One of the earliest manifestations of malignant progression in pathological biopsies is loss of organization, as seen by a disruption of the unity of form that exists between the epithelium and the stroma. I have argued that the microenvironment of luminal epithelial cells which includes the basement membrane (BM) is crucial for maintenance of normal breast function. My co-workers and I have created appropriate microenvironments using reconstituted basement membranes and other complex substrata in 3- dimensional cultures (3D) to study the molecular mechanisms involved in regulation of gene expression in normal and malignant mouse and human breast epithelial cells. We have shown that the BM and its most studied receptors, integrins, are essential regulators of function, and that a disruption in BM, or an imbalance in integrins, leads to inappropriate morphogenesis, apoptosis and cancer. In the last granting period, we characterized a phenotypically normal human breast epithelial cell line (HMT3522- S1) and its malignant derivative (T4-2), which overexpresses beta1 integrin (>5 folds) and EGFR (>10 folds) on its cell surface. Treatment with either inhibitory antibodies (ab) or Fab fragments to beta1 integrin, or abs against BGFR or treatment with compounds that inhibit BGFR activity, reverted the malignant behavior in 3D cultures and sharply reduced growth in agar and tumorigenicity in nude mice. Whereas reciprocal cross-modulation of beta1 integrin and BGFR (i.e. inhibition of one receptor causes inhibition of the other) was observed in 3D growth in BM, growth factor and adhesion signaling were not cross modulated in monolayers. The hypotheses examined in this proposal are that the integrity of breast unit structure (referred to here as acini in culture; and TDLU or terminal ductal lobular unit in vivo) is crucial both for maintenance of breast-specific gene expression and for integration of adhesion and growth factor signalling. Further, that in vivo, it is the myoepithelial cells (myoeps) that provide structural cues for the luminal cells, and thus are the ultimate """"""""tumor suppressors"""""""" in the breast. We propose to analyze the kinetics and the mechanism of reversion and integration in 3D and perform a genetic screen for the discovery of intermediate signaling molecules that connect the two pathways. Since EGFR, alpha6beta4 integrin and a number of other markers that are expressed on epithelial cells in culture but in vivo are expressed essentially in myoeps and not in luminal cells, we will explore how the two cell types communicate and integrate these signals. Finally, we will define conditions for formation of a TDLU in culture using both primary luminal and myoeps and those immortalized with human papilloma type 16 E6/E7. We will also assess the consequences of myoep loss using conditional ablation of these cells in culture and in vivo in mammary gland fat pads of nude mice. These experiments address fundamental and poorly studied aspects of breast physiology, i.e. those of the importance and dominance of tissue structure, as well as the role of myoeps in regulation of normal breast function. Since the loss of proper interaction and integration of these pathways could lead to malignant conversion, the results may form the basis of new intervention and therapies in breast cancer.
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