Nanog maintains stemness of Lkb1 ‐deficient lung adenocarcinoma and prevents gastric differentiation
2021; Springer Nature; Volume: 13; Issue: 3 Linguagem: Inglês
10.15252/emmm.202012627
ISSN1757-4684
AutoresXinyuan Tong, Yueqing Chen, Xinsheng Zhu, Yi Ye, Yun Xue, Rui Wang, Yijun Gao, Wenjing Zhang, Wei‐Qiang Gao, Lei Xiao, Haiquan Chen, Peng Zhang, Hongbin Ji,
Tópico(s)Pancreatic and Hepatic Oncology Research
ResumoArticle13 January 2021Open Access Source DataTransparent process Nanog maintains stemness of Lkb1-deficient lung adenocarcinoma and prevents gastric differentiation Xinyuan Tong Xinyuan Tong State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yueqing Chen Yueqing Chen State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xinsheng Zhu Xinsheng Zhu Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yi Ye Yi Ye School of Life Science and Technology, Shanghai Tech University, Shanghai, China Search for more papers by this author Yun Xue Yun Xue State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Rui Wang Rui Wang Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Yijun Gao Yijun Gao State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Wenjing Zhang Wenjing Zhang State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Weiqiang Gao Weiqiang Gao State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China Search for more papers by this author Lei Xiao Lei Xiao College of Animal Science and Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Haiquan Chen Haiquan Chen Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Peng Zhang Corresponding Author Peng Zhang [email protected] orcid.org/0000-0003-1771-7545 Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Hongbin Ji Hongbin Ji orcid.org/0000-0003-0891-6390 State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China School of Life Science and Technology, Shanghai Tech University, Shanghai, China Search for more papers by this author Xinyuan Tong Xinyuan Tong State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yueqing Chen Yueqing Chen State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xinsheng Zhu Xinsheng Zhu Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yi Ye Yi Ye School of Life Science and Technology, Shanghai Tech University, Shanghai, China Search for more papers by this author Yun Xue Yun Xue State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Rui Wang Rui Wang Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Yijun Gao Yijun Gao State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Wenjing Zhang Wenjing Zhang State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Weiqiang Gao Weiqiang Gao State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China Search for more papers by this author Lei Xiao Lei Xiao College of Animal Science and Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Haiquan Chen Haiquan Chen Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Peng Zhang Corresponding Author Peng Zhang [email protected] orcid.org/0000-0003-1771-7545 Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Hongbin Ji Hongbin Ji orcid.org/0000-0003-0891-6390 State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China School of Life Science and Technology, Shanghai Tech University, Shanghai, China Search for more papers by this author Author Information Xinyuan Tong1, Yueqing Chen1,2, Xinsheng Zhu3, Yi Ye4, Yun Xue1,2, Rui Wang5,6, Yijun Gao1, Wenjing Zhang1, Weiqiang Gao7,8, Lei Xiao9, Haiquan Chen5,6, Peng Zhang *,3 and Hongbin Ji1,2,3,4 1State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China 2University of Chinese Academy of Sciences, Beijing, China 3Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China 4School of Life Science and Technology, Shanghai Tech University, Shanghai, China 5Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China 6Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China 7State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China 8Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China 9College of Animal Science and Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China *Corresponding author. Tel: +86 21 65115006; E-mail: [email protected] *Corresponding