest Osei Bonsu, Erica Proctor , Sofia D. Merajver, Max Wicha, Azadeh Stark A version of Chapter IV, ALDH expression characterizes tumor initiating stem cells in Triple Negative breast cancers across different ethnicities is being submitted to Cancer Research. The author list is as follows: Evelyn Jiagge, Shukmei Wong , Rabia Gilani, Sean McDermott PhD, Qingxuan Song, Tahra Luther, Jungsoo Chang, Jessica Bensenhaver, Michele Dziubinski, Joseph Kwaku Oppong, Francis Aitpillah, Ishmael Kyei, Ernest Osei-Bonsu, Ernest Adjei, Awuah Baffour, Jessica Aldrich, Ahmet Kurdoglu, David Craig, Jeff Trent, Jun Li, Lisa Newman, Max Wicha, John Carpten, Sofia Merajver, v Table of Contents Acknowledgement ............................................................................................... ii Preface ................................................................................................................ iv List of Figures ...................................................................................................... x List of Tables ..................................................................................................... xii Abstract ............................................................................................................. xiv Chapter 1 : Introduction ...................................................................................... 1 Breast Cancer Burden .................................................................................................................. 1 Breast Cancer Incidence and Mortality ........................................................................................ 2 Breast Cancer Sub-types ............................................................................................................. 8 Triple Negative Breast Cancer ................................................................................................... 10 Hereditary Breast Cancer and Breast Cancer in Blacks ............................................................ 12 Breast Cancer Treatment ........................................................................................................... 14 Cancer Stem Cells ..................................................................................................................... 15 Breast Cancer Stem Cells .......................................................................................................... 17 Breast Cancer Ctem cells and Breast Cancer in Blacks ............................................................ 18 Implications of CSCs for Cancer Therapy .................................................................................. 19 Models for Breast Cancer Research .......................................................................................... 20 References .......................................................................................................... 22 vi Chapter 2 : Breast Cancer and African Ancestry: Importance of International Collaborations ............................................................................. 33 Abstract ............................................................................................................... 33 Background ......................................................................................................... 34 Breast Cancer and African Ancestry- Patterns identified through the IBR ................................. 37 Breast Cancer in Ghana- Diagnostic and Treatment Advances Promoted by the Ghana- Michigan Partnership .................................................................................................................. 44 Breast Cancer and African Ancestry: The Ghana-Michigan Partnership as a Model for Expansion ................................................................................................................................... 47 References .......................................................................................................... 52 Chapter 3 : Correlation Between African Ancestry, Triple Negative Breast Cancer and BRCA Gene Expression ............................................................... 57 Abstract ............................................................................................................... 57 Introduction .......................................................................................................... 60 Methods ............................................................................................................... 61 Ethics .......................................................................................................................................... 61 Pathology and Immunohistochemistry ....................................................................................... 62 Exome Sequencing and Data Analysis ...................................................................................... 63 Ascertainment of Cases ............................................................................................................. 64 Statistical Analysis ...................................................................................................................... 65 Results ................................................................................................................ 66 Expression of BRCA1 and BRCA2 protein in Ghanaian TNBC patients. ................................... 74 Recurrently Mutated Genes ....................................................................................................... 78 vii Discussion ........................................................................................................... 80 References .......................................................................................................... 88 Chapter 4 : ALDH Expression Characterizes Tumor Initiating Stem Cells in Triple Negative Breast Cancers across Different Ethnicities. ....................... 91 Abstract ............................................................................................................... 91 Introduction .......................................................................................................... 93 Methods ............................................................................................................... 96 Patient Derived Xenograft Generation ....................................................................................... 96 Tissue Dissociation and Flow Cytometry ................................................................................... 97 Breast Cancer Stem Cells Whole Transcriptome Library Construction and Paired End NGS: .. 98 Data Analysis ............................................................................................................................. 99 Tumorsphere Formation Assay ................................................................................................ 100 Results .............................................................................................................. 100 Creating PDX based TNBC Models from Tumors of Women of Different Ancestries. ............. 100 Isolating Breast Cancer Stem Cells and Bulk Cell Sub-populations from Established PDX .... 104 ALDH+ Stem Cell Population has a Distinct Gene Expression Profile from CD44+/CD24- and BULK Population, in TNBC PDXs from all Ethnicities. ............................................................. 107 ALDH+ not CD44+/C24- Cells Exhibit Stem Cell Characteristics in Primary TNBCs. .............. 110 In Primary TNBC ALDH+ Stem Cells are Characterized by a Distinct Gene Expression Profile. .................................................................................................................................................. 110 Significant signaling interactions exist between the genes that are differentially altered in ALDH+ stem cells in primary TNBC. ..................................................................................................... 114 Discussion ......................................................................................................... 116 References ........................................................................................................ 121 viii Chapter 5 : Summary and Discussion ........................................................... 126 Introduction ........................................................................................................ 126 Summary of Chapters ....................................................................................... 129 Future Directions. .............................................................................................. 133 Final Remarks ................................................................................................... 135 References ........................................................................................................ 137 ix List of Figures Figure 1-1: GLOBOAN 2012 global breast cancer incidence to mortality ratio ..... 4 Figure 1-2 : Trends in Female Breast Cancer Incidence and Mortality Rates by Race/Ethnicity, United States, 1975 to 20121 ....................................................... 6 Figure 1-3: Female breast incidence trends, select countries, 1973–2012 ........... 7 Figure 2-1: International variation in breast cancer incidence, mortality, and mortality-incidence ratios. Data from Jemal et al7, Globocan 20088, Newman et al 9 and Jiagge et al10. ....................................................................................... 35 Figure 2-2: Population migration patterns; the African diaspora; adapted from Campbell, et al20 .................................................................................................. 42 Figure 3-1: A comparison of breast cancer phenotypes across different ethnicities: ........................................................................................................... 72 Figure 3-2: BRCA immunostaining ...................................................................... 75 Figure 3-3: Results of BRCA1 and BRCA2 IHC staining in Ghanaian BC patients ............................................................................................................................. 76 Figure 3-4: Pipeline for somatic variant calling. ................................................... 77 Figure 4-1: A. processing and stem cell characterization from the patient derived xenograft ........................................................................................................... 101 Figure 4-2: schematic for flow sorting for bulk and stem cell sub-populations .. 103 x