Discovery of novel amplified genes in primary breast cancer with copy number and gene expression analysis of whole exome and transcriptome sequencing data

Abstract

Introduction: Copy number alteration (CNA) of genome is common in breast cancer and tend to have more driver role than single point mutations. Traditionally, genome-wide analysis of DNA copy number changes were done by array CGH or SNP array method. Here, we did DNA whole exome sequencing (WES) and RNA-seq using Next Generation Sequencing (NGS) technology to find common genes or chromosomal regions of which DNA copy was highly amplified and at the same time RNA expression was also upregulated. Materials and Methods: RNA and DNA were extracted from fresh frozen tissues of 93 breast cancer patients. WES and RNA-seq were done using NGS technology (Illumina HiSeq 2000). As a control, normal DNA from all matched patients were also sequenced. GATK was used to gain mean depth and coverage data for targeted regions. CNAs were calculated with Exome CNA, a statistical method to detect somatic CNAs using depth-of-coverage information from mapped short sequence reads. To estimate expression levels, the relative transcript abundances were measured in FPKM using Cufflinks. FISH was performed for amplification validation of target genes and siRNA transfection for target genes role in breast cancer cell lines. After siRNA transfection, stem cell marker assay and mammosphere formation count were used for the target genes effect on stem cell. Results: DNA of 1,737 genes were highly amplified (log R>1.0) in two or more samples. The two most commonly amplified chromosomes were chromosome 8 and 17. We applied a cut-off for higher gene expression as relative FPKM >1.5. ERBB2 amplifications and high expression were most common (21.5%) of all genes and it was in agreement with HER-2 IHC and FISH result. Among previously reported amplified genes, FGFR1 (5.4%) and PVT1 (8.6%) in chromosome 8, CCND1, PAK1 (3.2%) and EMSY (4.3%) in chromosome 11, CCNE1 (4.3%) in chromosome 19 were also identified in this study. IGF1R high amplification and expression was found in two samples, and ESR1, MDM2, KIT was found in only one sample each. We found uncommon but novel and recurrent highly amplified and expressed genes: CLK4 in 5q (3.2%), AHI/MYB in 6q (3.2%), MMP7 (2.2%) and MALAT1 in 11q (1.1%), and NEK8 in 17q (4.3%) We designed FISH probe for this 5 new genes and confirmed the high amplifications in each sample with FISH. Real-time PCR results showed that mRNA expression of 4 genes was significantly down regulated after interfered with siRNA for each of 4 genes( (NEK8, CLK4, MMP7 and MALAT1). Proliferation, migration and invasion were inhibited in various breast cancer cell lines after knock-down by siRNA for 4 genes and inhibition of NEK8 down-regulated stem cell properties in MDA-MB-157 cell line. Conclusion: Our results revealed the amplification and the role in breast cancer of 4 new genes. These genes will be candidates for new therapeutic targets for breast cancer.