Oncogene (2005) 24, 869–879 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc A network of clinically and functionally relevant genes is involved in the reversion of the tumorigenic phenotype of MDA-MB-231 breast cancer cells after transfer of human chromosome 8 Susanne Seitz*,1, Renate Frege1, Anja Jacobsen2,Jo¨ rg Weimer2, Wolfgang Arnold3, Clarissa von Haefen4, Dieter Niederacher5, Rita Schmutzler6, Norbert Arnold2 and Siegfried Scherneck1 1Department of Tumor Genetics, Max Delbrueck Center for Molecular Medicine, Robert Roessle Str. 10, 13125 Berlin, Germany; 2Oncology Laboratory, Gynecology and Obstetrics Clinic, University Hospital Schleswig-Holstein Campus Kiel, Michalisstr. 16, 24105 Kiel, Germany; 3atugen AG, Wiltbergstr. 50, 13125 Berlin, Germany; 4Department of Hematology, Oncology and Tumor Immunology, Charite-Campus Berlin-Buch, Humboldt University, Lindenberger Weg 80, 13125 Berlin-Buch, Germany; 5Department of Gynecology and Obstetrics, University of Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany; 6Department of Molecular Gynecology and Oncology, Gynecology and Obstetrics Clinic, Kerpener Str. 34, 50931 Ko¨ln, Germany Several investigations have supposed that tumor suppres- and 17p are detected frequently in more than 20–25% of sor genes might be located on human chromosome 8. We primary BC, suggesting the presence of tumor suppres- used microcell-mediated transfer of chromosome 8 into sor genes (TSG) in these regions of the genome MDA-MB-231 breast cancer cells and generated inde- (Lerebours and Lidereau, 2002). The regions of LOH pendent hybrids with strongly reduced tumorigenic in BC are usually large and complex and it is difficult to potential. Loss of the transferred chromosome results in identify candidate genes relying on allelic loss alone. reappearance of the malignant phenotype. Expression Furthermore, dissection of genetic events in LOH analysis identified a set of 109 genes (CT8-ps) differen- regions is complicated by factors such as tumor tially expressed in microcell hybrids as compared to the heterogeneity and contamination with nonmalignant tumorigenic MDA-MB-231 and rerevertant cells. Of material (Murakami, 2002). It is therefore important to these, 44.9% are differentially expressed in human breast obtain functional evidence for a TSG before laborious tumors. The expression pattern of CT8-ps was associated positional cloning attempts are made. with prognostic factors such as tumor size and grading as Microcell-mediated chromosome transfer (MMCT) is well as loss of heterozygosity at the short arm of an alternative approach to identify TSGs in sporadic chromosome 8. We identified CT8-ps networks suggesting cancers. Cancer cells showing deletions at the target that these genes act cooperatively to cause reversion of chromosomal region are preferentially used as recipient, tumorigenicity in MDA-MB-231 cells. Our findings working on the assumption that both alleles of a TSG provide a conceptual basis and experimental system to have been inactivated. The altered phenotype exhibited identify and evaluate genes and gene networks involved in by MMCT hybrids, as compared to the parental cancer the development and/or progression of breast cancer. cells, allows conclusions regarding the location and the Oncogene (2005) 24, 869–879. doi:10.1038/sj.onc.1208260 function of genes relevant for neoplastic development Published online 6 December 2004 under physiological conditions (Kugoh et al., 2002). Several lines of evidence suggest that chromosome 8 Keywords: breast tumorigenesis; microcell-mediated harbors one or more TSGs in BC. Various laboratories, chromosome transfer; expression difference analysis including ours, have reported up to 86% LOH in both familial and sporadic BC as well as in ductal carcinoma in situ (Seitz et al., 1997, 2000; Suzuki et al., 1999). LOH on chromosome 8p is also associated with higher Introduction grade and invasive behavior of tumors (Yaremko et al., 1996; Seitz et al., 2000) and has been proposed as a Breast Cancer (BC) is thought to result from the prognostic indicator to guide postoperative manage- accumulation of a number of genetic alterations, ment of patients (Tsuneizumi et al., 2002). Studying 305 including inactivation of tumor suppressors. Loss of breast tumors by CGH Rennstam et al. (2003) found heterozygosity (LOH) analysis and comparative geno- that combined loss of 8p and gain of 8q, one of the most mic hybridization (CGH) indicate that allelic losses of common coupled aberrations in BC, is correlated with human chromosomes 1p, 3p, 6q, 8p, 9p, 11p, 13q, 16q high DNA nondiploidy, high histological grade, lymph node positivity and low survival rate. *Correspondence: S Seitz; E-mail: [email protected] Functional evidence for one or more TSGs on Received 18 June 2004; revised 1 October 2004; accepted 7 October 2004; chromosome 8p stems from MMCT of chromosome 8 published online 6 December 2004 into rat prostate- and colorectal cancer cells, indicating