Identification and Functional Activation of Peroxisome Proliferator-Activated Receptor in Human Upper Aerodigestive Cancer A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Simon K. Wright, MD IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Frank G. Ondrey, MD., Ph.D November 2012 © Simon K. Wright, MD, 2012 Acknowledgements Supported by NCI/NIH P30 CA77598-07 Cancer Center Support Grant (FGO), The Translational Biomarkers Initiative and other funding from the Lions 5M Foundation (FGO), Iowa Health Systems Research Grant (SKW) and American Cancer Society Institutional Research Grant IRG-58-001-40IRG44 (PMG). i Abstract Squamous cell carcinoma of the head and neck (SCCHN) is an aggressive malignancy whose carcinogenesis occurs in multiple stages years to decades after carcinogen exposure. In spite of continued advances in the understanding of molecular biology of SCCHN and the introduction of a multitude of multi-modality treatment protocols, the 20-50% survival of stage III and IV disease has not changed appreciably in over twenty years. Efforts to treat or prevent recurrence have predominantly involved the use of cytotoxic chemotherapy, however the use of retinoids as a chemoprevention agent has been clinically assessed. Success has been limited by toxicity of retinoids and reversal of differentiation changes upon cessation of treatment. Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors belonging to the steroid/thyroid/retinoic acid nuclear receptor superfamily. PPARs primarily target genes involved in lipid homeostasis; one isoform, PPARγ, directs the differentiation of precursor cells into adipocytes. PPARγ heterodimerizes with RXRα to form a functional transcription factor. The recognition of ETYA as a PPAR activator along with the previously-discovered anti-cancer and differentiation effects of this agent invoked the possibility that a differentiation strategy could be used in the treatment in SCCHN. This study examined PPARγ in SCCHN. PPARγ protein was expressed in SCCHN cell lines. Treatment of two cell lines with three chemically distinct ligands of PPARγ caused dose- dependent inhibition of proliferation. Cells treated with these agents caused cytoplasmic lipid vacuole accumulation consistent with adipogenic phenotype shift. Electromobility supershift analysis demonstrated DR-1 consensus sequence binding activity in SCCHN nuclear extracts. The more-specific cyp4a1 oligonucleotide also demonstrated binding, the amount of which was upregulated with treatment. Supershift analysis demonstrated presence of PPARγ in nuclear extracts. Functional activation was assessed using dual transfection reporter gene assays, which demonstrated dose dependent increased PPRE activation with treatment using each agent. We conclude that PPARγ ligands may represent a class of drugs which have value in the treatment of SCCHN. ii Table of Contents List of Tables ...................................................................................................................... v List of Figures .................................................................................................................... vi 1.0. INTRODUCTION ................................................................................................... 1 2.0. LITERATURE REVIEW ........................................................................................ 3 2.1. Peroxisomes ......................................................................................................... 3 2.2. Peroxisome Proliferator-Activated Receptors (PPARs) ...................................... 3 2.3. DNA Binding Properties of PPARs ..................................................................... 5 2.4. PPAR Isoforms and Tissue Distribution .............................................................. 5 2.5. PPARγ and Apipose Differentiation .................................................................... 6 2.6. Regulation of PPARγ ........................................................................................... 8 3.0. DETAILED REVIEW ........................................................................................... 11 3.1. Epidemiology ..................................................................................................... 11 3.2. Anatomy and Clinical Presentation .................................................................... 15 3.3. Diagnosis and Treatment .................................................................................... 16 3.4. Concurrent Chemoradiation by Subsites ............................................................ 19 4.0. REVIEW OF PPARS AND CANCER .................................................................. 28 4.1. Solid and Hematologic PPARγ Activity ............................................................ 28 4.3. Mechanisms of Anti-neoplastic Effects ............................................................. 32 4.4. Mutations of PPARγ........................................................................................... 38 5.1. Colorectal Cancer ............................................................................................... 40 5.2. Gastric Cancer .................................................................................................... 43 5.3. Hepatic Cancer ................................................................................................... 44 5.4. Pancreatic Cancer ............................................................................................... 45 5.5. Lung Cancer ....................................................................................................... 45 5.6. Breast Cancer ..................................................................................................... 47 5.7. Ovarian Cancer ................................................................................................... 50 5.8. Prostate Cancer ................................................................................................... 50 5.9. Renal Tumors ..................................................................................................... 51 5.10. Urinary Bladder .............................................................................................. 51 5.11. Sarcoma .......................................................................................................... 52 5.12. Hematologic Malignancies ............................................................................. 53 5.13. Neurological Neoplasms ................................................................................. 54 5.14. Thyroid Neoplasms......................................................................................... 55 6.0. NUCLEAER HORMONE RECEPTORS IN HNSCC .......................................... 55 6.1. Thyroid Hormone Receptor-like Receptors ....................................................... 55 6.2. Lotan and Chemoprevention .............................................................................. 57 6.3. RA and Diseases, Treatments, and Chemoprevention ....................................... 57 6.4. Peroxisome-Proliferator-Activated Receptors ................................................... 60 6.5. Peroxisom Proliferator-activated Receptors ....................................................... 62 7.0. MATERIAL AND METHODS ............................................................................. 64 7.1. Cell Culture ........................................................................................................ 64 7.2. Preparation of Nuclear Extracts ......................................................................... 64 iii 7.3. Aerodigestive Cancer Cell Line Western Blotting ............................................. 65 7.4. Electromobility Shift Assays .............................................................................. 65 7.5. Transient Transfections/Luciferase Assays ........................................................ 66 7.6. Cell Proliferation Assays .................................................................................... 67 7.7. Clonogenic Assays ............................................................................................. 67 7.8. Oil Red O Studies ............................................................................................... 67 7.9. Genomics Analysis ............................................................................................. 68 7.10. Extraction of Total RNA and Probe Preparation ............................................ 68 7.11. Analysis of Microarray Data .......................................................................... 69 7.12. Tumor Specimen Western Blotting ................................................................ 70 7.13. Biochemicals .................................................................................................. 70 7.14. Statistical Analysis ......................................................................................... 70 7.15. Apoptosis Assays ............................................................................................ 71 8.0. RESULTS .............................................................................................................. 71 8.1. PPARγ Protein Expression and Induction Squamous Aerodigestive Cancer .... 71 8.2. Ligands of PPARγ Induce Growth Arrest and Decrease Clonogenicity