DNA amplification is a ubiquitous mechanism of oncogene activation in lung and other cancers

Abstract

Chromosomal translocation is the best-characterized genetic mechanism for oncogene activation. However, there are documented examples of activation by alternate mechanisms, for example gene dosage increase, though its prevalence is unclear. Here, we answered the fundamental question of the contribution of DNA amplification as a molecular mechanism driving oncogenesis. Comparing 104 cancer lines representing diverse tissue origins identified genes residing in amplification 'hotspots' and discovered an unexpected frequency of genes activated by this mechanism. The 3431 amplicons identified represent approximately 10 per hematological and approximately 36 per epithelial cancer genome. Many recurrently amplified oncogenes were previously known to be activated only by disease-specific translocations. The 135 hotspots identified contain 538 unique genes and are enriched for proliferation, apoptosis and linage-dependency genes, reflecting functions advantageous to tumor growth. Integrating gene dosage with expression data validated the downstream impact of the novel amplification events in both cell lines and clinical samples. For example, multiple downstream components of the EGFR-family-signaling pathway, including CDK5, AKT1 and SHC1, are overexpressed as a direct result of gene amplification in lung cancer. Our findings suggest that amplification is far more common a mechanism of oncogene activation than previously believed and that specific regions of the genome are hotspots of amplification.

Figure 1

Hotspots of amplification in cancer genomes. A histogram summarizing the regions of amplification across all 104 samples with the resulting values scaled to the segment with the highest count (28) and plotted against their corresponding genomic position. Hotspots are denoted by the dark blue shading, whereas the light blue shading represents regions amplified ≤5 times. Triangles mark common fragile sites. Detailed genomic position of hotspots and common fragile sites are provided in Supplementary Tables 5 and 17.

Figure 2

Impact of amplification on gene transcription levels. The relative expression values for samples with amplification and those with neutral copy number status are plotted as heatmaps for overexpressed genes from representative hotspots. The expression values for each gene have been normalized and scaled across the samples from 0 to 100.

Figure 3

Frequent amplification and overexpression of multiple EGFR-family-signaling components in non-small cell lung cancer (NSCLC). Diagram highlighting the interaction of EGFR, SHC1, CDK5, SHC1 and MYC in the EGFR family-signaling pathway. Altered components are shaded grey. The table summarizes the number and specific samples with amplification and overexpression of each pathway component. The total represents the number of samples with at least one pathway component amplified and overexpressed.

Figure 4

SHC1disruption in NSCLC cell lines and clinical tumors. (a) Representative array CGH profiles for samples with and without SHC1 amplification. Vertical lines denote log₂ signal ratios from −1 to 1 with copy number increases to the right (red lines) and decreases to the left (green lines) of 0 (purple line), with amplified region shaded orange. Red and green arrows mark clones used in subsequent Fluorescence in situ hybridization (FISH) analysis. (b) SHC1 expression in NSCLC cell lines. The normalized fold change of expression compared to a normal lung reference is plotted for samples with amplification (red) and those with neutral copy number status (black). (c) Overexpression of SHC1 in clinical tumors. The log₂ fold change in expression levels of SHC1 relative to their matched normal lung tissue is plotted for each tumor. The P-value from the Wilcoxon sign-rank test is indicated. (d and e) FISH confirmation of SHC1 amplification in H1395. FISH was performed using BAC clones mapping to SHC1 (RP11-624P9) and to an adjacent neutral copy number region (RP11-313J15).