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. 2020 Feb;14(2):277-293.
doi: 10.1002/1878-0261.12608. Epub 2019 Dec 15.

Comparative Analysis of TTF-1 Binding DNA Regions in Small-Cell Lung Cancer and Non-Small-Cell Lung Cancer

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Free PMC article

Comparative Analysis of TTF-1 Binding DNA Regions in Small-Cell Lung Cancer and Non-Small-Cell Lung Cancer

Satoshi Hokari et al. Mol Oncol. .
Free PMC article

Abstract

Thyroid transcription factor-1 (TTF-1, encoded by the NKX2-1 gene) is highly expressed in small-cell lung carcinoma (SCLC) and lung adenocarcinoma (LADC), but how its functional roles differ between SCLC and LADC remains to be elucidated. Here, we compared the genome-wide distributions of TTF-1 binding regions and the transcriptional programs regulated by TTF-1 between NCI-H209 (H209), a human SCLC cell line, and NCI-H441 (H441), a human LADC cell line, using chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq). TTF-1 binding regions in H209 and H441 cells differed by 75.0% and E-box motifs were highly enriched exclusively in the TTF-1 binding regions of H209 cells. Transcriptome profiling revealed that TTF-1 is involved in neuroendocrine differentiation in H209 cells. We report that TTF-1 and achaete-scute homolog 1 (ASCL1, also known as ASH1, an E-box binding basic helix-loop-helix transcription factor, and a lineage-survival oncogene of SCLC) are coexpressed and bound to adjacent sites on target genes expressed in SCLC, and cooperatively regulate transcription. Furthermore, TTF-1 regulated expression of the Bcl-2 gene family and showed antiapoptotic function in SCLC. Our findings suggest that TTF-1 promotes SCLC growth and contributes to neuroendocrine and antiapoptotic gene expression by partly coordinating with ASCL1.

Keywords: ASCL1; ChIP-seq; NKX2-1; SCLC; TTF-1; lung cancer.

Conflict of interest statement

KM and SE were partly supported by Eisai, Co., Ltd. The remaining authors declare no conflict of interest.