Chromosome transfer and gene expression analysis S Seitz et al 870 the existence of a metastasis suppressor gene at 8p12– Restoration of human chromosome 8 in MDA-MB-231 p21 and a TSG at 8p22–p23, respectively (Gustafson breast cancer cells et al., 1996; Nihei et al., 2002). The critical regions defined for these putative suppressor genes encompass To identify a functional role for chromosome 8 in breast the regions most commonly deleted in BC. cancer, we introduced an intact copy of chromosome 8 Few if any studies have linked functional changes to into the MDA-MB-231 cell line. Following transfer, the presence or absence of chromosome 8 in breast eight microcell hybrid clones were isolated, expanded as tumor cells. Recently, Wilson et al. (2003) reported distinct cell lines and characterized. First, the hybrid R gene by transfer of chromosome 8 into two BC cell lines and cells were screened for the presence of the neo PCR using specific primers and the presence of the localized three chromosome 8p regions harboring transferred chromosome 8 using 19 informative MS putative in vitro growth suppressor genes. However, markers. In all hybrid cells, the presence of the these authors did not provide evidence for suppression neoR gene was detected, thus confirming that the of tumorigenicity in vivo. G418-resistant colonies arose as a result of microcell In the present study, we provide an experimental fusion (data not shown). MS analyses revealed that system to identify and evaluate genes and gene net- all hybrid clones carried the donor allele (Figure 1a works involved in BC genesis. For the first time we and c). These data are consistent with the results provide functional evidence for the presence of tumor suppressor(s) on chromosome 8(p) and show of cytogenetic analyses. As shown by FISH-MD CT60/4 hybrid cells contain an entire chromosome 8 that the suppression of the tumorigenic phenotype (donor chromosome), whereas in MDA-MB-231 cells of the BC cell line MDA-MB-231 is mediated by a only fragments of this chromosome were detected specific set of genes (CT8-ps)/networks regulated by one (Figure 1d, Weimer et al., in preparation). In addition, or more genes on chromosome 8. It is of particular CGH analyses demonstrated amplification of chromo- interest that the CT8-ps signature is also reflected in a some 8 in the hybrid clones (CT60/4, CT60/6) as panel of breast tumors and is correlated with worse compared to the parental MDA-MB-231 cells patient prognosis. (Figure 1e). In parallel, hybrids (CT60/4, CT60/6) were screened with FISH to determine whether any mouse chromosomes had inadvertently been transferred along Results with the neoR-tagged chromosome 8. Both cell lines were found to be negative for the presence of contaminating Homozygosity mapping of deletions (HOMOD) analysis mouse DNA. of breast cancer cell lines We analysed a panel of 11 breast cancer cell lines with 24 Consequences of chromosome 8 transfer in microsatellite (MS) markers for extended regions of MDA-MB-231 breast cancer cells hemizygosity (X5 adjacent markers). These are indica- tive for allelic loss in the unmatched tumor cells The eight microcell hybrids were first tested for (Goldberg et al., 2000). These markers were concen- differences in cell morphology. In general, the hybrid trated in the 8p12–p21 region previously determined cells appeared flatter than the MDA-MB-231 parental to be frequently deleted in BC (Seitz et al., 2000). cell line (data not shown). All microcell hybrids showed We identified four cell lines (MDA-MB-231, CAMA-1, decreased anchorage-dependent growth. The doubling T-47D, BT-20) showing deletions of the 8p12–p21 times for exponentially growing MDA-MB-231 cells region, which are suitable as recipients of chromosome were 28.9 h, whereas the doubling times of the eight 8 by MMCT (data not shown). microcell hybrids varied between 33.4 h (CT60/4) and 44.6 h (CT60/8) (medium 37.7 h) (Figure 2a). Next, we Selection of MDA-MB-231 as recipient cells for MMCT determined the effect of transferred chromosome 8 on breast cancer cell invasion in vitro. As shown in As demonstrated by HOMOD –analysis, the MDA- Figure 2b, hybrid CT60/4 cells showed a clear reduction MB-231 cells were found to have a region of extended of invasive cells (>97%) compared with the parental hemizygosity of about 10 Mb at chromosomal region MDA-MB-231. We characterized two microcell 8p12–p21, encompassing the critical regions defined by hybrids, CT60/4 and CT60/6, in vivo to determine their LOH analysis (Figure 1a). CGH analysis of these cells tumorigenic potential. The CT60/4 cells (Figure 2c) revealed loss of chromosomal region 8pter–8q21and did not form tumors during a 3-month observation gain of 8q21–qter (Figure 1b). In addition, we compared period and CT60/6 cells exhibited a clear reduction in the gene expression profiles of MDA-MB-231 cells with the ability to form tumors in nude mice in comparison MCF-10A breast epithelial cells and found a high to the parental cell line.