The Role of Heme Oxygenase in Metastatic Melanoma
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THE ROLE OF HEME OXYGENASE IN METASTATIC MELANOMA TUMORIGENICITY ________________________________________________________________ A Dissertation Presented to The Faculty of the Graduate School Of the University of Missouri ________________________________________________________________ In Partial Fulfillment Of The Requirements for the Degree Doctor of Philosophy ________________________________________________________________ By KIMBERLY J. JASMER Dr. Mark Hannink and Dr. Steve Alexander, Dissertation Advisors July 2015 The undersigned, appointed by the Dean of the Graduate School, Have examined the dissertation entitled THE ROLE OF HEME OXYGENASE IN METASTATIC MELANOMA TUMORIGENICITY Presented by Kimberly J. Jasmer A candidate for the degree of Doctor of Philosophy And hereby certify that, in their opinion, it is worthy of acceptance. ________________________________________________________________ Dr. Mark Hannink. Dissertation Co-Advisor ________________________________________________________________ Dr. Steve Alexander, Dissertation Co-Advisor ________________________________________________________________ Dr. David Setzer ________________________________________________________________ Dr. Troy Zars In Loving Memory of Ruby Christiansen and Elmer “Al” Jasmer ACKNOWLEDGEMENTS First, I’d like to thank my doctoral co-advisors, Dr. Mark Hannink and Dr. Steve Alexander, who have supported me in countless ways. My graduate career had a bit of an unorthodox start as I tried to balance obtaining a graduate degree with competitive swimming. Dr. Alexander took on the challenge, brought me into his lab, and patiently introduced me to many molecular biology techniques, which I’ve used throughout my time as a graduate student. He, Dr. Hannah Alexander, and Dr. David Setzer even came to watch one of my swim meets. Steve, the insights you shared with me, your honesty, your belief in my abilities as a scientist and support of me as a whole person will be forever appreciated. Dr. Mark Hannink has provided endless opportunities for me to collaborate on projects, mentor students, and experience aspects of a young researchers career that are not necessarily included in ones graduate education. Mark, you hold high expectations, thank you for the opportunity and the tools to live up to them. Thank you for the encouragement when I needed it and for creating an environment in which I was able to work independently, pursuing my own ideas, while receiving the benefits of your expertise. I’d also like to thank Dr. David Setzer and Dr. Troy Zars for serving on my committee: providing support, encouragement, and invaluable feedback over the past five years. I’d like to acknowledge my labmates, in particular, Jordan Wilkins, Cy McConnell, and Rich Sachdev for introducing me to techniques, troubleshooting my experiments, and eating Taj Mahal for lunch more times than I can count. I’d like to extend my gratitude to the Division of Biological Sciences for becoming my home away from home. I’d particularly like to thank Nila Emerich for constantly answering my questions and providing support throughout this process and Josh Hartley ii" for rescuing my crashed hard drive, recovering important files. I am grateful for the GAANN fellowship I received which financially supported me for four years and gave me the opportunity to gain teaching experience. Next, I’d like to acknowledge my parents who have provided love and support throughout this journey. Dad, you are my biggest fan. Thank you, for constantly checking in on me and making sure I am holding it together, especially these past few months. Mom, you have been my rock. Thank you for celebrating each milestone throughout this and lending an ear to vent about every frustration. Without both of you, I could not have made it through the struggles of graduate school and I most definitely could not have planned a wedding while simultaneously finishing my degree. I need to thank Sean Hutchison, my swim coach in Seattle, and his thoughtful input and encouragement to pursue graduate school. I also would like to thank Patrick Rowan and Phil Garverick, two Missouri swim coaches, who gave me a sanctuary to escape to as well as love and support over these past few years. I’d like to thank Mizzou Swimming for funding my first year of graduate school and bringing me to the Midwest. I’d also like to acknowledge my grandmother, Mel. Thank you, grandma, for your support, your worried emails every time there is a tornado in or near Missouri, and for canning Oregon tuna so that I can have a piece of home, even 2,000 miles away. Thank you for sending your love every day of this process. Finally, I have to thank my (soon-to-be) husband for being a sounding board to bounce ideas off, for reading this entire dissertation and providing valuable feedback, and, perhaps most impressively, dealing with last minute wedding preparations so that I could focus on finishing my dissertation. Alex, thank you for picking up my slack, talking me through my panic attacks, and being proud of me, despite my overuse of the word ‘which’ in my writing. iii" TABLE OF CONTENTS Acknowledgements…………………………………..