author. Tel: +86 21 54921108; E-mail: [email protected] EMBO Mol Med (2021)13:e12627https://doi.org/10.15252/emmm.202012627 Correction(s) for this article Nanog maintains stemness of Lkb1-deficient lung adenocarcinoma and prevents gastric differentiation07 September 2021 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Growing evidence supports that LKB1-deficient KRAS-driven lung tumors represent a unique therapeutic challenge, displaying strong cancer plasticity that promotes lineage conversion and drug resistance. Here we find that murine lung tumors from the KrasLSL-G12D/+; Lkb1flox/flox (KL) model show strong plasticity, which associates with up-regulation of stem cell pluripotency genes such as Nanog. Deletion of Nanog in KL model initiates a gastric differentiation program and promotes mucinous lung tumor growth. We find that NANOG is not expressed at a meaningful level in human lung adenocarcinoma (ADC), as well as in human lung invasive mucinous adenocarcinoma (IMA). Gastric differentiation involves activation of Notch signaling, and perturbation of Notch pathway by the γ-secretase inhibitor LY-411575 remarkably impairs mucinous tumor formation. In contrast to non-mucinous tumors, mucinous tumors are resistant to phenformin treatment. Such therapeutic resistance could be overcome through combined treatments with LY-411575 and phenformin. Overall, we uncover a previously unappreciated plasticity of LKB1-deficient tumors and identify the Nanog-Notch axis in regulating gastric differentiation, which holds important therapeutic implication for the treatment of mucinous lung cancer. Synopsis This study reveals the plasticity of LKB1-deficient tumors, and identifies the Nanog-Notch axis in regulating gastric differentiation. Combinational treatment of γ-secretase inhibitor LY-411575 and phenformin effectively blocked invasive mucinous adenocarcinoma IMA formation. Invasive mucinous adenocarcinoma (IMA) was promoted by Nanog deficiency in the KrasLSL-G12D/+; Lkb1flox/flox KL mouse model. Concurrent loss of NANOG and LKB1 was frequent in human lung IMA. Mucinous differentiation was inhibited by perturbation of the Notch pathway. IMA was insensitive to phenformin treatment. IMA formation was blocked by a combinational treatment of LY-411575 and phenformin. The paper explained Problem Lineage plasticity has emerged as a mechanism allowing evasion in response to targeted therapies in several cancer types including non-small cell lung cancer (NSCLC). Recent studies have revealed an unexpected gastric differentiation program of lung adenocarcinoma, mimicking human invasive mucinous adenocarcinoma (IMA) with expression of gastric markers. However, the pathogenesis of IMA remains largely unknown. Moreover, the effect of this lineage switching on the therapeutic response is unclear. Results Our study reveals that Lkb1-deficient Kras tumors show significant up-regulation of Nanog expression, and knockout of Nanog in KrasLSL-G12D/+; Lkb1flox/flox (KL) tumors promotes gastric differentiation and occurrence of IMA. Importantly, we find that low or negative expression of NANOG or LKB1 is concurrent in about 89% of human IMA. We demonstrate that Notch signaling inactivation blocks mucin production in mice. Notably, this lineage switching desensitizes Lkb1-deficient lung tumors to phenformin treatment, which selectively targets KL tumors. Finally, we find that combinational treatment of γ-secretase inhibitor LY411575 and phenformin is sufficient to prevent mucinous differentiation as well as malignant progression in mice. Impact These results shed light on the gastric differentiation program involving the Nanog-Notch axis in an Lkb1-deficient context. They further provide novel therapeutic insights into human IMA treatment. Introduction Tissue-specific lineage trans-differentiation events such as gastric differentiation have been recently observed in human non-small cell lung cancer (NSCLC). The transcription factor NKX2-1 is generally expressed in human lung adenocarcinoma (ADC), but its expression decreases with poor differentiation and malignant progression (Winslow et al, 2011; Li et al, 2014; Cha & Shim, 2017; Boland et al, 2018). Previous