Involvement of Notch1 in the development of mouse mammary tumors

Abstract

The MMTV/neu transgenic (Tg) mice spontaneously develop mammary tumors stochastically after a long latent period, suggesting that the c-neu/erbB2 oncogene is not sufficient for tumor formation. To identify putative collaborator(s) of the c-neu/erbB2, we used the provirus insertional mutagenesis approach with mammary tumors arising in MMTV/neu Tg mice infected with the mouse mammary tumor virus (MMTV). The Notch1 gene was identified as a novel target for MMTV provirus insertional activation. In Notch1-rearranged tumors, the Notch1 gene was interrupted by the MMTV provirus insertion upstream of the exons coding for the TM domain. These insertions led to overexpression of novel 5′ truncated ∼7 kb RNA coding for 280 kDa mutant protein harboring only the Notch1 ectodomain, N(EC)^mut. These may be involved in tumor formation. Another consequence of these insertions was the expression of truncated 3′ Notch1 transcripts (3.5 – 4.5 kb) and proteins (86 – 110 kDa) deleted of most of the extracellular sequences (Notch1^intra). We found that 3′ truncated Notch1^intra can transform HC11 mouse mammary epithelial cells in vitro. Deletion analysis revealed that the ankyrin-repeats and the domain 1 (aa 1751 – 1821) are required, while a signal peptide, the two conserved cysteines (C¹⁶⁵² and C¹⁶⁸⁵) and the OPA and PEST sequences are dispensable for transformation. These results indicate that the N-terminally truncated Notch1^intra protein behaves as an oncogene in this system.