Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer

Abstract

Lung cancer is a leading cause of cancer death, where the amplification of oncogenes contributes to tumorigenesis. Genomic profiling of 128 lung cancer cell lines and tumors revealed frequent focal DNA amplification at cytoband 14q13.3, a locus not amplified in other tumor types. The smallest region of recurrent amplification spanned the homeobox transcription factor TITF1 (thyroid transcription factor 1; also called NKX2-1), previously linked to normal lung development and function. When amplified, TITF1 exhibited increased expression at both the RNA and protein levels. Small interfering RNA (siRNA)-mediated knockdown of TITF1 in lung cancer cell lines with amplification led to reduced cell proliferation, manifested by both decreased cell-cycle progression and increased apoptosis. Our findings indicate that TITF1 amplification and overexpression contribute to lung cancer cell proliferation rates and survival and implicate TITF1 as a lineage-specific oncogene in lung cancer.

Figure 1

TITF1 is focally amplified in lung cancer. (a) Frequency plot of cytobands harboring high-level DNA amplification in NSCLC cell lines and tumors (Supplementary Table 3). Cell lines were obtained from the Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center. Tumors were banked at the University Hospital Charité, Berlin, Germany, with patient consent and Institutional Review Board approval, and DNA was extracted from several 30 μm cryostat tissue sections containing ≥ 70% tumor cells. CGH was performed on cDNA microarrays from the Stanford Functional Genomics Facility containing 39 632 human cDNAs (representing 22 279 different mapped human genes/cDNAs), using our published protocol (Pollack et al., 2002). Map positions for arrayed cDNA clones were assigned using the NCBI genome assembly, accessed through the UCSC genome browser database (NCBI Build 36) (Kent et al., 2002). High-level DNA amplification was defined as tumor/normal aCGH ratios >3; selected cytobands with frequent amplification are indicated. The complete microarray data set is accessible from the GEO repository (GSE9995). (b) Genomic profiles by CGH on cDNA microarrays for NSCLC cell lines and tumors, histologies indicated (M = metastasis), for a segment of chromosome 14q13.1–q21.1. Genes are ordered by genome position. Red indicates positive tumor/normal aCGH ratios (scale shown), and samples called gained, using the fused lasso method (Tibshirani and Wang, 2008), at 14q13.3 are marked below by closed circles. Genes and ESTs (IMAGE clone ID shown) on the microarray residing within the amplicon core are highlighted by red text. (c) Genomic profiles by CGH on an Agilent (Santa Clara, CA, USA) high-definition custom microarray tiling 14q13.2–q21.1. The arrays comprised 10 614 probes tiling 3.3Mb (nt 34 457 000–37 750 000) at 14q13.2–q21.1 with an average inter-probe spacing of 310 nt, with an additional 32 451 probes spanning the remaining genome for data normalization. DNAs were labeled as above, then hybridized to the array following the manufacturer’s instructions. Arrays were scanned using an Agilent G2505B scanner and data extracted and normalized using Agilent Feature Extraction software (version 9.1) with default settings. Shown are two informative samples defining the amplicon boundaries, mapped onto the UCSC genome browser. The smallest region of recurrent amplification spans eight named genes. cDNA, complementary DNA; CGH, comparative genomic hybridization; NSCLC, non-small-cell lung cancer; UCSC, University of California Santa Cruz.

