Dissecting 3-D invasion mechanisms in lung cancer Público

Abstract

The process of metastasis is the leading cause of cancer-related deaths, killing about 90% of all cancer patients. An early step in the metastatic cascade is local invasion of tumor cells into the microenvironment, a process that arises due to aberrant cell polarity, reduction of cell-cell contacts, and ultimately increased motility. A known regulator of cell polarity is LKB1, a serine/threonine kinase that is frequently mutated in lung adenocarcinomas. Other known functions of LKB1 involve regulating cell metabolism, growth, and adhesion; thus, the loss of LKB1 in lung adenocarcinomas likely has a vast impact on the tumor cell biology. However, the role of the two known functional domains of LKB1, the kinase domain and C-terminal domain with a membrane-targeting farnesylation motif, in regulating early invasion has not been well studied, especially in the context of physiologically relevant 3-D matrices. Our work illustrates that each functional domain of LKB1 has an independent role. The farnesylation of LKB1 in the C-terminal domain is necessary for maintaining a polarized phenotype during 3-D invasion, and this is dependent on RhoA activity. Conversely, the kinase activity of LKB1 functions to regulate adhesion signaling, via repression of focal adhesion kinase (FAK). When the kinase activity of LKB1 is disrupted, FAK is hyperactive, which in turn increases the ability of the cell to reorganize and align the matrix during invasion. While the study of LKB1 in lung adenocarcinoma progression and metastasis is important, many patients do not harbor mutations in this gene; thus, taking a broader perspective on lung cancer invasion was warranted. In 3-D lung cancer models that utilize collective invasion, there is phenotypic heterogeneity with the collective invasion chain, with highly invasive leader cells that pioneer invasion and cells which immediately attach to and follow the leader cell, termed followers. In order to dissect these two subpopulations within the collective invasion chain, we created an image-guided genomics technique termed SaGA (spatiotemporal genomic and cellular analysis). With this technique, we were able to isolate, analyze, and amplify purified populations of leader and follower cells. We found that leader cells are highly stable, maintaining their invasive capabilities even when cultured as a purified population. Leader cells can also promote the motility and invasion of the noninvasive follower population. Transcriptome and molecular analyses revealed that the formation of the collective unit is dependent upon VEGF-based signaling, and that leader cells seem to hijack this canonical vasculogenic machinery to promote follower invasion. Additionally, leader cells themselves utilize cell-ECM interactions to create force on the microenvironment to promote fibrous highways to facilitate their invasion. Conversely, follower cells are a highly proliferative population, whereas leader cells struggle to grow and survive as a purified population. Follower cells rectify leader cell mitotic defects when grown in co-culture, and this is through an unknown soluble factor. Taken together, our data suggest that leader and follower cells within the lung cancer collective invasion chain possess a symbiotic relationship, wherein leaders provide followers with an escape mechanism while followers provide leaders with increased growth and survival. Collectively, these studies dissect 3-D lung cancer invasion mechanisms, providing novel insights into this early step in the metastatic cascade.

Table of Contents

Chapter 1. Introduction

1.1 Lung cancer pg 1

1.1.1 Lung cancer overview pg 1

1.1.2 Common lung cancer mutations pg 3

1.1.3 Heterogeneity in lung tumors pg 3

1.2 Tumor metastasis overview pg 5

1.2.1 Steps of the metastatic cascade pg 5

1.2.2 Factors that influence metastasis pg 8

1.3 LKB1 background pg 11

1.3.1 LKB1 in Peutz-Jeghers Sydrome and cancer pg 11

1.3.2 Cellular processes regulated by LKB1 pg 13

1.3.3 LKB1 in lung cancer invasion and metastasis pg 14

1.4 Phenotypic heterogeneity during invasion pg 15

1.4.1 Single cell invasion pg 15

1.4.2 Collective cell invasion pg 18

1.5 Dissertation goals pg 20

Chapter 2. LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion

2.1 Introduction pg 24

2.2 Methods pg 27

2.3 Results pg 36

2.4 Discussion pg 61

Chapter 3. Developing an image-guided genomics technique to probe the mechanisms regulating lung cancer collective invasion

3.1 Introduction pg 75

3.2 Methods pg 77

3.3 Results pg 88

3.4 Discussion pg 111

Chapter 4. Conclusions and Future Directions

4.1 LKB1 in regulation of 3-D lung cancer invasion pg 125

4.2 Cell symbiosis between invasive subpopulations in collective invasion pg 130

References

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Health Sciences, Oncology Biology, Cell
Palavra-chave	Lung cancer Collective invasion LKB1 Cancer biology
Committee Chair / Thesis Advisor	Marcus, Adam I, Emory University
Committee Members	Kowalczyk, Andrew, Emory University Boise, Lawrence, Emory University Zhou, Wei, Emory University Vertino, Paula M, Emory University

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