An In Vivo Inflammatory Loop Potentiates KRAS Blockade.

KRAS (KRAS proto-oncogene, GTPase) inhibitors perform less well than other targeted drugs in vitro and fail clinical trials. To investigate a possible reason for this, we treated human and murine tumor cells with KRAS inhibitors deltarasin (targeting phosphodiesterase-δ), cysmethynil (targeting isoprenylcysteine carboxylmethyltransferase), and AA12 (targeting KRASG12C), and silenced/overexpressed mutant KRAS using custom-designed vectors. We showed that KRAS-mutant tumor cells exclusively respond to KRAS blockade in vivo, because the oncogene co-opts host myeloid cells via a C-C-motif chemokine ligand 2 (CCL2)/interleukin-1 beta (IL-1ß)-mediated signaling loop for sustained tumorigenicity. Indeed, KRAS-mutant tumors did not respond to deltarasin in C-C motif chemokine receptor 2 (Ccr2) and Il1b gene-deficient mice, but were deltarasin-sensitive in wild-type and Ccr2-deficient mice adoptively transplanted with wild-type murine bone marrow. A KRAS-dependent pro-inflammatory transcriptome was prominent in human cancers with high KRAS mutation prevalence and poor predicted survival. Our findings support that in vitro cellular systems are suboptimal for anti-KRAS drug screens, as these drugs function to suppress interleukin-1 receptor 1 (IL1R1) expression and myeloid IL-1ß-delivered pro-growth effects in vivo. Moreover, the findings support that IL-1ß blockade might be suitable for therapy for KRAS-mutant cancers.

Validation of in vitro models for smoke exposure of primary human bronchial epithelial cells.

The bronchial epithelium is constantly challenged by inhalative insults including cigarette smoke (CS), a key risk factor for lung disease. In vitro exposure of bronchial epithelial cells using CS extract (CSE) is a widespread alternative to whole CS (wCS) exposure. However, CSE exposure protocols vary considerably between studies, precluding direct comparison of applied doses. Moreover, they are rarely validated in terms of physiological response in vivo and the relevance of the findings is often unclear. We tested six different exposure settings in primary human bronchial epithelial cells (phBECs), including five CSE protocols compared with wCS exposure. We quantified cell-delivered dose and directly compared all exposures using expression analysis of 10 well-established smoke-induced genes in bronchial epithelial cells. CSE exposure of phBECs was varied in terms of differentiation state, exposure route, duration of exposure, and dose. Gene expression was assessed by quantitative real-time PCR (qPCR) and Western Blot analysis. Cell type-specific expression of smoke-induced genes was analyzed by immunofluorescent analysis. Three surprisingly dissimilar exposure types, namely, chronic CSE treatment of differentiating phBECs, acute CSE treatment of submerged basal phBECs, and wCS exposure of differentiated phBECs performed best, resulting in significant upregulation of seven (chronic CSE) and six (acute wCS, acute submerged CSE exposure) out of 10 genes. Acute apical or basolateral exposure of differentiated phBECs with CSE was much less effective despite similar doses used. Our findings provide guidance for the design of human in vitro CS exposure models in experimental and translational lung research.

Prognostic phenotypes of early-stage lung adenocarcinoma.

BACKGROUND: Survival after curative resection of early-stage lung adenocarcinoma (LUAD) varies and prognostic biomarkers are urgently needed. METHODS: Large-format tissue samples from a prospective cohort of 200 patients with resected LUAD were immunophenotyped for cancer hallmarks TP53, NF1, CD45, PD-1, PCNA, TUNEL and FVIII, and were followed for a median of 2.34 (95% CI 1.71-3.49) years. RESULTS: Unsupervised hierarchical clustering revealed two patient subgroups with similar clinicopathological features and genotype, but with markedly different survival: "proliferative" patients (60%) with elevated TP53, NF1, CD45 and PCNA expression had 50% 5-year overall survival, while "apoptotic" patients (40%) with high TUNEL had 70% 5-year survival (hazard ratio 2.23, 95% CI 1.33-3.80; p=0.0069). Cox regression and machine learning algorithms including random forests built clinically useful models: a score to predict overall survival and a formula and nomogram to predict tumour phenotype. The distinct LUAD phenotypes were validated in The Cancer Genome Atlas and KMplotter data, and showed prognostic power supplementary to International Association for the Study of Lung Cancer tumour-node-metastasis stage and World Health Organization histologic classification. CONCLUSIONS: Two molecular subtypes of LUAD exist and their identification provides important prognostic information.

Non-canonical Wnt/PCP signalling regulates intestinal stem cell lineage priming towards enteroendocrine and Paneth cell fates.

