PTEN decreases NR2F1 expression to inhibit ciliogenesis during EGFRL858R-induced lung cancer progression

doi:10.21203/rs.3.rs-3244458/v1

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PTEN decreases NR2F1 expression to inhibit ciliogenesis during EGFRL858R-induced lung cancer progression

https://doi.org/10.21203/rs.3.rs-3244458/v1

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Lung cancer is the major cause of death worldwide. Activation of oncogenes or inhibition of tumour suppressors causes cancer formation. Previous studies have indicated that Pten, as a tumour suppressor, inhibits cancer formation. In this study, we studied the role of Pten in EGFR^L858R-induced lung cancer in vivo. Interestingly, loss of Pten increased bronchial cell hyperplasia but decreased alveolar cell hyperplasia in EGFR^L858R*Pten^−/−-induced lung cancer. Syatematic analysis of gene expression by RNA-seq showed that a number of genes related to ciliogenesis were upregulated in EGFR^L858R*Pten^−/−-induced lung cancer and subsequently showed that bronchial ciliated cells were hyperplastic. Several critical ciliogenesis-related genes, such as mucin5a, DNAI2 and DNAI3, were found to be regulated by NR2F1. Next, NR2F1 was found to be inhibited by overexpression of Pten, indicating that Pten negatively regulates NR2F1, thereby inhibiting the expression of ciliogenesis-related genes and leading to the inhibition of bronchial cell hyperplasia during EGFR^L858R-induced lung cancer progression. In addition, we also found that Pten decreased Akt phosphorylation in A549, Kras mutant, and H1299 cells but increased Akt phosphorylation in PC9, EGFR^L858R, and H1299^L858R cells, suggesting that Pten may function as a tumour suppressor and an oncogene in lung cancers with Kras mutation and EGFR mutation, respectively. PTEN acts as a double-edged sword that differentially regulates EGFR^L858R-induced lung cancer progression in different genomic backgrounds. Understanding the Pten in lung cancer with different genetic backgrounds will be beneficial for therapy in the future.

Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer

Biological sciences/Cell biology/Cell signalling/Nuclear receptors

Lung cancer, which includes non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), is the leading cause of cancer-related death worldwide. Lung cancer is classified into two main, non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC), based on their histological appearance and cellular origin. Epidermal Growth Factor Receptor (EGFR) and Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) mutations were associated with the development of NSCLC.

Pten, a well-known tumour suppressor gene, has been reported to be involved in inactivation of the PI3K/AKT signalling pathway, leading to inhibition of cell adhesion, migration, and invasion. It was observed that the absence of Pten protein expression resulted in increased phosphorylation of Akt and increased PI3K signalling in the breast cancer cell lines MDA-MB-468 and BT-549 (1). In lung cancer cells, overexpression of Pten suppressed cell proliferation and induced cell cycle arrest by reducing the Skp2 protein level (2). Recent studies have indicated that Pten may be a biomarker for the response to immunotherapy, suggesting that Pten regulates not only cancer cells but also other cell types in the tumour microenvironment (3). Understanding the role and mechanisms of Pten in cancer progression will be beneficial for the success of cancer therapies using precision medicine.

Ciliated airway epithelial cells have an elongated columnar structure and isolated contact with the basement membrane. Generally, cilia consist of a microtubule, a ciliary membrane, a basal body, and an axoneme (4, 5). In lung organs, ciliated cells are located in bronchioles with club-cells, goblet cells and basal cells. Previous studies have shown that PTEN interacts with Dishevelled (DVL) to inhibit WNT pathway-mediated ciliogenesis (6). Recently, studies have indicated that ciliogenesis in the mouse trachea and ependyma was requires Pten (7, 8). In Xenopus embryos with conditional knockout of Pten driven by the Foxj1 promoter, ciliogenesis was significantly decreased compared with that in control embryos. However, the role of Pten in ciliogenesis during cancer progression needs to be clarified. In addition, NR2F1 has been demonstrated to promote cancer cell dormancy in several malignant tumours, thereby leading to recurrence and metastasis during cancer progression (9). Numerous studies have investigated the role of NR2F1-AS1 as a sponge to inhibit the expression of many miRNAs in turn promote the formation of several cancer (10–12). In this study, we found that Pten can negatively regulate a transcription factor, NR2F1, to decrease the expression of genes related to ciliogenesis, thereby causing bronchial epithelial cell hyperplasia.

