Kit and PDGFR-alpha activities are necessary for Notch4/Int3 induced tumorigenesis.Transgenic mice over expressing Notch4 intracellular domain (Int3) under the control of the WAP or MMTV-LTR promoters, develop mammary tumors. Microarray analysis of these tumors revealed high levels of c-Kit expression. Gleevec is a tyrosine kinase inhibitor that targets c-Kit, platelet-derived growth factor receptors (PDGFRs) and c-Abl. This led us to speculate that tyrosine kinase receptor activity might be a driving force in the development of Int3 mammary tumors. WAP-Int3 tumor-bearing mice were treated with continuous release of Gleevec using subcutaneous implanted Alzet pumps. Phoshorylation of c-Kit, PDGFRs and c-Abl is inhibited in Int3 transgenic mammary tumors by Gleevec. Inhibition of these enzymes is associated with a decrease in cell proliferation and angiogenesis, and an induction of apoptosis. To examine the signaling mechanisms underlying Notch4/Int3 tumorigenesis, we employed siRNA to knockdown c-Kit, PDGFRs and c-Abl alone or in combination and observe the effects on soft agar growth of HC11 cells over-expressing Int3. Only siRNA constructs for c-Kit and/or PDGFR-alpha were able to inhibit HC11-Int3 colony formation in soft agar. Our data demonstrates an inhibitory effect of Gleevec on Int3 induced transformation of HC11cells and mammary tumors and indicates an oncogenic role for c-Kit and PDGFR- alpha tyrosine kinases in the context of Int3 signaling. This work is described in an article in Oncogene, 2006.Expression of Truncated Int6/eIF3e in Mammary Alveolar Epithelium Leads to Persistent Hyperplasia and Tumorigenesis.Int6/eIF3e was first identified in a screen for commonly mutated genes in mouse mammary tumor virus (MMTV)-induced mammary tumors. The MMTV insertion resided within an intron of one allele of Int6 producing a truncated mRNA. Expression of truncated Int6 (Int6sh) from the eEF1A promoter in stably-transfected human and mouse mammary epithelial cell lines lead to cellular transformation. To validate that Int6sh has transforming activity in vivo, a transgenic mouse line was constructed using the whey acidic protein (WAP) promoter to target expression of the truncated Int6 cDNA to the mammary gland. Mammary tumors developed in 40% of Wap/Int6sh heterozygous retired breeders at an average age of 18 months. The remaining glands without tumors contained widespread focal alveolar hyperplasia. Only 4% of Wap/Int6sh virgin females developed tumors by two years of age, an incidence that is consistent with the lower expression of the Wap promoter in mammary tissue of non-pregnant mice. Microarray analyses of Wap/Int6sh-expressing mammary tissues indicated that an early pattern of gene expression appeared during the first pregnancy and persisted in a more prominent fashion in the alveolar hyperplasia and adenocarcinomas. These observations provide the first in vivo evidence that mammary-specific expression of the Int6sh mutation leads to persistence of alveolar hyperplasia with the accompanying increased predisposition to mammary tumorigenesis. This work has been submitted to Cancer Research for publication.
