Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis.

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Publisher Correction: Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

In the originally published version of this Article, the positions of the final two authors in the author list were inadvertently inverted during the production process. This error has now been corrected in both the PDF and HTML versions of the Article.

Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

The transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call "entrapping". Our results support a key role for uc.339 in lung cancer.

Epigenome-wide analysis of DNA methylation in lung tissue shows concordance with blood studies and identifies tobacco smoke-inducible enhancers.

Smoking-associated DNA hypomethylation has been observed in blood cells and linked to lung cancer risk. However, its cause and mechanistic relationship to lung cancer remain unclear. We studied the association between tobacco smoking and epigenome-wide methylation in non-tumor lung (NTL) tissue from 237 lung cancer cases in the Environment And Genetics in Lung cancer Etiology study, using the Infinium HumanMethylation450 BeadChip. We identified seven smoking-associated hypomethylated CpGs (P < 1.0 × 10-7), which were replicated in NTL data from The Cancer Genome Atlas. Five of these loci were previously reported as hypomethylated in smokers' blood, suggesting that blood-based biomarkers can reflect changes in the target tissue for these loci. Four CpGs border sequences carrying aryl hydrocarbon receptor binding sites and enhancer-specific histone modifications in primary alveolar epithelium and A549 lung adenocarcinoma cells. A549 cell exposure to cigarette smoke condensate increased these enhancer marks significantly and stimulated expression of predicted target xenobiotic response-related genes AHRR (P = 1.13 × 10-62) and CYP1B1 (P < 2.49 × 10-61). Expression of both genes was linked to smoking-related transversion mutations in lung tumors. Thus, smoking-associated hypomethylation may be a consequence of enhancer activation, revealing environmentally-induced regulatory elements implicated in lung carcinogenesis.

Cross-Species Transcriptome Profiling Identifies New Alveolar Epithelial Type I Cell-Specific Genes.

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type-specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells' roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription-polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell-specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.

Data on isoaspartylation of neuronal ELAVL proteins.

This article contains experimental data examining the propensity of neuronal ELAVL proteins to become isoaspartylated. The data are related to the article "Isoaspartylation appears to trigger small cell lung cancer-associated autoimmunity against neuronal protein ELAVL4" (M.A. Pulido, M.K. DerHartunian, Z. Qin, E.M. Chung, D.S. Kang, A.W. Woodham, J.A. Tsou, R. Klooster, O. Akbari, L. Wang, W.M. Kast, S.V. Liu, J.J.G.M. Verschuuren, D.W. Aswad, I.A. Laird-Offringa, 2016) [1], in which it was reported that the N-terminal region of recombinant human ELAVL4 protein, incubated under physiological conditions, acquires a type of highly immunogenic protein damage. Here, we present Western blot analysis data generated by using an affinity-purified polyclonal rabbit antibody (raised against an N-terminal ELAVL4 isoaspartyl-converted peptide) to probe recombinant protein fragments of the other three members of the ELAVL family: the highly homologous neuronal ELAVL2 (HuB) and ELAVL3 (HuC), and the much less homologous ubiquitously expressed ELAVL1 (HuR).

Isoaspartylation appears to trigger small cell lung cancer-associated autoimmunity against neuronal protein ELAVL4.

Autoantibodies against SCLC-associated neuronal antigen ELAVL4 (HuD) have been linked to smaller tumors and improved survival, but the antigenic epitope and mechanism of autoimmunity have never been solved. We report that recombinant human ELAVL4 protein incubated under physiological conditions acquires isoaspartylation, a type of immunogenic protein damage. Specifically, the N-terminal region of ELAVL4, previously implicated in SCLC-associated autoimmunity, undergoes isoaspartylation in vitro, is recognized by sera from anti-ELAVL4 positive SCLC patients and is highly immunogenic in subcutaneously injected mice and in vitro stimulated human lymphocytes. Our data suggest that isoaspartylated ELAVL4 is the trigger for the SCLC-associated anti-ELAVL4 autoimmune response.

Pleiotropic Analysis of Lung Cancer and Blood Triglycerides.

Epidemiologically related traits may share genetic risk factors, and pleiotropic analysis could identify individual loci associated with these traits. Because of their shared epidemiological associations, we conducted pleiotropic analysis of genome-wide association studies of lung cancer (12 160 lung cancer case patients and 16 838 control subjects) and cardiovascular disease risk factors (blood lipids from 188 577 subjects, type 2 diabetes from 148 821 subjects, body mass index from 123 865 subjects, and smoking phenotypes from 74 053 subjects). We found that 6p22.1 (rs6904596, ZNF184) was associated with both lung cancer (P = 5.50x10(-6)) and blood triglycerides (P = 1.39x10(-5)). We replicated the association in 6097 lung cancer case patients and 204 657 control subjects (P = 2.40 × 10(-4)) and in 71 113 subjects with triglycerides data (P = .01). rs6904596 reached genome-wide significance in lung cancer meta-analysis (odds ratio = 1.15, 95% confidence interval = 1.10 to 1.21 ,: Pcombined = 5.20x10(-9)). The large sample size provided by the lipid GWAS data and the shared genetic risk factors between the two traits contributed to the uncovering of a hitherto unidentified genetic locus for lung cancer.

