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. 2014:5:3261.
doi: 10.1038/ncomms4261.

Transdifferentiation of lung adenocarcinoma in mice with Lkb1 deficiency to squamous cell carcinoma

Xiangkun Han  1 , Fuming Li  1 , Zhaoyuan Fang  2 , Yijun Gao  2 , Fei Li  2 , Rong Fang  2 , Shun Yao  2 , Yihua Sun  3 , Li Li  2 , Wenjing Zhang  2 , Huimin Ma  2 , Qian Xiao  2 , Gaoxiang Ge  2 , Jing Fang  4 , Hongda Wang  5 , Lei Zhang  2 , Kwok-kin Wong  6 , Haiquan Chen  3 , Yingyong Hou  7 , Hongbin Ji  2
Affiliations
Free PMC article

Transdifferentiation of lung adenocarcinoma in mice with Lkb1 deficiency to squamous cell carcinoma

Xiangkun Han et al. Nat Commun. 2014.
Free PMC article

Erratum in

Abstract

Lineage transition in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of non-small cell lung cancer, as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma, remains a fundamental yet unsolved question. Here we provide in vivo evidence showing the transdifferentiation of lung cancer from ADC to SCC in mice: Lkb1-deficient lung ADC progressively transdifferentiates into SCC, via a pathologically mixed mAd-SCC intermediate. We find that reduction of lysyl oxidase (Lox) in Lkb1-deficient lung ADC decreases collagen disposition and triggers extracellular matrix remodelling and upregulates p63 expression, a SCC lineage survival oncogene. Pharmacological Lox inhibition promotes the transdifferentiation, whereas ectopic Lox expression significantly inhibits this process. Notably, ADC and SCC show differential responses to Lox inhibition. Collectively, our findings demonstrate the de novo transdifferentiation of lung ADC to SCC in mice and provide mechanistic insight that may have important implications for lung cancer treatment.