Knockout of Pten enhances bronchial cell growth but inhibits alveolar cell growth in EGFR ^L858R -induced lung cancer

Pten function as a tumour suppressor by inhibiting the PI3K pathway (13). What is the role of Pten in bronchial and alveolar cells in EGFR^L858R-induced lung cancer? In this study, we established an animal model of induced lung cancer, the EGFR^L858R*Pten^-/- model, in which doxycycline treatment is used to induce the formation of a complex between TetO and scgb1a1-driven rtTA to express EGFR^L858R and tamoxifen treatment is used to activate scgb1a1-driven Cre to cut the Lox P sites in the 5th exon of the Pten gene, namely, its DNA binding motif (Fig. 1A). Genotyping was used to confirm the presence of EGFR^L858R/rtTA, Cre and Lox P (Fig. 1B). After 2 months of induction by doxycycline treatment, the levels of Pten in the lungs of EGFR^L858R*Pten^-/- mice (n = 8) were decreased compared to those in the lungs of EGFR^L858R mice (n = 4) (Fig. 1C and 1D). The lungs of EGFR^L858R*Pten^-/- and EGFR^L858R mice were larger in size than those of normal (RO) mice (Fig. 1E). In studying lung tissue pathology in EGFR^L858R*Pten^-/- and EGFR^L858R mice, we found that a larger lung area was occupied by cancer cells in EGFR^L858R mice than in EGFR^L858R*Pten^-/- mice (Fig. 1F, a). However, when we evaluated the lung tissues in detail (Fig. 1F, b), we found that knockout of Pten increased the hyperplasia of bronchial cells but inhibited the growth of alveolar cells in mice with EGFR^L858R-induced lung cancer, implying a differential effect of Pten on the tumour burden in bronchial and alveolar cells in mice with EGFR^L858R-induced lung cancer. Finally, the level of the hyperplasia marker CCSP was measured in EGFR^L858R*Pten^-/- and EGFR^L858R mice (Fig. 1G). The data indicated that the CCSP level was increased, primarily in bronchial cells, in EGFR^L858R*Pten^-/- mice compared to EGFR^L858R mice but was decreased in the alveolar region in the lungs. In summary, Pten might play different roles in bronchial and alveolar cells in EGFR^L848R-induced lung cancer.

Functional knockout of Pten promotes ciliogenesis in EGFR^L858R*Pten^-/--induced lung cancer

The molecular mechanism by which Pten regulates bronchial and alveolar cells in EGFR^L858R-induced lung cancer was next studied (Fig. 2). First, total RNA was isolated from lung tissues of EGFR^L858R*Pten^-/- and EGFR^L858R*Pten^+/+ mice to systematically study the gene expression, and bioinformatics analysis was then performed (Fig. 2). Quality control indicated that the distribution of gene expression was similar between EGFR^L858R*Pten^-/- and EGFR^L858R*Pten^+/+ mice, suggesting that the RNA-Seq data were of good quality (Suppl. Figure 1). In Pten knockout mice, 288 genes were upregulated and 97 genes were downregulated in EGFR^L858R-induced lung cancer tissues (Fig. 2A). In the Circus plot, most of the Pten-regulated genes can be seen to be involved in three pathways, namely, ciliary plasm, axoneme and cilium movement, implying that Pten might regulate ciliated bronchial cells in EGFR^L858R-induced lung cancer (Fig. 2B and Supplementary Table 1). The list of the top 30 terms identified ALL GO enrichment analysis shows that Pten was involved in most of the pathways, such as cilium movement, microtubule-based movement, and cilium organization, indicating that Pten inhibits hyperplasia of ciliated cells in EGFR^L858R-induced lung cancer (Fig. 2C). In addition, based on statistical significance, the dot plot of the ALL GO analysis results revealed that knockout of Pten resulted in enrichment of pathways related to ciliary organization and movement (Fig. 2D). Next, we also used Upset analysis to study the related genes in the different pathways (Fig. 2E). The data indicated that several genes were involved in most of the pathways related to cilium organization and movement (Fig. 2E). Next, the bar plot of the DEG All KEGG Enrichment Pathways analysis results suggested that loss of PTEN activated several pathways involved in circadian rhythms, drug metabolism, metabolism of xenobiotics, lysosomes and so on (Fig. 2F). Finally, the bar plot of the DEG All DisGeNET Enrichment analysis results indicated that loss of PTEN positively regulated myocardial ischaemia, lung diseases, preeclampsia, thrombosis, endothelial dysfunction and so on (Fig. 2G). In summary, loss of PTEN positively regulates a number of genes related to ciliogenesis, which may increase the hyperplasia of bronchial ciliated cells in EGFR^L858R-induced lung cancer.

