Doctor of Philosophy in Cell and Developmental Biology (PhD)
Characterisation of novel targets of metabolic acid stress resistance in malignant cells
Studies of genome heterogeneity and plasticity aim to resolve how genomic features underlie phenotypes and disease susceptibilities. Identifying genomic features that differ between individuals and cells can help uncover the functional variants that drive specific biological outcomes. For this, single cell studies are paramount, as characterizing the contribution of rare but functional cellular subpopulations is important for disease prognosis, management and progression. Until now, these studies have been challenged by our inability to map structural variants accurately and comprehensively. To overcome this, I employed the template strand sequencing method, Strand-seq, to preserve the organization and structure of individual homologues and visualize structural rearrangements in single cells. Using Strand-seq, I monitored homologue states in human genomes to quantify the degree of somatic rearrangements, and distinguished these from recurrent structural variants, such as inherited inversions. In so doing, I created an innovative tool to rapidly discover, map, and genotype structural polymorphisms with unprecedented resolution. Next, to facilitate systematic analyses of Strand-seq data, I developed novel bioinformatic software that locates putative genomic rearrangements in singles cells and identifies recurrent rearrangements across multiple cells. This provides an essential instrument for unbiased and non-targeted structural variant discovery in a high-throughput approach, helping to scale Strand-seq for population-based studies. Applying these tools, I explored the distribution and frequency of structural variation in a heterogeneous cell population to discover and genotype over 100 inversions in the human genome. I found significant structural heterogeneity resides in definable polymorphic domains and within complex and repetitive regions of our genome. Finally, I extended my strategy to comprehensively map the complete set of inversions in an individual’s genome and define their unique invertome. Comparing two invertomes, I found sets of inversions can be combined to make predictions about ancestry and health of an individual, and I characterized the architectural features of inversion breakpoints with base-pair resolution. Taken together, I describe a powerful new framework to study structural rearrangements and genomic heterogeneity in single cell samples, whether from individuals for population studies, or tissues for biomarker discovery.
The nucleation, polymerization, and depolymerization of actin filaments is spatially and temporally controlled in order to regulate cell motility, cell morphology, and protein organization within the plasma membrane. By limiting receptor diffusion, the submembrane actin cytoskeleton modulates the signaling output of receptors such as the B-cell antigen (Ag) receptor (BCR) that are activated by clustering. Restricting BCR mobility limits BCR-BCR collisions and the resultant ‘tonic’ signaling. Conversely, more dynamic actin filaments or F-actin clearance promotes BCR-BCR collisions and leads to a ‘primed’ state where the threshold for Ag-induced activation is reduced. In chapter 2, I show a mechanism of receptor cross-talk where microbial danger signals (TLR ligands) prime B cells for Ag-induced activation by enhancing actin dynamics. TLR signaling reduced BCR confinement, promoted BCR-BCR collisions and potentiated responses to low densities of membrane-associated Ags.The interaction of B-cells with antigen-presenting cells displaying membrane-Ags results in initial BCR signaling that promotes cell spreading and increases the probability of BCRs encountering Ag. This is coupled with increased BCR mobility and the formation of BCR microclusters that recruit and activate signaling enzymes. Cell spreading and BCR microcluster mobility require severing of cortical submembrane actin, a precursor to F-actin branching that drives cell spreading. In chapter 3, I show that BCR signaling increases actin dynamics and BCR microcluster formation by activating the actin-severing protein cofilin via a signaling pathway involving Rap GTPases.
No abstract available.
Circulating tumor cells become fully metastatic if they are able to extravasate from the microvasculature and move into microenvironmental niches within distant site organs where they survive and proliferate. To determine if inflammation facilitates this process, models of inflammatory asthma, hypersensitivity pneumonitis, or bleomycin-induced injury were used, followed by introduction of B16F0 melanoma cells into the microvasculature of the lungs. Strikingly, all three conditions increased overt metastasis without increasing extravasation and the number and size of early metastases were increased. Bleomycin induced inflammation led to the increased survival of B16F0 tumor cells and recruitment of monocyte derived macrophages (MoDM) to the lungs. These MoDM were located near the micrometastatic niche and their presence correlated with increased metastatic tumor cell burden. Inflammation also increased the deposition of the ECM component hyaluronan (HA) in the lung stroma and it was enriched in B16F0 containing metastatic nodules. HA binding through its cognate receptor CD44 correlates with an increase in the metastatic potential of B16 melanoma cells. However, deletion of CD44 using CD44-/- mice or CD44-/- B16F0 cells did not affect inflammation-driven increases in metastasis. Chondroitin sulphate (CS) was found to negatively regulate HA binding in B16 cells, and CS-null CD44 constitutively bound high levels of HA unlike parental B16F0 cells. Thus, high HA binding may be required for effects on metastasis, or HA may be priming the inflammatory premetastatic niche in a CD44 independent manner. Taken together these findings illustrate the importance of the microenvironment in distant site metastasis and they highlight inflammation as an important modifier of this microenvironment.
Cell-cell junctions regulate the form and function of epithelial tissues, in part, by mechanically coupling adjacent cells together. Unlike normal cells, pre-malignant cells are capable of mechanically uncoupling these junctions in response to motogenic factors such that the cells become invasive and, ultimately malignant. Therefore, I asked whether the mechanical responses of cell-cell junctions to increases in intracellular tension are altered in pre-malignant mammary epithelial cells in the absence of such motogenic factors. In an effort to answer this question I altered the intracellular tension on the cell-cell junctions of normal (EpH4) and pre-malignant oncogenic ras-transformed (EpRas) mammary epithelial cells either chronically, by altering the density of cells attached to a rigid substratum, or acutely, by physically extending (i.e. ‘stretching’) confluent monolayers of cells attached to a compliant silicone rubber substrate. When intracellular tension was chronically increased, the tension-sensitive protein zyxin relocalized to cell-cell junctions in normal, but not pre-malignant cells. The zyxin relocalization in normal cells was associated with a junctional increase in the phosphorylated form of myosin light chain 2 (MLC2) suggesting that it may involve actomyosin contractility. The same differential in zyxin relocalization and phosphorylated MLC2 accumulation occurred when the intracellular tension was acutely increased in the two cell types. This differential was blocked by Rho-ROCK inhibition which indicates that it may be dependent on actomyosin contractility. In addition, apical actin structure reorganization occurred when intracellular tension was acutely increased in the normal cells that did not occur in the pre-malignant cells. Taken together, these observations led me to conclude that the ability of cell-cell junctions to respond in a mechanosensory-appropriate manner to changes in intracellular tension is compromised in ras-transformed pre-malignant mammary epithelial cells. Acute pharmacologic inhibition of oncogenic Ras-mediated increases in MAPK and/or PI3K signalling did not correct this compromised response. Therefore, this compromised mechanosensitivity, which may functionally contribute to the ability of pre-malignant cells to become invasive in response to motogenic factors, may be initiated by long term epigenetic changes that occur under conditions of stable oncogenic transformation.