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Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O 2 consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis. Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1+ mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)–mediated epigenome editing. HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1+ myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1 -expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis -regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology. Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function. [Display omitted] This study identifies a transcription factor that is required for the normal production of the main inhibitory neurotransmitter in the gastrointestinal tract and demonstrates that increasing its levels using a drug that is already approved for humans can reverse diabetic gastroparesis in female mice. [ABSTRACT FROM AUTHOR]

The incidence of Crohn's disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. We aimed to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. We performed single-cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of patients with CD compared with healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signaling networks. We overlapped our differentially expressed genes with publicly available microarray data sets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD regulatory T (Treg) cells and to test predicted therapeutics. We identified 5 distinct FOXP3+ regulatory Treg subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to tumor necrosis factor–α signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid coculture system. As predicted in silico, the histone deacetylase inhibitor vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. We identified a novel, proinflammatory FOXP3+ T cell subpopulation in patients with CD and developed a pipeline to specifically target these cells using the US Food and Drug Administration–approved drug vorinostat. Crohn's intestinal lesions are enriched in a unique population of FOXP3+ cells that exhibit a pro-inflammatory transcriptional signature consistent with strong tumor necrosis factor–α signaling. [ABSTRACT FROM AUTHOR]

Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography–tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors–T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors–Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid–induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer–induced colitis. G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease. [Display omitted] [ABSTRACT FROM AUTHOR]

Summary: Caloric restriction (CR) mitigates many detrimental effects of aging and prolongs life span. CR has been suggested to increase mitochondrial biogenesis, thereby attenuating age-related declines in mitochondrial function, a concept that is challenged by recent studies. Here we show that lifelong CR in mice prevents age-related loss of mitochondrial oxidative capacity and efficiency, measured in isolated mitochondria and permeabilized muscle fibers. We find that these beneficial effects of CR occur without increasing mitochondrial abundance. Whole-genome expression profiling and large-scale proteomic surveys revealed expression patterns inconsistent with increased mitochondrial biogenesis, which is further supported by lower mitochondrial protein synthesis with CR. We find that CR decreases oxidant emission, increases antioxidant scavenging, and minimizes oxidative damage to DNA and protein. These results demonstrate that CR preserves mitochondrial function by protecting the integrity and function of existing cellular components rather than by increasing mitochondrial biogenesis. [ABSTRACT FROM AUTHOR]

Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) holds an important role in retaining lung function, but its association with lung cancer is unclear. A case-control study was conducted to determine the possible associations of the genetic variants in the CFTR gene with lung cancer risk. Genotypes of the most common deletion ΔF508, one functional SNP, and eight tag SNPs in the CFTR gene were determined in 574 lung cancer patients and 679 controls. A logistic regression model, adjusting for known risk factors, was used to evaluate the association of each variant with lung cancer risk, as confirmation haplotype and sub-haplotype analyses were performed. ΔF508 deletion and genotypes with minor alleles in one tag SNP, rs10487372, and one functional SNP, rs213950, were inversely associated with lung cancer risk. The results of haplotype and sub-haplotype analyses were consistent with single variant analysis, all pointing to deletion ΔF508 being the key variant for significant haplotypes and sub-haplotypes. Individuals with ‘deletion-T’ (ΔF508/rs10487372) haplotype had a 68% reduced risk for lung cancer compared to common haplotype ‘no-deletion-C’ (OR=0.32; 95% CI=0.15–0.68; p =0.01). Genetic variations in the CFTR gene might modulate the risk of lung cancer. This study, for the first time, provides evidence of a protective role of the CFTR deletion carrier in the etiology of lung cancer. [ABSTRACT FROM AUTHOR]

Objective: Our objective was to determine whether histologic grade independently contributes to the prognosis of non–small cell lung cancer. Methods: A total of 5018 hospital-based patients diagnosed from 1997 to 2003 and 712 population-based patients diagnosed from 1984 to 2003 were followed up through the end of 2004. The effect of histologic grade on postdiagnosis survival or postresection recurrence was evaluated by Cox proportional hazards models. Relative risks (RR) were estimated by comparing undifferentiated, poorly differentiated, and moderately differentiated carcinoma with well-differentiated carcinoma. Results: Histologic grade was significantly associated with survival after adjustment for the effects of age, gender, smoking history, tumor stage, histologic cell type, and treatment modality. Patients with undifferentiated carcinoma had an 80% elevated risk of death (RR = 1.83; 95% confidence interval [CI], 1.4-2.4) compared with those with well-differentiated carcinoma; 70% and 40% elevated risks were observed for patients with poorly and moderately differentiated carcinoma, respectively (RR, 1.7 [1.5-2.0] and 1.4 [1.2-1.6]). Similar results were observed for 718 incidence cases in which the relative risks were 1.6 (1.1-2.2) and 1.4 (1.0-1.9) for poorly/undifferentiated carcinoma and moderately differentiated carcinoma, respectively. Patients with less-differentiated carcinoma after tumor resection had a higher risk of recurrence, with adjusted hazard ratios of 2.1 (95% CI: 1.4-2.9) and 1.4 (1.0-1.9) for poorly/undifferentiated and moderately differentiated carcinoma compared with well-differentiated carcinoma. Conclusions: Histologic grade has significant prognostic value for survival of patients with non–small cell lung cancer. Histologic grade may provide useful information in defining the aggressiveness of tumors and should be considered as an independent factor affecting survival beyond TNM staging. [Copyright &y& Elsevier]

