Amplification of the receptor tyrosine kinase ErbB2 is frequently observed in breast cancer. Amplification of erbB2 is also associated with multiple genomic gains and losses; however, the importance of these associated changes is largely unknown. We demonstrate that Brk, a cytoplasmic tyrosine kinase, is coamplified and coexpressed with ErbB2 in human breast cancers. ErbB2 interacts with Brk and increases its intrinsic kinase activity. Expression of Brk enhances the ErbB2-induced activation of Ras/MAPK signaling and cyclin E/cdk2 activity to induce cell proliferation of mammary 3-dimensional acini in culture. In a murine model of breast cancer, expression of Brk was found to shorten the latency of ErbB2-induced tumors by promoting cell proliferation, with no effect on protection from apoptosis. Furthermore, overexpression of Brk conferred resistance to the ability of Lapatinib, an ErbB2 kinase inhibitor, to inhibit ErbB2-induced proliferation. Thus, we identified Brk as a drug target for ErbB2-positive cancers.amplification ͉ lapatinib ͉ tumorigenesis E rbB2 belongs to the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (1). Amplification of erbB2 occurs in approximately 25% of breast cancers and is correlated with poor clinical outcomes (2). Although ErbB2 is not activated by direct binding of a soluble ligand, it is activated by ligand-induced formation of heterodimers with other EGFR family members, namely, EGFR/ErbB1, ErbB3, and ErbB4. Overexpression of ErbB2 can also result in ligand-independent receptor homodimerization and activation (1, 3). Overexpression of neu (the murine homologue of human erbB2) under the control of mouse mammary tumor virus (MMTV) long terminal repeat (LTR) results in mammary tumorigenesis (4), suggesting that erbB2 can initiate tumorigenesis in vivo.ErbB2 is a drug target for human breast cancer. A humanized monoclonal antibody against the extracellular domain of ErbB2, Herceptin, in combination with chemotherapy, is used to treat patients expressing high levels of ErbB2 (5). In patients who respond to the treatment, Herceptin can delay mortality anywhere between 9 months and 3 years (5). However, almost 50% of patients with tumors that contain erbB2 amplification do not respond to Herceptin (6), and those who do respond develop resistance to the drug, highlighting the need for better options to treat patients with ErbB2-positive cancers. A better understanding of the ErbB2 signaling pathway can identify novel targets for combination therapy that will significantly aid our ability to treat patients with ErbB2-amplified breast cancers.Members of the ErbB2 signaling pathway can also be coamplified with ErbB2 in breast cancer (7-10). For example, Grb7, a Src homology domain (SH2) containing an adaptor molecule that associates with ErbB2 and amplifies signaling by ErbB2, is a component of the ErbB2 amplicon (10). In addition to the erbB2 amplicon, erbB2-amplified tumors also are characterized by several gains and losses located elsewhere in the genome (7,8)....