The erbB2 (or HER2, neu) gene-encoded receptor tyrosine kinase (RTK) belongs to the EGF receptor family. ErbB2 overexpression was previously shown to correlate with poor prognosis and the number of lymph node metastases in breast cancer patients. In our study, we found that stable tranfection of the human erbB2 gene into the low erbB2-expressing MDA-MD-435 human breast cancer cells (names 435.eB transfectants) enhanced the intrinsic metastatic potential of these cells. Contrary to the well established notion that erbB2 enhances metastasis of breast cancer cells, little is known about its underlying molecular mechanisms. Since erbB2 overexpression has been found in ~30% of breast tumors, it is very important to examine the molecular mechanisms underlying erbB2-mediate metastasis. During the current funding period, we found that erbB2 overexpression in 435.eB transfectants led to increased cell invasion, matrix metalloprotease (MMP)-2/MMP-9 activities, vascular endothelial growth factor (VEGF) secretion, and apoptosis resistance. We hypothesize that erbB2 overexpression can enhance the RTK signaling capacity that activates downstream signals to elicit these effects, which all can contribute to enhanced metastatic potential. This competitive renewal application outlines experimental approaches to elucidate the signaling events (from the erbB2 receptor to its downstream pathways) leading to these known effects of erbB2 related to metastasis. First, using various mutants of the erbB2 receptor, we will determine the structural requirements of the tyrosine kinase domain and tyrosine autophosphorylation sites in the erbB2 receptor for mediating signals leading to cell invasion, MMP-9 activity, VEGF secretion, angiogenesis (MMP-9 and VEGF are known to facilitate angiogenesis), survival, and ultimately, metastasis in an animal model of breast cancer metastasis. Second, we will investigate the involvement of erbB2 proximal-downstream signaling Shc-Grb2-Ras, PI3K-Akt) in the effects of erbB2 related to metastasis by biochemical and genetic manipulations of the signaling molecular in breast cancer cells. Similarly and finally, we will examine the contributions of erbB2 distal-downstream signaling (MEK-ERK, JNKK-JNK pathways) in erbB2-mediated metastasis. The completion of the experiments outlined in this proposal will provide the first detailed analysis of the relationship between erbB2 signal transduction and metastasis, which will identify critical points in the signaling pathways for future intervention of erbB2-mediated metastasis.
