Abstract
Wap-Int3 transgenic females expressing the Notch4 intracellular domain (designated Int3) from the whey acidic protein promoter exhibit two phenotypes in the mammary gland: blockage of lobuloalveolar development and lactation, and tumor development with 100% penetrance. Previously, we have shown that treatment of Wap-Int3 tumor bearing mice with Imatinib mesylate (Gleevec) is associated with complete regression of the tumor. In the present study, we show that treatment of Wap-Int3 mice during day 1 through day 6 of pregnancy with Gleevec leads to the restoration of their lobuloalveolar development and ability to lactate in subsequent pregnancies in absence of Gleevec treatment. In addition, these mice do not develop mammary tumors. We investigated the mechanism for Gleevec regulation of Notch signaling and found that Gleevec treatment results in a loss of Int3 protein but not of Int3 mRNA in HC11 mouse mammary epithelial cells expressing Int3. The addition of MG-132, a proteasome inhibitor, shows increased ubiquitination of Int3 in the presence of Gleevec. Thus, Gleevec affects the stability of Int3 by promoting the degradation of Int3 via E3 ubiquitin ligases targeting it for the proteasome degradation. Gleevec is a tyrosine kinase inhibitor that acts on c-Kit and PDGFR. Therefore, we investigated the downstream substrate kinase GSK3β to ascertain the possible role that this kinase might play in the stability of Int3. Data show that Gleevec degradation of Int3 is GSK3β dependent. We have expanded our study of the effects Gleevec has on tumorigenesis of other oncogenes. We have found that anchorage-independent growth of HC11-c-Myc cells as well as tumor growth in nude mice is inhibited by Gleevec treatment. As with Int3, Gleevec treatment appears to destabilize the c-Myc protein but not mRNA. These results indicate that Gleevec could be a potential therapeutic drug for patients bearing Notch4 and/or c-Myc positive breast carcinomas.