A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

Polyploidy and Mitotic Cell Death are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells

Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy.

Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.

From Polarity to Morphogenesis PAK Behaviors and Mechanism for Bud Sensing in Morphogenesis Checkpoint

Bud formation by Saccharomyces cerevisiae is a fundamental process for yeast proliferation. Bud emergence is initiated by the polarization of the cytoskeleton, leading to local secretory vesicle delivery and gulcan synthase activity. The master regulator of polarity establishment is a small Rho-family GTPase – Cdc42. Cdc42 forms a clustered patch at the incipient budding site in late G1 and mediates downstream events which lead to bud emergence. Cdc42 promotes morphogenesis via its various effectors. PAKs (p21-activated kinases) are important Cdc42 effectors which mediate actin cytoskeleton polarization and septin filament assembly. The PAKs Cla4 and Ste20 share common binding domains for GTP-Cdc42 and they are partially redundant in function. However, we found that Cla4 and Ste20 behaved differently during the polarization and this depended on their different membrane interaction domains. Also, Cla4 and Ste20 compete for a limited number of binding sites at the polarity patch during bud emergence. These results suggest that PAKs may be differentially regulated during polarity establishment.

Morphogenesis of yeast must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site prior to bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor”. Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed, and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.

Molecular Mechanisms of Airway Epithelial Progenitor Cell Maintenance and Repair.

The lungs are vital organs whose airways are lined with a continuous layer of epithelial cells. Epithelial cells in the distal most part of the lung, the alveolar space, are specialized to facilitate gas exchange. Proximal to the alveoli is the airway epithelium, which provides an essential barrier and is the first line of defense against inhaled toxicants, pollutants, and pathogens. Although the postnatal lung is a quiescent organ, it has an inherent ability to regenerate in response to injury. Proper balance between maintaining quiescence and undergoing repair is crucial, with imbalances in these processes leading to fibrosis or tumor development. Stem and progenitor cells are central to maintaining balance, given that they proliferate and renew both themselves and the various differentiated cells of the lung. However, the precise mechanisms regulating quiescence and repair in the lungs are largely unknown. In this dissertation, ionizing radiation is used as a physiologically relevant injury model to better understand the repair process of the airway epithelium. We use in vitro and in vivo mouse models to study the response of a secretory progenitor, the club cell, to various doses and qualities of ionizing radiation. Exposure to radiation found in space environments and in some types of radiotherapy caused clonal expansion of club cells specifically in the most distal branches of the airway epithelium, indicating that the progenitors residing in the terminal bronchioles are radiosensitive. This clonal expansion is due to an increase in p53-dependent apoptosis, senescence, and mitotic defects. Through the course of this work, we discovered that p53 is not only involved in radiation response, but is also a novel regulator of airway epithelial homeostasis. p53 acts in a gene dose-dependent manner to regulate the composition of airway epithelium by maintaining quiescence and regulating differentiation of club progenitor cells in the steady-state lung. The work presented in this dissertation represents an advance in our understanding of the molecular mechanisms underlying maintenance of airway epithelial progenitor cells as well as their repair following ionizing radiation exposure.

Role of Vinculin in Regulating Force-Sensitive Dynamics of Adherens Junctions During Collective Cell Migration

Dynamic processes such as morphogenesis and tissue patterning require the precise control of many cellular processes, especially cell migration. Historically, these processes are thought to be mediated by genetic and biochemical signaling pathways. However, recent advances have unraveled a previously unappreciated role of mechanical forces in regulating these homeostatic processes in of multicellular systems. In multicellular systems cells adhere to both deformable extracellular matrix (ECM) and other cells, which are sources of applied forces and means of mechanical support. Cells detect and respond to these mechanical signals through a poorly understood process called mechanotransduction, which can have profound effects on processes such as cell migration. These effects are largely mediated by the sub cellular structures that link cells to the ECM, called focal adhesions (FAs), or cells to other cells, termed adherens junctions (AJs).

Overall this thesis is comprised of my work on identifying a novel force dependent function of vinculin, a protein which resides in both FAs and AJs - in dynamic process of collective migration. Using a collective migration assay as a model for collective cell behavior and a fluorescence resonance energy transfer (FRET) based molecular tension sensor for vinculin I demonstrated a spatial gradient of tension across vinculin in the direction of migration. To define this novel force-dependent role of vinculin in collective migration I took advantage of previously established shRNA based vinculin knock down Marin-Darby Canine Kidney (MDCK) epithelial cells.

The first part of my thesis comprises of my work demonstrating the mechanosensitive role of vinculin at AJ’s in collectively migrating cells. Using vinculin knockdown cells and vinculin mutants, which specifically disrupt vinculin’s ability to bind actin (VinI997A) or disrupt its ability to localize to AJs without affecting its localization at FAs (VinY822F), I establish a role of force across vinculin in E-cadherin internalization and clipping. Furthermore by measuring E-cadherin dynamics using fluorescence recovery after bleaching (FRAP) analysis I show that vinculin inhibition affects the turnover of E-cadherin at AJs. Together these data reveal a novel mechanosensitive role of vinculin in E-cadherin internalization and turnover in a migrating cell layer, which is contrary to the previously identified role of vinculin in potentiating E-cadherin junctions in a static monolayer.

For the last part of my thesis I designed a novel tension sensor to probe tension across N-cadherin (NTS). N-cadherin plays a critical role in cardiomyocytes, vascular smooth muscle cells, neurons and neural crest cells. Similar to E-cadherin, N-cadherin is also believed to bear tension and play a role in mechanotransduction pathways. To identify the role of tension across N-cadherin I designed a novel FRET-based molecular tension sensor for N-cadherin. I tested the ability of NTS to sense molecular tension in vascular smooth muscle cells, cardiomyocytes and cancer cells. Finally in collaboration with the Horwitz lab we have been able to show a role of tension across N-cadherin in synaptogenesis of neurons.

Risk Prediction for Epithelial Ovarian Cancer in 11 United States-Based Case-Control Studies: Incorporation of Epidemiologic Risk Factors and 17 Confirmed Genetic Loci.

Previously developed models for predicting absolute risk of invasive epithelial ovarian cancer have included a limited number of risk factors and have had low discriminatory power (area under the receiver operating characteristic curve (AUC) < 0.60). Because of this, we developed and internally validated a relative risk prediction model that incorporates 17 established epidemiologic risk factors and 17 genome-wide significant single nucleotide polymorphisms (SNPs) using data from 11 case-control studies in the United States (5,793 cases; 9,512 controls) from the Ovarian Cancer Association Consortium (data accrued from 1992 to 2010). We developed a hierarchical logistic regression model for predicting case-control status that included imputation of missing data. We randomly divided the data into an 80% training sample and used the remaining 20% for model evaluation. The AUC for the full model was 0.664. A reduced model without SNPs performed similarly (AUC = 0.649). Both models performed better than a baseline model that included age and study site only (AUC = 0.563). The best predictive power was obtained in the full model among women younger than 50 years of age (AUC = 0.714); however, the addition of SNPs increased the AUC the most for women older than 50 years of age (AUC = 0.638 vs. 0.616). Adapting this improved model to estimate absolute risk and evaluating it in prospective data sets is warranted.