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. 2012 Dec;122(12):4388-400.
doi: 10.1172/JCI64048. Epub 2012 Nov 12.

Kras(G12D) and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung

Yutaka Maeda  1 , Tomoshi Tsuchiya, Haiping Hao, David H Tompkins, Yan Xu, Michael L Mucenski, Lingling Du, Angela R Keiser, Takuya Fukazawa, Yoshio Naomoto, Takeshi Nagayasu, Jeffrey A Whitsett
Affiliations

Kras(G12D) and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung

Yutaka Maeda et al. J Clin Invest. 2012 Dec.

Abstract

Mucinous adenocarcinoma of the lung is a subtype of highly invasive pulmonary tumors and is associated with decreased or absent expression of the transcription factor NK2 homeobox 1 (NKX2-1; also known as TTF-1). Here, we show that haploinsufficiency of Nkx2-1 in combination with oncogenic Kras(G12D), but not with oncogenic EGFR(L858R), caused pulmonary tumors in transgenic mice that were phenotypically similar to human mucinous adenocarcinomas. Gene expression patterns distinguished tumor goblet (mucous) cells from nontumorigenic airway and intestinal goblet cells. Expression of NKX2-1 inhibited urethane and oncogenic Kras(G12D)-induced tumorigenesis in vivo. Haploinsufficiency of Nkx2-1 enhanced Kras(G12D)-mediated tumor progression, but reduced EGFR(L858R)-mediated progression. Genome-wide analysis of gene expression demonstrated that a set of genes induced in mucinous tumors was shared with genes induced in a nontumorigenic chronic lung disease, while a distinct subset of genes was specific to mucinous tumors. ChIP with massively parallel DNA sequencing identified a direct association of NKX2-1 with the genes induced in mucinous tumors. NKX2-1 associated with the AP-1 binding element as well as the canonical NKX2-1 binding element. NKX2-1 inhibited both AP-1 activity and tumor colony formation in vitro. These data demonstrate that NKX2-1 functions in a context-dependent manner in lung tumorigenesis and inhibits Kras(G12D)-driven mucinous pulmonary adenocarcinoma.