studies have shown that loss of Nkx2-1 in Kras-driven lung cancer genetically engineered mouse model (GEMM) triggered the loss of pulmonary identity and promotes an unexpected gastric differentiation (Maeda et al, 2012; Snyder et al, 2013). Interestingly, these tumors mimic human lung invasive mucinous adenocarcinoma (IMA) expressing gastric markers such as Hnf4α, a master regulator of gastrointestinal differentiation (Snyder et al, 2013; Camolotto et al, 2018). Chromatin immunoprecipitation sequencing (ChIP-seq) analysis demonstrates that NKX2-1 transcriptionally regulates MUC5AC (mucin 5AC, oligomeric mucus/gel-forming) expression by binding to its promoter (Maeda et al, 2012). Meanwhile, HNF4α is suppressed by NKX2-1-mediated tissue-specific FOXA1/FOXA2 engagement (Minoo et al, 2007; Gao et al, 2008; Wederell et al, 2008; Snyder et al, 2013; Camolotto et al, 2018). In the context of NKX2-1 deficiency along with KRASG12D mutation, SPDEF and FOXA3 promote mucin genes expression and recapitulate human IMA histopathology (Park et al, 2007; Guo et al, 2017). IMA is genetically and therapeutically distinct from other lung ADC subtypes (Finberg et al, 2007; Li et al, 2014; Nakaoku et al, 2014; Shim et al, 2015; Luo et al, 2016; Cha & Shim, 2017; Boland et al, 2018). IMA frequently harbor "undruggable" KRAS mutations but rarely EGFR mutations and are unlikely responsive to tyrosine kinase inhibitors (TKIs) (Finberg et al, 2007; Boland et al, 2018). Although recent efforts have established several mouse models for mucinous tumors (Fisher et al, 2001; Maeda et al, 2012; Lee et al, 2013; Schuster et al, 2014; Skoulidis et al, 2015; Serresi et al, 2016; Guo et al, 2017), the pathogenesis of IMA and potential therapeutics still await further investigation. LKB1/STK11, encoding a serine/threonine kinase implicated in energy homeostasis, is one of the leading mutated genes in NSCLC (Sanchez-Cespedes et al, 2002; Shaw et al, 2004; Mahoney et al, 2009; Gao et al, 2010; Fang et al, 2014; Skoulidis et al, 2015). Accumulating evidences support that concurrent genetic alteration of KRAS and LKB1 defines a unique molecular subtype with potent cancer plasticity, exhibiting high metastatic competence, frequent therapeutic resistance and poor clinical prognosis, and thus represents a major challenge for lung cancer therapeutics (Mahoney et al, 2009; Skoulidis et al, 2015). This oncological genotype imposes a metabolic vulnerability related to the dependence on pyrimidine metabolism (Kim et al, 2017). GEMM studies highlight the strong plasticity of KrasLSL-G12D/+; Lkb1flox/flox (KL) lung tumors through lineage transition from adenocarcinomas (ADC) to squamous cell carcinomas (SCC), and its link with therapeutic resistance (Ji et al, 2007; Li et al, 2015). Although the mechanism remains unclear, we reason that LKB1 deficiency may confer strong stemness to the lung cancer cells, allowing trans-differentiation from ADC to SCC. We have previously found that excessive accumulation of oxidative stress induced by Lkb1 deletion and metabolic reprogramming during tumor progression plays important roles in this phenotypic transition. Moreover, the Hippo pathway and epigenetic factors including EZH2 also contribute to the squamous trans-differentiation (Gao et al, 2014; Li et al, 2015; Huang et al, 2017; Zhang et al, 2017). Nonetheless, the link between stemness of KL tumors and strong plasticity remains largely elusive. The roles of LKB1 in regulating self-renewal of skeletal muscle progenitor cells (Shan et al, 2014; Shan et al, 2017) and hematopoietic stem cell survival have been reported (Gan et al, 2010; Gurumurthy et al, 2010; Nakada et al, 2010). Investigating the molecular determinants governing the LKB1-deficient cancer stemness is of particular importance for better understanding of KL tumor plasticity as well as providing novel therapeutic strategy. NANOG is an important transcription factor maintaining the regulatory network responsible for embryonic stem cell self-renewal and pluripotency (Cavaleri & Scholer, 2003; Mitsui et al, 2003; Hyslop et al, 2005; Silva et al, 2009). Also, NANOG acts as a cancer stemness marker and promotes cancer tumorigenesis