Integrating Genomics and Molecular Biology: Identifying Transcriptional Targets for the Prostate Cancer Oncogene SOX4 And Evaluating the Efficacy of Aurora Kinase Inhibition in Chemotherapy-Resistant Ovarian Cancer Público

Abstract

The advent of genomic microarray technology has transformed molecular biology and genetics from single gene studies, allowing for the first time, experiments that analyze global regulatory networks and functions, giving birth to the fields of transcriptomics and genomics. Here, we have applied DNA expression-microarrays to discover global gene expression changes in cancer and transfected cell lines, tiling-microarrays to identify genomic binding sites for the SOX4 DNA binding protein, and unique double stranded DNA microarrays to determine sequence binding specificities for SOX4. In two independent studies, we built on knowledge gathered from genomic techniques to evaluate the efficacy of Aurora kinase inhibition in ovarian cancer and gain novel insights into the role of the transcription factor SOX4 in prostate cancer. In the first study, we investigated whether the Aurora family inhibitor VE-465 could induce apoptosis in a taxol-resistant and taxol-sensitive ovarian cancer cell line. We find that at Aurora- A specific doses, VE-465 synergized with taxol to induce apoptosis in ovarian cancer cell lines. These studies provide preliminary evidence that Aurora family inhibition may be beneficial in ovarian cancer patients whose tumors express high levels of Aurora-A. In the second study we applied two different array technologies to identify, for the first time, 282 high-confidence target genes for the SOX4 transcription factor. We also determined the sequence binding specificities for SOX4 using a novel double-stranded DNA microarray, and show that SOX4 binding sites are enriched in our ChIP-chip peaks. SOX4 influences key signaling pathways at the transmembrane and transcriptional level, as well as the small RNA pathway. These data provide novel insights into SOX4's role in development and cancer progression.

Table of Contents

Chapter I: Aurora Kinases: Mitotic Regulators and Cancer Agents .........1

1.1 Aurora-A and Family.....................................................3

1.2 Ovarian Cancer: Therapeutics and the Aurora Kinases ............7

Chapter II: Results: AURORA KINASE INHIBITORS SYNERGIZE WITH PACLITAXEL TO CAUSE APOPTOSIS IN OVARIAN CANCER CELLS .11

2.1 Expression Profiling Reveals Aurora-A Over Expression in Ovarian Cancer Patients .......12

2.2 Ovarian Cancer Tissue Microarray Analysis of Aurora-A .........15

2.3 Aurora Kinases are Expressed in Ovarian Cancer Cell Lines .....17

2.4 VE-465 Inhibits the Aurora Kinases ..................................19

2.5 VE-465 Induces Apoptosis in Ovarian Cancer Cell Lines .........20

2.6 VE-465 Promotes Apoptosis in a Paclitaxel-Resistant Ovarian Cancer Cell Line ...................22

2.7 VE-465 Synergizes with Paclitaxel to Induce Apoptosis at Low Doses Specific to Aurora-A .........25

Chapter III: Aurora Kinases: Discussion .........................................26