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Figures

Figure 1
Figure 1. Haploinsufficiency of Nkx2-1 increased tumorigenesis in KrasG12D mice.
(A) KrasG12D;Nkx2-1+/– mice (n = 10) lost weight compared with KrasG12D;Nkx2-1+/+ (n = 8) and control (n = 15) mice 2 months after Dox administration. (B) KrasG12D;Nkx2-1+/– mice (n = 5) had larger lungs than did KrasG12D;Nkx2-1+/+ (n = 5) and control (n = 10) mice 2 months after Dox administration. (C) KrasG12D;Nkx2-1+/– mice developed lung tumors (arrows), detected by microCT, 8 months after Dox administration. (D and E) Number (D) and volume (E) of lung tumors measured by microCT increased in KrasG12D;Nkx2-1+/– (n = 7) compared with KrasG12D;Nkx2-1+/+ (n = 9) and control (n = 11) mice 8 months after Dox administration. All results are mean ± SEM. *P < 0.05.
Figure 2
Figure 2. Haploinsufficiency of Nkx2-1 caused mucinous adenocarcinoma of the lung in KrasG12D mice.
(A) Lung sections were stained with H&E or with NKX2-1 and Alcian blue. Tumors in KrasG12D;Nkx2-1+/– mice (14 of 16) contained goblet cells with abundant intracytoplasmic mucin (Alcian blue positive, NKX2-1 negative). (B) CK7 and CK20, clinical biomarkers for human mucinous adenocarcinoma of the lung, were expressed in tumor cells of KrasG12D;Nkx2-1+/– mice. (C) Alcian blue–stained tumor cells in lungs of KrasG12D;Nkx2-1+/– mice 2 months after Dox administration (left; n = 6). Tumor cells in KrasG12D;Nkx2-1+/– mice regressed following withdrawal of Dox for 2 weeks after the 2-month administration (right; n = 8). Scale bars: 1 mm (A, top and middle); 100 μm (A, bottom, and B and C).
Figure 3
Figure 3. Tumor goblet cells are not dependent on the SPDEF/FOXA3 transcriptional program.
Sections from control, KrasG12D;Nkx2-1+/+, KrasG12D;Nkx2-1+/–, and HDM-challenged lungs and normal intestine were stained for mucins MUC2, MUC5AC, MUC5B, and AGR2 and the transcription factors NKX2-1, FOXA2, FOXA3, and SPDEF. FOXA3 and SPDEF, which were seen in goblet cells of HDM-challenged lungs, were not expressed in the tumor goblet cells of KrasG12D;Nkx2-1+/– mice. Scale bars: 100 μm.
Figure 4
Figure 4. NKX2-1 inhibits urethane- and KrasG12D-induced lung tumor formation in vivo.
1 week after Dox administration, urethane was injected intraperitoneally once weekly for 4 consecutive weeks. 16 weeks after the first urethane injection, lungs were harvested, and tumors were counted. (A) Lung sections of Scgb1a1-rtTA;[tetO]–Flag–Nkx2-1 mice were stained with anti-FLAG antibody. (B) The number of urethane-induced lung tumors on the lung surface was inhibited by NKX2-1. (C) Lung tumors (arrow) in Scgb1a1-rtTA;[tetO]-Kras4bG12D and Scgb1a1-rtTA;[tetO]-Kras4bG12D;[tetO]–Flag–Nkx2-1 mice, detected by H&E and microCT. (D) Number and volume of KrasG12D-induced lung tumors, measured by microCT, was reduced by NKX2-1. All results are mean ± SEM. *P < 0.05, **P < 0.01, Mann-Whitney test. Scale bars: 100 μm (A); 1 mm (C).
Figure 5
Figure 5. EGFRL858R mice did not develop mucinous adenocarcinoma of the lung regardless of Nkx2-1 expression.
(A) Number and volume of lung tumors, measured by microCT, were decreased in EGFRL858R;Nkx2-1+/– (n = 19) compared with EGFRL858R;Nkx2-1+/+ (n = 13) mice 4 months after Dox administration. n = 16 (control). (B) Lung sections were stained with Alcian blue, MUC5AC, and MUC5B. Mucinous tumors were not observed in EGFRL858R;Nkx2-1+/– or EGFRL858R;Nkx2-1+/+ mice. Tumor cells in lungs of EGFRL858R;Nkx2-1+/+ and EGFRL858R;Nkx2-1+/– mice did not stain with Alcian blue or MUC5AC, whereas tumor cells in lungs of EGFRL858R;Nkx2-1+/– mice stained with MUC5B. (C) MUC5AC mRNA was highly expressed in NKX2-1–negative KRAS mutant lung carcinoma cell lines (H2122 and A549), but not in EGFR mutant lung carcinoma cell lines. MUC5B mRNA was expressed in both KRAS mutant (H2122 and A549) and EGFR mutant (H3255) cell lines. Shown is fold induction compared with mRNA expression of H3255 cells. Results are mean ± SEM (A) and mean ± SD of triplicates for each group (C). N.D., not detectable. *P < 0.05. Scale bars: 100 μm.
Figure 6
Figure 6. A subset of genes induced in the mucinous tumor model and the asthma model was suppressed by NKX2-1.
