DNA amplification at band q13 of chromosome 11 is common in breast cancer, and CCND1 and EMS1 remain the strongest candidate genes. However, amplification patterns are consistent with the existence of four cores of amplification, suggesting the involvement of additional genes. Here we present evidence strongly suggesting the involvement of the recently characterized EMSY gene in the formation of the telomeric amplicon. EMSY maps at 11q13.5, 100 kb centromeric to the GARP gene, which has been mapped within the core of the distal amplicon. The EMSY protein was shown to interact with BRCA2 and has a role in chromatin remodeling. This makes EMSY a strong candidate oncogene for the 11q13.5 amplicon. DNA amplification was studied in a total of 940 primary breast tumors and 39 breast cancer cell lines. Amplification profiles were consistent with the EMSY-GARP locus being amplified independently of CCND1 and/or EMS1. EMSY RNA expression levels were studied along with those of five other genes located at 11q13.5 by real-time quantitative PCR in the 39 cell lines and a subset of 65 tumors. EMSY overexpression correlated strongly with DNA amplification in both primary tumors and cell lines. In a subset of 296 patients, EMSY amplification was found by both uni-and multivariate analyses to correlate with shortened disease-free survival. These data indicate that EMSY is a strong candidate oncogene for the 11q13.5 amplicon.

Rearrangements of chromosome 11q13 are frequently observed in human cancer. The 11q13 region harbors several chromosomal breakpoint clusters found in hematologic malignancies and exhibits frequent DNA amplification in carcinomas. DNA amplification patterns in breast tumors are consistent with the existence of at least 4 individual amplification units, suggesting the activation of more than 1 gene in this region. Two candidate oncogenes have been identified, CCND1 and EMS1/CORTACTIN, representing centrally localized amplification units. Genes involved in the proximal and distal amplicons remain to be identified. Recently we reported on a putative transforming gene, MYEOV, mapping 360 kb centromeric to CCND1. This gene was found to be rearranged and activated concomitantly with CCND1 in a subset of t(11;14)(q13;q32)-positive multiple myeloma (MM) cell lines. To evaluate the role of the MYEOV gene in the proximal amplification core, we tested 946 breast tumors for copy number increase of MYEOV relative to neighboring genes or markers. RNA expression levels were studied in a subset of 72 tumors for which both RNA and DNA were available. Data presented here show that the MYEOV gene is amplified in 9.5% (90/946) and abnormally expressed in 16.6% (12/72) of breast tumors. Amplification patterns showed that MYEOV was most frequently coamplified with CCND1 (74/ 90), although independent amplification of MYEOV could also be detected (16/90). Abnormal expression levels correlated only partially with DNA amplification. MYEOV DNA amplification correlated with estrogen and progesterone receptorpositive cancer, invasive lobular carcinoma type and axillary nodal involvement. In contrast to CCND1 amplification, no association with disease outcome could be found. Our data suggest that MYEOV is a candidate oncogene activated in the amplification core located proximal to CCND1.

E2F transcription factors (E2F1 to 6) are central players in the control of animal cell proliferation as regulators of genes involved in cell cycle progression and in transformation. In this report, we have investigated the potential involvement of the E2F5 gene in tumorigenesis. We show that E2F5 can promote the formation of morphologically transformed foci in primary baby rat kidney cells (BRK) when it is overexpressed in the presence of its heterodimeric partner DP1 and activated RAS. This suggests that E2F5 behaves like a MYC-type cooperating oncogene in functional assays, prompting us to monitor potential amplifications of the E2F5 gene in primary human tumors. We mapped the human E2F5 gene to 8q21.1-21.3 equidistant from the MOS (8q12) and MYC (8q24) oncogenes. Since the long arm of chromosome 8 is frequently the site of increased gene copy number (ICN) in breast cancer, we screened 442 breast tumor DNAs for gains of E2F5, MOS, and MYC genes. The three genes showed ICN, albeit at variable incidence and levels of amplification, with the ICN of E2F5 occurring concomitantly with those of MOS and/or MYC in almost half of the cases. Moreover, a marked increase of the 2. 5-kb E2F5 transcript was also detected in some tumors and tumor cell lines. In conclusion, the evidence that sustained unregulated expression of E2F5 can cooperate with other oncogenes to promote cell transformation in functional assays, together with the detection of chromosomal amplifications and overexpressions of the E2F5 gene in breast tumors, provides the first indications that E2F5 deregulation may have a role in human tumor development.