We describe a survey of genetic changes by comparative genomic hybridization (CGH) in 11 human breast cancer cell lines recently established in our laboratory. The most common gains took place at 8q (73%), 1q (64%), 7q (64%), 3q (45%) and 7p (45%), whereas losses were most frequent at Xp (54%), 8p (45%), 18q (45%) and Xq (45%). Many of the cell lines displayed prominent, localized DNA amplifications by CGH. One-third of these loci affected breast cancer oncogenes, whose amplifications were validated with specific probes: 17q12 (two cell lines with ERBB2 amplifications), 11q13 (two with cyclin-D1), 8p11–p12 (two with FGFR1) and 10q25 (one with FGFR2). Gains and amplifications affecting 8q were the most common genetic alterations in these cell lines with the minimal, common region of involvement at 8q22–q23. No high-level MYC (at 8q24) amplifications were found in any of the cell lines. Two-thirds of the amplification sites took place at loci not associated with established oncogenes, such as 1q41–q43, 7q21–q22, 7q31, 8q23, 9p21–p23, 11p12–p14, 15q12–q14, 16q13–q21, 17q23, 20p11–p12 and 20q13. Several of these locations have not been previously reported and may harbour important genes whose amplification is selected for during cancer development. In summary, this set of breast cancer cell lines displaying prominent DNA amplifications should facilitate discovery and functional analysis of genes and signal transduction pathways contributing to breast cancer development. © 1999 Cancer Research Campaign
Breast cancer remains one of the most common malignant diseases in women in North America and Western Europe, yet therapies for the more aggressive estrogen independent tumors are limited and few model systems are available for the study of this type of breast cancer. In these studies, we characterized a novel estrogen independent breast cancer cell line, SUM-159PT. SUM-159PT cells are epithelial in origin, demonstrated by expression of cytokeratin 18. SUM-159PT cells are estrogen independent, demonstrated by lack of estrogen receptor (ER) protein and ER ligand binding studies. Furthermore, SUM-159PT cells injected subcutaneously or orthotopically are tumorigenic in ovariectomized athymic nude mice in the absence of estradiol supplementation. SUM-159PT cells are capable of invading through an 8 microm Matrigel membrane and display a stellate morphology in Matrigel, indicative of a metastatic phenotype. Correlating with this phenotype, we have detected secondary tumors upon inoculation of SUM-159PT cells into the mammary fat pad. To further investigate the metastatic potential of the SUM-159PT cells, we examined the expression of two proteins, vimentin and E-cadherin, implicated in the transition of carcinoma cells to a metastatic phenotype. Western blot and immunohistochemical analysis demonstrated that both SUM-159PT cells and xenografts express vimentin. No expression of E-cadherin was detected in SUM-159PT cells. Our data indicate that despite estrogen independence, SUM-159PT cells are growth inhibited in vitro by compounds such as 1,25(OH)2D3, transforming growth factor beta (TGF-beta), and the phorbol ester TPA. These studies indicate that SUM-159PT cells represent a good model system for the study of late stage estrogen independent, invasive breast cancer.
To better understand the mechanisms of transformation by the oncogene HER-2, we transduced the human mammary epithelial (HME) cell line MCF-10A with HER-2 and developed a cell line that appeared to moderately overexpress HER-2. These MCF-10HER-2 cells were unable to grow in the absence of epidermal growth factor (EGF). However, coexpression of HER-2 with the HPV-16 oncoproteins E6 and E7 resulted in EGF-independent cells that expressed very high levels of constitutively activated HER-2. Interestingly, coexpression of E7 with HER-2 resulted in cells that were EGF-independent for growth but did not express HER-2 to high levels, and coexpression of E6 with HER-2 resulted in cells expressing higher levels of HER-2, which were still dependent on EGF for growth and survival. The MCF-10HER-2E7 and HER-2/E6E7 cells exhibited constitutive activation of a form of epidermal growth factor receptor (EGFR) that had a faster electrophoretic mobility than EGFR activated by exogenous growth factors. Exposure of cells with EGFR activation to ZD1839 (Iressa), at concentrations specific for EGFR, had little or no influence on proliferation of cells with amplified HER-2 but little or no EGFR. These results indicate that HER-2, E6, and E7 cooperate with endogenous EGFR to yield fully transformed cells.
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