Aurora Kinase A Promotes Oncogenic Signaling Through Novel Protein-Protein Interactions Open Access

Umstead, MaKendra (2016)

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Cancer is a collection of diseases driven by genomic changes that alter normal protein-protein interactions, induce aberrant cellular signaling, and drive cellular transformation. Aurora Kinase A (Aurora A), a mitotic kinase that is amplified in several cancer types, has emerged as a compelling target for cancer therapy. While increased expression of Aurora A correlates to a worse prognosis for cancer patients, the impact of Aurora A overexpression on protein-protein interactions, oncogenic signaling, and cancer development remains unclear. This work describes the discovery and characterization of the novel interaction of Aurora A with two important mediators of cancer growth and development: H-Ras and Forkhead box transcription factor, FOXO1. The Ras-mitogen activated protein kinase (MAPK) signaling cascade is a critical pathway for sustained cell growth and proliferation in cancer. We validated the interaction of Aurora A and H-Ras and determined that the kinase domain of Aurora A and the N-terminal Switch I and II domains of H-Ras are involved in binding. Aurora A positively regulates this pathway by forming a protein complex with H-Ras and Raf-1, the Ras effector that mediates MAPK signaling. Aurora A stabilizes the H-Ras/Raf-1 protein complex and enhances MAPK signaling in a H-Ras-dependent manner. We also determined that the kinase activity of Raf-1 also functions to enhance binding of the Aurora A/H-Ras/Raf-1 protein complex. Aurora A also promotes oncogenic signaling through negative regulation of the tumor suppressor, FOXO1. In response to cell stress, FOXO1 localizes to the nucleus to initiate transcription of pro-apoptotic genes. Aurora A was found to interact with FOXO1, promoting exclusion of FOXO1 from the nucleus and inhibition of cell death. Overall, this work demonstrates that through novel protein-protein interactions, Aurora A functions as a positive regulator of oncogenic Ras-MAPK signaling and as a negative regulator of the tumor suppressive activity of FOXO1. This provides two potential therapeutic protein-protein interaction targets for cancers with Aurora A overexpression, as inhibition of either the Aurora A/H-Ras or Aurora A/FOXO1 interactions may reduce pro-growth signaling and induce cell death. Ultimately, understanding the role of Aurora A in cellular signaling will provide new opportunities to develop targeted therapies for cancer.

Table of Contents

Chapter 1: Introduction 1

1.1 Cancer and its characteristics 2

1.2.1 The role of protein-protein interactions (PPIs) in cancer 3

1.2.2 PPI network mapping to unravel tumor biology 7

1.3.1 Aurora kinases 7

1.3.2 Aurora A protein structure 8

1.3.3 Aurora A regulation 11

1.3.4 Aurora A in tumorigenesis 12

1.4.1 Non-canonical functions of Aurora A 13

1.4.1.i Aurora A PPIs with GTPases and their regulators 13

1.4.1.ii Aurora A PPIs with transcription factors 14

1.5.1 Novel Aurora A interaction partners 14

1.5.2 Ras 16

1.5.2.i Ras protein structure 16

1.5.2.ii Ras regulation 18

1.5.2.iii Ras in tumorigenesis 20

1.6 Forkhead box protein of the O class (FOXO1) 22

1.8.1 Aurora A and MAPK in cancer 25

1.8.2 Aurora A and FOXO1 in cancer 25

1.9 Scope of the dissertation 25

Chapter 2: Aurora Kinase A interacts with H-Ras and potentiates Ras-MAPK signaling 27

2.1 Abstract 28

2.2 Introduction 28

2.3 Materials and methods 30

2.4 Results 36

2.4.1 Aurora A is a novel H-Ras binding partner 36

2.4.2 Aurora A interacts with H-Ras through the switch I and II regions 40

2.4.3 The kinase domain of Aurora A mediates the H-Ras interaction 40

2.4.4 Aurora A enhances ERK phosphorylation 44

2.4.5 Aurora A-induced ERK phosphorylation requires Ras-MAPK signaling 45

2.4.6 Aurora A forms a protein complex with H-Ras and Raf-1 and acts through H-Ras to enhance MAPK signaling 49

2.5 Discussion 54

Chapter 3: The impact of kinase activity on the Aurora A/H-Ras/Raf-1 protein complex 57

