The role of LKB1 in regulating actin and cellular polarization during motility 公开

Wilkinson, Scott Christopher (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/n009w2917?locale=zh
Published

Abstract

Lung cancer is the leading cause of cancer related deaths in the United States, with an estimated 158,080 deaths in 2016. Importantly, 90% of all cancer related deaths are due to metastasis, highlighting the importance of understanding the biological basis of cancer cell invasion and metastasis.

LKB1 is the 3rd most-commonly mutated gene in lung adenocarcinoma, with the majority of mutations being truncations disrupting kinase activity and removing its C-terminal domain (CTD). Since LKB1 inactivation drives cancer metastasis in mice, we examined how LKB1 inactivation impacts cytoskeletal structure, single cell motility, and cell polarization during invasion. Cells re-expressing wildtype LKB1 colocalize strongly with actin in a farnesylation-dependent but kinase-independent manner. These cells also exhibit actin stress fiber assembly, which we show is through RhoA-ROCK signaling. Further, LKB1 kinase activity regulates cell membrane ruffling, through the regulation of FAK activity. A combination of the LKB1 farnesylation and kinase activity coordinate to spatially regulate focal adhesion deposition and size during cell motility.

While examining 3D invasion, we show that cells re-expressing wildtype LKB1 or CTD alone exhibited mesenchymal polarity with strong directional persistence, which is completely abrogated upon farnesylation loss. Since the CTD is kinase-dead, these data highlight a farnesylation-dependent but kinase-independent regulation of polarity and directionality during invasion. We then examined how farnesylation regulates cellular polarity, and show rescuing RhoA activity in farnesylation-compromised cells restores this polarized phenotype. Importantly, activating RhoA in the absence of LKB1 fails to restore mesenchymal polarity, indicating a region of LKB1 is necessary for the regulation of polarity. Inverse of polarity, LKB1 signals to MARK1 in a farnesylation-independent but kinase-dependent manner to repress FAK, which represses collagen remodeling during 3D invasion. Since LKB1 frequently undergoes truncations affecting farnesylation and kinase activity, cancer cells with LKB1-loss would lose regulation of the actin cytoskeleton, exhibit unique amoeboid morphologies with hyperactive FAK, and acquire the ability to remodel collagen. Together, these data suggest that a combination of kinase-dependent and -independent defects create a uniquely invasive cell upon LKB1 inactivation.

Table of Contents

Table of Contents

Abstract ii

Acknowledgements iv

Table of Contents v

List of Figures vii

List of Abbreviations x

Chapter 1: Introduction 1

1.1 Lung Cancer 1

1.1.1 Lung Cancer Invasion and Metastasis 3

1.1.2 Common Lung Cancer Mutations 5

1.2 Cellular Polarization in Lung Cancer 5

1.2.1 Rho-GTPases and Polarity 6

1.2.2 Epithelial-to-Mesenchymal Transition and Cancer 9

1.3 Cell Motility During Invasion 10

1.3.1 Focal Adhesions 10

1.3.2 Actin Dynamics During Motility 11

1.4 LKB1 as a Tumor Suppressor in Lung Cancer 14

1.4.1 Identification of LKB1 through Peutz-Jeghers Syndrome 15

1.4.2 LKB1 Regulates Cell Polarity 16

1.4.3 LKB1 Drives Adhesion Dynamics 19

1.5 Identifying LKB1-mediated Cancer Cell Polarization and Invasion 22

Chapter 2: Coordinated cell motility is regulated by a combination of LKB1 farnesylation

and kinase activity 24

2.1 Author's Contribution and Acknowledgement of Reproduction 24

2.2 Abstract 25

2.3 Introduction 26

2.4 Methods 29

2.5 Results 37

2.6 Discussion 57

2.7 Acknowledgements

Chapter 3: LKB1 kinase-dependent and -independent defects disrupt polarity and

adhesion signaling to drive collagen remodeling during invasion 68

3.1 Author's Contribution and Acknowledgement of Reproduction 68

3.2 Abstract 69

3.3 Introduction 70

3.4 Methods 74

3.5 Results 88

3.6 Discussion 110

3.7 Acknowledgements 125

Chapter 4: General Discussion and Future Directions 126

4.1 LKB1 in Lung Cancer 126

4.2 LKB1 Localization 127

4.3 LKB1 and Actin Dynamics During Cell Motility 128

4.4 LKB1 Drives Cellular Polarization During Invasion 132

4.5 LKB1 Regulates Adhesion Signaling 134

4.6 Conclusions 136

Chapter 5: References 140

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