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Figures

Figure 1
Figure 1. Lkb1-deficient Lung ADC progressively transdifferentiates into SCC.
(a) Representative histology for AAH, adenoma, ADC and SCC lesions in Kras/Lkb1 mice at a serial of time points post Ad-Cre treatment. Scale bar, 150 μm. (b,c) Quantification of average tumour number (b) and tumour size (c) per mouse for ADC and SCC in Kras/Lkb1 mice at 6 weeks (n=6), 8 weeks (n=12) and 12 weeks (n=8) post Ad-Cre treatment. Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01, ***P<0.001. (d) Quantification of individual tumour size for ADC and SCC in Kras/Lkb1 mice at 10–12 weeks post Ad-Cre treatment. n=12, Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01, ***P<0.001. (e) Representative mAd-SCC shown with haematoxylin and eosin staining, p63 and SP-C immunohistochemical staining on serial sections. Scale bar, 150 μm. (f) Quantification of tumour percentage for mAd-SCC and SCC in Kras/Lkb1 mice at indicated time points post Ad-Cre treatment. n=8 for each time point. Scale bar, 150 μm. Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2. Type II pneumocyte lineage-derived lung ADC with Lkb1 deficiency transdifferentiates into SCC.
(a) Representative photos for SP-C and X-gal double staining in lung sections of SP-C-CreERT2/Rosa26R-LacZ mice at 2 weeks post tamoxifen administration. Scale bar, 10μm. (b,c) Quantification of average tumour number (b) and tumour size (c) per mouse for ADC and SCC in SP-C-CreERT2/Kras/Lkb1 (SKL) mice at 4–8 weeks (n=8), 9–12 weeks (n=8), 13–16 weeks (n=10) post tamoxifen administration. Data are shown as mean±s.e.m. t-test, **P<0.01, ***P<0.001. (d) Quantification of individual tumour size for ADC and SCC in SKL mice at 12 weeks post tamoxifen administration (n=6). Data are shown as mean±s.e.m. t-test, ***P<0.001. (e) Representative SCC in SKL mice with haematoxylin and eosin staining and immunohistochemical staining for p63, Krt5, Sox2 and SP-C. Scale bar, 10 μm.
Figure 3
Figure 3. Deregulation of Lox and ECM in lung SCC transdifferentiated from ADC with LKB1 deficiency.
(a,b) Heat maps showing significantly upregulated (red) and downregulated (green) pathways (a) and gene signatures (b) between ADC and SCC from Kras/Lkb1 mice. (c) Representative photos showing Massons Trichrome staining of collagen as well as immunohistochemical staining for Lox, p63 and SP-C in lung ADC and SCC from Kras/Lkb1 mice. Scale bar, 50 μm. (d) Real-time PCR detection of mRNA levels for p63, SP-C and Lox in lung ADC (n=8) and SCC (n=8) from Kras/Lkb1 mice. Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01. (e) Western blot detection of protein levels for p63, SP-C and Lox in lung ADC (n=6, A1–6) and SCC (n=7, S1–7) from Kras/Lkb1 mice. Actin serves as control, see also Supplementary Fig. 9a.
Figure 4
Figure 4. Involvement of hypoxia and collagen deposition in ADC to SCC transdifferentiation.
(a) Comparison of hypoxia levels in lung ADC and SCC from Kras/Lkb1 mouse model. Scale bar, 150 μm. (b) Representative photos for haematoxylin and eosin and p63 immunohistochemical staining in Kras/Lkb1 and Kras/Lkb1/Hif2a mice at 10 weeks post Ad-Cre treatment, Scale bar, 150 μm. (c) Quantification of average tumour number per mouse for ADC and SCC from Kras/Lkb1 mice (n=8) and Kras/Lkb1/Hif2a mice (n=7). Data are shown as mean±s.e.m. t-test, **P<0.01. (d) Massons Trichrome staining of lung ADC from Kras/Lkb1 mice virally infected by either Lenti-Cre or Lenti-Cre-MC. Scale bar, 150 μm. (e,f) Quantification of average tumour number per mouse for ADC and SCC (e) and mAd-SCC (f) from Kras/Lkb1 mice virally infected with Lenti-Cre (n=8) or Lenti-Cre-MC (n=7). Data are shown as mean±s.e.m. t-test, *P<0.05, ***P<0.001. (g) Quantification of average tumour size per mouse for ADC and SCC from Kras/Lkb1 mice virally infected with Lenti-Cre (n=8) or Lenti-Cre-MC (n=7). Data are shown as mean±s.e.m.
Figure 5
Figure 5. Ectopic expression of lox inhibits ADC to SCC transdifferentiation.
(a) Scheme of viral infection strategy for ectopic Lox expression in Kras/Lkb1 mouse model. (b) Lox immunohistochemical staining and Massons Trichrome staining in lung ADC in Kras/Lkb1 mice at 13 weeks post viral infection with Lenti-Cre or Lenti-Cre-Lox. Scale bar, 150 μm. (c) Representative photos for lung SCC formation and p63 immunohistochemical staining. Scale bar, 150 μm. (d,e) Quantification of average tumour number (d) and tumour size (e) per mouse for ADC and SCC from Kras/Lkb1 mice virally infected with Lenti-Cre(n=11) or Lenti-Cre-Lox (n=11). Data are shown as mean±s.e.m. t-test, *P<0.05.
Figure 6
Figure 6. Pharmacological inhibition of Lox enzymatic activity promotes ADC to SCC transdifferentiation.
(a) A scheme for BAPN and DPA treatment in Kras/Lkb1 mice. (b) Representative photos for haematoxylin and eosin and p63 immunohistochemical staining in Kras/Lkb1 mice treated with BAPN or saline. Scale bar, 150 μm. (c,d) Quantification of average tumour number per mouse for ADC and SCC (c) and mAd-SCC (d) from Kras/Lkb1 mice treated with BAPN (n=14) or saline (n=18). Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01, ***P<0.001. (e,f) Quantification of average tumour size per mouse for ADC and SCC (e) and individual tumour size for SCC (f) from Kras/Lkb1 mice post BAPN (n=14) or saline (n=18) treatment. Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01, ***P<0.001.
Figure 7
Figure 7. Lox inhibition activates p63 to promote ADC to SCC transdifferentiation.
(a) Representative photos for haematoxylin and eosin staining and p63 immunohistochemical staining of lung tumours from Kras/Lkb1 mice treated with BAPN or saline for 2 weeks. Scale bar, 150 μm. (b) Representative photos for haematoxylin and eosin staining and p63 immunohistochemical staining on tumours derived from A549 xenografts in nude mice with or without BAPN treatment. Scale bar, 150 μm. (c) Real-time PCR analysis of relative expression of SCC signature genes in lung ADC from Kras/Lkb1 mice treated with BAPN (n=5) or saline (n=7) for 2 weeks. Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01, ***P<0.001. (d) Real-time PCR analysis shows that the SCC signature genes can transcriptionally upregulate p63 in human NSCLC cell lines A549 and CRL5800 (three replicates for each sample). Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01, ***P<0.001. (e) Real-time PCR analysis of relative expression of a serial of SCC signature genes in Kras/p53 ADC cells with or without ectopic p63 expression (three replicates for each sample). Data are shown as mean±s.e.m. t-test, *P<0.05, **P<0.01, ***P<0.001. (f) Representative histology, immunohistochemical staining for p63, Krt5 and Ttf1 in lung tumours derived from Kras/p53 ADC cells with or without ectopic DNp63α expression in nude mice lung seeding assay. Scale bar, 150 μm.
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