Stromal activation in breast cancer
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Epithelial-derived cancers such as breast cancer consist of transformed epithelial cells surrounded by a stroma that contains numerous cell types. Understanding the kinetics of stromal activation and characterizing both the cell types involved in the cancer stroma as well as their secreted proteins may lead to future therapies to stop breast cancer progression. It is unknown how stromal cells communicate with transformed epithelial cells to promote tumor growth and metastasis. The transforming growth factor β (TGFβ) pathway promotes epithelial to mesenchymal transition (EMT). Our lab has identified a secreted protein, aortic carboxypeptidase-like protein (ACLP) that is expressed in the breast cancer stroma and can exert effects by activating the TGFβ pathway. The role of ACLP in EMT during cancer progression is not known. We hypothesize that changes in the breast cancer stroma drive cancer progression and correlate with increased expression of α-smooth muscle actin (α-SMA) and collagen I (Col I) in stromal fibroblasts and that ACLP promotes EMT in the cancerous mammary epithelium. The first goal of this project was to develop an in vivo mouse model to first track and then isolate activated cells in the breast cancer stroma. The second goal was to test whether ACLP induces gene expression changes consistent with EMT. To identify changes in the breast cancer stroma we crossed mice expressing the Her2/neu receptor, which is part of the epidermal growth factor receptor family and occurs in 20-30% of all human breast cancers, with transgenic mice harboring α-SMA (SMA-mCherry) and collagen (Col I-Tpz) promoters driving red and green variants of green fluorescent protein (GFP) respectively. Initial experiments characterized the expression of these reporters at baseline and in tumors. Both the tumor and mammary gland tissue were isolated and a new method to detect fluorescent reporter activity was developed. Whole mount and sections of the tumors and the surrounding stroma defined at least two distinct cell populations: those that express SMA and those that express Col I, and a possibility of a third population expressing both. In contrast to the stroma of a normal mammary duct, which contained dispersed Col I+ cells and not cells of the other two populations, the stroma in the tumor sections contained a population of SMA+ cells and an increase in Col I+ cells dispersed on the periphery of the tumor. Aligned streaks of Col I+ cells were identified. Normal murine mammary gland (NMuMG) cells treated with ACLP showed a decrease in E-cadherin mRNA levels and increase in vimentin mRNA levels. Immunofluorescence staining revealed decreased E-cadherin on the cell borders after ACLP treatment, and, when also treated with TGFβ results in increased detected intracellular vimentin. In summary, we developed a new model to detect changes in the stroma associated with breast cancer development as well as a procedure for preserving fluorescence in mouse tissue for analysis. We also found evidence to support the idea that ACLP promotes EMT. Ongoing work will characterize the stromal cells by flow cytometry and expression profiling and develop strategies to target stroma-derived proteins to treat breast cancer.
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