and stemness (Noh et al, 2012a; Noh et al, 2012b; Chen et al, 2016; Lu et al, 2018; Zhang et al, 2018). Aberrant NANOG expression is commonly found in multiple cancer types including lung ADC (Watanabe, 2009; Du et al, 2013; Vaira et al, 2013; Li et al, 2013a; Li et al, 2013b; Liu et al, 2014; Park et al, 2016; Zhao et al, 2018). Moreover, NANOG is known for critically contributing to tumor initiation and epithelial–mesenchymal transition (Chiou et al, 2010; Yin et al, 2015). High NANOG expression is associated with poor tumor differentiation and advanced tumor stage (Du et al, 2013; Li et al, 2013a; Li et al, 2013b; Liu et al, 2014; Park et al, 2016). A recent study also demonstrates that NANOG elicits a lineage-restricted mitogenic function in squamous cell carcinomas from stratified epithelia (Piazzolla et al, 2014). Understanding the roles of NANOG in cancer stemness and lineage switch may benefit therapeutic development for a wide range of human diseases. We find here that Lkb1-deficient lung tumors from the KL model harbor stronger stemness and display significant up-regulation of Nanog expression, in contrast to KrasG12D/+ or KrasG12D/+; P53-/- tumors. Genetic ablation of Nanog in the KL model triggers gastric differentiation and produces mucinous lung tumors through Notch signaling activation. Such lineage transition renders lung tumors resistant to phenformin treatment, which could be overcome via combinational treatments with γ-secretase inhibitor LY-411575. Results Nanog as an important factor in maintaining KL tumor stemness To investigate the stemness property in relation to LKB1 deficiency, we comparatively analyzed the RNA-Seq data from lung ADC from KrasLSL-G12D/+ (K), KrasLSL-G12D/+; Lkb1flox/flox (KL), and KrasLSL-G12D/+; P53flox/flox (KP) mouse models, in which the oncogenic KrasG12D allele is conditionally activated following Ad-Cre infection by nasal inhalation as described previously (Li et al, 2015). We found that stem cell pluripotency expression pattern was significantly enriched in KL tumors in contrast to K or KP tumors (Fig 1A and B). Notably, Nanog, the well-established factor maintaining stem cell property (Mitsui et al, 2003), was remarkably increased in KL tumors (Fig 1C). We further confirmed this observation through IHC analysis (Fig 1D). Figure 1. Identification of Nanog as the stemness factor in KL lung tumors A, B. Gene Set Enrichment Analysis (GSEA) identifies stem cell-like expression signature in KL tumors compared with KrasG12D/+ (K) lung tumor (A) or KrasG12D/+; P53-/- (KP) lung tumors (B). Significance was calculated by permutation test. C. Heat map demonstration of genes that were mostly enriched in the pathway of pluripotency of stem cells. The color bar showed the relative RNA-Seq signal (Z-score of normalized FPKM). D. The protein abundance of NANOG was up-regulated in KL tumors compared with K and KP mice. Scale bar, 50 μm. E. Scheme illustrator of mouse model. F–H. Histological examinations of tumor types in (F) K versus KrasG12D; NanogL/L (KN), (G) KP versus KrasG12D; P53L/L; NanogL/L (KPN), and (H) KL versus KrasG12D; Lkb1L/L; NanogL/L (KLN). Tumor tissues collected form indicated mice analyzed using hematoxylin and eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom). Download figure Download PowerPoint We hypothesized that Nanog might be a crucial factor for maintaining KL tumor stemness. We thus generated Nanog conditional knockout mice (Fig EV1) and crossed them with various Kras models to obtain KrasLSL-G12D/+; Nanogflox/flox (KN), KrasLSL-G12D/+; P53flox/flox; Nanogflox/flox (KPN), and KrasLSL-G12D/+; Lkb1flox/flox; Nanogflox/flox (KLN) cohorts (Fig 1E). We treated the mice with Ad-Cre and analyzed lung tumor incidence. There were no significant alterations on either the tumor burden or tumor number following Nanog deletion in K, KL, or KP mice compared with control mice (Fig EV2A–C). Detailed pathological analyses revealed no impact of Nanog deletion in K or KP lung tumors, both of which displayed classical ADC pathology (Fig 1F and G). Interestingly, the deletion of Nanog in KL model promoted the formation of mucinous lung tumors (Fig 1H). In KLN mice, we found more