Neutrophil elastase and α1-antitrypsin are a pair of protease and protease inhibitor counterparts. The imbalance between the two counterparts is generally thought to cause tissue damage, which could create a favourable tissue environment for carcinogens and tumour progression. Laboratory research and clinical findings have indicated that a deficiency in α1-antitrypsin is associated with increased risk of liver cancer, bladder cancer, gall bladder cancer, malignant lymphoma, and lung cancer. Conversely, raised concentrations of neutrophil elastase might promote the development, invasion, and metastasis of many cancers. Several mechanisms of carcinogenesis have been postulated. Excess neutrophil elastase might facilitate cancer development by causing tissue damage and air trapping, which foster longer carcinogen exposure, might promote cancer progression by degrading the intercellular matrix barrier, and might directly lead to cancer development through the tumour-necrosis-factor signalling pathway. [Copyright &y& Elsevier]

Abstract: Lung adenocarcinoma is the most common type of primary lung cancer. The purpose of this study was to delineate gene expression patterns for survival prediction in lung adenocarcinoma. Gene expression profiles of 82 (discovery set) and 442 (validation set 1) lung adenocarcinoma tumor tissues were analyzed using a systems biology-based network approach. We also examined the expression profiles of 78 adjacent normal lung tissues from 82 patients. We found a significant correlation of an expression module with overall survival (adjusted hazard ratio or HR=1.71; 95% CI=1.06–2.74 in discovery set; adjusted HR=1.26; 95% CI=1.08–1.49 in validation set 1). This expression module contained genes enriched in the biological process of the cell cycle. Interestingly, the cell cycle gene module and overall survival association were also significant in normal lung tissues (adjusted HR=1.91; 95% CI, 1.32–2.75). From these survival-related modules, we further defined three hub genes (UBE2C, TPX2, and MELK) whose expression-based risk indices were more strongly associated with poor 5-year survival (HR=3.85, 95% CI=1.34–11.05 in discovery set; HR=1.72, 95% CI=1.21–2.46 in validation set 1; and HR=3.35, 95% CI=1.08–10.04 in normal lung set). The 3-gene prognostic result was further validated using 92 adenocarcinoma tumor samples (validation set 2); patients with a high-risk gene signature have a 1.52-fold increased risk (95% CI, 1.02–2.24) of death than patients with a low-risk gene signature. These results suggest that a network-based approach may facilitate discovery of key genes that are closely linked to survival in patients with lung adenocarcinoma. [Copyright &y& Elsevier]

Lung cancer occurs in never-smokers. Epigenetic changes in lung cancer potentially represent important diagnostic, prognostic, and therapeutic targets. We compared DNA methylation profiles of 28 adenocarcinomas of the lungs of never-smokers with paired adjacent nonmalignant lung tissue. We correlated differential methylation changes with gene expression changes from the same 28 sample pairs. Using principal component analysis, we observed a distinct separation in methylation profiles between tumor and adjacent nonmalignant lung tissue. Tumors were generally hypomethylated compared with adjacent nonmalignant tissue. Of 1,906 CpG sites differentially methylated between tumor and nonmalignant tissue, 1,198 were within classically defined CpG islands where tumors were hypermethylated compared with nonmalignant tissue. A total of 708 sites were outside CpG islands where tumors were hypomethylated compared with nonmalignant tissue. There were significant differences in expression of 351 genes (23%) of the 1,522 genes matched to the differentially methylated CpG sites. Genes that were not significantly differentially expressed and were hypermethylated within CpG sites were enriched for homeobox genes. These results suggest that the methylation profiles of lung adenocarcinomas of never-smokers and adjacent nonmalignant lung tissue are significantly different. Despite the differential methylation of homeobox genes, no significant changes in expression of these genes were detected. [ABSTRACT FROM AUTHOR]

