CEBPD modulates the airway smooth muscle transcriptomic response to glucocorticoids.

BACKGROUND: CCAAT/Enhancer Binding Protein D (CEBPD), a pleiotropic glucocorticoid-responsive transcription factor, modulates inflammatory responses. Of relevance to asthma, expression of CEBPD in airway smooth muscle (ASM) increases with glucocorticoid exposure. We sought to characterize CEBPD-mediated transcriptomic responses to glucocorticoid exposure in ASM by measuring changes observed after knockdown of CEBPD and its impact on asthma-related ASM function. METHODS: Primary ASM cells derived from four donors were transfected with CEBPD or non-targeting (NT) siRNA and exposed to vehicle control, budesonide (100 nM, 18 h), TNFα (10 ng/ml, 18 h), or both budesonide and TNFα. Subsequently, RNA-Seq was used to measure gene expression levels, and pairwise differential expression results were obtained for exposures versus vehicle and knockdown versus control conditions. Weighted gene co-expression analysis was performed to identify groups of genes with similar expression patterns across the various experimental conditions (i.e., CEBPD knockdown status, exposures). RESULTS: CEBPD knockdown altered expression of 3037 genes under at least one exposure (q-value < 0.05). Co-expression analysis identified sets of 197, 152 and 290 genes that were correlated with CEBPD knockdown status, TNFα exposure status, and both, respectively. JAK-STAT signaling pathway genes, including IL6R and SOCS3, were among those influenced by both TNFα and CEBPD knockdown. Immunoblot assays revealed that budesonide-induced IL-6R protein expression and augmented IL-6-induced STAT3 phosphorylation levels were attenuated by CEBPD knockdown in ASM. CONCLUSIONS: CEBPD modulates glucocorticoid responses in ASM, in part via modulation of IL-6 receptor signaling.

Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface ß2AR (ß2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces ß2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases ß-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

Insights into glucocorticoid responses derived from omics studies.

Glucocorticoid drugs are commonly used in the treatment of several conditions, including autoimmune diseases, asthma and cancer. Despite their widespread use and knowledge of biological pathways via which they act, much remains to be learned about the cell type-specific mechanisms of glucocorticoid action and the reasons why patients respond differently to them. In recent years, human and in vitro studies have addressed these questions with genomics, transcriptomics and other omics approaches. Here, we summarize key insights derived from omics studies of glucocorticoid response, and we identify existing knowledge gaps related to mechanisms of glucocorticoid action that future studies can address.

A functional splice variant associated with decreased asthma risk abolishes the ability of gasdermin B to induce epithelial cell pyroptosis.

BACKGROUND: Genetic variants in the chromosomal region 17q21 are consistently associated with asthma. However, mechanistic studies have not yet linked any of the associated variants to a function that could influence asthma, and as a result, the identity of the asthma gene(s) remains elusive. OBJECTIVES: We sought to identify and characterize functional variants in the 17q21 locus. METHODS: We used the Exome Aggregation Consortium browser to identify coding (amino acid-changing) variants in the 17q21 locus. We obtained asthma association measures for these variants in both the Genetic Epidemiology Research in Adult Health and Aging (GERA) cohort (16,274 cases and 38,269 matched controls) and the EVE Consortium study (5,303 asthma cases and 12,560 individuals). Gene expression and protein localization were determined by quantitative RT-PCR and fluorescence immunostaining, respectively. Molecular and cellular studies were performed to determine the functional effects of coding variants. RESULTS: Two coding variants (rs2305480 and rs11078928) of the gasdermin B (GSDMB) gene in the 17q21 locus were associated with lower asthma risk in both GERA (odds ratio, 0.92; P = 1.01 × 10-6) and EVE (odds ratio, 0.85; joint PEVE = 1.31 × 10-13). In GERA, rs11078928 had a minor allele frequency (MAF) of 0.45 in unaffected (nonasthmatic) controls and 0.43 in asthma cases. For European Americans in EVE, the MAF of rs2305480 was 0.45 for controls and 0.39 for cases; for all EVE subjects, the MAF was 0.32 for controls and 0.27 for cases. GSDMB is highly expressed in differentiated airway epithelial cells, including the ciliated cells. We found that, when the GSDMB protein is cleaved by inflammatory caspase-1 to release its N-terminal fragment, potent pyroptotic cell death is induced. The splice variant rs11078928 deletes the entire exon 6, which encodes 13 amino acids in the critical N-terminus, and abolishes the pyroptotic activity of the GSDMB protein. CONCLUSIONS: Our study identified a functional asthma variant in the GSDMB gene of the 17q21 locus and implicates GSDMB-mediated epithelial cell pyroptosis in pathogenesis.