Next, we prepared more mice to study the effect of Pten on these cilia-related genes (Fig. 3). Indeed, the results indicated that Pten knockout significantly increased the mRNA levels of most of the ciliated genes, namely, Cep126, Dcdc2a, Dnaic2, Dnah5, FOXJ1, Hydin, Tctex1d4, Spag16, and Wdr63, implying that loss of Pten may be involved in ciliogenesis during EGFR^L858R-induced lung cancer progression (Fig. 3A). Next, we used Alcian blue staining to study the ciliated cells among bronchial epithelial cells from mice with EGFR^L858R*Pten^+/+- and EGFR^L858R*Pten^-/--induced lung cancer (Fig. 3B). The data indicated that Pten knockout strongly increased the Alcian blue signal in bronchial epithelial cells, suggesting that loss of Pten stimulates hyperplasia of bronchial epithelial cells in the EGFR^L858R*Pten^-/- mouse model of lung cancer (Fig. 3B). Finally, we also used a marker of cilia, mucin5a, to study the effect of Pten on ciliogenesis in EGFR^L858R-induced lung cancer (Fig. 3C). The data indicated that mucin5a was more abundant in the bronchial of EGFR^L858R*Pten^-/- mice compared to EGFR^L858R mice (Fig. 3C). In summary, Pten inhibits most of cilia-related genes to regulate ciliogenesis, thereby suppressing bronchial epithelial cell hyperplasia in EGFR^L858R-induced cancer.

Pten inhibits NR2F1 expression to decrease ciliogenesis-related gene expression in EGFR^L858R-induced lung cancer

Next, we studied how Pten regulates the expression of cilia-related genes (Fig. 4). We investigated the transcription factors possibly recruited to the promoters of all the Pten-regulated cilia-related genes and then checked the read numbers of these transcription factors in the RNA-seq data. The results showed that NR2F1 can be recruited to the promoters of most cilia-related genes and increased their read numbers in mice with EGFR^L858R*Pten^-/--induced lung cancer, implying that Pten may inhibit NR2F1 to in turn inhibit the expression of cilia-related genes. First, we examined the effect of Pten on the expression of cilia-related genes in BEAS-2B (Fig. 4A). The data indicated that Pten knockout increased the mRNA levels of DNAI2, DNAI3 and MUC5AC in BEAS-2B cells, a bronchial cell line. Knockdown of NR2F1 abolished the effect of Pten on DNAI2 and DNAI3 expression but not MUC5AC expression (Fig. 4A). Next, the protein and mRNA levels of NR2F1 were found to be increased in Pten knockdown BEAS-2B cells (Fig. 4B). The NR2F1 level was also increased in EGFR^L858R*Pten^-/- mice compared to that of EGFR^L858R-mice (Fig. 4C), indicating that Pten negatively regulates NR2F1 in vitro and in vivo. Pten negatively regulates the Akt signalling pathway, which is involved in cancer progression (14). Thus, knockout of Pten increased the levels of phosphor-Akt in EGFR^L858R*Pten^-/- mice compared to EGFR^L858R mice (Fig. 4D). Additionally, inhibition of Akt phosphorylation by MK2206 treatment abolished the effect of Pten on the expression of DNAI2, DNAI3 (Fig. 4E) and NR2F1 (Fig. 4F). Finally, loss of Pten increased the recruitment of NR2F1 to the promoter of DNAI2 (Fig. 4G, a and b) and increased the luciferase activity driven by the promoter of NR2F1 (2K bp), indicating that NR2F1 can regulate DNAI2 directly. In summary, Pten suppresses Akt activation, thereby inhibiting NR2F1 expression and subsequently decreasing cilia-related gene expression, leading to inhibition of bronchial epithelial hyperplasia in EGFR^L858R-induced lung cancer.

