Identification of Regulators Associated with Androgen Deprivation Therapy Response and Metastatic Progression in Prostate Cancer Open Access

Sharma, Nitya (Summer 2018)

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While some patients will benefit from androgen deprivation therapy (ADT), there is another subset of patients for whom therapy is not sufficient. These patients will acquire castration resistant prostate cancer that is typically associated with metastasis. Changes in tumor biology reflect the increasingly aggressive stages of prostate cancer, and drive differential clinical responses. Complex variations in tumor transcriptional profiles depend on a precise interplay of multiple regulators and cofactors. However, the transcription factor dynamics that promote aggressive prostate cancer and metastasis are not fully understood. Here, we identified transcription factor coordinated groups that may reflect activation of compensatory regulatory mechanisms that mediate ADT response and metastatic progression. Furthermore, our data suggest that these transcription factor interactions are maintained after ADT and in metastatic tumors, despite predicted genomic re-localization. Transcription factors exhibit context dependent re-localizations to drive multiple phases of cancer. SOX4 is a transcription factor that can transform prostate cancer. We investigated a continued role of the oncogene SOX4 after transformation in the epithelial to mesenchymal transition (EMT), a critical step in the metastatic cascade. Our studies suggest that SOX4 functions downstream of the important EMT-promoting signaling pathways, TGF-β and EGF, and is sufficient to promote the EMT program. We also shed light on the potentially interdependent relationships between SOX4 and the histone acetyltransferase complex PCAF to transcriptionally activate pro-EMT genes. The analyses described in this dissertation demonstrate how regulatory activities that drive aggressive prostate cancer depend on precise combinatorial transcription factor relationships.

Table of Contents

Chapter 1. Introduction pg. 1

1.1 Prostate Cancer Introduction pg. 1

1.1.1 Overview pg. 1

1.1.2 Evaluation and Classification of Prostate Cancer pg. 2

1.2 Androgen receptor: A brief introduction pg. 4

1.2.1 Androgens and the androgen receptor pg. 4

1.2.2 AR signaling pg. 8

1.3 AR-signaling and prostate cancer pg. 10

1.3.1 Androgen deprivation therapy as a prostate cancer therapeutic pg. 10

1.3.2. Aberrant AR-signaling in castration resistant prostate cancer pg. 13

1.4 The transition to CRPC and metastasis pg. 15

1.5 SOX4 is a developmental transcription factor that is important for prostate cancer metastasis pg. 16

1.5.1 SOX4 background pg. 16

1.5.2 SOX4 in cancer pg. 18

1.5.3 SOX4 in the epithelial to mesenchymal transition, and metastasis pg. 20

1.6 Concerted action of multiple transcription factors drives complex changes in transcriptional profiles pg. 23

 1.7 Dissertation objectives pg. 25

Chapter 2. The biology of castrate resistant prostate cancer  pg. 26             

2.1 Introduction pg. 28

2.2 The androgen receptor pg. 29

2.3 PI3K-AKT-mTOR pathway pg. 31

2.4 Receptor tyrosine kinase (RTK) growth factor pathways pg. 32

2.5 Analysis of CRPC genomes pg. 33

2.6 Epigenetic pathways pg. 34

2.7 EMT and SOX family genes pg. 36

2.8 WNT signaling pg. 38

2.9 Notch and Hedgehog pg. 39

2.10 lncRNA-mediated pathways pg. 40

2.11 Angiogenic pathways pg. 41

2.12 Biomarkers pg. 42

2.13 Conclusion pg. 44

Chapter 3. Identification of transcription factor relationships associated with androgen deprivation therapy response and metastatic progression in prostate cancer pg. 47

3.1 Introduction pg. 49

3.2 Materials and methods pg. 51

3.3 Results pg. 55

3.4 Discussion pg. 99

Chapter 4. The Role of SOX4 in Epithelial to Mesenchymal Transition in Prostate Cancer pg. 104

4.1 Introduction pg. 105

4.2 Materials and methods pg. 108

4.3 Results pg. 111

4.4 Discussion pg. 117

Chapter 5. TFBSET: A New Tool for the Identification of Enriched Transcription Factor Binding Sites in the Promoter Regions of Human Genes pg. 121

