Tumorigenesis and Neoplastic Progression Overexpression of Activated Murine Notch 1 and Notch 3 in Transgenic Mice Blocks Mammary Gland Development and Induces Mammary Tumors

The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1 gene, producing insertional mutations in mammary tumors of MMTV/ neu transgenic (Tg) mice. In these mammary tumors, the Notch1 gene is truncated upstream of the transmembrane domain, and the resulting Notch1 intracellular domain (Notch1), deleted of most extracellular sequences, is overexpressed. Although Notch1 transforms mammary epithelial cells in vitro , its role in mammary gland tumor formation in vivo was not studied. Therefore, we generated MMTV/ Notch1 Tg mice that overexpress murine Notch1 in the mammary glands. We observed that MMTV/Notch1 Tg females were unable to feed their pups because of impaired ductal and lobuloalveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of -casein. Notch1 repressed expression of the -casein gene promoter, as assessed in vitro with a -casein/luciferase reporter construct. The MMTV/Notch1 Tg females developed mammary gland tumors, confirming the oncogenic potential of Notch1 in vivo. Furthermore, MMTV/ Notch3 Tg mice exhibited a very similar phenotype. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland. (Am J Pathol 2006, 168:973–990; DOI: 10.2353/ajpath.2006.050416) The mammalian Notch genes are the homologues of the Drosophila Notch, Caenorhabditis elegans lin-12 and glp-1 and Xenopus Xotch genes. The mammalian Notch1 belongs to the family of Notch genes (Notch1 to Notch4) that play critical roles in cell fate determination in many developmental systems. Notch1 encodes a large 330-kd transmembrane protein. Its extracellular domain harbors 36 EGF-repeats, 3 Notch-Lin12 repeats, and two conserved cysteines (C and C) close to the transmembrane domain. The intracellular Notch1 domain (Notch1) contains the domain I (RAM 23), six CDC10/ankyrin repeats, and OPA and PEST sequences at the C-terminus. Truncated versions of Notch1 protein, containing only the intracellular part of the receptor (Notch1), behave as constitutively active gain-of-function mutants in Drosophila, in C. elegans, in Xenopus embryos, and in mammalian cells. –10 The Notch3 gene has been detected only in mammals. Its structure is similar to that of Notch1, although its intracellular domain is shorter and lacks the OPA region. In addition, several deletions, insertions, and substitutions of amino acid residues distinguish Notch1 from Notch3. Moreover, its intracellular domain is a poor activator of HES-1 and HES-5, in contrast to that of Notch1. The Notch4 protein intracellular domain is significantly shorter than that of Notch1 at the C-terminus. A number of deletions, insertions, and substitutions of amino acid residues also differ between the Notch1 and Notch4 domains. To date, two members of the murine Notch gene family, Notch1 and Notch4, have been implicated in mammary gland development and neoplasia. The Notch4/int3 gene was initially identified as a common