Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer

Abstract

We used high-resolution array analysis to discover a recurrent lung cancer amplicon located at 14q13.3. Low-level gain of this region was detected in 15% of lung cancer samples, and high-level amplification was detected in an additional 4% of samples. High-level focal amplification appears to be specific to lung cancers, because it was not detected in >500 samples of other tumor types. Mapping of the commonly amplified region revealed there are three genes in the core region, all of which encode transcription factors with either established lung developmental function (TTF1/NKX2-1, NKX2-8) or potential lung developmental function (PAX9). All three genes were overexpressed to varying degrees in amplified samples, although TTF1/NKX2-1 was not expressed in the squamous cancer subtype, consistent with previous reports. Remarkably, overexpression of any pairwise combination of these genes showed pronounced synergy in promoting the proliferation of immortalized human lung epithelial cells. Analysis of human lung cancer cell lines by both RNAi and ectopic overexpression further substantiates an oncogenic role for these transcription factors. These results, taken together with previous reports of oncogenic alterations of transcription factors involved in lung development (p63, CEBPA), suggest genetic alterations that directly interfere with transcriptional networks normally regulating lung development may be a more common feature of lung cancer than previously realized.

Fig. 1.

The commonly amplified core region of lung cancer 14q13.3 amplicons. (A) The segmented array CGH DNA copy number values (y axis) for 11 samples containing the 14q13.3 amplicon are plotted against the chromosome 14 nucleotide position (x axis) from the March 2006 (hg18) assembly of the International Human Genome Sequencing Consortium (http://genome.ucsc.edu). The commonly amplified core region (chr14:35,820,860-36,348,636), which maximally spans a 530-kb region, is shaded in blue. The 11 amplified samples consist of eight primary tumors (700, 457, 1242, 1041, 817, 1308, 531, and 647) and three cell lines (NCI-H661, NCI-H2170, and NCI-H146). NCI-H661 is from a large-cell carcinoma, both NCI-H2170 and 647 are from SCC, NCI-H146 is from an SCLC, and the remaining samples are from AC. (B and C) Determination by real-time PCR of the centromeric boundary of the amplicon found in primary tumor sample 647 (B) and of the telomeric boundary of the amplicon found in large-cell carcinoma cell line NCI-H661 (C). The DNA copy numbers measured by both array CGH and real-time PCR are as plotted. This analysis defined the two boundaries (chr14:35,897,833-36,311,043) of the commonly amplified 413-kb region.

Fig. 2.

Effects of overexpressing individually or combinatorially the 14q13.3 amplicon genes on the proliferation of premalignant lung epithelial cells. (A) Colony formation of BEAS-2B retroviral transfectants stably expressing individual genes (single transfectant) or pairwise combinations of the three candidate driver genes (double transfectant). The assay was initiated by plating 1,000 cells per well in six-well plates. After 12 days, cells were fixed and stained with crystal violet. Representative pictures of experiments performed in triplicate are shown. (B) The crystal violet stain in A was dissolved in 0.1% SDS solution and quantified by absorbance at 595 nm according to Scragg and Ferreira (20). V, vector only; N, NKX2-8; T, TTF1; P, PAX9; V-V, vector-vector; T-P; TTF1-PAX9; N-P, NKX2-8-PAX9; T-N, TTF1-NKX2-8.

Fig. 3.

Continuous expression of NKX2-8 and PAX9 is essential to the tumor maintenance of amplified SCC cells. (A) Western blot analysis indicates the absence of Ttf1 protein expression in two lung SCC cell lines (H2170 and HCC15), as expected from the literature (–24). Two additional SCC cell lines (EPLC-272H and CHAGO-K-1) were also shown by immunoblotting to lack the expression of Ttf1 protein (data not shown). Analysis of the large-cell lung cancer cell line (H661) was included as a positive control. Twenty micrograms of whole-cell extracts were loaded. The 14q13.3 amplicon status and tumor subtype are as indicated. (B and D) Effective shRNAs (indicated by an asterisk) in knocking down the endogenous protein expression of NKX2-8 or PAX9 were identified by immunoblotting of whole-cell extracts prepared from stable transfectants based in the amplified H2170 cells. Immunoblottings of β-actin or tubulin were performed to control for total amounts of proteins analyzed. (C and E) Stable H2170 transfectants expressing individual shRNAs of NKX2-8 or PAX9 were evaluated for tumorigenicity in athymic nude mice. Approximately 24 h before injections, the mice were gamma-irradiated at 400 rad to minimize residual immune responses. Subsequently, five million cells of individual transfectant populations were injected into a group of five athymic nude mice s.c., and these animals were observed weekly for tumor formation. Tumor take rate was calculated as of the last weekly tumor measurement, and the averaged tumor size (y axis) was plotted against days after injection (x axis).