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. 2009 Nov;8(21):2042-50.
doi: 10.4161/cbt.8.21.9764. Epub 2009 Nov 7.

Amplification of chromosomal segment 4q12 in non-small cell lung cancer

Alex H Ramos  1 , Amit Dutt, Craig Mermel, Sven Perner, Jeonghee Cho, Christopher J Lafargue, Laura A Johnson, Ann-Cathrin Stiedl, Kumiko E Tanaka, Adam J Bass, Jordi Barretina, Barbara A Weir, Rameen Beroukhim, Roman K Thomas, John D Minna, Lucian R Chirieac, Neal I Lindeman, Thomas Giordano, David G Beer, Patrick Wagner, Ignacio I Wistuba, Mark A Rubin, Matthew Meyerson
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Amplification of chromosomal segment 4q12 in non-small cell lung cancer

Alex H Ramos et al. Cancer Biol Ther. 2009 Nov.

Abstract

In cancer, proto-oncogenes are often altered by genomic amplification. Here we report recurrent focal amplifications of chromosomal segment 4q12 overlapping the proto-oncogenes PDGFRA and KIT in non-small cell lung cancer (NSCLC). Single nucleotide polymorphism (SNP) array and fluorescent in situ hybridization (FISH) analysis indicate that 4q12 is amplified in 3-7% of lung adenocarcinomas and 8-10% of lung squamous cell carcinomas. In addition, we demonstrate that the NSCLC cell line NCI-H1703 exhibits focal amplification of PDGFRA and is dependent on PDGFRalpha activity for cell growth. Treatment of NCI-H1703 cells with PDGFRA-specific shRNAs or with the PDGFRalpha/KIT small molecule inhibitors imatinib or sunitinib leads to cell growth inhibition. However, these observations do not extend to NSCLC cell lines with lower-amplitude and broader gains of chromosome 4q. Together these observations implicate PDGFRA and KIT as potential oncogenes in NSCLC, but further study is needed to define the specific characteristics of those tumors that could respond to PDGFRalpha/KIT inhibitors.

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Figures

Figure 1
Figure 1
Recurrent genomic amplifications of PDGFRA and KIT in NSCLC samples. (A) Smoothed copy number estimates within chromosome arm 4q in top 200 NSCLC samples (columns; ordered by amplification of 4q12). The color scale ranges from blue (deletion) to red (amplification) with estimated copy numbers shown. Grey regions represent the absence of SNP copy number data. Plotted GISTIC G-scores on the right are from all available samples. The green line on the GISTIC plot represents a significance threshold of 0.25 false discovery rate q-value. (B) Magnified view of smoothed copy number estimates from the centromere to 61 Mb on chromosome 4 from 31 NSCLC samples having amplification greater than 2.46 copies (log2 ratio of 0.3) at 4q12. Samples are sorted according to the maximum copy number estimate for PDGFRA and KIT. Solid and dashed lines indicate positions of PDGFRA and KIT, respectively. Color scale as in (A). (C) FISH for PDGFRA (red) and chromosome 4 reference probe (green) displaying low-level and high-level gain of PDGFRA in two different lung squamous cell carcinoma samples, Case 1 and Case 2 respectively. A lung adenocarcinoma sample, Case 3, with no amplification at PDGFRA is shown on the right for reference. Nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI; blue).
Figure 2
Figure 2
NCI-H1703 cells are addicted to PDGFRα activity. (A) PDGFRα expression in NCI-H1703 and HCC15 was confirmed by immunoblotting using actin as a loading control (left). shRNA constructs used to knockdown PDGFRA expression were packaged into lentivirus and used to infect NCI-H1703 and HCC15 cells. Anti-PDGFRα immunoblot shows that hairpins #1, #3 and #5 efficiently knock down endogenous PDGFRα expression in NCI-H1703 cells. Actin is included as a loading control. NI, no infection. shGFP, control hairpin specific for green fluorescent protein used as a negative control (right). (B and C) Infection with three independent hairpins (#1, #3 and #5) did not inhibit cell survival of HCC15 cells as assessed by WST assay (B) but did inhibit survival of NCI-H1703 cells overexpressing PDGFRA (C). All results normalized to survival of cells infected with shGFP.
Figure 3
Figure 3
PDGFRα tyrosine kinase activity is essential in NCI-H1703 cells. (A) PDGFRα is constitutively phosphorylated, with or without PDGF ligand, in NCI-H1703 cells, as compared with HCC15 cells. Stimulation with PDGF was carried out for 20 minutes at 37°C with 100 ng/ml of PDGF. Treatment of these cell lines for 40 minutes with 2 µM PDGFR kinase inhibitor imatinib and sunitinib inhibits basal phosphorylation, as evidenced by immunoblotting with anti-phospho-PDGFRα (upper). Similar levels of expression of PDGFRA are confirmed by immunoblotting with anti-PDGFRα (middle) using actin as a loading control (lower). (B and C) Treatment with the indicated concentrations of imatinib and sunitinib inhibited survival of NCI-H1703 cells, but not of HCC15 cells, as determined by WST assay performed after 4 days treatment. IC50s are indicated.
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