Multiregion sequencing reveals the intratumor heterogeneity of driver mutations in TP53-driven non-small cell lung cancer.

Intratumor heterogeneity (ITH) in non-small cell lung cancer (NSCLC) may account for resistance after a period of targeted therapies because drugs destroy only a portion of tumor cells. The recognition of ITH helps identify high-risk patients to make effective treatment decisions. However, ITH studies are confounded by interpatient heterogeneity in NSCLC and a large amount of passenger mutations. To address these issues, we recruited NSCLC patients carrying TP53 mutations and selected driver mutations within recurrently mutated genes in NSCLC. A total of 12-paired normal-tumor tissues were subjected to whole-genome/whole-exome sequencing. From these, 367 non-silent mutations were selected as driver mutations and deeply sequenced in 61 intratumoral microdissections. We identified a universal prevalence of heterogeneity in all 12 tumors, indicating branched evolution. Although TP53 mutations were observed in single biopsy of all 12 tumors, most tumors consist of both TP53 mutated and non-mutated cells in separate regions within the same tumor. This suggests the late molecular timing of the acquisition of TP53 mutations; therefore, the detection of TP53 mutations in a single biopsy may simply not reflect the early malignant potential. In addition, we identified regions of loss of heterozygosity surrounding TP53 and CDKN2A mutations in tumor 711, which also exhibited heterogeneity in different regional samples. Because the ITH of driver mutations likely has clinical consequences, further efforts are needed to limit the impact of ITH and to improve therapeutic efficiency, which will benefit NSCLC patients receiving targeted treatments.

Leukocyte telomere length is associated with advanced age-related macular degeneration in the Han Chinese population.

Telomeres located at the ends of chromosomes are involved in genomic stability and play a key role in various cancers and age-related diseases. Age-related macular degeneration (AMD) is a late-onset, age-associated progressive neurodegenerative disease, which includes the geographic atrophy (GA) subtype and the choroidal neovascularization (CNV) subtype. To better understand how leukocyte telomere length (LTL) is related to AMD, we conducted an association study in 197 AMD patients and 259 healthy controls using the established quantitative PCR technique. Logistic regression was performed to evaluate the association of LTL and AMD with the age-adjusted ratio of the telomere length to the copy number of a single-copy gene (T/S). Notably, we found a significant association between AMD and LTL (OR=2.24; 95% CI=1.68-3.07; P=0.0001) after adjusting for age and sex. Furthermore, the results showed a strongly significant association between the GA subtype and the LTL (OR=4.81; 95% CI=3.15-7.82; P=0.0001) after adjusting for age and sex. Our findings provide evidence of the role that LTL plays in the pathological mechanisms of AMD, mainly in the GA subgroup but not the CNV subgroup.

Hypermethylated Epidermal growth factor receptor (EGFR) promoter is associated with gastric cancer.

Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinases ErbB family and it is found to be overexpressed in gastric cancer. However, the mechanism of the regulation of the EGFR expression is still unknown. We used the Sequenom EpiTYPER assay to detect the methylation status of the EGFR promoter in normal and tumour tissues of 30 patients with gastric cancer. We also carried out quantitative real time PCR (qPCR) to detect the expression level of EGFR in our 30 patients. Notably, increased methylation level at EGFR promoter was found in tumour tissues than the corresponding adjacent noncancerous. In both Region I DMR and Region II DMR detected in our study, tumor tissues were significantly hypermethylated (P=2.7743E-10 and 2.1703E-05, respectively). Region I_⊿CpG_2 was also found to be associated with the presence of distant metastasis (P=0.0323). Furthermore, the results showed a strongly significant association between the relative EGFR expression and the EGFR methylation changes in both Region I and Region II (P=0.0004 and 0.0001, respectively). Our findings help to indicate the hypermethylation at EGFR promoter in gastric cancer and it could be a potential epigenetic biomarker for gastric cancer status and progression.