Pten promotes alveolar cell growth in EGFR^L858R-induced lung cancer

Thus far, we have elucidated the mechanism by which Pten negatively regulates hyperplasia of bronchial cells in EGFR^L858R-induced lung cancer. However, what is the molecular mechanism by which Pten positively regulates alveolar cellular growth in EGFR^L858R-induced lung cancer (Fig. 5)? In addition, is the positive effect of Pten in EGFR^L858R mice is EGFR^L858R dependent? We used two alveolar lung cancer cell lines, A549 and PC9, which have Kras and EGFR mutations, respectively, to study the role of Pten in cell proliferation (Fig. 5A). Overexpression of GFP-Pten inhibited A549 cell growth but promoted PC9 cell growth (Fig. 5A, a and b). Knockdown of Pten increased A549 cell growth and decreased PC9 cell growth (Fig. 5A, b and c), indicating that Pten negatively regulates Kras-mutant cell growth but positively regulates EGFR^L858R-mutant cell growth. In addition, overexpression of GFP-Pten increased the level of phosphorylated Akt (p-Akt) in PC9 and H1299^L858R cells but not in A549 and H1299 cells, suggesting that the EGFR^L858R mutation is critical for the effect of Pten on alveolar cell growth (Fig. 5B and Fig. 5C). Finally, the cell cycles in PC9 and A549 cells with or without GFP-PTEN overexpression was studied by flow cytometry (Fig. 5D). The data indicated that overexpression of GFP-Pten increased the percentage of G1/S-phase PC9 cells (71.1%/78.6%) but decreased the percentage of G1/S-phase A549 cells (60.8%/54.9%), indicating that Pten may have differential effects on alveolar cells with different mutation backgrounds, a finding that is expected to be beneficial for precision therapy in the future (Fig. 5D). In summary, differential effects of PTEN were found on EGFR-mut and Kras-mut lung cancer cell lines, implying that the PTEN status might be important for clinical lung cancer patients with EGFR mutations or Kras mutations.

The clinical relationships between the survival rate and the expression of Pten and ciliogenesis-related proteins

Next, we used online data, animal samples and clinical specimens to study the relationship among survival, Pten, NR2F1 and cilia-related proteins (Fig. 6 and Fig. 7). First, the levels of NR2F1 and DNAI2 in EGFR^L858R*Pten^-/- and EGFR^L858R*Pten^+/+ mice were measured (Fig. 6A). The data indicated that the levels of both NR2F1 and DNAI2 were dramatically higher in EGFR^L858R*Pten^-/- mice, implying that loss of PTEN increases NR2F1 expression, thereby promoting DNAI2 expression and leading to cilium formation (Fig. 6A). Next, the related information of non-small cell lung cancer (NSCLC) cohorts from TCGA was used to study the relationships between the survival rate and the expression of cilium-related genes (Fig. 6B-6E). First, the survival rate in the lung cancer cohorts with EGFR mutations was found to be lower than that in the cohort with wild-type EGFR (Fig. 6B). MUC5B expression was decreased in the EGFR-mut and PTEN-mut cohorts (Fig. 6C). In addition, SCGB1A1 expressionwas also inhibited in the PTEN-mut cohort (Fig. 6D, a). NR2F1 was highly expressed in the PTEN-mut cohorts (Fig. 6D, b). There was a positive correlation between NR2F1 and DNAI2 expression in all the NSCLC patients (Fig. 6D, c). Finally, the Kras level was different in the PTEN-WT and PTEN-mut cohorts, but no difference was found in the EGFR level, implying that the effect of PTEN on Kras-mutant and EGFR-mutatnt cancers is different (Fig. 6E).

