KrasG12D and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung
Yutaka Maeda, 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
Yutaka Maeda, 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
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Research Article Oncology

KrasG12D and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung

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 KrasG12D, but not with oncogenic EGFRL858R, 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 KrasG12D-induced tumorigenesis in vivo. Haploinsufficiency of Nkx2-1 enhanced KrasG12D-mediated tumor progression, but reduced EGFRL858R-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 KrasG12D-driven mucinous pulmonary adenocarcinoma.

Authors

Yutaka Maeda, 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

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Figure 8

Identification of NKX2-1 binding sites by ChIP-seq.

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Identification of NKX2-1 binding sites by ChIP-seq. (A) NKX2-1 associate...
(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.