Chapter IV: SOX4: The Diversity of Mammalian SOXs ......................32

4.1 A Collection of SOXs ..................................................34

4.2 SOX4 .....................................................................41

4.2.1 Developmental Regulator ....................................42

4.2.2 Molecular Functions ..........................................44

4.2.3Carcinogenesis ................................................47

Chapter V: Results: GENOME-WIDE PROMOTER ANALYSIS OF THE SOX4 TRANSCRIPTIONAL NETWORK IN PROSTATE CANCER CELLS .....50

5.1 SOX4 Transcriptionally Regulates EGFR, ERBB2, TLE1 and PUMA .........................51

5.2 Genome-wide Localization Analysis ................................54

5.3 Target Gene Expression Analysis ....................................59

5.4 Novel SOX4 Position-Weight Matrix ...............................62

5.5 SOX4 Peaks Contain SOX4 Binding Sites .........................64

5.6 Ontology and IPA Analysis ..........................................69

Chapter VI: SOX4: Discussion .........................................71

6.1 SOX4 Direct Target Genes ...........................................73

6.2 Modulation of Input/Output Network Signals .....................76

6.3 DNA Binding and Transcriptional Cofactors ......................79

6.4 SOX4 and Cancer ......................................................81

6.5 SOX4 Regulates Components of RISC and the Small RNA Pathway .............84

6.6 Conclusions and Future Directions .................................85

Appendix A: SOX4 Binding Sites and Regulatory Genes .....................90

Appendix B: Illumina Genes with Significant Expression Changes ........90

Appendix C: 282 SOX4 High Confidence Genes ...............................91

Appendix D: Enrichment Scores of K-mers Bound by SOX4 ...............94

Appendix E: Primers and Oligos ...................................................94

Appendix F: Materials and Methods .............................................95

I. AURORA KINASE INHIBITORS SYNERGIZE WITH PACLITAXEL TO INDUCE APOPTOSIS IN OVARIAN CANCER CELLS .......95

1.1 Tumor samples, RNA isolation, Microarray Hybridization and Normalization .....95

1.2 Cell Culture and Drug Treatment ..........................95

1.3 Fluorescence Activated Cell Sorting (FACS) Analysis..95

1.4 Drug Treatment and Caspase Assay .......................96

1.5 Immunofluorescence .........................................97

1.6 Western Blot ..................................................97

1.7 Tissue Microarray Analysis .................................98

II. GENOME-WIDE PROMOTER ANALYSIS OF THE SOX4 TRANSCRIPTIONAL NETWORK IN PROSTATE CANCER CELLS .....98

2.1 Cell Culture and Stable Cell Line Construction .........98

2.2 ChIP Assay ...................................................98

2.3 ChIP-chip Analysis ..........................................99

2.4 Plasmid Luciferase Assays..................................100

2.5 Quantitative Real Time PCR ...............................100

2.6 Microarray Analysis .........................................100

2.7 Beta-catenin Luciferase Assay .............................101

2.8 Electromobility Shift Assays .............................101

2.9 Immunoblotting ............................................102

Appendix G: Literature Cited ....................................................103

List of Tables

TABLE 1:

Eukaryotic Aurora kinase family members...................3

TABLE 2:QPCR Verification of Aurora-A and Selected Aurora-A Network Genes ...............14

TABLE 3: IPA Analysis of The Aurora-A Network in Ovarian Cancer..15

TABLE 4: Summary of Patient Data and TMA Staining with Aurora-A Antibody ...............................16

TABLE 5: Co-occurring Transcription Factor Binding Sites ..............65

TABLE 6: Transcription Factors Regulated by SOX4 .....................66

TABLE 7: TGF Genes Regulated by SOX4 as Detected by

GSEA Leading Edge Analysis ..............................................70

List of Figures

FIGURE 1: Domain Structure of the Human Aurora Kinases ....................4

FIGURE 2: Aurora-A and the Aurora-A Network is Over Expressed

in Ovarian Cancer Patients at the mRNA and Protein Level..13

FIGURE 3: VE-465 Inhibits the Aurora Kinases in

Ovarian Cancer Cell Lines ........................................18

FIGURE 4: VE-465 Causes Apoptosis in Ovarian Cancer Cell Lines .....21

FIGURE 5: VE-465 Synergizes with Paclitaxel in Taxol-Sensitive Ovarian Cancer Cell Lines ...........23

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Bioinformatics Biology, Molecular
Palabra Clave	ChIP Genomics Cancer SOX4
Committee Chair / Thesis Advisor	Moreno, Carlos, Emory University
Committee Members	Moberg, Kenneth H, Emory University Boss, Jeremy, Emory University Vertino, Paula M, Emory University Caspary, Tamara, Emory University

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