small adenomas but there was no significant change in ADC tumorigenesis compared with KL mice (Fig EV2D and E). Meanwhile, there was no impact on squamous differentiation (Ji et al, 2007; Li et al, 2015) (Fig EV2F). Considering the specific up-regulation of Nanog in KL tumors, these observations suggest that Nanog might serve as an important factor for maintaining cancer stemness triggered by LKB1 deficiency. Click here to expand this figure. Figure EV1. Schematic illustration of Nanogflox/flox mouse model To insert LOXP sites in Nanog, a homologous recombinant vector was constructed. Targeting vector contained two arms homologous to the genome sequence of the Nanog gene, exon2 with LOXP sites on both sides and a drug-screening marker PGK NEO. Mutant allele showing the insertion of LOXP sites on both sides of exon2. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Nanog deletion has little effects upon lung tumorigenesis in KrasG12D-driven models A–C. Quantification of average tumor number (no.) and tumor burden from indicated mice. Mice were sacrificed after 16 weeks (A, n = 6 per group), 12 weeks (B, n = 6 per group), or 8 weeks (C, n = 6 per group) for pathological analysis. D. Statistical analysis of numbers of tumors with different tumor grade in KL (n = 6) and KLN (n = 6) mice. E. Statistical analysis of ratio of small tumor ( 0.1mm2) in KL (n = 6) and KLN (n = 6) mice. F. Statistical analysis of numbers of ADC and SCC in KL (n = 6) and KLN (n = 6) mice. Download figure Download PowerPoint Nanog deletion in KL tumors triggers gastric differentiation IHC staining and qPCR analysis verified the efficient knockout of Nanog in KL mice (Fig EV3A and B). We then pathologically and molecularly characterized the mucinous lung tumors from KLN mice. Human IMA tumor cells contained abundant intracytoplasmic mucin admixed with invasive adenocarcinoma patterns (Fig 2A). Similarly, KLN mucinous tumors were composed of columnar or goblet cell morphology with mucus in apical cytoplasm and showed small basal oriented nuclei (Fig 2B). We observed that these tumors showed the same heterogeneous mixture of acinar or papillary growth patterns as in non-mucinous tumors. Human and murine IMA were both invasive and lacked a circumscribed border with miliary spread into adjacent lung parenchyma. Consistent with the histological features, the reactivity of tumor tissues with Alcian blue (AB) and Periodic acid–Schiff (PAS) dyes confirmed the presence of acidic polysaccharides, mucopolysaccharides, and neutral mucins, typical of human mucinous carcinomas (Hollingsworth & Swanson, 2004). Nanog deletion in KL mice significantly promoted the formation of IMA (Fig 2C). The mucinous differentiation was not detectable at 4 weeks of Ad-Cre treatment but was clearly present in around 15% tumors at 8 weeks (Fig 2D and E). The precursor lesions such as atypical alveolar hyperplasia (AAH) and adenoma (AD) were occasionally present with mucin, whereas most ADC were evidently mucinous (Fig 2F). Non-mucinous ADC in KLN mice expressed type II pneumocyte marker pro-surfactant protein C (proSPC) or Club cell 10-kDa protein (CC10), whereas mucinous tumors only expressed CC10 (Fig EV3C). TP63, the basal cell marker for SCC, was also absent in mucinous ADC (Fig EV3C). These data indicate that Club cells might serve as the cellular origin for mucinous ADC. Click here to expand this figure. Figure EV3. Simultaneous deletion of Nanog and Lkb1 is indispensable for mucinous tumor emergence in KrasG12D mice A, B. Verification of Nanog knockout in mice using IHC staining (A) and qRT–PCR (B) in KL, KLN mice. C. HE and IHC staining of proSPC, p63, and CC10 in mucinous tumors and non-mucinous tumors of KLN mice. Representative images were shown. Scale bar, 50 μm. Download figure Download PowerPoint Figure 2. Nanog deletion triggers gastric differentiation in KL model§§ Correction added on 7 September 2021, after first online publication: Panel B of this figure has been corrected; see the associated Corrigendum at https://doi.org/10.15252/emmm.202114795. A, B. Human (A) and KLN murine (B) lung sections were stained with H&E or with Periodic acid–Schiff (PAS) and Alcian blue (AB). Scale bar, 50 μm. C. Statistical analysis of mucinous tumor numbers (no.) in KL (n = 7) or KLN (n = 7) mice. