RBPJK Conditional Knockout Mice Reveal Distinct Functions For NOTCH4 Intracellular Domain (Int3) on Mammary Development and Tumorigenesis. We have conditionally knocked out the RBP-J gene in the mammary gland of mice expressing the WAP-Int3 transgene. WAP-CRE/ RBP-Jf/+ /WAP-Int3 mice are phenotypically the same as WAP-Int3 mice with respect to mammary gland development and tumorigenesis. In WAP-CRE/ RBP-Jf/f/WAP-Int3 mammary gland development is normal and the mice lactate. However, these mice also develop mammary tumors at a frequency similar to WAP-Int3 mice but with a slightly longer latency. These results are consistent with the conclusion that the arrest of mammary gland development in WAP-Int3 mice is a consequence of RBP-J -dependent Notch signaling and mammary tumor development is a consequence of a RBP-J -independent component of Notch4 signaling or the simple derepression of RBP-J target genes. If the latter possibility were true then HC11 mammary epithelial cells the stable expression of RBP-J shRNA should confer on the cells the capability of anchorage independent growth in soft agar. However this result was not obtained. Furthermore, the stable expression of RBP-J shRNA did not block the ability of HC11-Int3 cells to grow in soft agar. This is consistent with Notch4/Int3 induced mammary tumor growth as being a consequence of Notch4/Int3 - RBP-J independent signaling. This work is currently being prepared for publication. R-spondin2/Int7 and Wnt1 collaborate to induce anchorage independent growth by mammary epithelial cells. R-spondin2/Int7 was identified as a novel common integration site for the mouse mammary tumor virus (MMTV) in five independently arising mouse mammary tumors. MMTV integration, which occurred in 5flanking region of the 2610028F08RIK gene (Lowther et al., 2005) results in over expression of the R-spondin2/Int7 gene which usually is silent or expressed at very low levels during mammary gland development. It is of interest that tumors with an MMTV insertion at Int7 were also found to have additional viral insertions flanking a member of the Wnt (Wnt3a, Wnt10b) and/or FGF (Fgf3, Fgf4) gene family. Reverse transcriptase PCR demonstrated that each of the viral insertions led to elevated expression levels of the presumed target flanking genes. These results suggested that there may be a collaboration between R-spondin2/Int7 and Wnt/Fgf genes during mammary tumorigenesis. R-spondins are described as secreted proteins with ligand-type activities that partially overlap canonical Wnt ligands. In fact several reports in the last 2 years have demonstrated activation of beta-catenin transcription activity by R-spondin family members in a range of species, using both in vitro and in vivo systems. We hypothesize that R-spondin2/Int7 may collaborate with Wnt/Fgf family members in the deregulation of normal control of growth and differentiation to stimulate the malignant transformation of mammary epithelial cells. To confirm the transforming potential of R-spondin2/Int7 we transfected two mouse mammary epithelial cell lines, HC11 and C57MG, with R-spondin2/Int7 under the control of the elongation factor-1alpha (EF-1α) promoter. Stable over expression of R-spondin2/Int7 dramatically changed morphological features and growth rate of the cell lines, but the cells did not exhibit the capacity for anchorage-independent growth in soft agar, indicative of a transformed phenotype. To test a putative collaboration of R-spondin2/Int7 with other pathways we introduced R-spondin2/Int7 to a C57MG Wnt1 cell line. Assays showed that C57MG cells over expressing Wnt1 alone were also not able to form colonies in soft agar whereas double transfectants had this capability. Preliminary microarray data confirmed synergistic activities between R-spondin2/Int7 and Wnt1 genes as well as changes in gene expression that were unique to Wnt1 and R-spondin2/Int7, respectively. This work is currently being prepared for publication. New common integration sites (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. We have found 81 CIS for MMTV in 255 viral induced mouse mammary tumors from CzechII, BALB/cfCZ and BALB/cfSpretus mice. Twelve of the CIS were identified as CIS in MMTV induced tumors in previous studies. The distribution of CIS was dependent on the host genetic background (BALB/c or CzechII) and on the strain of virus (MMTV-Cz or MMTV-Sp). Thirty of The CIS occurred within a gene. The target of the other CIS is the gene closest to the CIS. Currently we are validating the CIS target genes for activated expression using microarray analysis of tumor RNA