Finally, we collected lung cancer clinical specimens to compare the level of DNAI2 in tissues of different stages (Fig. 7A). The results indicated that DNAI2 was increased in the early stage of NSCLC but was decreased in the late stage, indicating that DNAI2 might be involved in lung tumorigenesis (Fig. 7A).

In this study, we found that loss of Pten increases NR2F1 expression and subsequently increases DNAI2 expression, leading to bronchial cell hyperplasia but inhibiting alveolar cell hyperplasia during EGFR^L858R-induced lung cancer progression, a finding expected to be beneficial for the precision medicine in the treatment of lung cancer in the future (Fig. 7B).

Interestingly, Pten is well known as a tumour suppressor in the various cancer types. However, we found that Pten may act as a double-edged sword in lung cancer progression. In this study, we found that Pten knockout induced the hyperplasia of bronchial epithelial cells but inhibited the growth of alveolar cells in EGFR^L858R-induced lung cancer. Previous studies revealed that Pten knockout also induced the hyperplasia of bronchial epithelial cells but did not change the growth of alveolar epithelial cells in Kras^G12D-induced lung cancer, in which Pten inactivation cooperated with oncogenic Kras^G12D to promote lung cancer progression (15). First, loss of Pten was found to increase the hyperplasia of bronchial epithelial cells in EGFR^L858R- and Kras^G12D-induced lung cancer. In this study, we clarified that Pten negatively regulates NR2F1 to inhibit cilia-related gene expression in EGFR^L858R-induced lung cancer, a mechanism that is involved in bronchial epithelial cell hyperplasia. In addition, numerous studies have indicated that NR2F1-AS1 sponges many miRNAs, subsequently inducing the expression of several oncogenes (10–12). Therefore, Pten may also inhibit NR2F1-AS1 to suppress lung cancer progression. In addition, previous studies have indicated that NR2F1 may regulate dormancy and metastasis in the late-stage of lung cancer (16, 17). In this study, we used EGFR^L858R mice to study the initiation of lung cancer, not the late-stage of lung cancer. Based on previous studies and this study, NR2F1 may have differential effects on cancer in the early and late stages. In addition, although Pten also induces hyperplasia in bronchial epithelial cells of Kras^G12D-induced lung cancer mice (15), the molecular mechanism is not yet clarified. Second, Pten plays different roles in bronchial and alveolar epithelial cells of mice with EGFR^L858R-induced lung cancer but not in mice with Kras^G12D-induced lung cancer. This is the first study to identify the dual roles of Pten as a tumour suppressor and an oncogene in bronchial and alveolar epithelial cells, respectively, in EGFR^L858R-induced lung cancer. Overexpression of Pten increased cell growth in the EGFR-mutant cell lines PC9 and H1299^L858R but not in the Kras-mutant cell line A549, indicating that EGFR mutation is critical for Pten-mediated cell growth. In analysing the RNA-seq data, we found that Pten knockout increased Akt1 and Akt2 expression but decreased Akt3 expression (data not shown). Previous studies also indicated that, unlike Akt1 and Akt2 phosphorylation, Akt3 phosphorylation inhibits cancer cell proliferation (18, 19). However, why the EGFR mutation is required for the enhancement of cell proliferation by Pten in alveolar cells needs to be addressed in the future. Finally, EGFR is one of the most well-known tyrosine kinase receptor domain-containing proteins and is commonly mutated in tumour, with uncontrolled cell growth, proliferation, and migration documented in approximately 33% of NSCLCs (20–22). The five-residue deletion (746ELREA750) in exon19 accounts for 47% of EGFR mutations, and the exon 21 (L858R) substitution accounts for 41% (23). Herein, we used mice with EGFR^L858R-induced lung cancer and cancer cells to study the role of Pten in bronchial and alveolar cells. Whether other EGFR mutation types also have the same effects needs to be clarified in the future.