Background Extracellular microRNAs (miRNAs) embedded in circulating exosomes may serves as prognostic biomarkers in cancer. Objective To identify and evaluate plasma exosomal miRNAs for prognosis in castration-resistant prostate cancer (CRPC). Design, setting, and participants RNA sequencing was performed to identify candidate exosomal miRNAs associated with overall survival in a screening cohort of 23 CRPC patients. Candidate miRNAs were further evaluated for prognosis using quantitative real-time polymerase chain reaction in a follow-up cohort of 100 CRPC patients. Outcome measurements and statistical analysis Cox regression and Kaplan-Meier survival analyses were used to evaluate survival association using candidate miRNAs along with clinical prognostic factors. Results and limitations RNA sequencing in screening cohort generated approximately 6.80 million mappable reads per patient. Of those with normalized read counts ≥5, 43% were mapped to miRNAs for a total of 375 known and 57 novel miRNAs. Cox regression analysis identified an association of miR-1290, -1246, and -375 with overall survival (false discover rate < 0.05). Of those, higher levels of miR-1290 and -375 were significantly associated with poor overall survival ( p < 0.004) in the follow-up cohort. Incorporation of miR-1290/-375 into putative clinical prognostic factors-based models in CRPC stage significantly improved predictive performance with a time-dependent area under the curve increase from 0.66 to 0.73 ( p = 6.57 × 10 −6 ). Conclusions Plasma exosomal miR-1290 and miR-375 are promising prognostic biomarkers for CRPC patients. Prospective validation is needed for further evaluation of these candidate miRNAs. Patient summary In this study, we evaluated whether small RNAs circulating in blood could be used to predict clinical outcomes in late-stage prostate cancer patients. We identified two blood-based small RNAs whose levels showed significant association with survival. Our results warrant further investigation because the noninvasive blood-based test has great potential in the management of late-stage prostate cancer. [ABSTRACT FROM AUTHOR]

Summary: Background: Lung cancer in individuals who have never smoked tobacco products is an increasing medical and public-health issue. We aimed to unravel the genetic basis of lung cancer in never smokers. Methods: We did a four-stage investigation. First, a genome-wide association study of single nucleotide polymorphisms (SNPs) was done with 754 never smokers (377 matched case-control pairs at Mayo Clinic, Rochester, MN, USA). Second, the top candidate SNPs from the first study were validated in two independent studies among 735 (MD Anderson Cancer Center, Houston, TX, USA) and 253 (Harvard University, Boston, MA, USA) never smokers. Third, further replication of the top SNP was done in 530 never smokers (UCLA, Los Angeles, CA, USA). Fourth, expression quantitative trait loci (eQTL) and gene-expression differences were analysed to further elucidate the causal relation between the validated SNPs and the risk of lung cancer in never smokers. Findings: 44 top candidate SNPs were identified that might alter the risk of lung cancer in never smokers. rs2352028 at chromosome 13q31.3 was subsequently replicated with an additive genetic model in the four independent studies, with a combined odds ratio of 1·46 (95% CI 1·26–1·70, p=5·94×10−6). A cis eQTL analysis showed there was a strong correlation between genotypes of the replicated SNPs and the transcription level of the gene GPC5 in normal lung tissues (p=1·96×10−4), with the high-risk allele linked with lower expression. Additionally, the transcription level of GPC5 in normal lung tissue was twice that detected in matched lung adenocarcinoma tissue (p=6·75×10−11). Interpretation: Genetic variants at 13q31.3 alter the expression of GPC5, and are associated with susceptibility to lung cancer in never smokers. Downregulation of GPC5 might contribute to the development of lung cancer in never smokers. Funding: US National Institutes of Health; Mayo Foundation. [Copyright &y& Elsevier]

Abstract: Combined modality therapy is the standard care for limited stage-small cell lung cancer (LS-SCLC) and has led to a significant improvement in patients’ survival. This study sought to investigate and define the importance of prognostic effects of known and controversial factors especially the impact of smoking status and treatment strategies. A total of 284 patients with LS-SCLC were diagnosed and prospectively followed from 1997 to 2008 at Mayo Clinic; their characteristics and survival outcome were assessed on the basis of age, gender, smoking history, performance status (PS), tumor recurrence or progression, and treatment using Cox proportional hazards models. Our main results are as follows: (1) Although neither smoking status (former or current smokers) nor intensity (pack-years smoked) at the time of SCLC diagnosis were significant survival predictors, compared to continued smokers (who never quit smoking), patients who quit at or after diagnosis cut the risk of death by 45% (HR=0.55, 95% CI 0.38–0.79); patients who quit before lung cancer diagnosis also experienced survival benefit (HR=0.72, 95% CI 0.52–1.00). (2) Thoracic radiotherapy and platinum-based chemotherapy could significantly improve survival but the timing (within or after one month of diagnosis) of starting chemotherapy or radiation therapy did not. (3) After adjusting for other known factors, a lower PS did not predict poorer survival, suggesting that PS should not be the only factor for making treatment decisions. In conclusion, this study demonstrated the negative impact of continued cigarette smoking on survival; therefore, clinicians and all care providers should strongly encourage smoking cessation at diagnosis of LS-SCLC. [Copyright &y& Elsevier]