Neu differentiation factor (NDF/heregulin) is secreted by ras-transformed cells and elevates tyrosine phosphorylation of the neu-encoded receptor kinase. Evidence from animal model systems and clinical observations suggest that overexpression of the neu/erbB-2 proto-oncogene may contribute to carcinoma development in humans. In vitro, NDF, as well as certain tumor-inhibitory monoclonal antibodies to p185neu, induce growth-arrest and differentiation of certain mammary tumor cells. This raises the possibility that mAb- or ligand-mediated effects may reverse the transformed phenotype through interaction with the oncogene product. Here we propose to experimentally test this intriguing possibility on the basis of a comprehensive biological study of NDF. Ectopic expression of wild-type and mutant NDF molecules will test the mitogenic versus differentiation potential of the factor, and correlate the domain structure of the precursor molecule with specific cellular functions. Preliminary in situ hybridization analysis and chromosomal mapping will be extended in order to guide later experiments that will address the physiological role of NDF by using gene targeting and inhibitory mAbs. The latter will be used also to assay NDF in body fluids in an attempt to correlate its expression with physiological disorders. Lastly, the mechanism of signal transduction by NDF will be analyzed. This includes the identification of a co-receptor of Neu whose existence was inferred by recent experiments, and characterization of the intracellular targets and processing of NDF-Neu complexes. Vectors that direct the synthesis of anti-sense RNA, or negative dominant mutants of p185neu, will be expressed in human tumor cells in order to test the role of Neu in maintaining the malignant state. Independent of this genetic approach, the effect of NDF and mAbs to Neu will be tested on cultured tumor cells and their xenografts in athymic mice. If successful, our later experiments will employ growth inhibitory combinations of NDF with various modified mAbs. Combinely, these studies are expected to shed light on the physiology of NDF and the neu gene, and their relevance to cancer development. They may further yield novel strategies and molecular tools that will help to control the growth of human carcinoma cells.
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