DNA amplification is a frequent occurrence in cancer genomes. While tumor amplicons may harbor known oncogenes “driving” amplification, amplicons rarely comprise only single genes. The potential functional contribution of coamplified genes remains largely unexplored. In breast cancer, 20–30% of tumors exhibit amplification within chromosome band 17q12, containing theERBB2oncogene. Analysis of array-based comparative genomic hybridization and expression profiling data indicate that the minimum region of recurrent amplification (i.e., the amplicon “core”) at 17q12 includes two other genes,GRB7 and STARD3, which exhibit elevated expression when amplified. Western blot analysis confirms overexpression of each at the protein level in breast cancer cell lines SKBR3 and BT474 harboring amplification. In these cell lines (but not in control MCF7 breast cancer cells lacking 17q12 amplification), targeted knockdown of ERBB2 expression using RNA interference (RNAi) methods results in decreased cell proliferation, decreased cell-cycle progression, and increased apoptosis. Notably, targeted knockdown of either GRB7 or STARD3 also leads to decreased cell proliferation and cell-cycle progression, albeit to a lesser extent compared with ERBB2 knockdown. We conclude that the amplification and resultant overexpression of genes coamplified withERBB2at 17q12 can contribute to proliferation levels of breast cancer cells. Our findings validate the utility of RNAi in the functional interrogation of tumor amplicons, and provide evidence for a contribution of coamplified genes to tumor phenotypes. © 2006 Wiley-Liss, Inc.