In cancer, gene amplification represents a type of genetic alteration that results in increased copy number of a gene or genes and subsequent increases in protein expression. When the target of amplification is a cellular oncogene, the corresponding increases in oncoprotein expression often result in tumor development and/or progression. To date, all comprehensively studied regions of amplification have been found to contain oncogenes, suggesting that characterization of novel amplicons will lead to identification of novel oncogenes that contribute directly to development of breast cancer. Over the last two years we have characterized the structure of an amplicon on chromosome 17q23 in breast cancer cell lines and breast tumors. We have identified seven independently amplified regions within the amplicon suggesting that as many as seven different oncogenes may reside in this amplicon. We have identified a total of twelve highly amplified genes in these seven amplified regions, including the TBX2 candidate oncogene that functions as an oncogene by inhibiting senescence and inducing immortalization. We have also noted that at least one of these twelve genes is amplified in 42 percent of all breast tumors, including DCIS. Together, these data strongly suggest that the amplicon contains several other genes with oncogenic properties, perhaps one from each of the seven amplification peaks, and that these oncogenes may have an important role in early progression of breast tumors. In this study we propose to follow up on these observations by identifying and characterizing the oncogenes in the amplicon. In order to achieve this objective we aim to: 1) determine the level and frequency of expression of the genes in the amplified regions in breast tumors to verify that the selected candidates are overexpressed as a result of amplification; 2) characterize the oncogenic activity of the overexpressed candidate genes using a series of oncogenicity assays; 3) investigate the prognostic potential of the oncogenes from the region. To address these aims, we will measure the expression level of the twelve highly and frequently amplified genes from the amplicon in tumors and cell lines by microarray analysis and quantitative RT-PCR. The candidate oncogenes with the best correlation between amplification and overexpression will be assessed for oncogenic activity in a series of immortalization, transformation, tumorigenesis, invasion, and metastasis assays. Finally, the prognostic relevance of amplification of the validated oncogenes from the region will be studied by correlating patient outcome with amplification, as measured by fluorescence in situ hybridization, in node negative, node positive, and DCIS breast tumors. The importance of this project derives from the potential for identification of novel oncogenes that will further our understanding of breast tumor progression. It must be noted that we are taking a comprehensive approach to the identification of oncogenes in the region so that a full understanding of the relevance of amplification of the region to tumor progression can be developed. The study is also important because it may result in discovery of clinically useful molecular markers of prognosis that may lead to individualized treatment regimens. Thus, the project may involve a complete transition from benchtop to bedside. Finally, the amplified and overexpressed genes may prove useful as important targets of gene, pharmacological, and immunological therapy in the future.
