Distinct tyrosine autophosphorylation sites mediate induction of epithelial mesenchymal like transition by an activated ErbB-2/Neu receptor.

Tight control of cell proliferation and morphogenesis is required to ensure normal tissue patterning and prevent cancer formation. Overexpression of the ErbB-2/Neu receptor tyrosine kinase is associated with increased progression in human breast cancer, yet in breast explant cultures, the ErbB-2/Neu receptor contributes to alveolar differentiation. To examine the consequence of deregulated ErbB-2/Neu activation on epithelial morphogenesis, we have expressed a constitutively activated mutant of ErbB-2/Neu in a Madin-Darby canine kidney (MDCK) epithelial cell model. Using two-dimensional cultures we demonstrate that activated ErbB-2/Neu induces breakdown of cell-cell junctions, increased cell motility and dispersal of epithelial colonies. This correlates with reorganization of the actin cytoskeleton and focal adhesions and loss of insoluble cell-cell junction complexes involving E-cadherin. Interestingly, a constitutively activated ErbB-2/Neu receptor promotes an invasive morphogenic program in MDCK cells in a three-dimensional matrix. We show that two tyrosines in the carboxy-terminal tail of ErbB-2/Neu, involved in the phosphorylation of the Shc adapter protein, are each sufficient to promote epithelial-mesenchymal like transition and enhanced cell motility in two-dimensional culture and cell invasion rather than a morphogenic response in matrix culture. This provides a model system to investigate ErbB-2/Neu induced signaling pathways required for epithelial cell dispersal and invasion versus morphogenesis.

Signal transduction in mammary tumorigenesis: a transgenic perspective.

A number of genes have been implicated in breast cancer development, yet few have been demonstrated to play causative roles in mammary tumor formation. The advent of transgenic mouse and embryonic stem cell technologies now permits manipulation of the mouse genome in such a way as to temporally and spatially control a gene product's expression. Thus, the basic researcher now can directly assess the involvement of particular genes in tumorigenesis and disease progression and, in the process, to develop mouse models of human genetic disease. The utility of such technologies is emphasized in transgenic mice expressing genes thought to play important roles in the initiation and progression of mammary carcinomas. As these transgenic strains have been the subject of several reviews, here we focus on two mouse mammary tumor models, Polyomavirus middle T antigen and the Neu/ErbB-2 receptor tyrosine kinase, which are most amenable to study specific signaling pathways in process of mammary tumorigenesis.

Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation.

A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Ras-dependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites.

Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors.

Amplification of the Neu/c-erbB-2 receptor tyrosine kinase has been implicated as an important event in the genesis of human breast cancer. Indeed, transgenic mice bearing either an activated form of neu or the wild-type proto-oncogene under the transcriptional control of the mouse mammary tumor virus promoter-enhancer frequently develop mammary carcinomas (L. Bouchard, L. Lamarre, P. J. Tremblay, and P. Jolicoeur, Cell 57:931-936, 1989; C. T. Guy, M. A. Webster, M. Schaller, T. J. Parson, R. D. Cardiff, and W. J. Muller, Proc. Natl. Acad. Sci. USA 89:10578-10582, 1992; W. J. Muller, E. Sinn, R. Wallace, P. K. Pattengale, and P. Leder, Cell 54:105-115, 1988). Induction of mammary tumors in transgenic mice expressing the wild-type Neu receptor is associated with activation of the receptor's intrinsic tyrosine kinase activity (Guy et al., Proc. Natl. Acad. Sci. USA 89:10578-10582, 1992). Here, we demonstrate that activation of Neu in these transgenic mice occurs through somatic mutations located within the transgene itself. Sequence analyses of these mutations revealed that they contain in-frame deletions of 7 to 12 amino acids in the extracellular region proximal to the transmembrane domain. Introduction of these mutations into a wild-type neu cDNA results in an increased transforming ability of the altered Neu tyrosine kinase. These observations suggest that oncogenic activation of Neu in mammary tumorigenesis frequently occurs by somatic mutation.

Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity.

Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30% of human breast cancers. Although the activation of Neu receptor tyrosine 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.

Isolation of chicken phosphotyrosyl phosphatase cDNA sequences and identification of a brain-specific species related to human PTP zeta.

The first example of a chicken cDNA sequence encoding a phosphotyrosyl phosphatase (PTPase) has been identified and found to contain coding sequences for the entire cytoplasmic and membrane spanning domains as well as a portion of the extracellular region of a transmembrane PTPase resembling human PTP zeta. Like HPTP zeta, chicken PTP zeta contained two phosphatase domains (D1 and D2), and D2 lacked a critical cysteine residue required for catalytic activity. The entire intracellular portion of CPTP zeta was expressed in bacteria and shown to be capable of dephosphorylating both p-nitrophenylphosphate and reduced carboxyamidomethylated and maleyated lysozyme but not phosphoseryl casein. Genetic analysis indicated that the presence of D2 was required for full activity. CPTP zeta mRNA was identified as a single large transcript expressed exclusively in the brain of chick embryos at both early and late stages of embryogenesis. These results suggested that CPTP zeta may perform a brain-specific function and have a role in development.