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Regulation and Function of the Cancer Stem Cell Transcription Factors GLI1 and Kruppel-Like Factor 4

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Regulation and Function of the Cancer Stem Cell Transcription Factors GLI1 and Kruppel-Like Factor 4 Graduate Theses, Dissertations, and Problem Reports 2015 Regulation and Function of the Cancer Stem Cell Transcription Factors GLI1 and Kruppel-like Factor 4 Daniel B. Vanderbilt Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Vanderbilt, Daniel B., "Regulation and Function of the Cancer Stem Cell Transcription Factors GLI1 and Kruppel-like Factor 4" (2015). Graduate Theses, Dissertations, and Problem Reports. 6863. https://researchrepository.wvu.edu/etd/6863 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Regulation and Function of the Cancer Stem Cell Transcription Factors GLI1 and Krüppel-like Factor 4 Daniel B. Vanderbilt Dissertation submitted to the School of Medicine at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cancer Cell Biology Elena N. Pugacheva, Ph.D., Chair Erik A. Bey, Ph.D. Karen H. Martin, Ph.D. Mohamad A. Salkeni, Ph.D. Scott A. Weed, Ph.D. J. Michael Ruppert, M.D., Ph.D., Mentor Cancer Cell Biology Program Morgantown, West Virginia 2015 Key Words: Breast cancer, DDR1, GLI, Hedgehog, KLF4, metastasis, pancreatic cancer, SOX9, β-TrCP Copyright 2015 Daniel B. Vanderbilt ABSTRACT Regulation and Function of the Cancer Stem Cell Transcription Factors GLI1 and Krüppel-like Factor 4 Daniel B. Vanderbilt Transcription factors are crucial to the normal and pathologic biology of cells. Aberrant genetic regulation underlies all facets of cancer, including metastasis, therapeutic resistance, and other clinically relevant manifestations. Krüppel-like factor 4 (KLF4) and GLI1, transcription factors that interact with DNA through conserved zinc finger domains, are implicated in human malignancy. The stability of GLI1 protein is a central determinant of Hedgehog pathway signaling output, and is regulated through interaction with the F-box protein β-TrCP. In pancreatic ductal adenocarcinoma, GLI1 may be activated by oncogenic mechanisms. While KLF4 is not specific to any discrete signal pathway, KLF4 is well-characterized in numerous biological processes, including pluripotency and stress response. The functions of KLF4 are highly context-dependent, and the role of KLF4 in metastasis and other emergent phenotypes remains unclear. This dissertation addresses two distinct topics: Mechanisms underlying GLI1 stabilization in pancreatic cancer, and the function of KLF4 in the metastasis of breast cancer cells. As an effector protein of the Hedgehog (Hh) signal pathway, GLI1 is implicated in pancreatic ductal adenocarcinoma (PDA) and other cancer types. Like other GLI proteins, GLI1 stability is regulated by the ubiquitin-proteasome system through the E3 ligase SKP1/CUL1/F- box (SCF) protein SCFβ-TrCP. While Hh signaling is known to induce expression of the high mobility group box protein SOX9 in several contexts, the function of this signaling mechanism in PDA is poorly understood. In Chapter 2, we report the stabilization of GLI1 protein by SOX9 in PDA cells, finding that SOX9 inhibited SCFβ-TrCP-mediated protein degradation. In the presence of SOX9, the association of GLI1 and β-TrCP was reduced. The discovery that SOX9 interacts with the F-box domain of β-TrCP led us to question whether SOX9 could inhibit SCFβ- TrCP complex assembly, as this region is known to function in binding the SKP1 adaptor protein. Indeed, we observed enhanced association between SKP1 and β-TrCP upon suppression of SOX9, whereas overexpression of SOX9 led to drastically reduced SKP1-β-TrCP interaction. Additionally, SOX9 functioned to promote the nuclear tethering and degradation of β-TrCP. In PDA cells, deficiency of SOX9 resulted in loss of malignant properties and cancer stem cell (CSC) traits, effects that could be rescued by suppression of β-TrCP. We also provide evidence that additional substrates of β-TrCP may be similarly regulated by SOX9. These results identify a positive feedback mechanism of GLI1 stability in PDA cells, revealing that SOX9 can inhibit SCFβ-TrCP activity to suppress degradation of oncoproteins. While KLF4 inhibits cell proliferation and is downregulated in some tumor types, KLF4 promotes survival and therapeutic resistance in breast cancer cells. Metastasis remains a significant clinical problem, yet a comprehensive understanding of the metastatic process remains elusive. In