Cell-Surface Interactions in Pathogenesis

我们在这个项目中的主要研究目标是描述细胞与细胞外基质和外源性添加的细胞外分子相互作用在发病机制中的作用。一个主要的焦点是肿瘤细胞的侵袭和转移，目标是识别新的分子调节剂和介质。癌症生物学中的一个基本问题是局部基质环境与癌症进展的关系。例如，晚期癌的一个共同特征是在结缔组织形成过程中，肿瘤周围会形成致密的胶原基质。在完成了确定致密纤维胶原蛋白在诱导蛋白水解活性侵过体中的作用的研究后，我们转向了基质底物物理刚性的改变如何改变各种人类肿瘤细胞系细胞迁移的性质的问题。为了量化细胞迁移的各种参数，已经开发了一种无偏差的自动化计算机系统，该系统使用视频荧光显微镜算法跟踪各种肿瘤细胞系的hoechst标记细胞核，以提供快速量化的速度，持久性和多定向迁移。这个名为“FastTracks”的程序是使用MATLAB软件开发的；然而，在最终出版之后，它可以在没有专有软件的普通计算机上下载使用。

Our major research goals in this project are to characterize the roles of cell interactions with the extracellular matrix and exogenously added extracellular molecules in pathogenesis. One major focus is on tumor cell invasion and metastasis, with a goal of identifying novel molecular regulators and mediators. A fundamental question in cancer biology is the relationship of the local matrix environment to cancer progression. For example, a common feature of advanced carcinomas is the induction of a dense collagenous matrix surrounding tumors during the process of desmoplasia. After completing studies establishing the role of dense fibrillar collagen in induction of proteolytically active invadopodia, we have turned to the question of how alterations in the physical rigidity of the matrix substrate can alter the nature of cell migration by a variety of human tumor cell lines. For quantifying a variety of parameters of cell migration, a non-biased, automated computer-based system has been developed that uses algorithms for video fluorescence microscopy tracking of Hoechst-labeled nuclei of a variety of tumor cell lines to provide rapid quantification of speed, persistence, and durotactic migration. This program termed "FastTracks" was developed using MATLAB software; after final publication, however, it can be downloaded for use on regular computers without proprietary software. Tumor cell invasiveness may be promoted by epithelial-mesenchymal transition (EMT). In studies of normal tissue morphogenesis, we identified the novel regulator Btbd7 as an inducer of the EMT transcription factor Snail2 and a disruptor of E-cadherin mediated cell-cell adhesion. We are currently evaluating whether Btbd7 or fibronectin, its upstream extracellular matrix regulator, can play a role in the invasiveness of certain tumor cells. Molecular interactions at the cell surface with soluble or matrix factors are likely to play important roles in many diseases. We have continued a long-term collaboration with Dr. Subhash Dhawan in CBER, FDA to characterize cell-surface and extracellular interactions involved in the pathogenesis or suppression of infectious diseases. Some molecules are found to enhance infectivity, while others can suppress this process. For example, the host cellular response to extracellularly provided hemin, an inducer of heme oxygenase-1 (HO-1), elicits host protective responses against viral infections including HIV and poxvirus. This collaborative project has recently been extended to explore and to document the effectiveness of inducing HO-1 mediated host responses to suppress Ebola and Zika virus infections. Since hemin is an FDA-approved drug, its induction of HO-1 might provide a potential general host-defense therapeutic strategy against a variety of pathogens. Specifically, we established that Zika virus can infect primary human monocyte-derived macrophages. Hemin dramatically reduced Zika virus replication in vitro. Hemin induced HO-1 expression and produced major reductions of >90% in Zika virus replication in human macrophages and LLC-MK2 monkey kidney cells with minimal toxicity. RNAi silencing of the expression of HO-1, or of its upstream regulatory gene Nrf2, attenuated hemin-induced suppression of Zika virus infection. Hemin, an FDA-approved drug for an unrelated disease (porphyria) may provide a useful therapeutic approach based on its ability to stimulate an innate cellular response against Zika virus infection. In a multi-site collaboration that extended beyond the FDA to include the Molecular and Translational Sciences Division of the United States Army Medical Research Institute of Infectious Diseases at Frederick and the Department of Medicine at Howard University, we tested whether hemin could inhibit Ebola virus replication through induction of HO-1. Treatment of not only primary monocyte-derived macrophages (MDM), but also Vero cells, HeLa cells, and human foreskin fibroblasts, reduced Ebola virus infection by >90% with minimal toxicity to infected cells. Inhibition of HO-1 enzymatic activity and RNAi silencing of HO-1 expression prevented this hemin-mediated suppression of Ebola virus infection. Consequently, re-purposing hemin to stimulate the innate HO-1 cellular response may provide an alternative approach to help alleviate Ebola virus infection. Because we have extensive expertise in real-time imaging of cell behavior in vitro and in organ explants, we have initiated a collaboration with Dr. Ashok Kulkarni's laboratory to use the GCaMP6 mouse system for direct visualization of calcium signaling for characterizing pain signaling in the mouse trigeminal ganglion.