Whole DNA methylome profiling in lung cancer cells before and after epithelial-to-mesenchymal transition.

BACKGROUND: Metastatic lung cancer is one of the leading causes of cancer death. In recent years, epithelial-to-mesenchymal transition (EMT) has been found to contribute to metastasis, as it enables migratory and invasive properties in cancer cells. Previous genome-wide studies found that DNA methylation was unchanged during EMT induced by TGF-ß in AML12 cells. In this study, we aimed to discover EMT-related changes in DNA methylation in cancer cells, which are poorly understood. METHODS: We employed a next-generation sequencing-based method, MSCC (methyl-sensitive cut counting), to investigate DNA methylation during EMT in the A549 lung cancer cell line. RESULTS: We found that methylation levels were highly correlated to gene expression, histone modifications and small RNA expression. However, no differentially methylated regions (DMRs) were found in A549 cells treated with TGF-ß for 4 h, 12 h, 24 h and 96 h. Additionally, CpG islands (CGIs) showed no overall change in methylation levels, and at the single-base level, almost all of the CpGs showed conservation of DNA methylation levels. Furthermore, we found that the expression of DNA methyltransferase 1, 3a, 3b (DNMT1, DNMT3a, DNMT3b) and ten-eleven translocation 1 (TET1) was altered after EMT. The level of several histone methylations was also changed. CONCLUSIONS: DNA methylation-related enzymes and histone methylation might have a role in TGF-ß-induced EMT without affecting the whole DNA methylome in cancer cells. Our data provide new insights into the global methylation signature of lung cancer cells and the role of DNA methylation in EMT. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1112892497119603.

A study of ethnic differences in TGFß1 gene polymorphisms and effects on the risk of radiation pneumonitis in non-small-cell lung cancer.

BACKGROUND: To investigate whether Chinese non-small-cell lung cancer (NSCLC) patients with risk of radiation pneumonitis (RP) (grade ≥ 3) caused by radiotherapy display reliable single-nucleotide polymorphism (SNP) marker(s) located on the transforming growth factor-beta-1 (TGFß1) gene. METHODS: DNA was isolated from blood samples obtained from NSCLC patients (n = 167) treated with radiotherapy alone (n = 23) or chemoradiation (n = 144) with the median total radiation dose of 56 Gy between 2007 and 2010. A comprehensive approach toward characterizing the TGFß1 SNPs in Chinese patients was carried out in this study. Eight SNPs of the TGFß1 gene (rs1800469, rs1800471, rs1982073, rs4803455, rs11466345, rs12983047, rs10417924, and rs10980942) were finally genotyped by the direct DNA sequencing method. Kaplan-Meier cumulative probability was used to assess the risk of RP of grade 3 or higher. Cox proportional hazard analysis was used to evaluate the effect of TGFß1 genotypes on RP risk. RESULTS: A total of 46 patients (27.5%) developed RP of grade 3 or higher, based on Common Terminology Criteria for Adverse Events version 3.0, with a median follow-up of 29.5 months (range, 16-58). The rs1982073 SNP, associated with RP risk in whites, was not found to be associated with the risk of RP of grade 3 or higher in Chinese NSCLC patients. Multivariate analysis found AG/GG genotypes of the novel TGFß1 SNP rs11466345 to be associated with a significantly higher risk of RP of grade 3 or higher compared with the AA genotypes, after adjustment for age, smoking status, and dosimetric parameters (for mean lung dose, adjusted hazards ratio = 2.295, 95% confidence interval: 1.101-4.783, p = 0.027; for volume of normal lung receiving 20 GY or more radiation, adjusted hazards ratio = 2.142, 95% confidence interval: 1.033-4.441, p = 0.041). CONCLUSION: The AG/GG genotypes of novel TGFß1 rs11466345 were associated with a higher risk of RP in Chinese NSCLC patients treated with radiotherapy. The rs1982073 SNP, associated with RP risk in whites, was not correlated with higher RP risk in the Chinese patients studied. Further studies are warranted to confirm these findings in different ethnic populations.