3.1 Introduction 58

3.2 Materials and methods 59

3.3 Results 63

3.3.1 The kinase domains of Aurora A and Raf-1 mediate their interaction 63

3.3.2 The Aurora A/H-Ras interaction is phosphorylation independent 67

3.3.3 Constitutively active Raf-1 enhances Aurora A/Raf-1 binding 69

3.3.4 Kinase activity is required for Raf-1 to enhance the Aurora A/H-Ras binding 70

3.3.5 Sorafenib attenuates the Aurora A/H-Ras interaction 70

3.4 Discussion 74

Chapter 4: Aurora Kinase A interacts with FOXO1 and inhibits FOXO1-induced apoptosis 78

4.1 Introduction 79

4.2 Materials and methods 81

4.3 Results 85

4.3.1 Validation of the Aurora A/FOXO1 interaction 85

4.3.2 Aurora A interacts with FOXO1 independently of AKT phosphorylation 85

4.3.3 Aurora A binds nuclear and cytoplasmic FOXO1 in a distinct pattern 88

4.3.4 Aurora A inhibits FOXO1-induced apoptosis 91

4.3.4 Aurora A suppresses FOXO1-induced apoptosis by inhibiting the nuclear localization of FOXO1 93

4.4 Discussion 99

Chapter 5: Discussion 103

5.1 Novel functions for Aurora Kinase A (Aurora A) are revealed through protein-protein interactions 104

5.2 Aurora A interacts with H-Ras, forming a positive feedback loop that sustains enhanced oncogenic MAPK signaling 107

5.3 Structural domain characterization provides insight into functional outcomes of Aurora A interactions 109

5.3.1 Aurora A domains 109

5.3.2 H-Ras domains 110

5.4 Dual roles for the Aurora A/Raf-1 interaction 111

5.5.1 Functional impacts of the Aurora A/FOXO1 interaction 112

5.5.2 Postulated mechanisms for FOXO1 deregulation by Aurora A 113

5.6 Future directions and translational implications 113

References 116

List of Figures

Figure 1-1. Protein-protein interactions and cancer. 6

Figure 1-2. The structure of Aurora A. 10

Figure 1-3. Aurora A signaling in cancer. 15

Figure 1-4. The structure of Ras proteins. 17

Figure 1-5. Ras activation by receptor tyrosine kinases (RTKs). 19

Figure 1-6. The mitogen-activated protein kinase (MAPK) signaling pathway. 21

Figure 1-7. FOXO1 signaling in cancer. 24

Figure 2-1. Detection of the Aurora A/H-Ras interaction. 38

Figure 2-2. Interactions between Aurora and Ras proteins are mediated through conserved domains. 42

Figure 2-3. Aurora A potentiates ERK activation via H-Ras. 47

Figure 2-4. Aurora A forms a complex with H-Ras and Raf-1, acting through H-Ras to enhance ERK activation. 51

Figure 2-5. Proposed model for the role of Aurora A in the Aurora A/H-Ras/Raf-1 oncogenic signaling complex. 53

Figure 3-1. The kinase domains of Aurora A and Raf-1 are involved in the interaction. 65

Figure 3-2. The Aurora A/Raf-1 interaction is phospho-independent. 68

Figure 3-3. The role of Raf-1 in the Aurora A/H-Ras/Raf-1 protein complex. 72

Figure 3-4. Proposed model for the role of Raf-1 in the Aurora A/H-Ras/Raf-1 signaling complex. 77

Figure 4-1. Validation of the Aurora A/FOXO1 interaction. 87

Figure 4-2. Localization of the Aurora A/FOXO1 interaction. 90

Figure 4-3. Aurora A inhibits FOXO1-induced cell death in non-transformed cells. 92

Figure 4-4a. Aurora A inhibits FOXO1 nuclear translocation and rescues FOXO1-induced cell death. 96

Figure 4-4b. Aurora A inhibits FOXO1 nuclear translocation. 98

Figure 4-5. Potential mechanisms for negative regulation of FOXO1 nuclear translocation and inhibition by Aurora A protein-protein interactions. 102

Figure 5-1. Novel Aurora A interactions and their function in cancer. 106

Figure A-1. Schematic representation of TR-FRET and Venus PCA assays. 129

Figure A-2. The Aurora A/H-Ras interaction localizes to the plasma membrane and possibly the Golgi apparatus in HEK 293T and Cos7 cells. 130

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