ii Dysregulation of the Wnt signaling pathway in the development of colorectal cancer in HIV-infected patients By Jennifer Marcy August 2018 A Dissertation Presented to the Faculty of Drexel University College of Medicine in partial fulfillment of the Requirements for the Degree of Masters in Science in Molecular and Cellular Biology and Genetics _________________________ _________________________ Vanessa Pirrone, PhD Brian Wigdahl, PhD Assistant Professor Professor and Chair Microbiology and Immunology Microbiology and Immunology _________________________ Todd Strochlic, PhD, VMD Assistant Professor Biochemistry and Molecular Biology iii Copyright by Jennifer Marcy 2018 iv DEDICATION This master’s thesis is dedicated to the strong individuals who are responsible for creating brilliant minds and to populate a future of caring and responsible adults. As a stay at home mom for 20 years, it has been a long journey to get to this stage. I had a turning point when my kids were infants where I would say to myself “This is so boring hanging out with basically two pieces of Jello.” I gave myself a job title: Brain Grower. My business plan involved finding new stimuli in the world every day so they could cultivate new synaptic connections and develop into contributing members of society. I did this for them, I did this for me. Thank you to my children for pushing me to be a better person and role model. You have truly enriched my life in ways you will never know. If there was a single person who was responsible for showing me the ability to have a different life, it would be my ex-husband. He has given me the opportunity and kick in the ass that was needed so I could go into the world, reinvent, and discover myself. In the end however, I have to dedicate this portion of my new life to my faithful companion and best friend, Nutmeg who has saved my life in more ways than one. I truly appreciate every single one of you and I hope I have made you proud. “What lies behind you and what lies before you are small matters compared to what lies within you”. – Ralph Waldo Emerson v ACKNOWLEDGEMENTS Thank you to all those who have, directly or indirectly, influenced the completion of this thesis. I would like to express my sincere gratitude to my mentor, Dr. Vanessa Pirrone, for the continuous support of my studies and related research, for her patience, motivation, and immense knowledge. The door to Dr. Pirrone’s office was always open whenever I ran into a trouble spot or had a question. She consistently allowed this paper to be my own work but rescued me many times in these deep waters whenever I needed it. Besides my mentor, I would like to thank the rest of my thesis committee: Dr. Brian Wigdahl and Dr. Todd Strochlic, for their insightful comments, encouragement, and questions which incented me to widen my research from various perspectives. I would like to thank my friends for accepting nothing less than excellence from me. I thank my fellow lab mates for stimulating discussions and all the fun we have had in the last two years. Last but not least, I would like to thank my family: my children, my brother, sister, and parents for supporting me throughout these studies and my life in general. vi TABLE OF CONTENTS Chapter I: Gastrointestinal cancers in patients infected with HIV ........................... 1 1.1. Abstract ................................................................................................. 2 1.2. Introduction ............................................................................................. 3 1.3. Human Immunodeficiency virus (HIV) ................................................... 4 1.4. Mechanism of cancer initiation ............................................................ 10 1.5. Cancers of the gastrointestinal tract .................................................... 13 1.6. Interplay between oncogenic pathways within the GI tract and potential HIV interacts within these pathways ..................................... 19 1.6.1 BMP ......................................................................................... 20 1.6.2 Notch ....................................................................................... 22 1.6.3 EGF pathway........................................................................... 28 1.6.4 Hedgehog signaling ................................................................ 30 1.6.5 Wnt .......................................................................................... 31 1.7. ART penetration ................................................................................... 34 1.8. Conclusion ............................................................................................ 37 1.9. List of References ................................................................................ 39 Chapter II: Dysregulation of the Wnt signaling pathway in the development of colorectal cancer in HIV-infected patients ............................................................. 52 2.1. Abstract ............................................................................................... 53 2.2 Introduction ............................................................................................ 54 vii 2.3. Materials and Methods ......................................................................... 59 2.3.1. Plasmid growth and purification ............................................. 59 2.3.2. Cell culture ............................................................................. 61 2.3.3. Transient transfection ............................................................ 61 2.3.4. Microscope pictures ............................................................... 62 2.3.5. Lentiviral production and transduction................................... 62 2.3.6. Protein isolation ...................................................................... 62 2.3.7. Bicinchoninic acid assay ........................................................ 62 2.3.8. RNA extraction ....................................................................... 63 2.3.9. Western blot ........................................................................... 63 2.3.10. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) ............................................................................ 63 2.3.11. TOP-/FOP-FLASH Wnt reporter .......................................... 64 2.4. Results ............................................................................................... 64 2.4.1. Transfection of HCT116 cells ................................................ 65 2.4.2. Downstream gene product detection..................................... 67 2.4.3. Gene product detection utilizing quantitative reverse transcription PCR (RT-qPCR) .............................................. 71 2.4.4. TOP/FOP Flash -catenin expression in HCT116 cells ....... 71 2.4.5. Nef affects downstream Wnt pathway protein expression in HEK293T cells ....................................................................... 73 2.5. Discussion ............................................................................................ 77 2.6. List of References ................................................................................ 83 viii LIST OF FIGURES Figure 1.1. Structures of polyanionic compounds assessed as systemic inhibitors ................................................................................................................................ 24 Figure 1.2. Structures of polyanionic compounds assessed as microbicidal compounds ............................................................................................................. 27 Figure 1.3. Combination of inhibitors with different mechanisms of action to provide full protection from HIV-1 infection through sexual transmission .......................... 35 Figure 2.1. Proof of transfection efficiency ............................................................ 66 Figure 2.2. Cyclin D1 is not detected in HCT 116 colorectal cancer cells following transfection with HIV-1 Nef .................................................................................... 68 Figure 2.3. Downstream Wnt target gene c-myc expression ................................ 70 Figure 2.4. C-myc and CyclinD1 expression in HCT116 cells .............................. 72 Figure 2.5. -catenin expression in HCt116 and HEK293T cells.......................... 74 Figure 2.6. Wnt target gene expression in HEK293T cells ................................... 76 ix Abstract Dysregulation of the Wnt signaling pathway in the development of colorectal cancer in HIV-infected patients Jennifer J. Marcy Vanessa Pirrone With the advent of antiretroviral therapy (ART), patients with human immunodeficiency virus-1 (HIV-1) have experienced an improvement in morbidity and life expectancy. As this patient population ages, there has been evidence of an elevated risk and earlier onset of colonic neoplasia. The average age of developing colon cancer in HIV-1-infected patients is 48 compared to 60 in the general population. Moreover, the stage of colorectal cancer diagnosed in HIV-1- infected patients is more advanced compared to uninfected individuals. During primary HIV-1 infection, an observed loss of CD4+ T cells is seen in the gastrointestinal (GI) tract along with subsequent systemic immune hyperactivation. Additionally, increased expression of proinflammatory cytokines in the GI tract during HIV-1 infection results in chronic mucosal inflammation.