Shown are results from 3 sets of mRNA microarray analysis (for simplicity, symbols for the human genes only are shown). For analysis of genes induced in mucinous tumors, mRNAs were isolated from lungs of control and KrasG12D;Nkx2-1+/– mice and subjected to mRNA microarray analysis (n = 3 per group). For analysis of genes regulated by NKX2-1 in A549 human lung carcinoma cells, mRNAs were isolated from lentiviral Nkx2-1–expressing and control A549 cells and subjected to mRNA microarray analysis (n = 3 per group). For analysis of genes induced in asthma, mRNAs were isolated from lungs of control and HDM-challenged mice and subjected to mRNA microarray analysis (n = 3 per group) (23). Several mRNAs associated with mucous genes — AGR2, MUC5AC, and MUC5B — were induced in both the mucinous tumor and the asthma mouse models and suppressed by NKX2-1 in A549 human lung carcinoma cells. HNF4A, a non–lung lineage transcription factor, was suppressed by NKX2-1, a lung lineage transcription factor. SOX2, expressed in conducting airways but not in alveolar regions, was also suppressed by NKX2-1, and expressed in both conducting airways and alveoli.
Figure 7
Figure 7. NKX2-1 inhibited mRNAs associated with tumorigenesis and induced those related to apoptosis.
(A) mRNAs were isolated from lentiviral Nkx2-1–expressing and control A549 cells and subjected to mRNA microarray analysis. A heat map of the mRNAs is shown. Green and red denote mRNAs decreased and increased, respectively, by NKX2-1. mRNAs associated with mucus (MUC5AC, MUC5B, MUC13, and AGR2) and oncogenes (FGFR1 and CDK6) were suppressed by NKX2-1. NKX2-1 induced surfactant protein genes (SFTPA1 and SFTPB) and apoptotic genes (FAS and TIMP3). (B) Lentiviral Nkx2-1–expressing and control A549 cells were treated with cisplatin (final concentration, 0–20 μM). 72 hours after treatment, MTS assay was used to measure cell viability (n = 3 per group). Cisplatin treatment reduced Nkx2-1–expressing A549 carcinoma cell viability significantly more than that of control A549 cells. (C) Phase-contrast images of control and Nkx2-1–expressing A549 cells 24 hours after 20 μM cisplatin (original magnification, ×20). (D) NKX2-1 sensitized A549 cells to cisplatin-induced apoptosis. 24 hours after cisplatin treatment (20 μM), proteins were extracted from control or Nkx2-1–expressing A549 cells. Induction of cleaved PARP, an apoptosis marker, was confirmed by IB. Results are mean ± SD. *P < 0.001 vs. control.
Figure 8
Figure 8. Identification of NKX2-1 binding sites by ChIP-seq.
(A) NKX2-1 associated with the promoter and first intron of SFTPA1 at sites that contain canonical NKX2-1 binding motifs (CAAG and CTTG). (B) NKX2-1 associated with the promoter of MUC5AC that contains an AP-1 binding motif (TGACTCA). (C) NKX2-1 associated with the first and second introns of FGFR1. (D) Distribution of peaks of NKX2-1 binding sites in genes up- or downregulated by NKX2-1. (E) Motifs present in NKX2-1 ChIP-seq peaks. (F) H441 lung adenocarcinoma cells were transfected as described in Methods. Results are presented as fold activation of light unit, normalized to β-galactosidase activity, relative to control constructs. NKX2-1 inhibited AP-1 activity in H441 cells (n = 3 per group). (G) Lentiviral Nkx2-1–expressing and control A549 cells were treated with PMA, an AP-1 inducer, at a final concentration of 10 ng/ml. FGFR1 mRNA was measured as described in Methods. PMA rescued the inhibitory effect of NKX2-1 on FGFR1 mRNA expression. (H) A549 cells stably expressing Nkx2-1, FOSL1, or both were developed using lentiviral vectors. Protein expression of NKX2-1 and FOSL1 was confirmed by IB. FOSL1-induced colony formation was inhibited by NKX2-1, as determined by soft agar assays (see Methods) performed using the stably infected cells and A549 control cells. n = 3 per group. Results are mean ± SD of biological triplicates for each group. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 9
Figure 9. Inhibitory role of NKX2-1 in mucinous adenocarcinoma of the lung.
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