In this study, we found that loss of Pten significantly induced the proliferation of ciliated cells among bronchial epithelial cells of mice with EGFR^L858R-induced lung cancer. Furthermore, we clearly clarified the molecular mechanism by which PTEN regulates ciliogenesis in bronchial epithelial cells during lung cancer progression. During lung morphogenesis, the ciliated cell differentiation pathway is activated (24, 25). Geminin coiled-coil containing protein 1 (GMNC), the "master regulator" of ciliated cell fate, and the MYB proto-oncogene are induced (24). Forkhead box protein J1 (Foxj1), a transcription factor required for cilia formation and motility, is also expressed (26, 27). Chronic obstructive pulmonary disease (COPD) develops in smokers. Patients with COPD have a considerable reduction in the number of ciliated cells (28, 29). Ciliated cell dysfunction not only causes this disorder but is also found in various cancers. A primary cilium is frequently present on cells of human differentiated thyroid tumours. The relationship between primary cilia and thyroid cancer was uncovered by (30, 31). In that study, the researchers established a mouse model of thyrocyte- specific loss of the primary cilium, and the results indicated that the lack of primary cilia resulted in increased apoptosis in thyroid cancer cells, possibly reveling a new therapeutic target for thyroid cancers (31). In addition, in a study on pancreatic cancer, the authors analysed survival and outcome in 100 patients and drew on conclusion that primary cilia can be formed on pancreatic cancer cells and that their presence is strongly correlated with the prognosis of pancreatic ductal adenocarcinoma (32, 33). In contrast, primary cilia are also positively regulated in oral squamous cell carcinoma (OSCC). In OSCC, a significant reduction in the percentage of ciliated cells was found in oral leukoplakia (OLK), especially in patients with OSCC, and EGFR was a target, suggesting that loss of cilia induced oral tumour growth (34). Because of Pten is a well-known as tumour suppressor that regulates the expression of numerous genes involved in cell biogenesis, the link between Pten and cilia needs to be discussed. However, another study showed the conflicting result that Pten negatively regulates dendritogenesis. Loss of Pten is related to autism spectrum disease (ASD) and causes excessive neuronal development, including the formation of lengthened and branched total dendritic spines. Microtubules are an important part of the ciliary structure that are highly polymerized in primary hippocampal culture (35). Ciliated cells are progeny of club cells, such as goblet cells, and a complicated transcriptional network including Notch signalling controls how these cells differentiate (36, 37). Mucins are glycoproteins that are secreted by gel-forming mucin-producing goblet cells.

In this study, we found that Pten knockout induced mucin-related genes. Two high-molecular-weight secreted mucins that are expressed mainly in the mucus layer, which contains electrolytes, metabolites, fluids, and antimicrobial substances, are MUC5AC and MUC5B (26, 38). The mucus layer serves as the first line of innate protection in the respiratory tract against inhaled pathogens and particles (39–41). Ciliated cells facilitate mucin trafficking to trap particles and as components of the ciliary escalator that drives mucus into the oropharynx for eventual removal by expectoration or swallowing. Pten regulates mucin-related genes but not through NR2F1. The detailed mechanism by which Pten regulate mucin gene expression will be addressed in the future.

Dynein Axonemal Intermediate Chain 2 (DNAI2) is part of the dynein complex of respiratory cilia and sperm flagella (42, 43). Mutations in DNAI2 are involved in the development of primary ciliary dyskinesia type 9. Recent studies have also shown that various isoforms encoded by alternatively spliced transcript variants are involved in primary ciliary dyskinesia (PCD), which may be as a new genetic risk factor for PCD (44, 45). There is no reported study about DNAI2 in cancer. This is the first study to determine the role of PTEN-mediated DNAI2 expression in the hyperplasia of bronchial epithelial cells in EGFR^L858R-induced lung cancer. Recent studies have also revealed that PTEN mutations might be involved in mediating drug resistance and immunotherapy efficacy during cancer therapy (46–48). Pten mutation induced ciliogenesis in bronchial epithelial cells might be a critical mechanism. We found that more lymphocytes were recruited to the region with hyperproliferation of bronchial epithelial cells in EGFR^L858R mice, implying that Pten mutation regulates not only cancer cells but also other cells in the surrounding environment of cancer cells (data not shown). Several genes (Gsn/Cd24a/Igfbp2/Foxj1/Adam8/Zbtb16/Hsph1/Efnb2/Gpam/Vcam1/Ctla2a) related to positive regulation of lymphocyte activation were upregulated in EGFR^L858R*Pten^−/− mice. The detailed function of Pten in regulating the tumour microenvironment in lung cancer and the associated mechanism need to be explored in the future. In addition to cilia-related genes, other Pten-regulated genes were also found in our RNA-seq analysis. For example, ACE2, which has been reported to be overexpressed in different cell subsets of NSCLC, was also upregulated in EGFR^L858R*Pten^−/− mice (49, 50). In addition, previous studies indicated that Pten inactivation in mice with Kras^G12D-induced lung cancer increased the immune response (15, 51, 52). In this study, we also found that several genes related to the inflammatory response, lymphocyte activation and innate immune response in the mucosa (Hp/Reg3g/Nupr1/Adam8/Cd55/Cfh/Ednrb) were upregulated in mice with EGFR^L858R*Pten^−/− induced lung cancer. Finally, many genes related to epithelial cell migration, extracellular matrix assembly and cellular extravasation were upregulated by Pten knockout, implying that Pten may significantly promote cancer metastasis. The effect of the Pten-regulated tumour microenvironment on cancer metastasis in vivo might be evaluated in vivo by using models of EGFR^L858R- or Kras^G12D-induced lung cancer in the future.

