ERBB-4 RIBOZYMES ABOLISH NEUREGULIN-INDUCED MITOGENESIS

The epidermal growth factor-like receptor tyrosine kinase (ErbB) famil y is frequently overexpressed in a variety of human carcinomas, includ ing breast cancer. To assist in characterizing the role of ErbB-4 in b reast cancer, we generated three specific hammerhead ribozymes targete d to the ErbB-4 mRNA, These ribozymes, Rz6, Rz21, and Rz29, efficientl y catalyzed the specific cleavage of ErbB-4 message in a cell-free sys tem. We demonstrated that the neuregulin-induced mitogenic effect was abolished in ribozyme Rz29- and Rz6-transfected 32D/ErbB-4 cells, Inhi bition of mitogenesis was characterized by ribozyme-mediated down-regu lation of ErbB-4 expression. In addition, we provide the first evidenc e that different threshold levels of ErbB-4 expression and activation correlate with different responses to nenregulin stimulation, High lev els of ErbB-4 expression, phosphorylation, and homodimerization are ne cessary for neuregulin-stimulated, interleukin 3-independent cell prol iferation in the 32D/E4 cells, In the case of RzZ9-transfected 32D/E4 cells, low levels of ErbB-4 expression allowed neuregulin-induced phos phorylation but were insufficient to couple the activated receptor to cellular signaling. Furthermore, expression of the functional ErbB-4 r ibozyme in T47D human breast carcinoma cells led to a down-regulation of endogenous ErbB-4 expression and a reduction of anchorage-independe nt colony formation. These studies support the use of ErbB-4 ribozymes to define the role of ErbB-4 receptors in human cancers.