The Role of SOX4 In Prostate Cancer Open Access

Lai, Yu-Heng (2011)

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SOX4 is a developmental transcription factor that is required for differentiation and proliferation in multiple tissues. SOX4 is overexpressed in many human malignancies, but the precise role of SOX4 in cancer progression is still not well understood.

The first part of my study identified proteins that interact with SOX4 via a one-step affinity purification method that enables rapid purification of SOX4 complexes to perform large-scale proteomics analysis. We discovered that junction plakoglobin (JUP) interacts with SOX4 in both the cytosol and the nucleus, and that the interaction between SOX4 and plakoglobin is significantly increased when prostate and breast cancer cells are stimulated with WNT3A. The SOX4-plakoglobin complex affected the expression of Wnt pathway target genes and SOX4 downstream targets, such as AXIN2, DICER1, and DHX9. In addition, SOX4 DNA binding activity to the promoters of DICER1, AXIN2, DHX9 and SOX4 itself was reduced by conditions that promote SOX4-plakoglobin complex formation. Conditions that enhanced SOX4-plakoglobin interactions resulted in reduced transcriptional activity of β-catenin luciferase reporters. These data suggest that this newly identified interaction between SOX4 and plakoglobin is inhibitory and provides new insights into the role of SOX4.

In the second part of my research, we performed miRNA profiling from 70 tumor samples and identified miRNAs that are regulated by SOX4, including miR-103, miR-339, miR-182 and miR-221. We also discovered that SOX4 regulates miR-16 and miR-196 in response to Wnt signals and also searched for miRNAs that might regulate SOX4. We identified the 1100-2350 nt region of the SOX4 3' UTR as a potential target for miRNAs that translationally inhibit SOX4 expression in prostate cancer cells. These studies provide new insights in to the function of SOX4 and its interaction with the miRNA pathway.

Table of Contents

Chapter I: SOX4: transforming Oncogene......................1

1.1 The SOX Family...3

1.1.1 Molecular properties of SOX proteins...3

1.1.2 Function and mechanism...5

1.2 SOX4...8

1.2.1 Developmental regulation...9

1.2.2 Carcinogenesis...10

1.2.3 Wnt signaling in prostate cancer.....12

Chapter II: Results: SOX4 Interacts with Plakoglobin in a Wnt3a-dependent Manner in Prostate Cancer Cells...18

2.1 Indentification of proteins that interact with SOX4...19

2.2 Plakoglobin interacts with SOX4...19

2.3 Interaction between SOX4 and plakoglobin in the nucleus responds to Wnt signaling...20

2.4 Wnt signaling and SOX4-associated pathway are affected by SOX4-plakoglobin interaction...22

2.5 SOX4-plakoglobin complex modulates β-catenin-mediated transcriptional activity...23

Chapter III: Plakoglobin: Discussion...36

Chapter IV: Network Between SOX4 and MiRNA in Prostate Cancer...42

4.1 MicroRNA...43

4.1.1 Biogenesis...43

4.1.2 Developmental regulation...44

4.1.3 Carcinogenesis...47

Chapter V: Results: The network between SOX4 and miRNA...54

5.1 SOX4 mediates the miRNA aberrant expression in prostate cancer...56

5.2 Wnt-induced SOX4 mediates miRNA expression in prostate cancer cells...58

5.3 The 1100-2350 region of SOX4 3'UTR mediates repression of SOX4 expression...58

5.4 High-throughput selection system for identification of miRNA regulators of SOX4 3'UTR...59

Chapter VI: MiRNA: Discussion...69

6.1 MiRNA regulators on SOX4...70

6.2 MiRNA biomarker in prostate cancer...72

6.3 SOX4-mediated miRNAs in Wnt signaling...73

Appendix A Materials and Methods...76


1.1 Reagents and cell culture...76

1.2 Biotinlyated HA-tagged SOX4 expression construct...76

1.3 Purification of biotinlyated HA-tagged SOX4...76

1.4 Sample preparation form mass spectrometry...77

1.5 Co-immunoprecipitation and Western blot...77

1.6 Immunofluorescent antibody staining...78

1.7 Cell fractionation into nuclear and cytoplasmic lysates...79

1.8 Quantitative real-time PCR...79

1.9 Chromatin immunoprecipitation (ChIP) assay...79

1.10 Luciferase reporter assay...80

1.11 siRNA transfection...80


2.1 Reagents and cell culture...81

2.2 Patient samples...81

2.3 SOX4 3'UTR construct...82

2.4 Luciferase reporter assay...83

2.5 Cold fusion cloing and miR-selection stable cell line establishment...83

2.6 Chromatin immunoprecipitation (ChIP) assay...83

2.7 Quantitative real-time PCR...84

2.8 pLemiR-miRNA stable cell line establishment...84

Appendix B Primer lists...85

Appendix C Literature Cited...87

List of Tables

TABLE 1: LC-MS/MS analysis of SOX4 binding partners in LNCaP cells...26

TABLE 2: Twelve-gene predictors of prostate cancer recurrence following surgery...65

List of Figures

FIGURE 1: SOX4 family subgroups and protein domain architecture...14

FIGURE 2: Summary of expression patterns and biological function of SOX proteins...16

FIGURE 3: The TAP-TAG SOX4 clone...25

FIGURE 4: Plakoglobin binds to SOX4 in LNCaP cells...27

FIGURE 5: Wnt signaling induceds nuclear colocalization of SOX4 and plakoglobin...29

FIGURE 6: Interaction between SOX4 and plakoglobin in affected by Wnt3A signaling...31

FIGURE 7: SOX4 transcriptional activity is modulated by Wnt-induced interaction with plakoglobin...33

FIGURE 8: Wnt signaling is downregulated by SOX4-plakoglobin complex...35

FIGURE 9: Model depicting the role of SOX4 and plakoglobin in Wnt signaling regulation...41

FIGURE 10: The schematic shows the major steps in miRNA processing...52

FIGURE 11: The pleiotropic functions of miR-17-92 achieved by repressing specific targets..53

FIGURE 12: Validation of recurrence-associated miRNA...62

FIGURE 13: Establishment of pri-miR-103 and pri-miR-182 stable cell lines...63

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