Figures

Figure 1
Figure 1
High expression of NKX2‐1 in a subset of SCLC. (A) Expression of NKX2‐1 mRNA (encoding TTF‐1) in various cancers from the CCLE database. Normalized expression of the microarray data was calculated by robust multichip analysis (RMA). (B) Lung cancer cell datasets from CCLE were divided into SCLC (n = 52), LADC (n = 73), and squamous cell carcinoma (n = 28). *P < 0.05, one‐way ANOVA with Dunnett’s test. (C) Expression of NKX2‐1 in 23 clinical SCLC tumors and 42 normal tissues (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43346). Red dotted bar indicates the average expression of NKX2‐1 in the normal tissues. ***P < 0.001, unpaired t‐test. (D) Comparison of NKX2‐1 expression in the clinical SCLC samples (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43346) between the classic (n = 12) and variant (n = 11) subtypes. Bars indicate the mean and S.E. ***P < 0.001, unpaired t‐test. (E) Relationship between NKX2‐1 expression and overall survival in SCLC patients (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43346) (Sato et al., 2013) analyzed by the Kaplan–Meier plot. Patients were divided into NKX2‐1 low (GeneChip score < 250, n = 13) and NKX2‐1 high (score > 250, n = 10). P‐value was calculated by log‐rank test. (F) qRT‐PCR analysis of NKX2‐1 mRNA in lung cancer cells used in this study. Data represent means of the two biological replicates. Error bars, SE. (G) IB for TTF‐1 in the lung cancer cell lines.
Figure 2
Figure 2
Distinct properties of TTF‐1 binding regions in SCLC. (A) Motif centrality analysis of TTF‐1 binding regions using CentriMo. The known TTF‐1 binding motif from HOCOMOCOv11 (NKX21_HUMAN.H11MO.0.A, upper panel) was used for calculation. The x‐axis indicates the relative position (bp) of the best site from the peak summit of each binding region. (B) Venn diagram showing the overlap of TTF‐1 ChIP‐seq peaks between H209 and H441. (C) The upper or lower two lanes show TTF‐1 binding signals of the two biological replicates (Rep 1 and Rep 2) obtained from H441 (blue) or H209 (magenta) cells, respectively. Arrows show the position of ChIP‐qPCR analysis evaluated in (D). kb Sizes denote the ranges shown in the panels. (D) ChIP‐qPCR analysis of TTF‐1 binding in H441 and H209 cells. Data represent the result of two biological replicates. %input values at the target genomic loci were normalized to those at HBB locus. (E) Charts showing the proportion of overlaps with TTF‐1 binding regions in NSCLC cell lines. The binding regions of TTF‐1 were identified by ChIP‐seq data using H441 and H209 cells (this study) or other NSCLC cell lines (SRP045118). The proportions of overlaps with the TTF‐1 binding regions in H441 and H209 cells were compared. ***P < 0.0001, chi‐square test. (F) Motif enrichment and centrality analysis using DREME and CentriMo. The top four de novo‐calculated motifs with the smallest E‐values identified by DREME are shown.
Figure 3
Figure 3
Interaction between TTF‐1 and ASCL1 proteins in SCLC. (A) Charts showing the overlaps with TTF‐1 binding regions in ASCL1 or NEUROD1 binding regions. The binding regions of ASCL1/NEUROD1 were identified by ChIP‐seq data of SCLC cell lines (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69398). (B) A chart showing the overlaps with TTF‐1 binding regions in ASCL1 binding regions in H209 cells. (C) A heat map representation of TTF‐1 and ASCL1 binding regions (two biological replicates, Rep 1 and Rep 2) in H441 and H209 cells. The vertical blue or magenta line indicates the TTF‐1 binding regions in H441 or H209 cells, respectively. (D) Motif centrality analysis using CentriMo in the overlapping regions between TTF‐1 and ASCL1 ChIP‐seq peaks. The 500‐bp sequences flanking the summit position of each TTF‐1 or ASCL1 binding region were used for the analysis. The known NKX‐homeodomain binding motif (Nkx2‐5, MA0503.1) and ASCL1 binding motif (ASCL1, MA1100.1) were used for calculation. The x‐axis indicates the relative position (bp) of the best site from the peak summit of each binding region. (E) Co‐IP assay of HEK293T cells transfected with expression plasmids for 6xMyc‐TTF‐1 and FLAG‐ASCL1. (F) In situ PLA using TTF‐1 and ASCL1 antibodies in H209 cells to show their proximity in the nucleus. H209 cells treated only with the TTF‐1 antibody were used as a control. Proximity