Kruppel-Like Factor 9 Inhibits Glioblastoma Stemness

KRUPPEL-LIKE FACTOR 9 INHIBITS GLIOBLASTOMA STEMNESS THROUGH GLOBAL TRANSCRIPTION REPRESSION AND INHIBITION OF INTEGRIN ALPHA 6 AND CD151 By Jessica Tilghman A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland October, 2015 Abstract Glioblastoma (GBM) stem cells (GSCs) represent tumor-propagating cells with stem-like characteristics (stemness) that contribute disproportionately to GBM drug resistance and tumor recurrence. Understanding the mechanisms supporting GSC stemness is important for developing novel strategies that target tumor propagation to inhibit cancer progression and improve patient survival. Krüppel-like factor 9 (KLF9) has emerged as a regulator of cell differentiation, neural development, and oncogenesis; however, the molecular basis for KLF9’s diverse contextual functions has been unclear. We establish for the first time a genome-wide map of KLF9-regulated targets in human glioblastoma stem-like cells, and show that KLF9 functions as a transcriptional repressor and thereby regulates multiple signaling pathways involved in oncogenesis and regulation of cancer stem-like phenotype. A detailed analysis of two novel KLF9 targets suggests that KLF9 inhibits glioma cell stemness by repressing expression of integrin α6 and CD151. The expression of one candidate KLF9 target gene ITGA6 coding for integrin α6 was verified to be downregulated by KLF9 in GSCs. ITGA6 transcription repression by KLF9 altered GBM neurosphere cell behavior as evidenced by reduced cell adhesion to and migration through membrane coated with the integrin α6 ligand laminin. Forced expression of integrin α6 partially rescued GBM neurosphere cells from the differentiating and adhesion/migration-inhibiting effects of KLF9. Using GBM derived neurospheres, we identified cell surface tetraspanin family member CD151 as a novel regulator of GSC stemness and tumorigenicity. CD151 was found to be ii overexpressed in GBM tumors and GBM neurospheres enriched in GSCs. Silencing CD151 inhibited neurosphere self-renewal and cell proliferation and attenuated expression of stem cell markers and drivers. Conversely, forced CD151 expression promoted neurosphere self-renewal, cell migration and expression of stemness-associated transcription factors. Additionally, targeting CD151 inhibited glioma angiogenesis and growth of GBM neurosphere derived xenografts. CD151 was found to form complexes with integrins α3, α6 and β1 in neurosphere cells and blocking the CD151-intgerin α3/α6 interaction impaired sphere formation, migration and phosphorylation of AKT, a marker of integrin signaling. These findings identify CD151 and its interactions with integrins α3 and α6 as potential therapeutic targets for depleting stem cell populations and stemness-driving mechanisms in GBM. Thesis Advisor: Dr. John Laterra Thesis Readers: Dr. John Laterra Dr. Angelika Doetzlhofer iii Acknowledgements I would like to acknowledge my thesis advisor Johns Laterra for his support and direction with this project. I thank my colleagues in the laboratory; it was a pleasure to work alongside you during my thesis research. Gratitude goes to Mingyao Ying who was involved in many facets of this project and who assisted with the design and execution of some of these experiments. I would also like to thank Paula Schiapparelli for conducting some of the migration experiments, and Hongkai Ji and Yingying Wei for conducting the bioinformatics analyses. I would like to acknowledge the following people who contributed to the project in one way or another: Han Sun, Shuli Xia, Yunqing Li, Hernando Lopez-Bertoni and Bachuchu Lal, Kathryn Wagner, Adam Moyer, Mary Blue, Mary Ann Wilson, Joseph Bressler, John Coulter, Paul Watkins, and Johnathan Pevsner. Thanks to the faculty, staff and students in the Neuroscience Department, the Neuroscience graduate program and the faculty and staff at the Kennedy Krieger Research Institute. I would also like to express thanks to the Molecular Neuroscience Research. I acknowledge the advice from the faculty who served on my thesis committee: Angelika Doetzlhofer, Alfredo Quinones-Hinojosa, Charles Eberhart and John Laterra. Thanks in particular to John Laterra and Angelika Doetzlhofer for comments on the thesis. This project was supported by predoctoral awards from the National Science Foundation and Ford Foundation Fellowship. Portions of this work have been published previously and reprinted with permission from the Journal of Biological Chemistry. Other parts are being prepared for publication. iv Table of Contents Abstract…………………………………………………...……………………….....……………ii Acknowledgements..…...………………………………………………………….…………...…iv Table of Contents….…………………………………………………………………………..…..v Table of Figures…...