The transcriptional regulation and novel functions of the filopodia motor protein MYO10 during collective lung cancer invasion Restricted; Files Only

Summerbell, Emily (Summer 2020)

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Tumor heterogeneity drives disease progression, treatment resistance, and patient relapse, yet many questions remain regarding its contributions to tumor invasion and metastasis. Here, we investigated how heterogeneous DNA methylation and gene expression promote specialized myosin-X (MYO10)-expressing filopodia in leader cells to direct collective cancer cell invasion.

We investigated heterogeneity within collective cancer invasion by integrating DNA methylation and gene expression analysis in rare purified lung cancer leader and follower cells. Our results showed global DNA methylation rewiring in leader cells and phenotype-specific gene expression that differentiated both leader cells and follower cells from their parental population. Integration of DNA methylation and transcriptome analyses revealed that promoter hypermethylation silenced the tumor suppressor gene HTATIP2 in leader cells. Analysis also identified the filopodial motor MYO10 as a critical gene at the intersection of epigenetic heterogeneity and 3D collective invasion, being hypomethylated and overexpressed in leader cells. We further identified the Notch ligand JAG1 as a previously unknown upstream activator of MYO10 expression in leader cells. Using 3D live cell imaging of invading leader cells, we discovered that MYO10 drives filopodial persistence necessary for micropatterning extracellular fibronectin into linear tracks at the edge of 3D collective invasion. We further show that filopodia-directed fibronectin alignment is dependent upon the MYO10 cargo protein integrin b1. Our data fit a model where epigenetic heterogeneity and JAG1 signaling jointly drive collective cancer invasion through MYO10 upregulation in epigenetically permissive leader cells, which induces filopodia dynamics and long-term stability necessary for linearized fibronectin micropatterning.

In addition to localizing at the tips of long-lived leader cell filopodia at the front of collective invasion, MYO10 also localizes to additional subcellular compartments. Here, we show that MYO10 also localizes to filopodia-like structures including regions of cell-cell contact, retraction fibers, and the cleavage midbody in mitotic cells, but it does not localize with spindle poles. Thus, our data suggest that MYO10 acts not only within filopodia at the front of collective invasion and ECM remodeling but also regulates filopodia and filopodia-like structures in multiple cellular contexts necessary to coordinate collective cancer invasion.

Table of Contents

Chapter 1: Introduction

1.1 Cancer metastasis: A complex biological conundrum

1.1.1 Cancer by the numbers

1.1.2 Lung cancer

1.1.3 The process of metastasis

1.1.4 Single cell invasion

1.1.5 Collective cell invasion

1.1.6 Diversity and plasticity in migration modalities

1.1.7 Epigenetic regulation of phenotype plasticity and its implications for cancer invasion and metastasis

1.1.8 Heterogeneity in cancer invasion and metastasis

1.1.9 Leader and follower cells in lung cancer

1.2 The filopodia and its motor protein MYO10

1.2.1 The actin cytoskeleton: many forms and many functions

1.2.2 Filopodia: The fingers of the cell

1.2.3 Structure and function of MYO10

1.2.4 MYO10 regulates filopodia initiation and elongation

1.2.5 MYO10 regulates collective migration during development

1.2.6 Filopodia and MYO10 in cancer

1.3 Dissertation Goals

Chapter 2: Epigenetic heterogeneity between leader and follower cells reveals functional regulators of collective cancer invasion

2.1 Author’s Contribution and Acknowledgement of Reproduction

2.2 Abstract

2.3 Introduction

2.4 Methods

2.5 Results

2.6 Discussion

Chapter 3: MYO10 directs collective invasion through filopodia-driven fibronectin micropatterning by leader cells

3.1 Author’s Contribution and Acknowledgement of Reproduction

3.2 Abstract

3.3 Introduction

3.4 Methods

3.5 Results

3.6 Discussion

Chapter 4: Looking beyond the leading edge: Additional roles of MYO10 in regulating leader cell function

4.1 Author’s Contribution and Acknowledgement of Reproduction

4.2 Abstract

4.3 Introduction

4.4 Methods

4.5 Results

4.6 Discussion

Chapter 5: Discussion and Future Directions

5.1 Epigenetic heterogeneity functionally contributes to distinct leader and follower phenotypes

5.2 The uncharted waters of the genome: Future avenues for examining how chromatin regulation contributes to tumor phenotypic heterogeneity

5.3 MYO10 promotes collective invasion through long, stable filopodia in leader cells

5.4 JAG1/Notch regulates MYO10 expression

5.5 MYO10 promotes FN micropatterning at the invasive front of 3D collective invasion by inducing filopodial stability

5.6 MYO10 utilizes additional functions beyond the leading edge to regulate mitotic integrity and cell-cell adhesion in leader cells

5.7 Conclusions

Chapter 6: References

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