Genomic Instability of Human Mammary Epithelial Cells Overexpressing a Truncated Form of EMSY

Afshin Raouf 0 1 2 Lindsay Brown 0 1 2 Nikoleta Vrcelj 0 1 2 Karen To 0 1 2 Winnie Kwok 0 1 2 David Huntsman ) 0 1 2 Connie J. Eaves ceaves@bccrc 0 1 2 0 Journal of the National Cancer Institute , Vol. 97, No. 17, September 7, 2005 1 Affiliations of authors: Terry Fox Laboratory, British Columbia Cancer Agency , Vancouver , British Columbia , Canada (AR , KT, WK, CJE); Genetic Pathology Evaluation Center, Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver General Hospital, and the University of British Columbia , Vancouver , British Columbia , Canada (LB, NV , DH); Prostate Center , Vancouver General Hospi- tal, Vancouver , British Columbia , Canada (LB, NV , DH); Department of Medical Genetics, University of British Columbia , Vancouver , British Columbia, Canada (CJE). Fox Laboratory , 675 West 10th Ave., Vancouver , BC V5Z 1L3, Canada ( 2 Foundation and the CIHR. D. Huntsman is a Michael Smith Foundation for Health Research Scholar. We thank Luke Hughes-Davies (University of Cambridge, United Kingdom ) for the EMSY cDNA and helpful comments , Philippe Leboulch (Harvard Medical School , Boston, MA ) for the KA391 lentiviral vector, Sandra Dunn (Department - The EMSY gene encodes a protein that interacts with Brca2 and is amplified in some sporadic cases of human breast cancer. To examine whether overexpression of EMSY would mimic the chromosome instability phenotype that is associated with the loss of Brca2 function, we constructed a lentiviral vector (Lenti-EMSY/GFP) that encodes a truncated form of the Emsy protein, including its Brca2-interacting domain, and green fluorescent protein (GFP) and used it to transduce human telomerase-immortalized human breast epithelial (184-hTert) cells, which have a nearly normal karyotype. At passage 5 after transduction, 39 (26%) of 150 EMSY/GFP-transduced metaphase cells contained at least one structural chromosomal abnormality compared with 19 (13%) of 150 GFPtransduced metaphase cells (P = .003, chi-square test); at passage 10, the corresponding frequencies were 42% and 15%, respectively (P<.001). Mitomycin C also produced a severalfold higher frequency of chromosome breaks in the EMSY/GFP-transduced cells than in the control cells. These results support the hypothesis that EMSY overexpression can play a role in the genesis of human breast cancer. [J Natl Cancer Inst 2005;97:13026] The EMSY gene (1) encodes a protein whose amino-terminal region binds to the protein domain encoded by the third exon of the BRCA2 gene, which is deleted from the germline DNA of familial breast cancer kindreds (2). The aminoterminal region of the Emsy protein also contains separate domains that bind to the HP1 and BS69 proteins (Fig. 1, A), which suggests that Emsy may also play a role in chromatin remodeling (1). In irradiated cells, Emsy has been found to colocalize with -histone 2AX in response to DNA damage (1). BRCA2 gene mutations are not seen in sporadic breast cancer, but the EMSY gene is amplified in 13% of these cancers (1). In primary breast cancer samples and breast cancer cell lines, amplification of 11q13.5, the chromosomal region that contains the EMSY gene, is specifically correlated with an increase in levels of EMSY messenger RNA (mRNA) (3). Taken together, these findings have led to the suggestion that overexpression of the EMSY gene might be an alternative mechanism for suppressing Brca2 activity, which could lead to the emergence of malignant breast cell populations (46). BRCA2-null cells exhibit a chromosome instability phenotype that is characterized by the accumulation of structural chromosomal abnormalities in response to exposure to mitomycin C (79). In humans, loss of heterozygosity at the BRCA2 locus is associated with genomic instability early in breast cancer development and antedates the appearance of carcinoma in situ (10). We therefore hypothesized that forced overexpression of EMSY might cause a chromosome instability phenotype in human breast epithelial cells similar to that typical of BRCA2-null cells. To test this hypothesis, we isolated a 810-bp 5 fragment from the EMSY complementary DNA (cDNA) that encodes the Brca2-interacting domain as well as the HP1- and BS69-binding domains and inserted this fragment into a lentiviral vector that already contained an internal ribosomal entry site element and the cDNA for enhanced green fluorescent protein (eGFP) (11) (Fig. 1, A). We then used this vector or the control lentiviral vector expressing GFP only (Lenti-GFP) to infect log-phase 184hTert cells, a telomerase-immortalized line of human breast epithelial cells (12) generously supplied by S. Dunn, Department of Pediatrics and Experimental Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Two days later, we used a fluorescence-activated cell sorter to select EMSY/GFP- and control GFPtransduced cells that displayed high levels of green fluorescence (Fig. 1, B), and the selected cells were cultured further for up to five passages. By passage 5, the EMSY/GFP-transduced cells from three independent transduction experiments expressed 68- to 300-fold higher levels of EMSY mRNA that contained sequences from the 5 end of the EMSY gene compared with control GFPtransduced cells (Fig. 1, C). In the same EMSY/GFP-transduced cells, expression of the endogenous EMSY gene (i.e., EMSY mRNA that contained sequences from the 3 end of the gene) (Fig. 1, C) or genes encoding Brca1, Brca2, p21, or p53 (data not shown) was not different from the expression of any of these genes in the GFP-transduced control cells. The level of 5 EMSY transcripts in the transduced cells continued to increase with further cell passage (data not shown), suggesting overgrowth of the cultures by cells that expressed the highest levels of the EMSY transgene. The parental 184-hTert cells had a nearly diploid karyotype (48, XX, +20, +20) and were chromosomally stable. By contrast, in three separate experiments, cells that overexpressed mRNA from the EMSY transgene rapidly accumulated structural chromosomal abnormalities. These abnormalities included deletions, translocations, marker chromosomes, chromosome fragments, and dicentric chromosomes (Fig. 2, A), none of which were seen in metaphases from control GFP-transduced cells. For example, at passage 5 after transduction, 39 (26%) of 150 metaphases of EMSY/GFPtransduced cells contained at least one structural chromosomal abnormality compared with 19 (13%) of 150 metaphases from the GFP-transduced cells Fig. 1. The lentiviral expression vector, Lenti-EMSY-GFP, and isolation of transduced 184-hTert cells. (A) Design of the vector. An 810-bp region of the EMSY cDNA encoding the Brca2-binding domain (B2) and the HP1 (H) and BS69 (BS) binding domains was amplified by PCR using oligonucleotide primers EMSY-1810-F (5-GGCGCGCCCCACCATGCCTGTTGTGTGGCC-3; forward primer) and EMSY-1810-R (5-TTAATTAATGTCTGTGTTGATGGTT TAG-3; reverse primer) and clone (...truncated)