ONCOGENIC NEU ERBB-2 INCREASES ETS, AP-1, AND NF-KAPPA-B-DEPENDENT GENE-EXPRESSION, AND INHIBITING ETS ACTIVATION BLOCKS NEU-MEDIATED CELLULAR-TRANSFORMATION/

Overexpression of Neu (ErbB-2/HER2) is found in similar to 20% of brea st tumors. Activation of Neu by a point mutation (Neu(T)) causes const itutive tyrosine kinase activity of this transmembrane receptor and tr ansforming activity in fibroblasts. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. Expression of Neu(T), but not normal Neu, caused transcriptional activ ation of Ets, AP-1, or NF-kappa B-dependent reporter genes. Dominant i nhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for t his activation. Analysis with Ets2 mutants indicated that activation o f Ets2 transcriptional activity mediated by Neu(T) or oncogenic Ras re quired phosphorylation of the same Ets2 residue, threonine 72. Cotrans fection of dominant inhibitory Ets2 mutants specifically blocked Neu(T )-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of Neu(T) was inhibited 5-fold when Neu(T) was cotransfected with a d ominant negative Ets2 mutant. However, parallel colony formation assay s showed that the Ets2 dominant negative mutant did not inhibit the gr owth of normal cells. Together, these data show that Neu(T) activates a variety of transcription factor families via the Ras signaling pathw ay and that Ets activation is required for Neu(T)-mediated cellular tr ansformation. Thus, downstream targets of Neu, including Ets transcrip tion factors, may be useful points for therapeutic intervention in Neu /ErbB-2-associated cancers.