One of the earliest manifestations of malignant progression is loss of tissue organization. This proposal continues to examine the hypothesis that architectural integrity is crucial for maintenance of normal breast function as well as for suppression of neoplasia. We have postulated that elucidation of a "signaling integration plan" that establishes and maintains polarity and structure within the acini and ducts of breast tissue will uncover prognostic and therapeutically-relevant markers of intermediary steps in malignancy. As such, we postulated and provided evidence that myoepithelial cells (MEPs) provide crucial structural and functional cues to luminal epithelial cells (LEPs) partly via production of, and signaling through, laminin-111 (Ln-1). We further postulated, and now provide additional evidence, that in traditional cell culture conditions, purified LEPs almost immediately develop a hybrid LEP/MEP phenotype acquiring aspects of MEP gene expression and in particular developing the tumor suppressor functions normally conferred in vivo by MEPs and an ability to make acini, which normally requires the presence of MEPs. Consequently, in order to make relevant models for the study of human LEP-MEP interactions we must identify the conditions that instruct these cell types to retain their original functions. Accordingly, we have developed new and versatile microenvironmental arrays (MEArrays) to probe how MEPs and LEPs become, and remain, determined. We now propose to expand our findings in 3 specific aims. We will specifically: 1- identify pathways that allow retention of MEP- and LEP- specific functions in culture using designer MEArrays and designer media. We then could probe the importance of desmosomal proteins and other regulatory molecules in addition to Ln-1 produced by MEPs, in how MEPs and LEPs interact to retain polarity, architecture and function. 2- complete the identification of Ln-1 signaling cascade components for mammary specific functions using inhibitory peptides, blocking antibodies, mutant cells and shRNA ablation in high throughput assays using Cellomics and other devices and designer 3D microenvironments. 3- identify and characterize central players and connections in the 'signaling integration plan'for structural integrity of acini using bioinformatics analysis of gene expression arrays, genome-wide methylation profiles and other changes in chromatin structure, identification of miRNAs affecting cellular architecture, and by positioning the new genes we identified using a unique 3D screen, on our integration map. Collectively these experiments address the importance of MEP/LEP interactions in maintenance of polar acinar structures in breast tissue, and could also provide a proof of principle for other tissues. Since loss of appropriate balance and/or integration of these signals leads to malignancy, our results will both advance fundamental knowledge and yield novel markers for early diagnosis and therapeutic strategies to limit and/or reverse breast tumor progression.
This proposal seeks to define the influences of cellular microenvironments on human breast cells, with particular attention to the effects of myoepithelial and luminal epithelial cells on one another. Building on our studies characterizing the role of the microenvironment in normal human breast morphogenesis, we have repeatedly shown that changes in the microenvironment can cause malignant human breast tumor cells to re-acquire critical attributes of normal cells, and we have specifically demonstrated that myoepithelial cells play a central role in preventing human breast cells from becoming malignant. This proposal continues our research into this important field of inquiry;our results will further common understanding of the complex interactions which serve to contain and even reverse human breast tumors.
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