5.1 Introduction pg. 123

5.2 Materials and methods pg. 126

5.3 Usage and implementation  pg. 130

5.4 Results pg. 131

5.5 Discussion pg. 142

Chapter 6. Discussion and future directions  pg. 146

6.1 Predicting transcriptional regulators of ADT response and metastasis pg. 146

6.1.1 Early divergent transcriptional response to ADT could reflect an ADT mediated clonal selection pg. 146

6.1.2 Distinct transcription factor associations may drive transcriptional   programs that influence prostate cancer aggressiveness. pg. 148

6.1.3 Future Directions pg. 150

6.2 SOX4 promotes EMT in prostate cancer cells pg. 153

Figures and Tables List

Figure 1.1. Prostate cancer incidence and mortality statistics. pg. 3

Figure 1.2. Synthesis of DHT and subsequent binding to the AR. pg. 6

Figure 1.3. AR locus, gene and protein structure. pg. 7

Figure 1.4. AR signaling axis. pg. 9

Table  1.1. Pharmacological agents that inhibit AR signaling in prostate cancer. pg. 11

Figure 1.5. Timing of first and second line drugs in CRPC. pg. 12

Figure 1.6. SOX4 domain structure. pg. 17

Figure 1.7. Increased SOX4 expression correlates with aggressive prostate cancer. pg. 19

Figure 1.8. Steps of the metastatic cascade. pg. 21

Figure 1.9. EMT induces molecular changes that allow for epithelial cells to acquire mesenchymal capabilities. pg. 22

Figure 2.1. Signaling pathways in castration resistant prostate cancer. pg. 45

Figure 3.1. Hierarchical clustering and PCA of 190 significantly differentially expressed genes in 20 matched Pre-ADT Bxs and Post-ADT RPs.  pg. 66

Figure 3.2. Divergent expression of PCS1 genes in the high impact group and common loss of PCS2 and PCS3 after ADT. pg. 69

Figure 3.3. Identification of TFCGs in the high impact group network. pg. 86

Table 3.1. 33 oTFCGs between the high impact ADT group and Met.PCS1 networks. pg. 97

Table S3.1. Patient and treatment characteristics. pg. 56

Table S3.2. Significantly differentially expressed genes identified using edgeR analysis of Pre-ADT Bxs and Post-ADT RPs. pg. 58

Table S3.3. Ingenuity Pathway Analysis identifies chemical agents associated with signaling pathways. Predicted upstream chemical agent regulators suggest inhibition of androgen driven genes, and increase in estrogen and PDGF-MAPK signaling. pg. 63

Table S3.4. Key TFs in the high impact network. pg. 71

Table S3.5. TFCGs in the high impact network. pg. 87

Table S3.6. Key TFs in the Met.PCS1 network. pg. 90

Table S3.7. TFCGs in the Met.PCS1 network. pg. 96

Figure S3.1. Hierarchical clustering of PCS genes of 20 matched pre-ADT Bxs and post-ADT RPs again segregates three groups. pg. 68

Figure 4.1. TGF-β and EGF initiate the EMT program and induce SOX4 expression in RWPE1 non-neoplastic cells. pg. 112

Figure 4.2. Over expression of SOX4 induces EMT in prostate cancer cell lines. pg. 113

Table 4.1. Identification of candidate SOX4 targets. pg. 115

Figure 4.3. SOX4 interacts with PCAF and TRRAP in prostate cancer cell lines. pg. 116

Figure 4.4. SOX4 expression returns after knockdown with CRISPR/Cas9. pg. 118

Figure 4.5. Proposed mechanism. pg. 120

Figure 5.1. TFBSET tool characteristics. pg. 127

Table 5.1. TFBSET was run on multiple gene sets to identify enriched TFBSs in proximal promotor regions pg. 135

Table 5.2. Comparison of TFBSET to other popular programs  pg. 139

Table S5.1 Optimal parameters for identification of Myc target sites in the Dang gene set pg. 132

Table S5.2 Comparison of results from 6 different gene sets upregulated by Myc. The  numbers in the table represent the FDR. pg. 133

Table S5.3 TFBSET produces better results at ±5000 bp than ±1000 bp around the TSS. pg. 134

Table S5.4 Comparison of TFBSET results of gene sets run excluding and including enhancer regions shows that the results are generally less accurate when the enhancer regions are included pg. 137

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