A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to treat chronic intestinal diseases. However, the different models of ISC lineage hierarchy1-6 and segregation7-12 are subject to debate. Here, we have discovered non-canonical Wnt/planar cell polarity (PCP)-activated ISCs that are primed towards the enteroendocrine or Paneth cell lineage. Strikingly, integration of time-resolved lineage labelling with single-cell gene expression analysis revealed that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7-12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells7. Finally, Wnt/PCP-activated Lgr5+ ISCs are molecularly indistinguishable from Wnt/ß-catenin-activated Lgr5+ ISCs, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we redefine the mechanisms underlying ISC lineage hierarchy and identify the Wnt/PCP pathway as a new niche signal preceding lateral inhibition in ISC lineage priming and segregation.

Proteasome activator PA200 regulates myofibroblast differentiation.

The proteasome is essential for the selective degradation of most cellular proteins and is fine-tuned according to cellular needs. Proteasome activators serve as building blocks to adjust protein turnover in cell growth and differentiation. Understanding the cellular function of proteasome activation in more detail offers a new strategy for therapeutic targeting of proteasomal protein breakdown in disease. The role of the proteasome activator PA200 in cell function and its regulation in disease is unknown. In this study, we investigated the function of PA200 in myofibroblast differentiation and fibrotic tissue remodeling. PA200 was upregulated in hyperplastic basal cells and myofibroblasts of fibrotic lungs from patients with idiopathic pulmonary fibrosis. Increased expression of PA200 and enhanced formation of PA200-proteasome complexes was also evident in experimental fibrosis of the lung and kidney in vivo and in activated primary human myofibroblasts of the lung in vitro. Transient silencing and overexpression revealed that PA200 functions as a negative regulator of myofibroblast differentiation of human but not mouse cells. Our data thus suggest an unexpected and important role for PA200 in adjusting myofibroblast activation in response to pro-fibrotic stimuli, which fails in idiopathic pulmonary fibrosis.

Cigarette smoke induces overexpression of active human cathepsin S in lungs from current smokers with or without COPD.

Cigarette smoking has marked effects on lung tissue, including induction of oxidative stress, inflammatory cell recruitment, and a protease/antiprotease imbalance. These effects contribute to tissue remodeling and destruction resulting in loss of lung function in chronic obstructive pulmonary disease (COPD) patients. Cathepsin S (CatS) is a cysteine protease that is involved in the remodeling/degradation of connective tissue and basement membrane. Aberrant expression or activity of CatS has been implicated in a variety of diseases, including arthritis, cancer, cardiovascular, and lung diseases. However, little is known about the effect of cigarette smoking on both CatS expression and activity, as well as its role in smoking-related lung diseases. Here, we evaluated the expression and activity of human CatS in lung tissues from never-smokers and smokers with or without COPD. Despite the presence of an oxidizing environment, CatS expression and activity were significantly higher in current smokers (both non-COPD and COPD) compared with never-smokers, and correlated positively with smoking history. Moreover, we found that the exposure of primary human bronchial epithelial cells to cigarette smoke extract triggered the activation of P2X7 receptors, which in turns drives CatS upregulation. The present data suggest that excessive CatS expression and activity contribute, beside other proteases, to the deleterious effects of cigarette smoke on pulmonary homeostasis.

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma.

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it's expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

Glutathione peroxidase 3 localizes to the epithelial lining fluid and the extracellular matrix in interstitial lung disease.

Aberrant antioxidant activity and excessive deposition of extracellular matrix (ECM) are hallmarks of interstitial lung diseases (ILD). It is known that oxidative stress alters the ECM, but extracellular antioxidant defence mechanisms in ILD are incompletely understood. Here, we extracted abundance and detergent solubility of extracellular antioxidant enzymes from a proteomic dataset of bleomycin-induced lung fibrosis in mice and assessed regulation and distribution of glutathione peroxidase 3 (GPX3) in murine and human lung fibrosis. Superoxide dismutase 3 (Sod3), Gpx3, and Gpx activity were increased in mouse BALF during bleomycin-induced lung fibrosis. In lung tissue homogenates, Gpx3, but not Sod3, was upregulated and detergent solubility profiling indicated that Gpx3 associated with ECM proteins. Immunofluorescence analysis showed that Gpx3 was expressed by bronchial epithelial cells and interstitial fibroblasts and localized to the basement membrane and interstitial ECM in lung tissue. As to human ILD samples, BALF of some patients contained high levels of GPX3, and GPX3 was upregulated in lung homogenates from IPF patients. GPX3 expression in primary human bronchial epithelial cells and lung fibroblasts was downregulated by TNF-α, but more variably regulated by TGF-ß1 and menadione. In conclusion, the antioxidant enzyme GPX3 localizes to lung ECM and is variably upregulated in ILD.