Chapter 3, we address the function of KLF4 in a mouse model of breast cancer metastasis. In triple-negative breast cancer cell lines, stable suppression of KLF4 resulted in enhanced spontaneous metastasis to the lungs and liver. Similarly, we observed fewer metastases in association with KLF4 overexpression. KLF4 had minimal impact on primary tumor initiation and growth. Although increased circulating tumor cells (CTCs) arose from KLF4-knockdown tumors, this effect was not predicted by function of KLF4 in 2D assessments of motility and invasion. Discoidin domain receptor 1 (DDR1) is a collagen-binding receptor tyrosine kinase associated with metastasis. We found that KLF4 suppresses expression of DDR1. Accordingly, suppression of KLF4 resulted in greater expression of DDR1 mRNA and protein, and increased adhesion to collagen. We propose that KLF4 inhibits metastasis of breast cancer cells through downregulation of DDR1. For Audrey iv ACKNOWLEDGEMENTS Throughout my work in preparing this doctoral dissertation, I am reminded of the many individuals who have helped me along the way. To family, friends, faculty, mentors, advisors, teachers, colleagues, and so many others, I am profoundly grateful. First, I thank my mentor and doctoral advisor, Dr. J. Michael Ruppert, for training me, and providing guidance and scientific insight throughout my years of predoctoral research. In gratitude I acknowledge other current and former lab members, including Mark Farrugia, Dr. Wentao Deng, Dr. Chen-Chung Lin, Michael Mckinstry, Jackie Metheny, and Sriganesh Sharma. All have provided expertise, advice, encouragement, and assistance. With greatest appreciation I thank Dr. Deng for his extraordinary efforts in our collaboration as co-authors. It has been a tremendous privilege to work with Dr. Deng. I also thank fellow students Mark Farrugia and Sriganesh Sharma for both research assistance and camaraderie, and offer congratulations for their recent success as doctoral candidates. Throughout my research, many faculty and staff have assisted in my projects, and I thank Drs. Amanda Ammer and Kathleen Brundage, Emily Ellis, Sarah McLaughlin, and Brandi Underwood for crucial instruction, training, and technical assistance. Many students have also provided valuable advice and support. These include those in the Cancer Cell Biology program, in other fields, and my fellow MD-PhD students. In particular, I thank Bridget Hindman, Colton Koontz, Audrey Jajosky, and Amanda Suchanek, who have all made significant contributions to my work. As a doctoral candidate, I have been aided, challenged, and encouraged by committee members Drs. Erik Bey, Karen Martin, Elena Pugacheva, Mohamad Salkeni, Scott Weed, and Robert Wysolmerski. I am especially grateful to Drs. Pugacheva and Weed, who took on additional responsibilities on short notice for my benefit. As Director of the Graduate Program in Cancer Cell Biology, Dr. Weed continues to work on behalf of myself and other students, and I am most thankful for his service. Forever I will be indebted to the many scientists who have served as teachers, mentors, and advisors. I offer my most sincere appreciation to my first mentor, Dr. Allison Calhoun, who has inspired many students to pursue science at the graduate level and beyond. At WVU, I especially thank Drs. Alexey Ivanov, Lisa Salati, and Robert Wysolmerski, who have directly aided my projects and provided critical advice. Finally, I am honored to thank Assistant Vice President for Graduate Education and former MD-PhD program director Dr. Fred Minnear, and current MD-PhD program director Dr. David Siderovsky, for their leadership and student advocacy. Without their efforts, my success and that of other students would not be possible. v TABLE OF CONTENTS Abstract .......................................................................................................................................... ii Acknowledgements ........................................................................................................................v List of tables.................................................................................................................................. xi List of figures ............................................................................................................................... xii List of abbreviations .................................................................................................................. xiv Chapter
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