The mouse mammary tumor virus (MMTV) induces mammary adenocarcinomas in the Czech II strain of feral Mus musculus musculus with predictable frequency. Roughly twenty percent of mice infected with the exogenously transmitted virus develop pregnancy independent tumors after 15 months. Compared to other inbred strains of mice, the Czech II mice develop tumors relatively slowly suggesting that these mice have not developed genetic predispositions to cancer. Unlike other inbred strains of mice such as C3H and RIII, which have been bred to have a 100% mammary tumor incidence among parous females, and an average latency of 250 days, Czech II mice have not undergone selection for increased tumor frequency or short latency. Furthermore, Czech II mice have no endogenous proviral genomes while most other inbred strains carry between 2 to 8. These two aspects of CzechII mice make it an attractive model in which to screen for new common integration sites (CIS) involved with mammary tumorigenesis. Both Int-3, the activated intracellular domain of the Notch-4 receptor, and Int-6, the p48 component of the eukaryotic translation initiation factor 3 complex (eIF3p48), were originally isolated as retroviral tags in tumors from Czech II mice. We have surveyed additional Czech II mammary tumors in search of other new CIS for MMTV. Using a PCR based approach we have identified a novel CIS from a panel of 40 independently arising mammary tumors. A novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) was identified in five independently arising mouse mammary tumors. The insertion sites all cluster within a 1 kb region that is 2-3 kb 5' of the transcription initiation site of a gene, 2610028F08RIK, whose expression is normally silent in the mammary gland. One consequence of MMTV integration at this site is the activation of expression of 2610028F08RIK. These five tumors were also found to have 2-3 additional viral insertions, one or two of which were members of either the Wnt and/or FGF gene family. RT-PCR results demonstrated that each of the viral insertions led to elevated expression of the presumed target flanking genes. These results, taken together, suggests that viral induced expression of 2610028f08rik collaborates with viral induced Wnt and FGF during tumor progression.In another study we have found that Notch4 intracellular domain (ICD) attenuates TGF-b signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth inhibitory effects of TGF-b. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-b/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we have demonstrated that ICD4 and dRAM23 ICD4 inhibits SBE and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4 and are resistant to the growth inhibitory effects of TGF-b. Blockage of Notch4 processing to ICD4 by g-secretase inhibitor (GSI) makes MCF-7 cells sensitive to growth inhibition by TGF-b. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.Recently we have identified a novel 1.8 Kb Notch4/Int3 RNA species (designated Int3sh) (Imatani and Callahan, 1999). Int3sh RNA encodes a protein that is missing the CBF1 binding region (Ram 23) of the Notch 4/Int3 intracellular domain (ICD). We have detected Int3sh RNA expression in human colon, mammary and lung tumor tissue culture cell lines. Expression of Int3sh in the MCF10A """"""""normal"""""""" human mammary epithelial cell line confers on it the capability of anchorage independent growth in soft agar. To investigate the consequences of Int3sh expression in vivo we have developed three independent founder lines of Whey acidic protein (WAP) promoter-Int3sh transgenic mice. Mammary gland development in each of these founder lines is normal and the females can lactate. This is in contrast to mammary gland development in transgenic mice expressing the entire ICD of Notch 4/Int3 from either the WAP promoter or the MMTV long terminal repeat (LTR). In these mice mammary lobular development does not occur nor do they lactate. This suggests that expression of the Notch4/Int3 ICD blocks mammary gland development through the CBF1-dependent component of the Notch signaling pathway. However, like WAP-Notch4/Int3 ICD mice, WAP-Int3sh females from each of the founder lines develop mammary tumors. Based on these observations we hypothesize that Notch4/Int3 ICD induced tumorigenesis is a consequence of a CBF1-independent component of Notch4/Int3 signaling.
