Tobacco smoking and somatic mutations in human bronchial epithelium.

Tobacco smoking causes lung cancer1-3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6-10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the pleura, with few available treatment options. We recently reported that loss of function of the nuclear deubiquitinase BRCA1-associated protein 1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptor 4 (DR4) and death receptor 5 (DR5). Using tissue microarrays of tumor samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptor expression. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wt-BAP1, but not catalytically inactive BAP1 mutant, reduced promoter activities of DR4 and DR5, suggesting deubiquitinase activity is required for the regulation of gene expression. Co-immunoprecipitation studies demonstrated direct binding of BAP1 to the transcription factor Ying Yang 1 (YY1), and chromatin immunoprecipitation assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Knockdown of YY1 also increased DR4 and DR5 expression and sensitivity to TRAIL. These results suggest that BAP1 and YY1 cooperatively repress transcription of TRAIL receptors. Our finding that BAP1 directly regulates the extrinsic apoptotic pathway will provide new insights into the role of BAP1 in the development of MPM and other cancers with frequent BAP1 mutations.

Lrig1 expression identifies airway basal cells with high proliferative capacity and restricts lung squamous cell carcinoma growth.

BACKGROUND: Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival. METHODS AND RESULTS: Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression. CONCLUSION: Together, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.

Investigating chromosomal instability in long-term survivors with glioblastoma and grade 4 astrocytoma.

Background: Only a small group of patients with glioblastoma multiforme (GBM) survives more than 36 months, so-called long-term survivors. Recent studies have shown that chromosomal instability (CIN) plays a prognostic and predictive role among different cancer types. Here, we compared histological (chromosome missegregation) and bioinformatic metrics (CIN signatures) of CIN in tumors of GBM typical survivors (≤36 months overall survival), GBM long-term survivors and isocitrate dehydrogenase (IDH)-mutant grade 4 astrocytomas. Methods: Tumor sections of all gliomas were examined for anaphases and chromosome missegregation. Further CIN signature activity analysis in the The Cancer Genome Atlas (TCGA)-GBM cohort was performed. Results: Our data show that chromosome missegregation is pervasive in high grade gliomas and is not different between the 3 groups. We find only limited evidence of altered CIN levels in tumors of GBM long-term survivors relative to the other groups, since a significant depletion in CIN signature 11 relative to GBM typical survivors was the only alteration detected. In contrast, within IDH-mutant grade 4 astrocytomas we detected a significant enrichment of CIN signature 5 and 10 activities and a depletion of CIN signature 1 activity relative to tumors of GBM typical survivors. Conclusions: Our data suggest that CIN is pervasive in high grade gliomas, however this is unlikely to be a major contributor to the phenomenon of long-term survivorship in GBM. Nevertheless, further evaluation of specific types of CIN (signatures) could have prognostic value in patients suffering from grade 4 gliomas.

Early human lung immune cell development and its role in epithelial cell fate.

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1ß drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1ß-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.

Toll-like receptor 2 orchestrates a tumor suppressor response in non-small cell lung cancer.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

Cell-intrinsic differences between human airway epithelial cells from children and adults.

The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells, and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium was similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony formation ability, sustained in vitro growth, and outcompeted adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states.

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non-small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. SIGNIFICANCE: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355.

Immune Surveillance in Clinical Regression of Preinvasive Squamous Cell Lung Cancer.

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.

Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions.

The molecular alterations that occur in cells before cancer is manifest are largely uncharted. Lung carcinoma in situ (CIS) lesions are the pre-invasive precursor to squamous cell carcinoma. Although microscopically identical, their future is in equipoise, with half progressing to invasive cancer and half regressing or remaining static. The cellular basis of this clinical observation is unknown. Here, we profile the genomic, transcriptomic, and epigenomic landscape of CIS in a unique patient cohort with longitudinally monitored pre-invasive disease. Predictive modeling identifies which lesions will progress with remarkable accuracy. We identify progression-specific methylation changes on a background of widespread heterogeneity, alongside a strong chromosomal instability signature. We observed mutations and copy number changes characteristic of cancer and chart their emergence, offering a window into early carcinogenesis. We anticipate that this new understanding of cancer precursor biology will improve early detection, reduce overtreatment, and foster preventative therapies targeting early clonal events in lung cancer.

Preinvasive disease of the airway.

Squamous cell carcinoma of the lung arises from preinvasive progenitors in the central airways. The archetypal model appears to be a stepwise morphological progression until there is invasion of the basement membrane. However, not every lesion appears to follow this course and many individuals can have stable disease, or indeed regress to normal epithelium. From our increased understanding of the molecular pathology it is becoming apparent that the respiratory epithelium accumulates progressive genetic and epigenetic insults in response to carcinogens. Still, little is known about how to predict those 'at risk' of progression, and it is likely that in the future molecular signatures will underpin prediction models of developing invasive lung cancer. Currently, autofluorescence bronchoscopy gives us the ability to follow the natural history of these lesions, with the prospect that detecting and treating lesions early may improve survival. However, treatment remains controversial, and radical therapies are offered to individuals with carcinoma in situ who may never develop invasive cancer. This has paved the way for the use of minimally invasive bronchoscopic treatments, which, while apparently effective, have not been tested in randomised controlled trials. In this paper we describe the known biology and natural history of preinvasive lesions and review the current treatment strategies.