Cell culture and reagents - Human lung bronchial epithelial cell lines BEAS-2B and adenocarcinoma human alveolar basal epithelial cells A549, PC9 were cultured with RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco™, Waltham, MA, USA), 100 µg/ml streptomycin and 100U/ml penicillin G (Gibco). All cells were maintained in a humidified incubator under 5% CO2 at 37^oC. For AKT inhibitor treatment, cells were seeded until reached confluence and treated with 1µM MK-2206 (Merk Millipore Corp, Billerica, MA, USA) for 24h. For transfecting plasmid, PolyJet (SignaGen Laboratories, Frederick, MD, USA) was used according to the manufacturer’s instructions.

Alcian blue staining - The paraffin embedded sections were incubated in xylene for dewaxing and a graded series of ethanol for hydration. Next, sections were incubated in acetic acid 3% for 5min and stained by Alcian blue (pH = 2.5) (Merk Millipore Corp, Billerica, MA, USA) solution for 30 min at room temperature. To remove exceed Alcian Blue and prevent non-specific staining, slides were rinsed briefly by acetic acid 3% for 10 second. The tissue samples were rinsed two times with distilled water and stained with Nuclear Fast Red solution (Merk Millipore Corp, Billerica, MA, USA) 0.1% for 5min to visualize histologic changes. All slices were dehydrated through graded alcohols before mounting.

Animal study - Transgenic mice were acquired from Jackson Lab and maintained at the National Laboratory Animal Center in Taiwan. Reverse tetracycline trans- activator (rtTA) protein was expressed under control of Scgb1a1 (secretoglobin, family 1A, member 1) promoter in Scgb1a1-rtTA transgenic mice. To generate the EGFR mutation, the TetO-EGFR^L858R transgenic mice, tetracycline- responsive promoter element (TRE; tetO) was utilized. TetO-EGFR^L858R mice were crossed with Scgb1a1-rtTA transgenic mice to generate bi-transgenic mice. After breeding Scgb1a1-rtTA/TetO-EGFR^L858R mice were used to study lung cancer. In order to induce lung cancer progression, doxycycline (0.5 g/l) was dissolved in drinking water for two month-old of transgenic mice orally administration. To engineer Pten conditionally knockout mice, two-month-old transgenic mice were administrated with intraperitoneal tamoxifen injection (1 mg/kg) and combined with tamoxifen in feed (400mg/kg) for one months. All mice were continuously maintained for two months and were sacrificed. Lung tissues were analyzed by immunohistochemistry staining. All treatments involving animals were conducted in accordance with the rules and regulation of Institutional Animal Care and Use Committee at National Cheng Kung University.

Genomic DNA extraction - Genomic DNA of human cell lines were extracted by using QIAamp DNA Mini kit (Qiagen, Germantown, MD, USA) according to manufactured protocol

Collection of specimens from lung cancer patients - All human study has been conducted in accordance with the guidelines and regulations. The study using human specimens was approved by the Clinical Research Ethics Committee at National Cheng Kung University Medical Center (Tainan, Taiwan; IRB: A-ER-107-039). After surgical resection at National Cheng Kung University Hospital, specimens of patients with lung adenocarcinomas were collected for Immunohistochemical analysis or western blotting. The pathological data were analyzed by clinical pathologists. Informed consent was obtained from all subjects.

Statistical analysis - All samples were used for statistical analysis. Data was analyzed using the GraphPad Prism software and western blot images were calculated by ImageJ software. The difference between two group was analyzed by Student’s t test; *p < 0.05, **p < 0.01, ***<0.001. The data analysis would be considered statistically significant if p value is lower than 0.05.

Ethics approval and consent to participate – This IRB (B-ER-108-368), Studying the Molecular Mechanisms and Drug Development in USP24-mediated Drug Resistance during Cancer Therapy, was approved by Institutional Review Board of National Cheng Kung University Hospital.

Consent for publication - No applicable

Availability of data and materials – All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests - The authors declare no potential conflicts of interest.

Funding Support - This work was supported by the grants (106-2320-B-006-065-MY3, 108-2320-B-006-019, and 109-2320-B-006-057) obtained from the National Science and Technology Council, Taiwan.

Author Contribution - Tran Thi Thanh Truc performed all the experiments of this study. Jan-Jong Hung organized whole of the experimental design and prepared manuscript.

Acknowledgement - We are grateful for the support of clinical specimens from the Human Biobank, Research Center of Clinical Medicine, National Cheng Kung University Hospital. This research was supported in part by Higher Education Sprout Project, Ministry of Education to the Headquarters of University Advancement at National Cheng Kung University(NCKU).

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There is NO conflict of interest to disclose.

Suppl.Fig.1.jpg
Suppl. Fig. 1. The global gene expression profile upon doxycycline-mediated induction of lung cancer with (Dox_1303) or without PTEN knockout by TAM injection (Dox_TAM_50) was studied by RNA-Seq. The distribution of gene expression levels is shown.
Suppl.Fig.2.jpg
Suppl. Fig. 2. The global gene expression profile upon doxycycline-mediated induction of lung cancer with (Dox_1303) or without PTEN knockout by TAM injection (Dox_TAM_50) was studied by RNA-Seq. The enrichment of various gene expression-related various pathways were analyzed by GSEA.
Supple.Table1.pdf
Suppl. Table 1. The GO pathways significantly enriched in the differentially expressed genes between control mice and PTEN^-/- mice
Supp.Table2.pdf
Suppl. Table 2. The primers used in this study.
Suppleinformation.docx

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PTEN decreases NR2F1 expression to inhibit ciliogenesis during EGFRL858R-induced lung cancer progression

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Version 1

Abstract

Figures

Introduction

Results

Functional knockout of Pten promotes ciliogenesis in EGFR^L858R*Pten^-/--induced lung cancer

Pten inhibits NR2F1 expression to decrease ciliogenesis-related gene expression in EGFR^L858R-induced lung cancer

Pten promotes alveolar cell growth in EGFR^L858R-induced lung cancer

The clinical relationships between the survival rate and the expression of Pten and ciliogenesis-related proteins

Discussion

Materials and methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

PTEN decreases NR2F1 expression to inhibit ciliogenesis during EGFRL858R-induced lung cancer progression

Status:

Version 1

Abstract

Figures

Introduction

Results

Functional knockout of Pten promotes ciliogenesis in EGFRL858R*Pten-/--induced lung cancer

Pten inhibits NR2F1 expression to decrease ciliogenesis-related gene expression in EGFRL858R-induced lung cancer

Pten promotes alveolar cell growth in EGFRL858R-induced lung cancer

The clinical relationships between the survival rate and the expression of Pten and ciliogenesis-related proteins

Discussion

Materials and methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Functional knockout of Pten promotes ciliogenesis in EGFR^L858R*Pten^-/--induced lung cancer

Pten inhibits NR2F1 expression to decrease ciliogenesis-related gene expression in EGFR^L858R-induced lung cancer

Pten promotes alveolar cell growth in EGFR^L858R-induced lung cancer