Cigarette smoke alters primary human bronchial epithelial cell differentiation at the air-liquid interface.

The differentiated human airway epithelium consists of different cell types forming a polarized and pseudostratified epithelium. This is dramatically altered in chronic obstructive pulmonary disease (COPD), characterized by basal and goblet cell hyperplasia, and squamous cell metaplasia. The effect of cigarette smoke on human bronchial epithelial cell (HBEC) differentiation remains to be elucidated. We analysed whether cigarette smoke extract (CSE) affected primary (p)HBEC differentiation and function. pHBEC were differentiated at the air-liquid interface (ALI) and differentiation was quantified after 7, 14, 21, or 28 days by assessing acetylated tubulin, CC10, or MUC5AC for ciliated, Clara, or goblet cells, respectively. Exposure of differentiating pHBEC to CSE impaired epithelial barrier formation, as assessed by resistance measurements (TEER). Importantly, CSE exposure significantly reduced the number of ciliated cells, while it increased the number of Clara and goblet cells. CSE-dependent cell number changes were reflected by a reduction of acetylated tubulin levels, an increased expression of the basal cell marker KRT14, and increased secretion of CC10, but not by changes in transcript levels of CC10, MUC5AC, or FOXJ1. Our data demonstrate that cigarette smoke specifically alters the cellular composition of the airway epithelium by affecting basal cell differentiation in a post-transcriptional manner.

Epigenetic mechanisms in COPD: implications for pathogenesis and drug discovery.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. The growing burden of COPD is due to continuous tobacco use, which is the most important risk factor of the disease, indoor fumes, occupational exposures and also aging of the world's population. Epigenetic mechanisms significantly contribute to COPD pathophysiology. AREAS COVERED: This review focuses on disease-relevant changes in DNA modification, histone modification and non-coding RNA expression in COPD, and provides insight into novel therapeutic approaches modulating epigenetic mechanisms. Recent findings revealed, among others, globally changed DNA methylation patterns, decreased levels of histone deacetylases and reduced microRNAs levels in COPD. The authors also discuss a potential role of the chromatin silencing Polycomb group of proteins in COPD. EXPERT OPINION: COPD is a highly complex disease and therapy development is complicated by the fact that many smokers develop both COPD and lung cancer. Of interest, combination therapies involving DNA methyltransferase inhibitors and anti-inflammatory drugs provide a promising approach, as they might be therapeutic for both COPD and cancer. Although the field of epigenetic research has virtually exploded over the last 10 years, particular efforts are required to enhance our knowledge of the COPD epigenome in order to successfully establish epigenetic-based therapies for this widespread disease.

Cigarette smoke-induced disruption of bronchial epithelial tight junctions is prevented by transforming growth factor-ß.

The airway epithelium constitutes an essential immunological and cytoprotective barrier to inhaled insults, such as cigarette smoke, environmental particles, or viruses. Although bronchial epithelial integrity is crucial for airway homeostasis, defective epithelial barrier function contributes to chronic obstructive pulmonary disease (COPD). Tight junctions at the apical side of epithelial cell-cell contacts determine epithelial permeability. Cigarette smoke exposure, the major risk factor for COPD, is suggested to impair tight junction integrity; however, detailed mechanisms thereof remain elusive. We investigated whether cigarette smoke extract (CSE) and transforming growth factor (TGF)-ß1 affected tight junction integrity. Exposure of human bronchial epithelial cells (16HBE14o(-)) and differentiated primary human bronchial epithelial cells (pHBECs) to CSE significantly disrupted tight junction integrity and barrier function. Specifically, CSE decreased transepithelial electrical resistance (TEER) and tight junction-associated protein levels. Zonula occludens (ZO)-1 and ZO-2 protein levels were significantly reduced and dislocated from the cell membrane, as observed by fractionation and immunofluorescence analysis. These findings were reproduced in isolated bronchi exposed to CSE ex vivo, as detected by real-time quantitative reverse-transcriptase PCR and immunohistochemistry. Combined treatment of 16HBE14o(-) cells or pHBECs with CSE and TGF-ß1 restored ZO-1 and ZO-2 levels. TGF-ß1 cotreatment restored membrane localization of ZO-1 and ZO-2 protein and prevented CSE-mediated TEER decrease. In conclusion, CSE led to the disruption of tight junctions of human bronchial epithelial cells, and TGF-ß1 counteracted this CSE-induced effect. Thus, TGF-ß1 may serve as a protective factor for bronchial epithelial cell homeostasis in diseases such as COPD.