The transcriptional consequences of somatic amplifications, deletions, and rearrangements in a human lung squamous cell carcinoma

Abstract

Lung cancer causes more deaths, worldwide, than any other cancer. Several histologic subtypes exist. Currently, there is a dearth of targeted therapies for treating one of the main subtypes: squamous cell carcinoma (SCC). As for many cancers, lung SCC karyotypes are often highly anomalous owing to large somatic structural variants, some of which are seen repeatedly in lung SCC, indicating a potential causal association for genes therein. We chose to characterize a lung SCC genome to unprecedented detail and integrate our findings with the concurrently characterized transcriptome. We aimed to ascertain how somatic structural changes affected gene expression within the cell in ways that could confer a pathogenic phenotype. We sequenced the genomes of a lung SCC cell line (LUDLU-1) and its matched lymphocyte cell line (AGLCL) to more than 50x coverage. We also sequenced the transcriptomes of LUDLU-1 and a normal bronchial epithelium cell line (LIMM-NBE1), resulting in more than 600 million aligned reads per sample, including both coding and non-coding RNA (ncRNA), in a strand-directional manner. We also captured small RNA (<30 bp). We discovered significant, but weak, correlations between copy number and expression for protein-coding genes, antisense transcripts, long intergenic ncRNA, and microRNA (miRNA). We found that miRNA undergo the largest change in overall expression pattern between the normal bronchial epithelium and the tumor cell line. We found evidence of transcription across the novel genomic sequence created from six somatic structural variants. For each part of our integrated analysis, we highlight candidate genes that have undergone the largest expression changes.

Figure 1

Two depictions of the lung SCC cell line (LUDLU-1) genome. (A) Visualization through multicolor fluorescence in situ hybridization. This image is the chromosomes from a single cell. The hybridized probes are colored according to their chromosome of origin, with each chromosome name being written in the corresponding color. (B) The genome averaged over all cells in the sample, visualized through a Circos plot. The outer track consists of chromosome ideograms with centromeres in red. Starting from the inner track, there is sequential depiction of structural rearrangements (intrachromosomal variants appear as short gray lines, whereas interchromosomal variants appear as arcs across the inner circle) and copy number changes (shaded gray is the most common, tetraploid, with dark and light orange showing gain and loss, respectively). Allelic ratios are represented by a black line, superimposed over copy number, with a return to baseline indicating loss of heterozygosity.

Figure 2

Dosage effects on expression. Correlation coefficients between copy number and either absolute tumor transcript expression (gray bars) or fold change between tumor and normal (black bars) for different functional transcript classes. Each coefficient is significant (Spearman P < .05).

Figure 3

Functional transcript expression. Mean expression levels for different functional transcript classes in both the normal bronchial epithelium cell line (gray bars) and lung squamous cell tumor cell line (black bars). Expression is quantified as RPKM.

Figure 4

Transcribed breakpoints in the lung SCC cell line (LUDLU-1). Each of A–F depicts a separate event. The reference genome is denoted by a gray, demarcated double line with a double-headed arrow above to indicate scale. Gene regions are indicated below the double line in blue with vertical blocks representing exons and horizontal lines: introns. The name of each gene is given where appropriate. Underneath the genes, the transcribed breakpoints are depicted; deletions are denoted by red blocks and inversions by blue arrows, indicating their reversed direction in the novel genome made by LUDLU-1 structural variants.