The Role of SOX4 in Bladder Cancer Cell Lines Pubblico

Abstract

In 2018, 81,190 patients will be diagnosed with bladder cancer and 17,240 will die of the disease. As many as 23\% of Bladder cancer patients harbor focal amplifi- cation of chromosome 6p.22, which contains several genes included the gene encoding SOX4. SOX4 is a developmental transcription factor that is overex- pressed in a variety of tumors including lymphomas, breast, and prostate cancers but is most highly altered in bladder cancer. Despite the high frequency of alterations, no clear consensus exists regarding SOX4’s role in bladder cancer. In order to determine the mechanisms by which SOX4 drives tumorigenesis, we have investigated SOX4 in two separate contexts. First, by way of determining novel SOX4 protein-protein interactions and second, determining high confidence SOX4 target genes as po- tential mechanisms to drive different hallmarks of bladder cancer. Our lab’s co- immunoprecipitation data demonstrate novel endogenous protein-protein interac- tions between CDKN2A, a tumor suppressor, and SOX4. We hypothesize SOX4 in- teracts with CDKN2A to promote cell cycle progression and tumorigenesis. More- over, our knockdown of SOX4 using CRISPR interference (CRISPRi) highlights a number of high-confidence SOX4 regulated genes. We have specifically identi- fied, a novel mechanism whereby SOX4 elicits tumor promoting ability by way of inhibiting, directly or indirectly, the tumor-suppressive arm of Wnt5a. Wnt5a is highly expressed in SOX4 knockdown cells and is positively correlated with decreased in- vasion. Restoring SOX4 levels drives down Wnt5a expression and concomitantly increases the invasiveness of T24 bladder cancer cell lines. In summary, our re- search suggests that SOX4 could promote various aspects of tumorigenicity via two distinct but not mutually exclusive pathways. The long term goal of this re- search could implicate SOX4 or it’s putative target genes as potential druggable targets or biomarkers for novel therapeutic approaches for bladder cancer patients.

Table of Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Bladder Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.2 Bladder Cancer Epidemiology . . . . . . . . . . . . . . . . . . 2

1.1.3 Bladder Cancer Staging. . . . . . . . . . . . . . . . . . . . . . 5

1.1.4 Genomic Landscape of Bladder Cancer. . . . . . . . . . . . . 7

1.1.4.1 Mutational Spectrum in NMIBC . . . . . . . . . . 7

1.1.4.2 Mutational Spectrum in MIBC . . . . . . . . . . . 12

1.1.5 The chr6p22.3 Amplification and CDKN2A Deletion . . . . . 15

1.1.5.1 SOX4 and Bladder Cancer – a brief introduction . 15

1.1.5.2 CDKN2A, RB, and E2F3 in Bladder Cancer . . . 16

1.2 Scope of this Dissertation. . . . . . . . . . . . . . . . . . . . . . . . . 17

2 SOX4 and the SOX gene Family . . . . . . . . . . . . . . . . . . . 19

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.1.1 Grouping of SOX Proteins gene family . . . . . . . . . . . . . 19

2.2 SOX4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.2.0.1 SOX4 and Development . . . . . . . . . . . . . . 26

2.2.0.2 SOX4 and Cancer . . . . . . . . . . . . . . . . . . 27

2.2.0.3 SOX4 - The Oncogene . . . . . . . . . . . . . . . . 27

2.2.0.4 SOX4 in Prostate Cancer . . . . . . . . . . . . . . 30

2.2.0.5 SOX4 in Bladder Cancer . . . . . . . . . . . . . . 30

2.2.1 SOX4 and EMT . . . . . . . . . . . . . . . . . . . . . . . . . . 32

2.2.1.1 History of EMT . . . . . . . . . . . . . . . . . . . 32

2.2.1.2 EMT in Embryonic Development andWound Healing ... 34

2.2.1.3 The Role of SOX4 in EMT . . . . . . . . . . . . . . 35

2.2.1.4 SOX4 – The Tumor Suppressor . . . . . . . . . . . 37

2.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3 SOX4 Regulates Invasion of Bladder Cancer Cells Via Repression of WNT5A. ...39

3.1 Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 43

3.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

3.4.1 Expression of SOX4 in bladder cancer patients and bladder cancer cell lines . .. 48

3.4.2 Wnt5a antagonist restores invasive ability in T24-SOX4-KD Cell line. . . . . . . . . 59

3.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

4 Novel Protein-Protein Interactions in Bladder Cancer Cell Lines . . . . . . . . . . . . . . . . 73

4.1 Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

4.3.1 Cell Lines , Reagents and Transfections. . . . . . . . . . . . . 78

4.3.2 High Throughput PPI Screening . . . . . . . . . . . . . . . . 79

4.3.3 In vivo mouse models . . . . . . . . . . . . . . . . . . . . . . 79

4.3.4 Cell Cycle Analysis . . . . . . . . . . . . . . . . . . . . . . . . 80

4.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

4.4.1 TR-FRET data Reveal Novel SOX4 Protein-Protein Interactions ... 80

4.4.2 Validating SOX4 Protein-Protein Interactions . . . . . . . . . 85

4.4.3 SOX4 knockdown and re-expression of p16 in shSOX4 cell line alters cellular morphology . . . . . 87

4.4.4 Re-expression of p16 in shSOX4 cell lines induces G1 arrest . 91

4.5 Orthotopic Bladder Injection of SW780 cells form tumors in mice . 93

4.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

5.1 SOX4/CDKN2A PPI Summary of Findings and Future Directions . 99

5.2 SOX4 and Wnt5A Summary of Findings and Future Directions. . . 102

5.3 Final Thoughts and Mechanism . . . . . . . . . . . . . . . . . . . . . 107

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Cell
Parola chiave	Bladder Cancer Cancer Biology
Committee Chair / Thesis Advisor	Moreno, Carlos S., Emory University
Committee Members	Marcus, Adam, Emory University Powers, Maureen, Emory University Kucuk, Omer, Emory University Santos, Carlos F., Frameshift LLC Fu, Haian, Emory University

Ultima modifica

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	The Role of SOX4 in Bladder Cancer Cell Lines ()	2018-11-18 17:47:23 -0500	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions