One of the earliest manifestations of malignant progression is loss of tissue organization. This proposal examines the hypothesis that structural integrity is crucial for maintenance of normal breast function and suppression of neoplasia. We propose that characterization of the """"""""signaling integration plans"""""""" which establish and sustain acini will drive the discovery of therapeutically-relevant markers of malignancy. Based on our progress in the previous funding period, we now postulate that myoepithelial cells (myoeps) provide crucial structural and polarity cues to luminal cells that enable formation of normal acini, cues likely responsible for myoep tumor suppressor functions. In the last funding period, we replicated acini in """"""""designer"""""""" 3D microenvironments and established double-layered acini from purified human myoeps and luminal cells. We immortalized and characterized several breast cell lines and showed that, in culture, luminal cells acquire myoep-specific survival markers (e.g. a6b4 integrin), permitting acinus formation in the absence of myoeps. Importantly, we also showed that myoep production of laminin 1 (Ln-1) is crucial in establishing acinar polarity, as tumor myoeps lacking Ln-1 fail to induce polarity. We expand upon these findings in 4 specific aims: 1-Probe the nature of the """"""""cross-talk"""""""" and formation of basement membrane between luminal cells and myoeps using mouse/human chimeras of primary cells or immortalized cell lines, and mouse/mouse chimeras from wild-type and genetically engineered mice. We will assess the roles of heterotypic cell-cell adhesion and morphogens in the cross talk; 2-Explore the genetic and epigenetic mechanisms behind Ln-1 silencing in tumor myoeps (deletions, rearrangements, methylation or acetylation); 3-Identify Ln-1 domains and Ln-1 receptors required for induction of polarity (using Ln-1 fragments, inhibitory peptides, blocking antibodies and cre-lox ablation of receptors); 4-Screen for genes and pathways affecting acinar polarity. Collectively, these experiments address the role of myoeps and the importance and dominance of polar acinar structure in breast function. As loss of appropriate integration of these signals could lead to malignancy, our results may yield new markers and therapeutic strategies to limit or reverse breast tumor progression.
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