Measuring signaling pathway dynamics during tissue growth in hydrogels
Principal Investigator: Lonnie D Shea
Abstract: DESCRIPTION (provided by applicant): Cancer initiation and metastatic progression are widely modeled in vitro, and biomaterials applied to these in vitro models can recapitulate the phenotypes that are observed in vivo. Our long-term goal is developing 3D systems for directed cell growth, and these studies are aimed at identifying pathways to target to prevent abnormal growth (e.g., cancer) or to promote the development of functional tissue replacements. We propose a cell array that reports on the large-scale dynamic activity of transcription factors (TFs for cells cultured in a hydrogel in order to investigate the relationship between oncogenes, material design (e.g., adhesion and degradation), and the active TFs, and the ultimate phenotype of the cells. Normal mammary epithelial cells (MECs) form round acini capable of milk production in permissive environments, whereas the combination of aberrant oncogene activity (e.g., ErbB2) and the presence of specific extracellular matrix environment can produce pre- invasive or invasive phenotypes. ErbB2 is overexpressed in 15-20% of patients with invasive breast cancer, and in 50% estrogen receptor (ER) negative and 12% of ER positive cases of ductal carcinoma in situ. The proposed studies focus on TFs that drive the transition from a normal to a pre-invasive phenotype (Aim 1), and subsequently from a pre-invasive phenotype to an invasive phenotype (Aim 2). TFs are key regulators of cell phenotype, as evidenced by their ability to generate pluripotent stem cells from fibroblasts, and they are the downstream targets of signaling pathways, which are the target of many pharmaceuticals. The quantification of TF activity is based on the parallel delivery of TF reporter constructs within an array, which is combined with bioluminescence imaging for large scale, dynamic quantification. Analysis of TF activity is distinct from the current genomics and proteomics approaches that quantify mRNA or proteins abundance respectively. These reporter constructs have typically been applied to few pathways at early time points, and our technology allows tracking of TF activity throughout development of normal and abnormal structures for cells cultured within hydrogels. We employ designer hydrogels to regulate adhesion and degradation in order to investigate the established dependence of phenotypic transitions on the matrix. In Aim 3, we investigate multiple drug therapies that target EGFR signaling, such as trastuzumab and lapatinib. While these compounds are being investigated clinically, their mechanism of action and the target patient population are unknown. Furthermore, many patients develop resistance to these compounds, and the dynamic analysis may identify compensatory pathways. The cellular response to therapeutics is dependent, in part, upon the microenvironmental context, and these studies are expected to identify conditions under which a therapeutic response is obtained. Taken together, we hypothesize that the dynamic TF activity will i) identify pathway signatures that correlate with normal and abnormal tissue growth, ii) provide fundamental design principles that relate the hydrogel design to active signaling pathways, and iii) identify mechanisms of drug action that may inform clinical trials. PUBLIC HEALTH RELEVANCE: In vitro models in biology and disease progression are an emerging opportunity, as 3D culture can provide more accurate models that recapitulate in vivo phenotypes. We apply a novel technology for large scale dynamic analysis of TF activity to investigate cancer initiation and progression resulting from aberrant activity of ErbB2, which is overexpressed in 15-20% of patients with invasive breast cancer, and invasion due to aberrant ErbB2 activity is dependent upon the properties of the extracellular matrix. Results from these studies are expected to identify which pathways are active in normal mammary epithelial cells and breast cancer cells to detect the transition from a normal to pre-invasive phenotype, or a pre-invasive to invasive phenotype, which can identify aberrant pathway activity that may lead to interventions that control the cell response.
Funding Period: 2012-09-10 - 2016-06-30
more information: NIH RePORT
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