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We have previously identified two genes (EMS) and PR4DI/cyclin DI) in the chromosome 11q13 region that are frequently coamplified and overexpressed in human breast cancer and in squamous cell carcinomas of the head and neck (E. Schuuring, E. Verhoeven, W. J. Mooi, and R. J. A. M. Michalides, Oncogene 7:355-361, 1992). We now report on the characterization of the 80/85-kDa protein that is encoded by the EMS) gene. Amino acid sequence comparison shows a high homology (85%) to a chicken protein that was recently identified as a substrate for the src oncogene (H. Wu, A. B. Reynolds, S. B. Kanner, R. R. Vines, and J. T. Parsons, Mol. Cell. Biol. 11:5113-5124, 1991). Immunocytochemistry reveals that in epithelial cells, the human EMS) protein is localized mainly in the cytoplasm and, to a very low extent, in protruding leading lamellae of the cell. However, in carcinoma cells that constitutively overexpress the protein as a result of amplification of the EMS) gene, the protein, except in cytoplasm, accumulates in the podosome-like adherens junctions associated with the cell-substratum contact sites. The protein was not found in intercellular adherens junctions. Our findings, and the previously reported observations in src-transformed chicken embryo fibroblasts, suggest that the EMS) protein is involved in regulating the interactions between components of adherens-type junctions. Since amplification of the 11ql3 region has been associated with an enhanced invasive potential of these tumors, overexpression and concomitant accumulation of the EMS) protein in the cell-substratum contact sites might, therefore, contribute to the invasive potential of these tumor cells.

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Purpose: Amplification of the 11q13 region is a frequent event in human cancer. The highest incidence (36%) is found in head and neck squamous cell carcinomas. Recently, we reported that the amplicon size in 30 laryngeal and pharyngeal carcinomas with 11q13 amplification is determined by unique genomic structures, resulting in the amplification of a set of genes rather than a single gene. Experimental Design: To investigate which gene(s) drive the 11q13 amplicon, we determined the smallest region of overlap with amplification and the expression levels of all genes within this amplicon. Results: Using array-based comparative genomic hybridization analysis, we detected a region of f1.7 Mb containing 13 amplified genes in more than 25 of the 29 carcinomas. Quantitative reverse transcription-PCR revealed that overexpression of 8 potential driver genes including, cyclin D1, cortactin, and Fas-associated death domain (FADD), correlated significantly with DNA amplification. FADD protein levels correlated well with DNA amplification, implicating that FADD is also a candidate driver gene in the 11q13 amplicon. Analysis of 167 laryngeal carcinomas showed that increased expression of FADD (P = 0.007) and Ser 194 phosphorylated FADD (P = 0.011) were associated with a worse disease-specific survival. FADD was recently reported to be involved in cell cycle regulation, and cancer cells expressing high levels of the Ser 194 phosphorylated isoform of FADD proved to be more sensitive to Taxol-induced cell cycle arrest. Conclusion: Because of the frequent amplification of the 11q13 region and concomitant overexpression of FADD in head and neck squamous cell carcinomas, we hypothesize that FADD is a marker to select patients that might benefit fromTaxol-based chemoradiotherapy.

Successive oncogenic steps are necessary to generate cancer. In many B-cell lymphomas, chromosomal translocations are considered to be an early oncogenic hit. We investigated whether the lymphoma-associated t(14;18) involving the BCL2 oncogene can occur outside the context of malignancy. To this end, we extensively screened blood cells from healthy blood donors by a very sensitive seminested polymerase chain reaction (PCR) for breakpoint junctions at JH1–5 on 14q32 and the major breakpoint region of BCL2 on 18q21. In each individual, mononuclear cells, granulocytes, flow-sorted B cells, and T cells were separately tested in five to seven independently performed PCRs (in total, 0.5 x 10(6) to 1.0 x 10(6) cells per fraction per individual). Amplification products that hybridized with an internal BCL2 probe and a JH probe were sequenced. Six of nine individuals harbored t(14;18) breakpoints. Translocations were restricted to B cells, with an estimated frequency of 1 in 10(5) or less circulating B cells. In total, 23 of 51 experiments on B cells were positive in contrast to 1 of 48 on T cells and 2 of 47 experiments on granulocytes. Consistent with the presence of 4.7% to 13.0% B cells in the mononuclear cell fractions, only very few (4 of 47) tests were positive in these fractions. Sequence analysis showed that four of six individuals harbored two to five unrelated t(14;18)-carrying B-cell clones. All breakpoints had a structure similar to that in follicular lymphoma. We propose that B cells with the t(14;18) translocation are regularly generated in normal individuals, but that only very few cells with the translocation will acquire the additional oncogenic hits necessary to establish the malignant phenotype.