The importance of detailed epigenomic profiling of different cell types within organs.

The human body consists of hundreds of kinds of cells specified from a single genome overlaid with cell type-specific epigenetic information. Comprehensively profiling the body's distinct epigenetic landscapes will allow researchers to verify cell types used in regenerative medicine and to determine the epigenetic effects of disease, environmental exposures and genetic variation. Key marks/factors that should be investigated include regions of nucleosome-free DNA accessible to regulatory factors, histone marks defining active enhancers and promoters, DNA methylation levels, regulatory RNAs, and factors controlling the three-dimensional conformation of the genome. Here we use the lung to illustrate the importance of investigating an organ's purified cell epigenomes, and outline the challenges and promise of realizing a comprehensive catalog of primary cell epigenomes.

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription.

Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low µM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5'-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents-small molecule, nucleic acid, or protein-reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition-and modulation-of disease-relevant RNA.

The 78-kD Glucose-Regulated Protein Regulates Endoplasmic Reticulum Homeostasis and Distal Epithelial Cell Survival during Lung Development.

Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated conditional knockout (KO) mice (cGrp78(f/f)) with lung epithelial cell-specific KO of Grp78, a gene encoding the ER chaperone 78-kD glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis, and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78(f/f) pups died immediately after birth, likely owing to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of the proapoptotic transcription factor CCAAT/enhancer-binding proteins (C/EBP) homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes encoding antioxidant enzymes suggest a role for oxidative stress in alveolar epithelial cell (AEC) apoptosis. Increased Smad3 phosphorylation and expression of transforming growth factor-ß/Smad3 targets Cdkn1a (encoding p21) and Gadd45a suggest that interactions among the apoptotic arm of the UPR, oxidative stress, and transforming growth factor-ß/Smad signaling pathways contribute to Grp78 KO-induced AEC apoptosis and developmental arrest. Chemical chaperone Tauroursodeoxycholic acid reduced UPR activation and apoptosis in cGrp78(f/f) lungs cultured ex vivo, confirming a role for ER stress in observed AEC abnormalities. These results demonstrate a key role for GRP78 in AEC survival and gene expression during lung development through modulation of ER stress, and suggest the UPR as a potential therapeutic target in BPD.

Combinations of differentiation markers distinguish subpopulations of alveolar epithelial cells in adult lung.

Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for some daughter AT2 cells, transdifferentiation into alveolar type I (AT1) cells. We investigated if subpopulations of alveolar epithelial cells (AEC) exist that represent various stages in transdifferentiation from AT2 to AT1 cell phenotypes in normal adult lung and if they can be identified using combinations of cell-specific markers. Immunofluorescence microscopy showed that, in distal rat and mouse lungs, ∼ 20-30% of NKX2.1(+) (or thyroid transcription factor 1(+)) cells did not colocalize with pro-surfactant protein C (pro-SP-C), a highly specific AT2 cell marker. In distal rat lung, NKX2.1(+) cells coexpressed either pro-SP-C or the AT1 cell marker homeodomain only protein x (HOPX). Not all HOPX(+) cells colocalize with the AT1 cell marker aquaporin 5 (AQP5), and some AQP5(+) cells were NKX2.1(+). HOPX was expressed earlier than AQP5 during transdifferentiation in rat AEC primary culture, with robust expression of both by day 7. We speculate that NKX2.1 and pro-SP-C colocalize in AT2 cells, NKX2.1 and HOPX or AQP5 colocalize in intermediate or transitional cells, and HOPX and AQP5 are expressed without NKX2.1 in AT1 cells. These findings suggest marked heterogeneity among cells previously identified as exclusively AT1 or AT2 cells, implying the presence of subpopulations of intermediate or transitional AEC in normal adult lung.

Molecular portraits of epithelial, mesenchymal, and hybrid States in lung adenocarcinoma and their relevance to survival.

