ErbB-4 ribozymes abolish neuregulin-induced mitogenesis

The epidermal growth factor-like receptor tyrosine kinase (ErbB) family is frequently overexpressed in a variety of human carcinomas, including breast cancer. To assist in characterizing the role of ErbB-4 in breast cancer, we generated three specific hammerhead ribozymes targeted to the ErbB-4 mRNA. These ribozymes, Rz6, Rz21, and Rz29, efficiently catalyzed the specific cleavage of ErbB-4 message in a cell-free system. We demonstrated that the neuregulin-induced mitogenic effect was abolished in ribozyme Rz29- and Rz6-transfected 32D/ErbB-4 cells. Inhibition of mitogenesis was characterized by ribozyme-mediated down-regulation of ErbB-4 expression. In addition, we provide the first evidence that different threshold levels of ErbB-4 expression and activation correlate with different responses to neuregulin stimulation. High levels of ErbB-4 expression, phosphorylation, and homodimerization are necessary for neuregulin-stimulated, interleukin 3- independent cell proliferation in the 32D/E4 cells. In the case of Rz29- transfected 32D/E4 cells, low levels of ErbB-4 expression allowed neuregulin- induced phosphorylation but were insufficient to couple the activated receptor to cellular signaling. Furthermore, expression of the functional ErbB-4 ribozyme in T47I) human breast carcinoma cells led to a down- regulation of endogenous ErbB-4 expression and a reduction of anchorage- independent colony formation. These studies support the use of ErbB-4 ribozymes to define the role of ErbB-4 receptors in human cancers.