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Involvement of Notch1 in the development of mouse mammary tumors
A Diévart1, N Beaulieu, P Jolicoeur
1Laboratory of Molecular Biology, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montréal, Québec, Canada H2W 1R7.
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 approximately 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 (Notch1intra). We found that 3' truncated Notch1intra 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 Notch1intra protein behaves as an oncogene in this system.
Insights
Mouse mammary tumor virus (MMTV) insertional mutagenesis identified Notch1 as a collaborator in c-neu/erbB2-driven mammary tumor formation. Truncated Notch1intra protein functions as an oncogene, transforming epithelial cells.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Transgenic mouse models are crucial for studying oncogene cooperation in cancer development.
- The c-neu/erbB2 oncogene alone is insufficient for spontaneous mammary tumor formation in MMTV/neu Tg mice.
- Identifying collaborating oncogenes is essential for understanding mammary tumorigenesis.
Purpose of the Study:
- To identify collaborating genes involved in c-neu/erbB2-driven mammary tumor formation using insertional mutagenesis.
- To investigate the role of Notch1 gene activation by MMTV provirus in mammary tumors.
- To characterize the oncogenic potential of truncated Notch1 proteins.
Main Methods:
- Provirus insertional mutagenesis in MMTV/neu transgenic mice infected with MMTV.
- Identification of MMTV integration sites in mammary tumors.
- Analysis of Notch1 gene rearrangements and transcript expression.
- In vitro transformation assays using HC11 mouse mammary epithelial cells with truncated Notch1 proteins.
Main Results:
- Notch1 was identified as a novel target for MMTV provirus insertional activation.
- MMTV insertions led to 5' truncated Notch1 transcripts encoding an ectodomain-only mutant (N(EC)mut).
- MMTV insertions also generated 3' truncated Notch1 transcripts (Notch1intra) lacking extracellular domains.
- The truncated Notch1intra protein transformed HC11 mouse mammary epithelial cells in vitro.
- Specific domains (ankyrin-repeats, domain 1) of Notch1intra were required for transformation, while others were dispensable.
Conclusions:
- The Notch1 gene is a collaborative oncogene cooperating with c-neu/erbB2 in mammary tumor development.
- N-terminally truncated Notch1intra protein acts as an oncogene by transforming mammary epithelial cells.
- These findings reveal a novel mechanism of oncogene activation and cooperation in mammary tumorigenesis.