Association of leukocyte telomere length with type 2 diabetes in mainland Chinese populations.

CONTEXT: Telomeres are structures at the ends of eukaryotic chromosomes. They help maintain genomic stability. High oxidative stress can lead to accelerated telomere shortening, which causes premature cell senescence. This is implicated in the development of type 2 diabetes (T2D). For this reason, we hypothesize that telomere shortening can characterize T2D. METHODS: We investigated the association between leukocyte telomere length (LTL) and T2D in a retrospective case-control study with a sample of 4016 Chinese Han subjects (1936 unrelated T2D cases and 2080 controls). Logistic regression analysis was performed to evaluate the association between LTL and T2D, adjusted for age and gender. Multivariate linear regression analysis was used to test for any association of LTL with a number of clinical, demographic, and diabetes-associated variables. RESULTS: Telomere repeat length (T)/copy number of a single-copy gene (S) ratios (T/S) of LTL were found to be significantly shorter in T2D cases [1.00 T/S, 95% confidence interval (CI) = 0.99-1.02] compared with controls (1.08 T/S, 95% CI = 1.06-1.09) over a wide age range (odds ratio of diabetes for a 1-U decrease in ln-transformed TL = 1.52; 95% CI = 1.23-1.88; P = 0.0001). CONCLUSION: Our research demonstrates association between shorter LTL and T2D in a population from mainland China. Our study suggests that shorter LTL might be associated with T2D in a manner independent of smoking and drinking habits or the time of T2D onset time.

GPC5 rs2352028 polymorphism and risk of lung cancer in Han Chinese.

rs2352028 in GPC5 has been reported to be associated with the risk of lung cancer in never-smokers. We performed a replication study in 1,045 lung cancer patients and 1,094 controls of Han Chinese origin. We found no association between rs2352028 and lung cancer/adenocarcinoma in never-smokers, but a p value of .04 (under the recessive model) was obtained between this SNP and overall lung cancer/adenocarcinoma. Our data and a recent meta-analysis suspected the possibility of rs2352028 being a risk variant of lung cancer risk in never-smokers. Our findings suggested that rs2352028 might confer a slight risk to lung cancer/adenocarcinoma.

Variations in/nearby genes coding for JAZF1, TSPAN8/LGR5 and HHEX-IDE and risk of type 2 diabetes in Han Chinese.

Several genetic loci (JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, ADAMTS9, VEGFA and HHEX-IDE) were identified to be significantly related to the risk of type 2 diabetes and quantitative metabolic traits in European populations. Here, we aimed to evaluate the impacts of these novel loci on type 2 diabetes risk in a population-based case-control study of Han Chinese (1912 cases and 2041 controls). We genotyped 13 single-nucleotide polymorphisms (SNPs) in/near these genes and examined the differences in allele/genotype frequency between cases and controls. We found that both IDE rs11187007 and HHEX rs1111875 were associated with type 2 diabetes risk (for both variants: odds ratio (OR)=1.15, 95% confidence interval (CI) 1.04-1.28, P=0.009). In a meta-analysis where we pooled our data with the three previous studies conducted in East Asians, we found that the variants of JAZF1 rs864745 (1.09 (1.03-1.16); P=3.49 × 10(-3)) and TSPAN8/LGR5 rs7961581 (1.11(1.05-1.17); P=1.89 × 10(-4)) were significantly associated with type 2 diabetes risk. In addition, the meta-analysis (7207 cases and 8260 controls) also showed that HHEX rs1111875 did have effects on type 2 diabetes in Chinese population (OR=1.15(1.10-1.21); P=1.93 × 10(-8)). This large population-based study and meta-analysis further confirmed the modest effects of the JAZF1, TSPAN8/LGR5 and HHEX-IDE loci on type 2 diabetes in Chinese and other East Asians.