Overexpression of the SARS-CoV-2 receptor ACE2 is induced by cigarette smoke in bronchial and alveolar epithelia.

Angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and a target for disease prevention. However, the relationship between ACE2 expression and its clinical implications in SARS-CoV-2 pathogenesis remains unknown. Here, we explored the location and expression of ACE2, and its correlation with gender, age, and cigarette smoke (CS), in a CS-exposed mouse model and 224 non-malignant lung tissues (125 non-smokers, 81 current smokers, and 18 ex-smokers) by immunohistochemistry. Moreover, the correlations of ACE2 with CS-induced oxidative stress-related markers, hypoxia-inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), and 4-hydroxynonenal (4-HNE) were investigated. Chromatin immunoprecipitation and luciferase reporter assays identified the cause of ACE2 overexpression in human primary lung epithelial cells. We demonstrated that ACE2 was predominantly overexpressed on the apical surface of bronchial epithelium, while reduced in alveolar epithelium, owing to the dramatically decreased abundance of alveolar type II pneumocytes in CS-exposed mouse lungs. Consistent with this, ACE2 was primarily significantly overexpressed in human bronchial and alveolar epithelial cells in smokers regardless of age or gender. Decreased ACE2 expression was observed in bronchial epithelial cells from ex-smokers compared with current smokers, especially in those who had ceased smoking for more than 10 years. Moreover, ACE2 expression was positively correlated with the levels of HIF-1α, iNOS, and 4-HNE in both mouse and human bronchioles. The results were further validated using a publicly available dataset from The Cancer Genome Atlas (TCGA) and our previous integrated data from Affymetrix U133 Plus 2.0 microarray (AE-meta). Finally, our results showed that HIF-1α transcriptionally upregulates ACE2 expression. Our results indicate that smoking-induced ACE2 overexpression in the apical surface of bronchial epithelial cells provides a route by which SARS-CoV-2 enters host cells, which supports clinical relevance in attenuating the potential transmission risk of COVID-19 in smoking populations by smoking cessation. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neutrophil extracellular traps induced by cigarette smoke contribute to airway inflammation in mice.

Neutrophil extracellular traps (NETs) were initially identified as an important antimicrobial barrier to capture and kill microorganisms. Emerging evidence suggests that NETs play a crucial role in chronic airway inflammation induced by cigarette smoke (CS). However, how NETs form and the mechanisms by which NETs function in CS-related airway diseases are still unclear. To explore NET formation and its potential role in CS-related airway diseases, we first established a CS-induced subacute airway inflammation model in mice and verified NET formation in the airways. Moreover, NETs degradation by aerosolized DNase I treatment significantly inhibited the airway inflammation induced by CS in mice. More importantly, by in vitro experiments, we found that cigarette smoke extract (CSE) induces NET formation in an NADPH oxidase-dependent manner, and that macrophages and human bronchial epithelial cells (HBEs) are important targets for the NETs-induced secretion of inflammatory cytokines. Therefore, NETs may represent a critical link among neutrophils, macrophages and HBEs under chronic inflammation conditions induced by CS.

Silencing ZIC2 abrogates tumorigenesis and anoikis resistance of non-small cell lung cancer cells by inhibiting Src/FAK signaling.

Aberrant expression of the zinc finger protein (ZIC) family has been extensively reported to contribute to progression and metastasis in multiple human cancers. However, the functional roles and underlying mechanisms of ZIC2 in non-small cell lung cancer (NSCLC) are largely unknown. In this study, ZIC2 expression was evaluated using qRT-PCR, western blot, and immunohistochemistry, respectively. Animal experiments in vivo and functional assays in vitro were performed to investigate the role of ZIC2 in NSCLC. Luciferase assays and chromatin immunoprecipitation (ChIP) were carried out to explore the underlying target involved in the roles of ZIC2 in NSCLC. Here, we reported that ZIC2 was upregulated in NSCLC tissues, and high expression of ZIC2 predicted worse overall and progression-free survival of NSCLC patients. Silencing ZIC2 repressed tumorigenesis and reduced the anoikis resistance of NSCLC cells. Mechanical investigation further revealed that silencing ZIC2 transcriptionally inhibited Src expression and inactivated steroid receptor coactivator/focal adhesion kinase signaling, which further attenuated the anoikis resistance of NSCLC cells. Importantly, our results showed that the number of circulating tumor cells (CTCs) was positively correlated with ZIC2 expression in NSCLC patients. Collectively, our findings unravel a novel mechanism implicating ZIC2 in NSCLC, which will facilitate the development of anti-tumor strategies in NSCLC.

Anti-tumour effects of Keratin 6A in lung adenocarcinoma.

BACKGROUND: To examine the effects of Keratin 6A (KRT6A) protein on the proliferation, migration and invasion abilities of lung adenocarcinoma cells, and to analyse the relationship between the expression level of KRT6A protein and the survival prognosis of lung adenocarcinoma patients. METHODS: Western Blot was used to detect the expression of KRT6A protein in lung adenocarcinoma cell lines. CCK-8 experiment and colony formation assays were performed to detect the proliferation ability. Wound healing assay and transwell migration assay were conducted to detect the migration ability. Transwell invasion assay was conducted to detect the invasion ability. Immunohistochemistry was used to detect the expression of KRT6A protein in lung adenocarcinoma tissues. RESULTS: We first found that the expression of KRT6A protein in lung adenocarcinoma cell lines was low. After overexpressed KRT6A protein in lung adenocarcinoma cells, we then found that KRT6A protein could not only inhibit the proliferation ability of lung adenocarcinoma cells but also inhibit them migration and invasion abilities. In addition, we also found that there had obvious difference in the expression of KRT6A protein in between patients. And through further analysis, we finally discovered that high expression of KRT6A protein was related to favourable prognosis in lung adenocarcinoma patients. CONCLUSIONS: KRT6A protein inhibits the proliferation, migration and invasion abilities of lung adenocarcinoma cells, and high expression of KRT6A protein is a predictor of good prognosis in patients with lung adenocarcinoma.

