A sequence-level map of chromosomal breakpoints in the MCF-7 breast cancer cell line yields insights into the evolution of a cancer genome - PubMed (original) (raw)

doi: 10.1101/gr.080259.108. Epub 2008 Dec 3.

Petra Den Hollander, Christopher A Miller, David A Delgado, Jian Li, Cristian Coarfa, Ronald A Harris, Stephen Richards, Steven E Scherer, Donna M Muzny, Richard A Gibbs, Adrian V Lee, Aleksandar Milosavljevic

Affiliations

A sequence-level map of chromosomal breakpoints in the MCF-7 breast cancer cell line yields insights into the evolution of a cancer genome

Oliver A Hampton et al. Genome Res. 2009 Feb.

Abstract

By applying a method that combines end-sequence profiling and massively parallel sequencing, we obtained a sequence-level map of chromosomal aberrations in the genome of the MCF-7 breast cancer cell line. A total of 157 distinct somatic breakpoints of two distinct types, dispersed and clustered, were identified. A total of 89 breakpoints are evenly dispersed across the genome. A majority of dispersed breakpoints are in regions of low copy repeats (LCRs), indicating a possible role for LCRs in chromosome breakage. The remaining 68 breakpoints form four distinct clusters of closely spaced breakpoints that coincide with the four highly amplified regions in MCF-7 detected by array CGH located in the 1p13.1-p21.1, 3p14.1-p14.2, 17q22-q24.3, and 20q12-q13.33 chromosomal cytobands. The clustered breakpoints are not significantly associated with LCRs. Sequences flanking most (95%) breakpoint junctions are consistent with double-stranded DNA break repair by nonhomologous end-joining or template switching. A total of 79 known or predicted genes are involved in rearrangement events, including 10 fusions of coding exons from different genes and 77 other rearrangements. Four fusions result in novel expressed chimeric mRNA transcripts. One of the four expressed fusion products (RAD51C-ATXN7) and one gene truncation (BRIP1 or BACH1) involve genes coding for members of protein complexes responsible for homology-driven repair of double-stranded DNA breaks. Another one of the four expressed fusion products (ARFGEF2-SULF2) involves SULF2, a regulator of cell growth and angiogenesis. We show that knock-down of SULF2 in cell lines causes tumorigenic phenotypes, including increased proliferation, enhanced survival, and increased anchorage-independent growth.

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Figures

Figure 1.

Figure 1.

(A) An illustration of the principle of the method. Breakpoints within a BAC containing segments from chromosomes 20, 3, and 17 are detected using a combination of “bridging” and “outlining” steps. The bridging step maps fosmid end-sequences onto the reference genome. The outlining step maps short tags (labeled “PyroSeqs”) using 454 technology from the BAC (in practice a pool of BACs) onto the reference genome. The results of bridging and outlining jointly allow precise mapping of breakpoints and reconstruction of rearranged BACs. (B) Organization of the mapping experiment. The nonredundant collection of 552 rearrangement containing BACs, 17 normal BAC negative controls, and seven positive controls was arrayed in six 96-well plates and pooled as indicated. Three 454 sequencing reactions (involving BACs pooled from plate pairs) produced tags for the purpose of outlining. Six fosmid libraries (one from each 96-well plate pool of BACs) were constructed for Sanger-based sequencing of fosmid ends and bridging. (C) Bar charts detailing the classification of detected MCF-7 breakpoints.

Figure 2.

Figure 2.

(A) Circular visualization of the MCF-7 genome obtained using Circos software. Chromosomes are individually colored with centromeres in white and LCR regions in black. MCF-7 BAC array comparative genome hybridization data (Jonsson et al. 2007) are plotted with gains in green and losses in red using log2ratio. The inner chromosome annotations depict 157 somatic MCF-7 breast tumor chromosomal rearrangements associated with LCRs (black) and breakpoints not associated with LCRs (green). Chromosomal rearrangements are depicted on each side of the MCF-7 breakpoints; intrachromosomal rearrangements (blue) are located outside and interchromosomal rearrangements (red) are located in the center of the circle. (B) Bar charts indicating classification of somatic breakpoints in MCF-7.

Figure 3.

Figure 3.

(A) Four clusters of breakpoints at 1p13.1-21.1, 3p14.1-p14.2, 17q22-q24.3, and 20q12-q13.33. (B) Low copy repeat (LCR) association with clustered and dispersed breakpoints. (C) The four clusters of breakpoints correspond exactly to the four highly amplified regions in MCF-7, as determined by array CGH.

Figure 4.

Figure 4.

Confirmation of the presence of predicted processed chimeric mRNA transcripts in MCF-7 using RT-PCR.

Figure 5.

Figure 5.

(A_–_C) Cells treated with SULF2 siRNA have an enhanced proliferation compared with cells treated with control siRNA. MCF-7B (A; Mao et al. 2005), MDA MD231 (B), and MCF-10A (C) cells were transfected with 50 nM SULF2 or control siRNA; 104 cells were plated in medium containing 10% FBS 48 h after transfection of the siRNA. Cells were counted on day 2, 4, 6, and 8. Experiments performed in triplicate; error bars show standard deviation. (D_–_F) Cells treated with SULF2 siRNA have an enhanced survival compared with cells treated with control siRNA. MCF-7B (D; Mao et al. 2005), MDA MD231 (E), and MCF-10A (F) cells were transfected with 50 nM SULF2 or control siRNA; 104 cells were plated in serum-free medium 48 h after transfection of the siRNA. Cells were counted on day 2, 4, and 6. Experiments performed in triplicate. Error bars, SD. (G,H) Treatment of MCF-7B and MDA MB231 cells with siRNA for SULF2 increases the anchorage-independent growth capabilities. After treatment with siRNA, 104 cells were plated in 0.3% agar in growth medium, MCF-7B colony formation is shown in G. Plates were incubated for 21 d, and colonies were counted; bar chart results shown in H. Experiments performed in triplicate. Error bars, SD.

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