The Notch signaling pathway is involved in cell fate decisions of tissues and organs in several different organisms. In mammals there are four members of the Notch gene family, some of which are targets for mutations that contribute to tumor development. Notch4 was originally identified as target for mouse mammary tumor virus (MMTV) induced insertional mutations in infected mouse mammary tumors . One consequence of MMTV integration into Notch4 is the transcription of a truncated Notch4 RNA species (designated Int3) that represents a gain of function mutation. The Int3 RNA species encodes the intracellular domain (ICD) of the receptor protein. Expression of Int3 as a transgene in mice from either MMTV long terminal repeat (LTR) or the whey acidic protein (WAP) promoter blocks female mammary lobular development and the ability to lactate. In addition all females develop mammary tumors. To identify genes that are differentially expressed in Int3 transgenic mammary tumors we have compared RNA from a WAP-Int3 mammary tumor and mammary tissue from a normal pregnant mouse. One of the genes that is expressed at high levels in all WAP-Int3 as well as MMTV LTR-Int3 mammary tumors relative to mammary gland RNA from normal pregnant females is the receptor tyrosine kinase c-Kit. At the present time we are determining whether the apparent up-regulation of c-Kit expression is a direct consequence of the Notch signaling pathway or whether it reflects the clonal expansion of a minority cell population within the mammary gland that forms the tumor. We are also interested in the role, if any c-Kit expression plays in Int3 induced mammary tumorigenesis. The drug STI571 is an inhibitor of cKit activity. To test the contribution of c-Kit activity on mammary tumor growth and development we have tested the effect of STI571 on WAP-Int3 mammary tumor development. Our preliminary results demonstrate that WAP-Int3 tumors regress to the point of being undetectable. When the drug is withdrawn tumor growth reappears. These results are consistent with the conclusion that cKit activity is both necessary and sufficient for tumor growth in the context of Notch4/Int3 signaling. 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.
