PMC3940063_01
Sample description and cytotoxic drug resistance of H2073. A) Cell line H1993 was derived from a metastatic site in patient's lymph nodes, while H2073 originated from the primary lung tumor after treatment with cisplatin and etoposide. BL1993 was derived from lymphoblastoid cells of the same patient, thus representing the matched normal blood sample. (B-D) In comparison to H1993, H2073 cells show higher viability upon treatment with etoposide, paclitaxel and vinorelbine. Error bars indicate standard deviation.
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Cell line H1993 was derived from the lymph nodes of a 47 year old female Caucasian with history of smoking and diagnosed with non-small cell lung cancer in 1988 (Figure 1A). As expected, H2073 shows resistance to etoposide (Figure 1B). Interestingly, the spectrum of drug resistance of H2073 cells encompasses a broader range of therapeutics including paclitaxel and vinorelbine (Figures 1C-D), which target mitotic division.
Integrative analysis of H1993 and H2037: a panomics approach. A) We applied an integrative analysis of H1993 and H2073 based on whole genome sequencing, RNA sequencing, DNA methylation quantification and copy number investigation. B) The genome of each cell line was sequenced at minimum 30x coverage. C) The panomics approach allowed us to analyze the landscape of single nucleotide variants, indels, differential gene expression, copy number changes, and structural variations. The numbers of detected aberrations are shown for these two cell lines.
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Illumina, to a minimum depth of 30x (Figure 2B). In addition, we constructed DNA libraries with variable insert sizes for both cell lines, performed Illumina-based paired-end sequencing, and used the resulting reads for de novo genome assembly, in order to identify genomic features missed by reference-based approaches. Results are summarized in Figure 2C.
Genomic landscapes and pathway alterations of H1993 and H2073. A) Multiple cancer related genes were somatically mutated in both cell lines (upper panel) or differentially expressed between the cell lines (lower panel). B) Integrating gene expression and focusing on instances of high mutant allele frequency enabled us to substantially reduce the set of candidate drivers. Known cancer related genes are highlighted. Genes with low expression (<0.5 RPKM) in both cell lines are shown in blue, while genes with low expression in one cell line are shown in green. Triangles indicate cell line-specific mutations, while circles correspond to common mutations.
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Both cell lines harbor non-synonymous mutations in genes known to be altered in lung cancer, including TP53, STK11, EPHB2, LRP1B, INHBA, ZNF458, and PRDM14 (Figure 3A). This expression based filtering reduced the number of common mutations from 313 to 106 (96 missense, 10 stop gain), eliminating a large fraction of candidate genes at the risk of possibly discarding low expressed drivers (Figure 3B). We then assessed mutant allele frequencies in DNA and RNA data and grouped the mutations into three frequency classes (Figure 3B): high (>0.9, class 1), medium (0.3 to 0.9, class 2), and low/inconsistent (class 3).
Differential gene expression analysis between H1993 and H2073. A) Volcano plot illustrating fold changes and false discovery rates for all human genes as calculated by differential gene expression analysis. B) Percentage of overexpressed genes with significant copy number gain or DNA hypomethylation. C) The sets of expressed genes with copy number amplification or promoter DNA hypomethylation were enriched for overexpressed genes. D) Treating both cell lines with an EGFR inhibitor Cetuximab reveals lower viability of H2073 in comparison to H1993. Treating the two cell lines with a MET inhibitor Criozotinib reveals lower viability in H1993. Error bars indicate standard deviation.
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Our RNA-Seq analysis identified 2,523 differentially expressed genes between H1993 and H2073 (Figure 4A), of which 1,668 (67%) were over-expressed in H2073. Similarly, we observed that 46 out of 885 (5.4%) genes overexpressed in H1993 are in genomic regions amplified in H1993 relative to H2073 (Figure 4B). Overall, regions amplified in H1993 and H2073 contained 100 and 114 expressed genes, respectively, out of which 46 (46%) and 39 (34%) were overexpressed according to our cutoffs, exhibiting higher rate of overexpression events than non-amplified regions (Figure 4C, Fisher exact test p-value <7x10-9 for both cell lines). The dependence of H1993 on MET is confirmed by its low viability in the presence of MET inhibitors (Figure 4D). Out of 1,668 genes overexpressed in H2073, 166 (9.9%) contained DMRs (Figure 4B). In total, hypomethylated DMRs were associated with 68 and 325 genes in H1993 and H2073, respectively, out of which 43 (63.2%) and 146 (44.9%) showed overexpression, exhibiting higher rate of overexpression events than hypermethylated or non-differentially methylated regions (Figure 4C, Fisher exact test p-values < 2x10-32 for both cell lines). H2073 than in H1993, and the dependence of H2073 on EGFR for survival and proliferation is strongly suggested by its higher sensitivity to EGFR inhibitors (Figure 4D). However, the observed overexpression of EGFR was not associated with either a copy number change or differential promoter DNA methylation in this study.
Overexpression of MET in H1993 is associated with copy number gain (A), while overexpression of PLAU in H2073 is associated with decreased promoter methylation (B). The panels show gene structure (top, individual transcript isoforms), expression normalized by sequencing depth (H1993: second from the top, H2073: third from the top), difference in DNA methylation (second from bottom, dashed lines correspond to differences of 0.5,0, and -0.5), and raw copy number (bottom, green line: H1993, yellow line : H2073, dashed black like: CN=2 (baseline)).
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Overall, regions amplified in H1993 and H2073 contained 100 and 114 expressed genes, respectively, out of which 46 (46%) and 39 (34%) were overexpressed according to our cutoffs, exhibiting higher rate of overexpression events than non-amplified regions (Figure 4C, Fisher exact test p-value <7x10-9 for both cell lines). One of the H2073 amplicons included transporter gene ABCC4, previously implicated in drug resistance and showing 3-fold region on chromosome 7, highly amplified (>10 copies) in H1993, contained oncogene MET (Figure 5A), which is known to be involved in tumor cell invasion and metastasis. Several of the genes with overexpression and promoter DNA hypomethylation in H2073 have been implicated in apoptosis evasion and drug resistance, including PLAU (Figure 5B), SNCG, BNIP3, GSTP1, ETS1, and MSLN.
Structural variations in H1993 and H2073. A) Illustration of genomic alterations in H1993 and H2073 using Circos plots. Candidate interchromosomal structural variations identified by the Complete Genomics Platform are shown as red lines. Copy number changes detected by Illumina SNP arrays are illustrated as bar plots. Loss of heterozygosity regions are shown in green. B) Structural variations, in particular smaller inversions (C), were more frequent in the cell line derived after chemotherapy (H2073)
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Overall, regions amplified in H1993 and H2073 contained 100 and 114 expressed genes, respectively, out of which 46 (46%) and 39 (34%) were overexpressed according to our cutoffs, exhibiting higher rate of overexpression events than non-amplified regions (Figure 4C, Fisher exact test p-value <7x10-9 for both cell lines).