between TTF‐1 and ASCL1 was detected as signals (red) in the nuclei (DAPI, blue). Scale bars, 50 µm. (G) Anti‐TTF‐1 and ASCL1 IHC on a tissue microarray of SCLC. The fraction of stained cancer cells was scored as shown in Fig. S4A. Representative images of TTF‐1‐ and ASCL1‐positive (upper panels, score 4) and negative (lower panels, score 0) tumors are shown. Scale bars, 50 µm. (H) Correlations of TTF‐1 and ASCL1 IHC staining scores in the SCLC tissue microarray. r, Spearman’s correlation coefficients. (I) Scatter plots of TTF‐1 (left) and ASCL1 (right) staining scores in a SCLC tissue microarray divided into two groups according to the SCLC stage. Data are represented as mean ± SE (*P < 0.05, **P < 0.01, Mann–Whitney U‐test).
Figure 4
Figure 4
Cooperative regulation of the expression of target genes by TTF‐1 and ASCL1 in SCLC. (A) Expression of NKX2‐1 (encoding TTF‐1; left) or ASCL1 (right) mRNA in H209 cells treated with negative control (siNC) or TTF‐1 siRNAs (siTTF‐1) by qRT‐PCR. (B) IB for TTF‐1 and ASCL1 in H209 cells treated with siNC or siTTF‐1. (C) Expression of NKX2‐1 and ASCL1 mRNAs in H209 cells treated with siNC and ASCL1 siRNAs (siASCL1). (D) IB for TTF‐1 and ASCL1 in H209 cells treated with siNC or siASCL1. (E) Visualization of the TTF‐1 and ASCL1 ChIP‐seq data at the CALCA and DLL3 gene loci. The upper two lanes show TTF‐1 binding signals in H441 cells. The middle or lower two lanes show TTF‐1 or ASCL1 binding signals in H209 cells, respectively. Rep 1 and Rep 2 indicate biological replicates 1 and 2. The kb sizes denote the ranges shown in the panels. Arrows show the position of ChIP‐qPCR analysis evaluated in (F). (F) ChIP‐qPCR analysis of TTF‐1 and ASCL1 at the genomic regions shown in (E). %input values at the target regions were normalized to those in HBB locus. (G) Fold change in the expression of CALCA and DLL3 mRNA in H209 cells treated with siTTF‐1, siASCL1, or both, relative to those treated with siNC. Expression of mRNAs was quantified by qRT‐PCR. (H) Genes down‐ or upregulated by both TTF‐1 and ASCL1 in H209 cells. The genes with overlapping peaks between TTF‐1 and ASCL1 ChIP‐seq data were selected from the commonly regulated genes of TTF‐1 and ASCL1 determined by RNA‐seq of TTF‐1‐ or ASCL1‐depleted cells. These genes are listed in the boxes. qRT‐PCR data represented as mean ± SE of the three independent experiments. **P < 0.01, ***P < 0.001, one‐way ANOVA with Dunnett’s test.
Figure 5
Figure 5
Regulation of cell growth and BCL2 by TTF‐1 in SCLC. (A) Correlations of NKX2‐1 and BCL2 gene expressions in SCLC cells of the CCLE database. r, Spearman’s correlation coefficients. (B) Relative expression of BCL2 mRNA in H209 cells treated with siTTF‐1 relative to those treated with siNC. (C) Immunofluorescence staining for TTF‐1 (red) and Bcl‐2 (green) in H209 cells treated with siNC (left) or siTTF‐1 #2 (right). Right panel shows the result of quantification of the Bcl‐2 expression. Data represented as mean ± S.E of randomly selected two microscopic fields. *P < 0.05, unpaired t‐test. Scale bars, 50 µm. (D) Anti‐TTF‐1 (left) and Bcl‐2 (right) IHC on a tissue microarray of SCLC. The intensity of staining was scored as shown in Fig. S4B. Representative images are shown. Scale bars, 50 µm. (E) Correlations of TTF‐1 and Bcl‐2 staining scores in the SCLC tissue microarray. r, Spearman’s correlation coefficients. (F) WST‐8 cell proliferation assay in H209 cells treated with siNC or siTTF‐1. (G) WST‐8 cell proliferation assay in Lu‐135 cells infected with adenovirus for LacZ (Ad‐LacZ) and TTF‐1 (Ad‐TTF1) expression. *P < 0.05, unpaired t‐test. (H) Induction of apoptosis by knockdown of TTF‐1 in H209 cells. Annexin V‐positive cells were evaluated by flow cytometry. *P < 0.05, one‐way ANOVA with Dunnett’s test. (I) Enrichment plot of the TTF‐1‐regulated gene set (fold‐changes ≤ 0.66) in H209 cells. GSEA was performed using the gene expression data of the clinical SCLC samples (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43346). Patients’ samples were divided into good (n = 12) and poor (n = 11) prognosis groups as in Fig. 1D. NES, normalized enrichment score. Data represented as mean ± S.E of the three (B, F, H) or two (G) independent experiments. *P < 0.05, **P < 0.01, one‐way ANOVA with Dunnett’s test.

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