Multiple Cooperating Oncogenes Drive Recurrent Breast Cancer-Associated Chromosomal Amplifications: Creation of Isogenic Human Cell Line Models.

摘要

Human breast cancers, like other cancers, frequently harbor amplifications of chromosomal regions containing multiple genes. Many such multi-gene amplifications are recurrent across many individual instances of breast cancer, suggesting they contain oncogenes critical for breast cancer development or progression. In some cases, traditional laboratory methods have identified more than one candidate driver oncogene within usuch amplified regions. This research proposal is designed to test the hypothesis that some recurrent amplicons actually function through the concerted action of multiple simultaneously overexpressed oncogenes. We are testing this hypothesis by attempting to create cell line models of such multigene amplifications, prospectively. We have designed an experimental strategy to target a dominantly selectable IMPDH gene to specific chromosomal regions using gene targeting technology. Targeted cells will then be selected in increasing concentrations of the IMPDH inhibitors mycophenolic acid and mizoribine to provide selective pressure for the targeted cells to amplify the targeted region, co-amplifying multiple genes simultaneously. Here we report the design of a functional, efficient gene targeting vector and the successful delivery of the IMPDH selection marker to two different chromosomal loci, ZNF703 and ERBB2, in two different human breast cancer cell lines, MCF-7, and T47D. In MCF-7, we have succeeded in creating subclones with focal, overlapping amplification of regions of chromosome 8p11-12, where ZNF703 is located. We demonstrate amplification by array CGH, digital PCR, and fluorescent in situ hybridization. Amplification is shown to cause coordinate overexpression of co-amplified genes at the mRNA and protein level.