Activated protein tyrosine kinases, which have been implicated in the genesis of a number of human cancers, rely on a variety of protein-protein interactions to transmit their proliferative signals within the cell. These interactions are often mediated by Src homology 2 and 3 (SH2 and SH3) domains. A class of proteins which are mainly composed of such domains, termed adaptor proteins, has been identified. The Growth factor receptor bound proteins Grb2 and Grb7 are SH2 domain adaptor proteins which have been shown to associate directly or in complex with many tyrosine kinases, including the c-ErbB-2/Neu receptor tyrosine kinase. While overexpression of either protein alone in rat fibroblasts is not transforming, human breast cancer cell lines exhibit Grb2 and Grb7 gene amplification, and mRNA and protein overexpression. The role of Grb2 in polyomavirus middle T antigen-mediated mammary tumorigenesis has been examined utilizing gene targeting and transgenic approaches. Initial characterization of the progeny of matings involving Grb2+/mice and MMTV/middle T transgenic mice indicated that delayed tumor kinetics may be the result of Grb2 dosage differences between mT+;Grb2+/-and mT+;Grb2+/+ animals. Transgenic animals expressing a dominant negative version of Grb2 in the mammary epithelium have been generated to explore an alternate method for disrupting signaling from middle T antigen. The role of Grb2 and Grb7 in Neu-mediated mammary tumorigenesis is also being examined. Both MMTV/Grb2 and MMTV/Grb7 transgenic mice that express the transgene in the mammary epithelium have been identified by ribonuclease protection analysis. Matings involving these strains and MMTV/neu mice should aid in determining the effects of overexpressing Grb2 or Grb7 on Neu-mediated mammary tumorigenesis. / Thesis / Master of Science (MS)

The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover. SLK activity is regulated by the LIM domain-binding proteins Ldb1/2. In addition to playing role in tumor initiation and progression, these proteins have been demonstrated to interact with LMO4. Therefore, this project assessed the ability of LMO4 to interact and regulate SLK activity. Results show that LMO4 can directly bind to SLK and activate its kinase activity. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding. Cre deletion of LMO4 inhibits cell migration and SLK activation, and impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Src/Yes/Fyn-deficient cells (SYF) express very low levels of LMO4 and do not recruit SLK to the leading edge. Src-family kinase inhibition impairs SLK recruitment to the leading edge, suggesting that both expression of LMO4 and the recruitment of SLK to the leading edge require c-Src activity. In conclusion, cell migration and activation of SLK requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. This study also investigated whether LMO4 deletion through MMTV-Cre-driven excision would impair mammary tumorigenesis in a PyMT mouse model of breast cancer. No difference in Overall Survival was observed between animals with and without LMO4 expression. Western blot analysis and IHC showed that tumors expressed LMO4 protein in animals genotyped as Cre-positive. This result suggests that expression of LMO4 is required for tumor initiation in the PyMT model of murine mammary carcinoma. This project has established a novel cytosolic role for the transcriptional co-activator LMO4 and validated it’s involvement in the regulation of SLK and cell migration. This pathway may provide a novel therapeutic strategy as LMO4 appears to be critical to the initiation and progression of breast cancer.