ERBB receptors and ligands have been implicated in breast development and tumorigenesis. Work in the applicant's laboratory has reinforced this view by showing that overexpression of an ERBB1/EGFR agonist, TGF-alpha, which is often elevated in human breast cancer, efficiently induces mammary tumors in mice. Glandular development and involution were also perturbed, suggesting not only that this might set the stage for subsequent tumorigenesis but also that these processes might be regulated normally via ERBB signaling as well. Indeed, his subsequent studies showed that the four ERBB receptors and their multiple ligands are all expressed in the virgin, pregnant, lactating, and involuting mouse mammary gland, albeit in different temporal patterns consistent with distinct roles. They also provided important evidence of functional interactions between ERBB receptors in vivo. Finally, his development of knockout mice lacking several EGFR ligands (amphiregulin (AR), TGF-alpha, EGF) individually or in combination established roles for ERBB signaling in mammary gland development and function. Thus, AR was required for pubescent ductal morphogenesis, while AR together with EGF and TGF-alpha was required for normal lobuloalveolar development and differentiation. Continuing this fruitful emphasis on mouse models, the applicant now wishes to address several issues raised by his previous work. First, he will investigate whether the requirement for AR in ductal morphogenesis reflects unique agonist properties as suggested by differences in the bioactivity of ERBB ligands in vitro. This will be accomplished by deriving mice harboring a knock-in mutation in which AR coding sequences have been replaced by those of TGF-alpha. Second, he will investigate the nature of the AR signal. He will begin by testing the hypothesis that paracrine activation of stromal EGFR is required for ductal morphogenesis; this will be accomplished by deriving mice that express only membrane-anchored, bioactive AR precursor. Using a combination of protein and RNA analyses, including microarray screening and subtractive cDNA cloning, he will then work to identify gene products whose AR-dependent expression or activation is critical for ductal morphogenesis. Third, using novel, knockout mouse models, he will determine whether three additional EGF family agonists that are expressed in the developing or differentiating and activate both EGFR and ERBB4, have roles as well. Fourth, he will investigate the physiological and pathological roles of ERBB4 signaling, and specifically test the hypothesis that ERBB4 promotes cellular differentiation rather than proliferation. This will be accomplished by generating transgenic mice that overexpress either wild type ERBB4 or a receptor chimera bearing the extracellular cellular domain of EGFR coupled to the signaling domain of ERBB4. The ability of ERBB4 to suppress TGF-alpha-induced mammary tumorigenesis will then be tested in bitransgenic mice harboring both receptor chimera and TGF-alpha transgenes.
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