Our analysis of large numbers of breast tumors by array CGH revealed variety in the numbers and types of copy number alterations in the tumor genomes. In the ductal invasive breast tumors reported here, three subtypes were distinguished by copy number alterations. The subtypes differed with respect to the numbers and types of aberrations, as well as patient survival. The1q/16q subtype with very few copy number alterations in addition to gain of 1q and loss of 16q was associated with the best patient outcome, consistent with other studies. Searches for tumor suppressor gene(s) on 16q have failed to find mutations in candidate genes in the region in ductal invasive breast cancer, although mutations in E cadherin and loss of 16q are characteristic of lobular breast tumors. Two genes involved in telomere maintenance, TERF2 and TERF2IP were among those ruled out as tumor suppressors on 16q, as was E2F4 [15–17]. The stability of the genome of these tumors also suggests that copy number alterations of these and other stability genes mapping within the aberrant regions, +1q and -16q are less likely to contribute to chromosomal level instability in breast cancer.
Complex tumors with extensive chromosomal level instability were associated with poor patient survival. They are similar to BRCA1 hereditary tumors in their copy number alterations [18, 19] (Figure 3). BRCA1 participates in a number of cell functions that maintain genome integrity either directly through double strand break repair or indirectly through maintenance of checkpoints at G1, S and mitosis [20–22]. Thus, it is possible that BRCA1 [23, 24] or the genes/pathways that interact with BRCA1 are defective in this subtype either through mutation, silencing or copy number mediated dosage effects. We note that the copy number loss on 17q associated with this subtype includes the BRCA1 locus (9/16 tumors, Figure 4).
The discrimination of breast tumor subtypes based on copy number aberrations led us to investigate possible associations of copy number aberration types with alterations in processes/genes involved in maintenance of genome stability. We observed shorter telomeres in tumors with greater numbers of amplifications, consistent with telomere attrition promoting this type of copy number aberration in breast tumors. Telomere dysfunction, often referred to as "telomere crisis" has been implicated in amplification, particularly by breakage-fusion-bridge processes. On the other hand, our analyses of stability gene expression in relation to copy number aberration types found that expression of genes in the functional classes; "mitosis," "cell cycle," "replication," and "DNA damage/repair" were associated with greater numbers of copy number transitions. Furthermore, a subsequent analysis found significant enrichment for these same classes among all GOA groups when analyzed with GOStats . The number of amplicons was associated with similar functional groups, "mitosis" and "cell cycle." Many of these genes are E2F targets [26–36] and therefore potentially coordinately deregulated due to Rb pathway defects . Abrogation of Rb pathway function is frequent in breast tumors by loss of expression of Rb or altered expression of inhibitors of Rb activity (e.g. loss/silencing of CDKN2A (p16) and amplification and/or over expression of CCND1, CDK4, CDK6) (Figure 7). It is interesting to note that whereas E2F1 is up-regulated in breast tumors, its expression is low in prostate tumors , which typically have genomes with fewer copy number changes than most ductal invasive breast cancers . For example, in an array CGH dataset of 64 primary prostate tumor samples , the median number of copy number transitions was 13 per tumor genome compared to 30 in our primary breast tumor samples (p < 5 × 10-9, Wilcoxon rank sum test). Mechanistic support for a central role of E2F1 in genomic instability comes from a recent report that elevated numbers of DNA double strand breaks are present in cell lines with deregulated E2F1 and Rb deficiency .
Chromosomal instability has been observed in vitro when many of these E2F target genes (Figure 7) associated with replication, DNA repair, cell cycle control and the mitotic checkpoint are mutated, knocked out or knocked down using siRNA [8, 41, 42]. Contrary to expectation, we observed that greater chromosomal instability in breast tumors is associated with increased expression levels of many of these genes, even though they have loss of function instability phenotypes. These assays further demonstrate that loss of a single copy of some of the genes results instability or cancer prone phenotypes. Genes that have been shown to be haploinsufficient in this way and that are among those we identified as showing significant association with the number of copy number aberrations in our tumors (FDR < 0.05) include RAD17, ATM and RB1, which are expressed at lower levels in tumors with more copy number changes. These genes are also negatively correlated with E2F1 expression. Other genes showing haploinsufficiency in vitro, MAD2L1, PLK4, BUB1B and CHEK1 show enhanced expression in association with number of chromosomal changes and are positively correlated with E2F1 expression (Supplementary Table 4, Additional file 5). As all seven of the above mentioned genes with haploinsufficiency phenotypes map to regions of frequent loss in breast tumors and genetic instability phenotypes are associated with deficiency in these genes, we asked whether loss of function might play a role in the subset of tumors in which there is a copy number loss of the locus. Specifically, we asked if their expression levels were down regulated when there is a copy number loss. Although 118 of the genome stability genes showed highly significant reduction in expression in tumors in which the locus was lost (FDR < 0.05, one-sided Wilcoxon rank sum test), we found little difference in expression level with copy number loss for MAD2L1, PLK4, ATM and RB1, whereas BUB1B was increased in expression in tumors with loss of the locus (Supplementary Table 4, Additional file 5). Only expression of RAD17 was significantly reduced when lost (unadjusted p = 8 × 10-4, Wilcoxon rank sum test), suggesting that RAD17 might be haploinsufficient in tumors with copy number loss of the locus at 5q13.
Our observations in tumors support the hypothesis that global alteration of expression of genes involved in processes such as chromosome segregation and maintenance of genome integrity, driven by deregulation of E2F, underlies much of the chromosomal instability in breast tumors. Furthermore gene expression appears to be relatively up-regulated. On the one hand, this observation seems contradictory in light of the phenotypes resulting from mutational analyses of genes involved in maintenance of genome stability. Such in vitro studies have generally assessed the consequences of functional deficiency one gene at a time and have found that individually many genes have loss of function instability phenotypes. On the other hand, as many of these genes participate in multi-protein complexes that depend on proper stoichiometry for function, alterations resulting in overproduction or deficiency are likely to have similar or related phenotypes (reviewed in ). Indeed, in mammalian cells, instability phenotypes have been reported in association with both up and down regulation of genes such as MAD2L1 [8, 41], ATR [44, 45], PLK4  and AURKA . Further studies will be required not only to assess instability phenotypes when expression levels are increased, but also how phenotypes might vary when multiple genes are up-regulated.
In tumors, changes in gene dosage due to low level copy number alterations may also lead to small alterations in expression of multiple genes, which together could contribute to dysfunction of processes manipulating the genome, resulting in more error prone cell division cycles. Thus, during tumor progression, genome instability may be enhanced not only by deregulation of E2F, but also by the acquisition of greater numbers of copy number changes encompassing more genes involved in genome maintenance. Since genetic instability is an on-going feature of tumors, allowing them to evolve resistance to therapy, the ability to recognize the active mechanisms of instability in tumors may help to guide therapeutic decisions.