Epithelial-to-mesenchymal transition (EMT) is a key process associated with tumor progression and metastasis. To define molecular features associated with EMT states, we undertook an integrative approach combining mRNA, miRNA, DNA methylation, and proteomic profiles of 38 cell populations representative of the genomic heterogeneity in lung adenocarcinoma. The resulting data were integrated with functional profiles consisting of cell invasiveness, adhesion, and motility. A subset of cell lines that were readily defined as epithelial or mesenchymal based on their morphology and E-cadherin and vimentin expression elicited distinctive molecular signatures. Other cell populations displayed intermediate/hybrid states of EMT, with mixed epithelial and mesenchymal characteristics. A dominant proteomic feature of aggressive hybrid cell lines was upregulation of cytoskeletal and actin-binding proteins, a signature shared with mesenchymal cell lines. Cytoskeletal reorganization preceded loss of E-cadherin in epithelial cells in which EMT was induced by TGFß. A set of transcripts corresponding to the mesenchymal protein signature enriched in cytoskeletal proteins was found to be predictive of survival in independent datasets of lung adenocarcinomas. Our findings point to an association between cytoskeletal and actin-binding proteins, a mesenchymal or hybrid EMT phenotype and invasive properties of lung adenocarcinomas.

Transcriptomic Profiling of Primary Alveolar Epithelial Cell Differentiation in Human and Rat.

Cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how changes in transcriptional activation during alveolar epithelial cell (AEC) differentiation determine phenotype. We performed transcriptomic profiling using in vitro differentiation of human and rat primary AEC. This model recapitulates in vitro an in vivo process in which AEC transition from alveolar type 2 (AT2) cells to alveolar type 1 (AT1) cells during normal maintenance and regeneration following lung injury. Here we describe in detail the quality control, preprocessing, and normalization of microarray data presented within the associated study [1]. We also include R code for reproducibility of the referenced data and easily accessible processed data tables.

Knockout mice reveal key roles for claudin 18 in alveolar barrier properties and fluid homeostasis.

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased ß-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase ß1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue.

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters and CpG islands (CGIs), while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF-binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, four of the five established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome.

CDKN2A/p16 inactivation mechanisms and their relationship to smoke exposure and molecular features in non-small-cell lung cancer.

INTRODUCTION: CDKN2A (p16) inactivation is common in lung cancer and occurs via homozygous deletions, methylation of promoter region, or point mutations. Although p16 promoter methylation has been linked to KRAS mutation and smoking, the associations between p16 inactivation mechanisms and other common genetic mutations and smoking status are still controversial or unknown. METHODS: We determined all three p16 inactivation mechanisms with the use of multiple methodologies for genomic status, methylation, RNA, and protein expression, and correlated them with EGFR, KRAS, STK11 mutations and smoking status in 40 cell lines and 45 tumor samples of primary non-small-cell lung carcinoma. We also performed meta-analyses to investigate the impact of smoke exposure on p16 inactivation. RESULTS: p16 inactivation was the major mechanism of RB pathway perturbation in non-small-cell lung carcinoma, with homozygous deletion being the most frequent method, followed by methylation and the rarer point mutations. Inactivating mechanisms were tightly correlated with loss of mRNA and protein expression. p16 inactivation occurred at comparable frequencies regardless of mutational status of EGFR, KRAS, and STK11, however, the major inactivation mechanism of p16 varied. p16 methylation was linked to KRAS mutation but was mutually exclusive with EGFR mutation. Cell lines and tumor samples demonstrated similar results. Our meta-analyses confirmed a modest positive association between p16 promoter methylation and smoking. CONCLUSION: Our results confirm that all the inactivation mechanisms are truly associated with loss of gene product and identify specific associations between p16 inactivation mechanisms and other genetic changes and smoking status.

Integrated transcriptomic and epigenomic analysis of primary human lung epithelial cell differentiation.

Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation.

Epigenetic therapy in lung cancer.

Epigenetic deregulation of gene function has been strongly implicated in carcinogenesis and is one of the mechanisms contributing to the development of lung cancer. The inherent reversibility of epigenetic alterations makes them viable therapeutic targets. Here, we review the therapeutic implications of epigenetic changes in lung cancer, and recent advances in therapeutic strategies targeting DNA methylation and histone acetylation.

Analysis of protein-RNA complexes involving a RNA recognition motif engineered to bind hairpins with seven- and eight-nucleotide loops.

U1A binds U1hpII, a hairpin RNA with a 10-nucleotide loop. A U1A mutant (ΔK50ΔM51) binds U1hpII-derived hairpins with shorter loops, making it an interesting scaffold for engineering or evolving proteins that bind similarly sized disease-related hairpin RNAs. However, a more detailed understanding of complexes involving ΔK50ΔM51 is likely a prerequisite to generating such proteins. Toward this end, we measured mutational effects for complexes involving U1A ΔK50ΔM51 and U1hpII-derived hairpin RNAs with seven- or eight-nucleotide loops and identified contacts that are critical to the stabilization of these complexes. Our data provide valuable insight into sequence-selective recognition of seven- or eight-nucleotide loop hairpins by an engineered RNA binding protein.