Figure 2

TITF1 is overexpressed when amplified in lung cancer lines. (a) FISH validation of TITF1 amplification in select NSCLC cell lines. FISH was performed using Vysis (Downers Grove, IL, USA) reagents according to the manufacturer’s protocols. A locus-specific BAC mapping to TITF1 at 14q13.3 (RP11-1083E2; BACPAC Resources Centre, Oakland, CA, USA) was labeled with SpectrumGreen, and co-hybridized with a SpectrumOrange-labeled telomere–14q probe (Vysis). Slides were counterstained with DAPI and imaged using an Olympus BX51 fluorescence microscope with Applied Imaging (San Jose, CA, USA) Cytovision 3.0 software. DNA amplification is evidenced by increased TITF1 (green)/telomere–14q (red) signals (HCC1195, right) or by TITF1 signal clusters (HCC1833, left). (b) mRNA levels of TITF1, measured by microarray, are elevated in NSCLC cell lines with TITF1 amplification and also in comparison to primary and immortalized (but non-tumorigenic) lung epithelial cultures (Ramirez et al., 2004). Gene expression profiling was performed as described (Lapointe et al., 2004). Reported fluorescence ratios for TITF1 are normalized to the average TITF1 expression level across all samples. Box plots show 25th, 50th and 75th percentiles of expression. P-values (Mann–Whitney U-test) are indicated. (c) Western blot analysis of representative NSCLC cell lines indicates that TITF1 is overexpressed at the protein level when amplified. Electrophoresis and blotting were performed as described (Kao and Pollack, 2006). TITF1 (~47 kDa) and GAPDH (loading control) were detected using anti-TITF1 rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-GAPDH rabbit polyclonal antibody (1:5000; Santa Cruz Biotechnology), followed by HRP-conjugated anti-rabbit IgG (1:20 000; Pierce, Rockford, IL, USA). Detection was carried out using the ECL kit (GE Healthcare, Piscataway, NJ, USA). FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NSCLC, non-small-cell lung cancer; TITF1, thyroid transcription factor 1.

Figure 3

TITF1 amplification/overexpression contributes to cell proliferation. (a) Confirmation of siRNA-mediated knockdown of target protein TITF1 by western blot. On-TARGETplus siRNAs targeting TITF1, along with a negative control siRNA pool (ON-TARGETplus siCONTROL Non-targeting Pool), were obtained from Dharmacon (Lafayette, CO, USA). Sequences of siRNAs are listed in Supplementary Table 4. Cell lines were maintained at 37 °C in RPMI-1640 with 10% fetal bovine serum. For transfection, 125 000–200 000 cells were seeded per well in a six-well plate and transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol, using a final concentration of 50 nM siRNA for 6 h. Cell lysates were harvested 72 h post-transfection; GAPDH served as a loading control. (b) TITF1 knockdown results in decreased cell proliferation in cells with (HCC1833, HCC1195) but not without (H1155, H1299) TITF1 amplification. At 24, 48, 72 and 96 h post-transfection, cell proliferation was quantified by colorimetry based on the metabolic cleavage of the tetrazolium salt WST-1 in viable cells, according to the manufacturer’s protocol (Roche, Indianapolis, IN, USA). Transfections were performed in replicate and mean (±1 s.d.) OD reported. (c) TITF1 knockdown reduces cell-cycle progression, evidenced by decreased S-phase fraction with G₁ block. At 72 h post-transfection, cell-cycle distribution analysis was performed using the BrdU-FITC Flow kit (BD Biosciences, San Jose, CA, USA) as per the manufacturer’s instructions. Cells were incubated with 10 μM BrdU at 37 °C for 4 h prior to processing for analysis. Anti-BrdU FITC and 7- aminoactinomycin D (for total DNA content) stainings were scored by FACSCalibur (BD Biosciences) and analysed using CellQuest software (BD Biosciences). Transfections were performed in triplicate and mean (±1 s.d.) cell-cycle fractions reported. Representative FAC S plots are also shown. (d) TITF1 knockdown leads to increased apoptosis. At 72 h post-transfection, apoptosis was assayed by annexin-V staining and quantified by flow cytometry using the Vybrant Apoptosis Assay kit (Invitrogen) as per the manufacturer’s instructions. Transfections were performed in triplicate, and mean (±1 s.d.) percent apoptosis reported. *P < 0.05. **P < 0.01 (Student’s t-test; TITF1 compared to control). BrdU, bromodeoxyuridine; siRNA, small interfering RNA; TITF1, thyroid transcription factor 1.