Characterizing the cancer genome in lung adenocarcinoma

Abstract

Somatic alterations in cellular DNA underlie almost all human cancers. The prospect of targeted therapies and the development of high-resolution, genome-wide approaches are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumours (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in approximately 12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.

Figure 1. Large-scale genomic events in lung adenocarcinoma

a, Smoothed copy number data for 371 lung adenocarcinoma samples (columns; ordered by degree of interchromosomal variation and divided into top, middle and bottom tertiles) is shown by genomic location (rows). The colour scale ranges from blue (deletion) through white (neutral; two copies in diploid specimens) to red (amplification). b, c, False discovery rates (q values; green line is 0.25 cut-off for significance) and scores for each alteration (x axis) are plotted at each genome position (y axis); dotted lines indicate the centromeres. Amplifications (red lines) and deletions (blue lines) are shown for large-scale events (b; ≥50% of a chromosome arm; copy number threshold = 2.14 and 1.87) and focal events (c; copy number threshold = 3.6 and 1.2). Open circles label known or presumed germline copy-number polymorphisms.

Figure 2. High-prevalence amplification of the MBIP/NKX2-1 locus on chromosome 14q

a, Copy number on chromosome 14q is shown for 371 lung adenocarcinomas (columns; ordered by amplification) from centromere (top) to telomere (bottom). Colour scale as in Fig. 1. b, Magnified view of the amplified region from a; grey bars represent the absence of SNPs on the array. c, Raw copy number data (y axis) for one sample defining the minimally amplified region are plotted according to chromosome 14 position (x axis; scale in megabases). Genomic positions of MBIP, NKX2-1, NKX2-8 and the BAC used for FISH (red bar) are shown along the x axis. d, FISH for NKX2-1 (red) and a chromosome 14 reference probe (green) on a lung adenocarcinoma specimen with high-level amplification of the NKX2-1 probe. Nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI; blue). The yellow box shows a single nucleus.

Figure 3. NKX2-1 RNAi leads to reduced anchorage-independent growth and viability of NCI-H2009 cells but not A549 cells

a, Anti-NKX2-1 and anti-vinculin control immunoblots of lysates from NCI-H2009 and A549 cells expressing shRNA against NKX2-1 (shNKX2-1a and shNKX2-1b) or GFP (shGFP) as control. b, Soft agar colony formation by NCI-H2009 cells is shown relative to the shGFP control as a mean percentage (± standard deviation in triplicate samples; P = 5.8 × 10⁻⁶ when comparing shGFP to shNKX2-1a and P = 5.1 × 10⁻⁴ when comparing shGFP to shNKX2-1b). c, Colony formation assays as in b for A549 cells (P > 0.5). d, Anti-MBIP and anti-vinculin immunoblots of lysates from shRNA-expressing NCI-H2009 cells. e, Colony formation of shMBIP NCI-H2009 cells relative to that of control shGFP cells (P = 0.0344).