……………………ii Table of Contents………………………………………….…………...…iv List of Figures……………..……………………………………………….v List of Tables………..………………………...………………………..…vii Chapter 1: Introduction: Bach1 Regulation of the Antioxidant Response Element (ARE): Implications for Melanoma Metastatic Tumorigenesis…..……....................................1 Chapter 2: Heme Oxygenase plays a role in the anchorage- independent melanosphere formation of B-Raf-active melanomas…..................................................................24 Chapter 3: A Novel Cell Line Model for Melanoma Development…..67 Chapter 4: Use of CHIP-qPCR to Characterize the Occupancy of Antioxidant Response Elements (AREs); Characterization of a Novel Bach1 Inhibitor………….……………………...84 Chapter 5: Concluding Remarks: Heme Oxygenase, a novel therapeutic target for metastatic melanoma………………………….................................105 Appendix Attucks, O.C., et al., Induction of heme oxygenase I (HMOX1) by HPP-4382: a novel modulator of Bach1 activity. PLoS One, 2014. 9(7): p. e101044.………..………………………………………………111 References………………………………………………………………131 VITA……………………………………………………………………...145 iv" LIST OF FIGURES Figure 1. Redox-dependent regulation of Nrf2………………………..19 Figure 2. The regulation of Nrf2 and the Antioxidant Response Element (ARE) is multi-faceted……………………………...20 Figure 3. Bach1 is an ARE-binding protein, which inhibits expression of ARE-dependent genes…………………………………….22 Figure 4. Heme Degradation……………………………………………23 Figure 5. Melanoma cell lines which harbor the B-RafV600E mutation form melanospheres………………………………………….51 Figure 6. Nrf2 contributes to melanosphere formation in SK-Mel-5 cells……………………………………………………………..52 Figure 7. Inhibition of HMOX1 diminishes melanosphere formation……………………………………………………….53 Figure 8. CoPP treatment enhances melanosphere formation……..55 Figure 9. Oncogenic B-Raf promotes melanosphere formation…….56 Figure 10. Oncogenic pBABE-B-RafV600E transformed HS936T cells demonstrate an increase in HMOX2 expression and protein levels……………………………………………………………57 Figure 11. CoPP treatment facilitates melanosphere formation in HS936T cells…………………………………………………..58 Figure 12. Pharmacological inhibition of HMOX1 and HMOX2 diminshes melanosphere formation in transformed HS936T cells……………………………………………………………..59 v" Figure 13. HS936T cells treated with CoPP or stably expressing the active form of B-Raf, B-RafV600E, have up-regulated expression of genes involved in focal adhesion and ECM- receptor interaction……………………………………………61 Figure 14. HMOX activity may promote metastatic tumorigenesis by modulating focal adhesions and ECM-receptor interactions…………………………………………………….65 Figure 15. Schematic of cell line generation protocol………………..80 Figure 16. NHEMTH14 cells express SV40 T antigen and hTERT, retain melanocyte specific markers, and remain proliferative…………………………………………………….81 Figure 17. Characterization of the NHEMTH14-N-RasQ61K cell line……..83 Figure 18. HPP-4382 induces HMOX1 expression in an Nrf2- dependent fashion…………………………………………….99 Figure 19. HPP-4382 does not behave as an electrophile…………101 Figure 20. HPP-4382 decreases Bach1 binding and increases Nrf2 binding at the HMOX1 EN2 ARE…………………………..103 Figure 21. HPP-4382 alters the binding of Nrf2 and Bach1 at the HMOX1 EN2 ARE in a similar fashion as CoPP but in a dissimilar manner as the electrophile, HPP-1014………..104 vi" LIST OF TABLES Table 1. FDA-Approved Therapies for the Treatment of Metastatic Melanoma……………………………………………………...18 Table 2. Nrf2 Regulatory Proteins……………………………………...21 Table 3. Sequencing of Melanoma Cell Lines………………………...50 Table 4. Nrf2-target gene expression changes in CoPP treated and B-RafV600E-transformed HS936T cells……………………63 Table 5. Primer Sequences for Chapter 2…………………………….66 Table 6. Primer Sequences for Chapter 4…………………………….92 vii" Chapter 1: Bach1 Regulation of the Antioxidant Response Element (ARE): Implications for Melanoma Metastatic Tumorigenesis Kimberly Jasmer Abstract: The transcription factor, Nrf2, is stabilized in response to oxidative stress, promoting expression of a battery of cytoprotective genes, which protect the cell from the insults of oxidative stress. Traditionally, this response has been considered inhibitory of cancer development, in part by eliminating damaging oxidative species. However, growing evidence suggests a role for these genes in cancer development and progression. Significant attention