Overexpression and amplification of the new (c-erbB2, ERBB2) protooncogene have been implicated in the development of aggressive human breast cancer. To directly assess the effect of mammary gland-specific expression of the neu protooncogene, transgenic mice carrying unactivated neu under the transcriptional control of the mouse mammary tumor virus promoter/enhancer were established. By contrast to the rapid tumor progression observed in several transgenic stra carrying the activated neu transgene, expression of unactivated neu in the mammary epithelium resulted in the development of focal mammary tumors after long latency. The majority of the mammar tumors analyzed expressed elevated levels of neu-encoded mINA anid protein. Overexpresslon of neu in the mammary tumors was also associated with elevated neu intrinsic tyrosine kinase activity and the de novo tyrosine phosphorylation of several cellular proteins. Interestingly, many of the tumor-baring transgenic mice developed secondary metastatic tumors in the lung. These observations suggest that overexpression of the unactivated neu protein can induce metastatic disease after long latency.

Transgenic mice expressing the polyomavirus (PyV) middle T antigen (MT) develop multifocal mammary tumors which frequently metastasize to the lung. The potent transforming activity of PyV MT is correlated with its capacity to activate and associate with a number of signaling molecules, including the Src family tyrosine kinases, the 85-kDa Src homology 2 subunit of the phosphatidylinositol 3 (PI-3) kinase, and the Shc adapter protein. To uncover the role of these signaling proteins in MT-mediated mammary tumorigenesis, we have generated transgenic mice that express mutant PyV MT antigens decoupled from either the Shc or the PI-3 kinase signaling pathway. In contrast to the rapid induction of metastatic mammary tumors observed in the strains expressing wild-type PyV MT, mammary epithelial cell-specific expression of either mutant PyV MT resulted in the induction of extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias expressing the mutant PyV MT defective in recruiting the PI-3 kinase were highly apoptotic, suggesting that recruitment of PI-3 kinase by MT affects cell survival. Whereas the initial phenotypes observed in both strains were global mammary epithelial hyperplasias, focal mammary tumors eventually arose in all female transgenic mice. Genetic and biochemical analyses of tumorigenesis in the transgenic strains expressing the PyV MT mutant lacking the Shc binding site revealed that a proportion of the metastatic tumors arising in these mice displayed evidence of reversion of the mutant Shc binding site. In contrast, no evidence of reversion of the PI-3 kinase binding site was noted in tumors derived from the strains expressing the PI-3 kinase binding site MT mutant. Tumor progression in both mutant strains was further correlated with upregulation of the epidermal growth factor receptor family members which are known to couple to the PI-3 kinase and Shc signaling pathways. Taken together, these observations suggest that PyV MT-mediated tumorigenesis requires activation of both Shc and PI-3 kinase, which appear to be required for stimulation of cell proliferation and survival signaling pathways, respectively.Mammary epithelial cell-specific expression of the polyomavirus (PyV) middle T (MT) oncogene in transgenic mice results in the induction of multifocal metastatic mammary tumors involving 100% of the transgene carriers (19). The potent oncogenic properties of the PyV MT are due to its ability to associate with and activate a number of cellular signaling proteins. One class of cellular enzymes activated by PyV MT consists of members of the Src family tyrosine kinases (c-Src and c-Yes) (8,12,27,30). Expression of PyV MT in mammary glands of Src-deficient mice rarely results in the induction of mammary tumors (20), suggesting that activation of Src by PyV MT is required for mammary tumorigenesis. Whereas activation of c-Src is required for the rapid induction of mammary tumors, this event is not sufficient, because expression of a constitutively active version of Src in the mammary ...

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Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30%o of human breast cancers. Although the activation of Neu receptor tmsine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu. To test whether c-Src was implicated in Neu-mediated signal transduction, we measured the activity of the c-Src tyrosine kinase in tissue extracts from either mammary tumors or adjacent mammary epithelium derived from transgenic mice expressing a mouse mammary tumor virus promoter/enhancer/unactivated neu fusion gene. The Neu-induced mammary tumors possessed six-to eightfold-higher c-Src kinase activity than the adjacent epithelium. The increase in c-Src tyrosine kinase activity was not due to an increase in the levels of c-Src but rather was a result of the elevation of its specific activity. Moreover, activation of c-Src was correlated with its ability to complex tyrosine-phosphorylated Neu both in vitro and in vivo. Together, these observations suggest that activation of the c-Src tyrosine kinase during mammary tumorigenesis may occur through a direct interaction with activated Neu.The neu (c-erbB2) proto-oncogene encodes a growth factor receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor (2,8,18,26,28,40). Oncogenic activation of Neu can occur through multiple molecular mechanisms, including point mutations in the transmembrane domain (3), deletion of the extracellular domain (4), and overexpression (9,10,16). Activation of the Neu kinase results in its association with and tyrosine phosphorylation of a number of downstream signalling proteins. Moreover, the activities of phospholipase C-yl, the GTPase-activating protein (GAP), and protein tyrosine phosphatase 1D are elevated following tyrosine phosphorylation by activated Neu (1,11,27,39).Elevated expression of Neu has been observed in 20 to 30% of primary breast cancers and has been inversely correlated with the survival of the patient (12,23,29,30 c-Src activity was attributed to an increase in the specific activity of c-Src (24). Consistent with these observations, activation of c-Src kinase activity in the mammary epithelium of transgenic mice by expression of the polyomavirus (PyV) middle-T antigen results in rapid induction of metastatic mammary tumors (13). Given the potential importance of c-Src in mammary tumorigenesis, we assessed whether c-Src would play a role in Neu-mediated mammary tumorigenesis. To this end, protein extracts from normal or tumor tissues derived from MMTV/unactivated neu transgenic mice were immunoprecipitated with Src-specific antibodies and subjected to in vitro kinase assays. The results of these analyses revealed that the mammary tumor extracts contained six-to eightfold higher levels...

The cyclin D1 gene is overexpressed in breast tumors and encodes a regulatory subunit of cyclin-dependent kinases that phosphorylate the retinoblastoma protein.