Project Summary/Abstract The activity proposed in this Research Specialist Award application is in line with the long-standing goals of Dr. Thea Tlsty (the Unit Director)'s currently NCI-funded research program which currently supports my activity/effort: i) understand dynamic mechanisms underlying cellular plasticity and tissue response to stress; and ii) explore the role of tissue microenvironment and how these multiple factors contribute to tumor initiation and progression. These efforts are particularly worthwhile when dealing with the most lethal type of breast cancer, metaplastic breast cancers (MBCs). Identifying tissue components and signaling pathways that contribute to the emergence of these cancers in order to design more successful therapies for patients diagnosed with MBCs is indeed of the outmost clinical relevance. This application is the result of my personal ‘twist’ on the long-term interest of the Tlsty laboratory on the role of stromal-epithelial interactions in breast tumorigenesis. Its core concept relies on the increasingly appreciated notion that cancer progression requires an instructive tumor microenvironment (TME). Generation of a TME results from: acquisition of a cancer- associated fibroblast signature, a shift in macrophage type, alterations of ECM characteristics that either facilitate or counteract tumor growth, a corrupted immune response, an expanded and leaky vasculature and as a correlate an hypoxic environment and increased epithelial cell plasticity. Such phenotypes are exemplified in aggressive tumors characterized by a desmoplastic stroma with extensive and altered ECM deposition typified by MBCs. Thus, the imbalance in ECM composition seen in MBCs is the result of extensive structural and functional stromal changes that result from alterations in both stromal and epithelial cell properties. Our provocative prediction that a specific ECM/stromal make-up may identify cancers with the poorest outcome across breast cancer subtypes (luminal, Her2-positive, basal-like) may be of high clinical relevance not only for MBCs and basal-like cancers, but also for luminal breast cancers with late recurrence. To gain valuable insights, I will: i) identify stromal drivers (and repressors) of progression of MBCs through comparative proteomic analysis of ECM from breast tumor (including MBC) and disease-free breast specimens with an emphasis on post-translational modification status; ii) interrogate gene expression databases for upregulation (or down-regulation) of these ECM proteins across breast cancer subtypes (luminal, Her2-positive, basal, claudin low) through data mining; iii) test the ability of some of these ECM proteins to drive (or repress) cell plasticity (multilineage commitment and epithelial mesenchymal transition) in vitro using 3D cell culture models; and iv) confirm in vivo the pro- (or anti-)metaplastic/tumorigenic activity of these ECM proteins in murine xenograft models. My extensive experience in protein ...