The mouse mammary tumor virus (MMTV) is a potent somatic mutagen, integrating within the host genome and disrupting normal gene expression. One of these genes is Notch4/Int3. We have used differential display analysis to compare mammary tissue from pregnant normal and transgenic WAP (whey acidic protein) promoter -Notch4/Int3 mice and a WAP-Notch4/Int3 mammary tumor for RNA expression. Eight genes were differentially expressed in these tissues. Analysis of RNA from ten additional WAP-Notch4/Int3 mammary tumors demonstrated that the tumors could be divided into three groups based on the milk protein transcripts they expressed. In contrast, all of the WAP-Notch4/Int3 mammary tumors were down regulated for the Wdnm1 (Westmead DMBA8 nonmetastatic cDNA1), Gp (glycogen phosphorylase) and a mouse UniGene related to Hs.24139 human UniGene and were up regulated for Pi3k (phosphoinositide 3-kinase). To determine whether the differentially expressed genes were downstream of Notch in the signaling pathway, their expression was also compared in the mammary epithelial cell line HC11 and HC11 cells expressing activated Notch4/Int3 (designated Int3). Consistent with our earlier results, Wdnm1, Casein, Wap and Hs.24139 were down regulated in HC11-Int3 cells, suggesting that these genes are negatively regulated by Int3 signaling. Pi3k and Gp were not expressed in these two cell lines. We speculate that differential expression of the eight genes identified above occurs as the result of two distinct mechanisms. First, as the direct result of Notch/Int3 signaling in the affected cells and second as the result of the origin of the tumor as a clonal outgrowth of a minor cell population in the mammary gland. To further understand the normal function of Notch4/Int3 we performed a yeast two-hybrid system screen to isolate protein-binding partners for its intracellular domain (ICD). We identified transforming acidic coiled-coil protein 3 (TACC3) as a binding partner for Notch4/Int3. Members of the TACC family of proteins are concentrated at the centrosomes and can interact with microtubules. TACC3 specifically interacts with the Notch4/Int3 CDC10 repeats in the ICD and to a lesser extent with residues C-terminal to the repeats. TACC3 also interacts with the homologous region of Notch1, 2, and 3 ICDs. TACC3 is most highly expressed in the suggesting tissue specific functions. The TACC3 gene is expressed in the spleen, lung, and testis as well as the mammary gland. In the mammary gland the highest level of expression occurs during pregnancy suggesting that it is hormonally regulated or that it has a specific developmental stage function. TACC3 inhibits Notch4/Int3 ICD induced growth of HC11 mouse mammary epithelial cells in soft agar. In contrast, TACC3 does not block Notch4/Int3 ICD suppression of milk protein expression in HC11 cells treated with lactogenic hormones. These results suggest that TACC3 may be a negative regulator of some components of the Notch signaling pathway.