Correlative analysis of gene mutation and clinical features in patients with non-small cell lung cancer.

BACKGROUND: Lung cancer is the main reason for death associated with cancer all over the world. In most cases of non-small cell lung cancer (NSCLC), patients only express one type of gene mutation, each gene mutation population has different clinicopathological features, and each is expressed differently in different regions of the population. At present, there are few studies on multiple driver genes and clinicopathological features of the population in Hunan, China. METHODS: From February 2016 to December 2017, the Department of Geriatric Respiratory Medicine of Xiangya Hospital of Central South University diagnosed 113 cases of NSCLC. Genetic testing of next-generation sequencing (NGS) was completed, and it conformed to the inclusion criteria. All cases were pathologically confirmed as NSCLC, with the tumor staging being based on the 8th edition of TNM classification. RESULTS: In this study, we included a total of 113 NSCLC cases, including 78 males and 35 females. Histological distributions were mainly adenocarcinoma (ADC, 78.76%) and squamous cell carcinoma (SCC, 21.24%). We found 71 cases had gene-mutations. There was one concurrent mutation of ALK and ROS1, one concurrent mutation of epidermal growth factor receptor (EGFR) and BRAF, one concurrent mutation of EGFR and MET, one concurrent mutation of MET and BRAF, and one concurrent mutation of EGFR and KRAS; there were two cases of concurrent mutation of EGFR and ERBB2. The distribution of each of the mutated genes was as follows: EGFR, 62.82%; ALK, 8.97%; ROS1, 5.13%; MET, 5.13%; ERBB2, 5.13%; RET, 0.00%; BRAF, 2.56%; KRAS, 10.26%. Our study found that in patients with EGFR mutation, the mutation rate of males was 32.05%, and the rate in females was 68.57% (P<0.01); the mutation rate in people aged 60 or above was 40.00% while for those aged lower than 60 it was 46.55% (P>0.05); the mutation rate of ADC was 52.81% and in SCC was 8.33% (P<0.01); the mutation rate in smokers was 32.84% and in non-smokers was 58.70% (P<0.05); the mutation rate in patients of IV stage was 47.37% and the rate in patients of non-IV stage was 22.22% (P>0.05). Our study found that among patients with ALK/ROS1/MET/ERBB2/BRAF/KRAS mutations, the mutation rate in men was 7.69%, 2.60%, 3.85%, 2.56%, 0.00%, and 8.97% respectively, and for females it was 2.86%, 5.71%, 2.86%, 5.71%, 5.71%, and 2.86% respectively; the mutation rate in patients aged 60 and older was 3.64%, 5.45%, 3.64%, 1.82%, 1.82%, and 9.09% respectively; the rate in patients aged lower 60 was 8.62%, 1.72%, 3.45%, 5.17%, 1.72%, and 5.17% respectively; the mutation rate of ADC was 6.74%, 3.37%, 3.37%, 4.49%, 2.25%, and 6.74% respectively, while for SCC, it was 4.17%, 4.17%, 4.17%, 0.00%, 0.00%, and 8.33% respectively; the mutation rate in smokers was 8.96%, 1.49%, 4.48%, 1.49%, 0.00%, and 10.45% respectively, while in non-smokers, it was 2.17%, 6.52%, 2.17%, 6.52%, 4.35%, and 2.17% respectively; the mutation rate in patients of IV stage was 7.37%, 4.21%, 2.11%, 4.21%, 2.11%, and 7.37% respectively, and in patients of non-IV stage, it was 0.00%, 0.00%, 11.11%, 0.00%, 0.00%, and 5.56% respectively. In ALK/ROS1/MET/ERBB2/BRAF/KRAS mutations, there was no statistically significant difference in gender, age, tissue type, smoking history, and stage. Our research shows that the distribution of each mutant type of EGFR mutation was as follows: exon 2, 1/74 (1.35%); exon 4, 1/74 (1.35%); exon 6, 1/74 (1.35%); exon 18, 1/74 (1.35%); exon 19, 25/74 (33.78%); exon 20, 12/74 (16.22%); exon 21, 19/74 (25.68%); exon 22, 1/74 (1.35%); and EGFR amplification, 13/74 (17.57%). CONCLUSIONS: (I) EGFR mutation was more common in non-smoking female patients with ADC and had no significant correlation with age and stage. (II) EGFR mutations were mainly concentrated in exon 19, 20, 21, and EGFR amplification. There was no significant statistical difference between mutations in exons 19, 20, 21, EGFR gene amplification and clinical features. (III) There was no statistically significant difference in the ALK/ROS1/MET/ERBB2/BRAF/KRAS mutations with gender, age, tissue type, smoking history, and tumor stage.