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. Results were shown as mean. **P = 0.0066. D. Representative pictures of lung tumors at 4 weeks and 8 weeks post-Ad-Cre infection in KLN mice. Scale bar, 50 μm. E. Statistical analysis of IMA ratio at 4 and 8 weeks post-Ad-Cre treatment (n = 5 per group). Results were shown as mean ± SEM. F. Statistical analysis of ratio of mucinous phenotype in AAH, AD, and ADC (n = 5 per group). Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. *P = 0.0215. G. qPCR analysis of stomach-restricted gene set between KL ADC and KLN mucinous tumor sections (n = 3 or 4 per group). Results were shown in a box and whisker plot with the minimum value, first quartile, median, third quartile, and maximum value. The box represented the 50% of the central data, with a line inside represented the median. The significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *P = 0.0260 (Hnf4α), *P = 0.0275 (Vsig1), *P = 0.0122 (Gkn3), *P = 0.0324 (Ctse), **P = 0.0084 (Onecut2). H. Representative histological and IHC staining for HNF4α, GKN1, NKX2-1, MUC5AC, CK20 in lung tumors derived from ADC and IMA. Scale bar, 50 μm. Download figure Download PowerPoint We next determined whether KLN tumors adopted the gastric cell fate conversion pattern as previously described (Maeda et al, 2012; Snyder et al, 2013; Camolotto et al, 2018). We evaluated a gene set correlated with gastric differentiation, including Hnf4α, Pdx1, Lgals4, Anxa8, Vsig1, Gkn1, Gkn3, Ctse, and Onecut2 (Snyder et al, 2013). We found that Hnf4α, Vsig1, Gkn3, Ctse, and Onecut2 were significantly up-regulated in mucinous KLN tumors (Fig 2G). We used IHC to evaluate the expression pattern of gastric and pulmonary proteins in mucinous and non-mucinous tumors (Fig 2H). Gastric proteins HNF4α and GKN1 were increased in mucinous tumors as previously reported (Snyder et al, 2013). On the contrary, NKX2-1 was decreased in mucinous tumors which was consistent with its inhibitory role in gastric differentiation (Maeda et al, 2012) (Fig 2H). These lines of evidence imply that Nanog deletion in KL tumors induces the expression of stomach-restricted genes and exhibits gastric differentiation. We also examined established biomarkers for mucinous lung tumors, such as CK20 (cytokeratin 20) and MUC5AC, which were specifically expressed with strong intensity in mucinous lung tumors in contrast to no expression in non-mucinous ADC from KL mice (Fig 2H). Frequent concurrent loss of NANOG and LKB1 expression in human mucinous lung tumors To determine the expression of NANOG in human lung tissues, we collected 175 human lung ADC and 76 IMA samples to perform IHC staining. In contrast to the nuclear location detected in the mouse model (Fig 1D), NANOG was mainly expressed in the cytoplasm of human lung cancer cells (Fig 3A), indicative of different expression patterns in different species. Although the majority of NANOG IHC staining were not strong, we classified NANOG expression into three groups with relative low, medium, high level as previously described (Gao et al, 2014) (Fig 3A). We next investigated the difference of NANOG expression between human lung ADC and IMA (Fig 3B and C). Statistical analysis showed that 21.7% (38/175) of ADC displayed relative high or medium expression of NANOG, whereas only 1.3% (1/76) of IMA showed high NANOG expression. While 2.6% (2/76) showed medium NANOG level, about 96% (73/76) of the IMA showed low NANOG level (Fig 3C). We observed that NANOG could be detected using Western blot in most of the human lung ADCs with relative medium or high level of IHC staining (Fig 3D). When we overexpressed full-length human NANOG cDNA in human lung cancer cell lines, we found that the exogenous NANOG expression produced 3 different bands, two around 40 kDa and another one around 25 kDa (Fig 3E). Western blot of one cell line H358 exhibited the 40 kDa lower band, which was also observed in human NANOG overexpression groups (Fig 3E). However, the endogenous NANOG band (about 40kDa) detected in human em
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