Structural and genic characterization of stable genomic regions in breast cancer: relevance to chemotherapy

Abstract

Background: Cancer genomes accumulate frequent and diverse chromosomal abnormalities as well as gene mutations but must maintain the ability to survive in vivo. We hypothesize that genetic selection acts to maintain tumour survival by preserving copy number of specific genes and genomic regions. Genomic regions and genes that remain unaltered in copy number and expression, respectively, may be essential for maintaining tumour survival.

Methods: We analyzed copy number data of 243 previously reported breast tumours and computationally derived stable copy number regions. To identify genes in stable copy number regions with nominal changes in expression, datasets for tumour and normal samples were compared. Results were replicated by analysis of a series of independent copy number, expression and genomic sequencing studies. A subset of stable regions, including stable paralogous regions, were confirmed by quantitative PCR and fluorescence in situ hybridization (FISH) in 5 breast cancer cell lines. We deduced a comprehensive set of dually stable genes (i.e. maintaining nominal copy number and expression) which were categorized according to pathway and ontology assignments. The stability of genes encoding therapeutic drug targets was also assessed.

Results and conclusion: Tumour genome analysis revealed 766 unstable (amplified and/or deleted) and 812 stable contiguous genomic regions. Replication analysis of an independent set of 171 breast tumours confirmed copy number stability of 1.3 Gb of the genome. We found that 5804 of these genes were dually stable. The composition of this gene set remained essentially unchanged (<2% reduction) after accounting for commonly mutated breast cancer genes found by sequencing and differential expression. The stable breast cancer genome is enriched for cellular metabolism, regulation of gene expression, DNA packaging (chromatin and nucleosome assembly), and regulation of apoptosis functions. Stable genes participating in multiple essential pathways were consistently found to be targets of chemotherapies. Preservation of stable, essential genes may be related to the effectiveness of certain chemotherapeutic agents that act on multiple gene products in this set.

Figure 1

Unstable and stable genomic regions mapped to chromosome 17. (A) Probes that detected frequent amplifications and deletions are displayed (orange) along the entire chromosome 17. Merging genomic intervals corresponding to these probes formed unstable regions (red). Stable regions (blue) were derived from the complement of unstable regions. (B) Unstable regions, stable regions, and genes (black) are displayed for chromosome 17(q12q21.1). The red arrow indicates the location of ERBB2, a gene within an unstable region and amplified in ∼25% of breast cancers.

Figure 2

Frequency and length distributions of unstable and stable genomic regions and protein‐coding genes. Histogram indicates that the majority of unstable regions (dark blue) are ≤250 kb in length (bin 250,000 bp) and stable regions (turquoise) are longer than 250 kb (bin 250,000 bp). The Y‐axis represents the number of stable or unstable regions, and genes as a percentage from each group. The X‐axis depicts genomic lengths binned in units of 1000 bp. In comparison to stable regions, protein‐coding genes (grey; consensus coding sequence project) have a smaller size distribution similar to that of unstable regions.

Figure 3

Length distribution of single‐copy (sc) stable genomic intervals per autosome. In the histogram, the lengths of stable regions including those within segmentally duplicated regions (red) are compared with overall chromosome length (black). While all chromosomes contain stable regions, the distribution of stable material is not related to chromosome length (r2 = 0.401). On a per nucleotide basis, there is a higher degree of preservation of sc‐genomic stability on chromosomes 2, 7, 9, 10, 15, 16, 17, 19 and 22. Stable regions of chromosome 13 and 18 appear to have more repetitive DNA (as represented by diminished length of sc‐intervals) than the other chromosomes.

Figure 4

MAPK signalling pathway with unstable and stable (copy number and expression) genes represented. The genes in the pathway are annotated as indicated in the figure. The preponderance of genes at the entry point (cell membrane) are either amplified and/or deleted, whereas stable genes are present downstream. This is consistent with abnormal signalling propagated through these downstream gene products. This figure was modified from the WikiPathways entry for MAPK. It used data from GENMAPP as indicated in the methods.