Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes

Abstract

Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1 Mb resolution on bacterial artificial chromosome (BAC) arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2 kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1 Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Second, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300 and FOXP4. Two gene fusions were demonstrated, TAX1BP1-AHCY and RIF1-PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.

Figure 1

Flow karyotype of chromosomes from the breast cancer cell line HCC1187. The axes show fluorescence intensities of Hoechst 33258 and Chromomycin A3 DNA staining on an arbitrary scale. The chromosome fractions or ‘peaks’ are designated A to k. Peak k was off the top of the scale at the gain shown: it contains the largest chromosome, approximately 1.5 times the size of the chromosome in peak A.

Figure 2

Summary of chromosome segments comprising the abnormal chromosomes in breast cancer cell lines, HCC1187, HCC1806 and ZR-75-30. Ideograms of chromosomes 1 to 22 and X are shown. To the right of each ideogram are lines representing the segments that constitute abnormal chromosomes and their approximate breakpoints in each of the three lines, HCC1187 (1187), HCC1806 (1806) and ZR-75-30 (ZR). The precise breakpoints are given in Supplementary Tables 1 and 2. Peak letters are shown at the top of each segment and correspond to the peak letters given in figure 1 and supplementary figures 1 and 2. Balanced breakpoints are indicated*. Normal copies of chromosomes are not shown.

Figure 3

Example hybridisations of chromosomes from reciprocal translocations to arrays of various resolutions. (a - c) Balanced chromosome 8 breakpoints in HCC1187. (a) Array CGH of genomic DNA from HCC1187 on 1Mb array, showing fluorescence log₂ ratios for clones on chromosome 8: note no copy number changes are detected, showing that all chromosome 8 rearrangements are balanced. (b) Hybridisation of flow-sorted chromosomes der(8)t(1;8)t(1;8) (peak E) (open circles) and der(1)t(1;8) (peak J) (closed circles) to a 1Mb BAC array, showing fluorescence log₂ ratios for chromosome 8. A change in the log₂ ratio from ∼0 to greater than 2.5 represents the 8q22.2 translocation breakpoint, indicated with a vertical line. (c) Hybridisation of sorted chromosomes der(8)t(1;8)t(1;8) (peak E) (open circles) and der(1)t(1;6)t(1;8) (closed circles) to an 8p tiling-path BAC array shows the balanced 8p22 breakpoint. (d - f) Examples of balanced breakpoints in HCC1806. (d) Hybridisation of sorted chromosomes der(4)t(4;6) (peak E) (open circles) and der(6)t(4;6) (peak N) (closed circles) to a chromosome 6 tiling path BAC array. (e) Hybridisation of sorted chromosome der(20)t(3;20;7) (peak M) to a custom Nimblegen oligonucleotide array. The breakpoint in TAX1BP1 on chromosome 7 is indicated with a black circle. (f) Hybridisation of sorted chromosome der(12)t(12;22) (peak k) to a custom Nimblegen oligonucleotide array. The breakpoint in P300 on chromosome 22 is indicated with a black circle. Nimblegen data shown is averaged over 300 bp.

Figure 4

Translocation of PKD1L1 results in fusion with RIF1 and upregulation of expression of the PKD1L1 3′ end. (a) Schematic representation of the products of the reciprocal translocation between chromosomes 2 and 7 in HCC1806. The approximate location of the RIF1 and PKD1L1 genes is shown on the der(2) where fusion occurs. The junction on the der(7) has not been mapped to the gene level on chromosome 2. (b) Hybridisation of der(2)t(13;11;2;7) to a custom Nimblegen oligonucleotide array covering specified regions on chromosomes 2 and 7. Breakpoints are indicated with a broken line. (c) Schematic representation of PKD1L1 (not to scale). Relevant exons are shown as numbered black boxes, the promoter is indicated with a black arrow and the breakpoint in HCC1806 is indicated with a broken line. Primer pairs used in quantitative RT-PCR are shown with black arrows below the relevant exons. (d and e) mRNA expression levels for PKD1L1 exons 25-26 (d) and exons 27-28 (e) in a panel of 21 breast cancer cell lines, and two ‘normal’ breast lines, HB4a (immortalised normal lumenal cells), and HMT3522 (breast epithelium from fibrocystic disease). Expression levels (y axis, mean of three replicates with standard error) are shown relative to the highest expressing cell line, MDA-MB-175, using GAPDH expression as a reference. Results for exons 18-19 were similar to (d) and for exons 48-49 similar to (e) (result not shown). (f) exons of RIF1 and PKD1L1 that flank the translocation junction. PCR primers used to amplify the cDNA junction are indicated with black arrows. The dotted lines show the junctions detected by these PCRs. PKD1L1 exon X is an undocumented alternative exon present in one of the junction PCR products. (g) RIF1 and PKD1L1 proteins. Both are large, of the order of 2,500 amino acids. The fusion consists of the first third of RIF1 joined to the transmembrane domains of PKD1L1.