……………………………………………………………………………...vi Chapter 1: Introduction……………..……………………………………………………………..1 Chapter 2: Materials and Methods………………………………...…………………………..…37 Chapter 3: Results: KLF9 Regulates a Transcriptional Network in GBM………………………58 Chapter 4: Results: KLF9 negatively regulates GSCs, in part, by suppressing integrin α6 gene expression and downstream functions…………………………………………………………...78 Chapter 5: Results: CD151 regulates glioblastoma stem cell stemness and tumorigenity in part through interactions with laminin-binding integrins……………………………………………..90 Chapter 6: Discussion………………………………………………..……...………………….118 References……………………………...……………………………………………………….127 Curriculum Vitae....…………………………………………………………………………….168 v Table of Figures Figure 1.1…………………………………………………………………………………………4 Figure 1.2…………………………………………………………………………………………5 Figure 1.3…………………………………………………………………………………………7 Figure 1.4………………………………………………………………………………………16 Figure 1.5………………………………………………………………………………………22 Figure 1.6………….……………………………………………………………………………24 Figure 1.7………………………………………………………………………………………30 Figure 3.1………………………..………………………………………………………………60 Figure 3.2………………………………………………………………………………………65 Figure 3.3……...…………………………………………………………………………………68 Figure 3.4………………………………………………………………………………………73 Figure 3.5………………………………………………………………………………………76 Figure 4.1………………………………………………………………………………………79 Figure 4.2………………………………………………………………………………………82 Figure 4.3………………………………………………………………………………………83 Figure 4.4.………………………………………………………………………………………..87 vi Figure 4.5..……………………………………………………………………………………89 Figure 5.1………………………………………………………………………………………93 Figure 5.2. ……………………………………………………………………………………94 Figure 5.3. ………………………………………………………………………………………98 Figure 5.4………………………………………………………………………………………100 Figure 5.5.………………………………………………………………………………………103 Figure 5.6………………………………………………………………………………………107 Figure 5.7………………………………………………………………………………………112 Figure 5.8………………………………………………………………………………..…….116 vii Chapter 1: Introduction 1.1 Glioblastoma Multiforme Approximately 70% of malignant primary brain tumors are gliomas. The most common glioma is glioblastoma multiforme (GBM), which makes up 50-70% of malignant brain tumors. GBM, or glioblastoma, is a grade IV astrocytoma and is the most aggressive primary brain tumor (Alifieris & Trafalis, 2015). There are two clinical GBM subtypes: primary and secondary (Ahmed, Oborski, Hwang, Lieberman, & Mountz, 2014). A majority (95%) of GBMs are primary tumors, which arise de novo, within 3-6 months. These tumors are highly aggressive, invasive, and typically occur in older patients. Secondary GBMs are less common and evolve from the progression of lower- grade astrocytomas (over 10-15 years). These typically occur in patients less than 45 years old (Crespo et al., 2015). GBMs can be further divided into four molecular subtypes: classical, mesenchymal, proneural and neural. These subtypes are distinguished by different mutation and transcriptional patterns and can also differ in patient survival, patient age and sensitivity to treatment (Verhaak et al., 2010). Each year, 2-3 cases of GBM per 100,000 adults and 1.1 to 3.3 cases per 100,000 children are reported in the United States and Europe. Males are more likely to develop the disease, with the incidence being 3 times higher in male children than female children (Urbanska, Sokolowska, Szmidt, & Sysa, 2014). Patients with GBM may experience headaches, vomiting, blurred vision, neurological deficits, memory loss, variations in personality or seizures (Alifieris & Trafalis, 2015). The current standard of treatment for newly diagnosed patients under 70 years of age is surgical resection followed by 1 radiotherapy and chemotherapy. Despite this aggressive treatment, recurrence is often seen. Patients with GBMs typically have a median survival time of only 12-15 months following treatment (Alifieris & Trafalis, 2015; Dolecek, Propp, Stroup, & Kruchko, 2012; McGirt et al., 2009). GBMs are typically located in the cerebral hemispheres, the brain stem or the cerebellum (Figure 1.1). They are composed of a heterogeneous, anaplastic population of small abnormal shaped cells, that also display a high nuclear to cytoplasmic ratio (Urbanska et al., 2014). GBMs also display some degree of necrosis. Primary tumors typically have a large area of necrosis in the center of the tumor due to insufficient blood supply. Smaller irregularly shaped necrotic foci surrounded by hypercellular zones called pseudopalisades are found in both primary and secondary tumors. Additionally, GBM is one of the most highly vascularized tumors. The tumor vessels are highly permeable with abnormal endothelial walls and discontinuous pericyte coverage(Soda, Myskiw, Rommel, & Verma, 2013). Activation of several signaling pathways is altered in GBM resulting in

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