Gene rearrangements in hormone receptor negative breast cancers revealed by mate pair sequencing

Abstract

Background: Chromosomal rearrangements in the form of deletions, insertions, inversions and translocations are frequently observed in breast cancer genomes, and a subset of these rearrangements may play a crucial role in tumorigenesis. To identify novel somatic chromosomal rearrangements, we determined the genome structures of 15 hormone-receptor negative breast tumors by long-insert mate pair massively parallel sequencing.

Results: We identified and validated 40 somatic structural alterations, including the recurring fusion between genes DDX10 and SKA3 and translocations involving the EPHA5 gene. Other rearrangements were found to affect genes in pathways involved in epigenetic regulation, mitosis and signal transduction, underscoring their potential role in breast tumorigenesis. RNA interference-mediated suppression of five candidate genes (DDX10, SKA3, EPHA5, CLTC and TNIK) led to inhibition of breast cancer cell growth. Moreover, downregulation of DDX10 in breast cancer cells lead to an increased frequency of apoptotic nuclear morphology.

Conclusions: Using whole genome mate pair sequencing and RNA interference assays, we have discovered a number of novel gene rearrangements in breast cancer genomes and identified DDX10, SKA3, EPHA5, CLTC and TNIK as potential cancer genes with impact on the growth and proliferation of breast cancer cells.

Figure 1

Validated somatic rearrangements in breast cancer genomes. Outer histogram of the Circos plot shows the number of deletions in each bin, and the inner histogram shows predicted insertions. Connections represent PCR validated (blue lines) and sequence validated (red lines) somatic translocations.

Figure 2

Gene knockdown results in cell growth inhibition and suppression of DDX10 leads to increased apoptosis. (A) Realtime quantitative PCR post-transfection of esiRNAs targeting CLTC, SKA3 and DDX10 showed efficient suppression of these three genes at the mRNA level. Suppression of EPHA5 and TNIK was not able to be assessed using this technique due to poor quality of primers. (B) siRNA targeting CLTC, EPHA5, SKA3, DDX10 and TNIK transfected MCF-10A and MCF-7 cell growth in vitro relative to controls 70 h and 120 h, respectively, after transfection is reported. Data from two independent experiments are shown with error bars representing standard deviations. Apoptosis (C) and micronuclei formation (D) of cell lines after transfection with siRNA targeting DDX10 (MCF-10A, N = 2486; MCF-7, N = 2838), SKA3 (MCF-10A, N = 2823; MCF-7, N = 5035) or both siRNAs (MCF-10A, N = 2390; MCF-7, N = 4701) was shown by numbers of cell nuclei that exhibit apoptotic nuclear morphology and micronuclei, respectively, per 1000 cells. siGFP transfected cells served as controls (MCF-10A, N = 2638; MCF-7, N = 3896). Data from a representative experiment.

Figure 3

Overlap of translocation breakpoints and array painting. BAC regions studied by Howarth et al. are shown as boxes on the ideogram. Black boxes indicate regions not overlapping with breakpoints from this study, blue boxes indicate regions in which one breakpoint was observed in this study, and red boxes indicate regions that contain more than one breakpoint. The gray shadows represent chromosome bands.