An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Augmented Lipocalin-2 Is Associated with Chronic Obstructive Pulmonary Disease and Counteracts Lung Adenocarcinoma Development.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator Lcn2 (lipocalin-2) was increased in normal airways from mice with knockout of the airway lineage gene Gprc5a (Gprc5a-/-) and that are prone to developing inflammation and LUAD. Yet, the role of LCN2 in lung inflammation and LUAD is poorly understood.Objectives: Delineate the role of Lcn2 induction in LUAD pathogenesis.Methods: Normal airway brushings, uninvolved lung tissues, and tumors from Gprc5a-/- mice before and after tobacco carcinogen exposure were analyzed by RNA sequencing. LCN2 mRNA was analyzed in public and in-house data sets of LUAD, lung squamous cancer (LUSC), chronic obstructive pulmonary disease (COPD), and LUAD/LUSC with COPD. LCN2 protein was immunohistochemically analyzed in a tissue microarray of 510 tumors. Temporal lung tumor development, gene expression programs, and host immune responses were compared between Gprc5a-/- and Gprc5a-/-/Lcn2-/- littermates.Measurements and Main Results:Lcn2 was progressively elevated during LUAD development and positively correlated with proinflammatory cytokines and inflammation gene sets. LCN2 was distinctively elevated in human LUADs, but not in LUSCs, relative to normal lungs and was associated with COPD among smokers and patients with LUAD. Relative to Gprc5a-/- mice, Gprc5a-/-/Lcn2-/- littermates exhibited significantly increased lung tumor development concomitant with reduced T-cell abundance (CD4+) and richness, attenuated antitumor immune gene programs, and increased immune cell expression of protumor inflammatory cytokines.Conclusions: Augmented LCN2 expression is a molecular feature of COPD-associated LUAD and counteracts LUAD development in vivo by maintaining antitumor immunity.

Pathogenesis of Tobacco-Associated Lung Adenocarcinoma Is Closely Coupled with Changes in the Gut and Lung Microbiomes.

Microbial dysbiosis has emerged as a modulator of oncogenesis and response to therapy, particularly in lung cancer. Here, we investigate the evolution of the gut and lung microbiomes following exposure to a tobacco carcinogen. We performed 16S rRNA-Seq of fecal and lung samples collected prior to and at several timepoints following (nicotine-specific nitrosamine ketone/NNK) exposure in Gprc5a-/- mice that were previously shown to exhibit accelerated lung adenocarcinoma (LUAD) development following NNK exposure. We found significant progressive changes in human-relevant gut and lung microbiome members (e.g., Odoribacter, Alistipes, Akkermansia, and Ruminococus) that are closely associated with the phenotypic development of LUAD and immunotherapeutic response in human lung cancer patients. These changes were associated with decreased short-chain fatty acids (propionic acid and butyric acid) following exposure to NNK. We next sought to study the impact of Lcn2 expression, a bacterial growth inhibitor, given our previous findings on its protective role in LUAD development. Indeed, we found that the loss of Lcn2 was associated with widespread gut and lung microbiome changes at all timepoints, distinct from those observed in our Gprc5a-/- mouse model, including a decrease in abundance and diversity. Our overall findings apprise novel cues implicating microbial phenotypes in the development of tobacco-associated LUAD.

Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer.

(1) The TBX2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) are markedly down-regulated in human non-small cell lung cancer (NSCLC) and exert tumor suppressor effects in lung malignancy. Yet, mechanisms underlying suppressed expression of the TBX2 subfamily in NSCLC are elusive. Here, we interrogated probable epigenetic mechanisms in suppressed expression of the TBX2 subfamily in human NSCLC. (2) TBX2 subfamily gene expression and methylation levels in NSCLC and normal lung tissues were surveyed using publicly available RNA-sequence and genome-wide methylation datasets. Methylation ß-values of the four genes were statistically compared between NSCLCs and normal lung tissues, correlated with gene expression levels, and interrogated with clinicopathological variables. Expression and methylation levels of TBXs were quantified in NSCLC cells using real-time PCR and methylation-specific PCR assays, respectively. Effects of the DNA methyltransferase inhibitor 5-azacytidine (Aza) on TBX2 subfamily expression were assessed in NSCLC cells. Impact of TBX2 subfamily expression on Aza-treated cells was evaluated by RNA interference. (3) All four TBXs were significantly hypermethylated in NSCLCs relative to normal lung tissues (p < 0.05). Methylation ß-values of the genes, with exception of TBX2, were significantly inversely correlated with corresponding mRNA expression levels (p < 0.05). We found no statistically significant differences in hypermethylation levels of the TBX2 subfamily by clinicopathological features including stage and tobacco history. Expression levels of the TBX genes were overall suppressed in NSCLC cells relative to normal alveolar cells. Members of the subfamily were significantly hypermethylated in all tested NSCLC cell lines relative to normal alveolar cells. Treatment with Aza induced the expression of the TBX2 subfamily concomitant with NSCLC cell growth inhibition. Further, simultaneous knockdown of the four TBX genes markedly reduced anti-growth effects of Aza in NSCLC cells. (4) Our study sheds light on new epigenetic profiles in the molecular pathogenesis of human NSCLC.

Somatic Mutations in Normal Tissues: Calm before the Storm.

We explore the phenomenon of somatic mutations, including those in cancer driver genes, that are present in healthy, normal-appearing tissues and their potential implications for cancer development. We also examine the landscape of these somatic mutations, discuss the role of clonal cell competition and external factors like inflammation in enhancing the fitness of mutant clones, and conclude by considering how understanding these mutations will aid in prevention and/or interception of cancer.

Inflammation mediated by gut microbiome alterations promotes lung cancer development and an immunosuppressed tumor microenvironment.

Accumulating evidence indicates that the gut microbiome influences cancer progression and therapy. We recently showed that progressive changes in gut microbial diversity and composition are closely associated with tobacco-associated lung adenocarcinoma (LUAD) in a human-relevant mouse model. Furthermore, we demonstrated that the loss of the antimicrobial protein Lcn2 in these mice, exacerbates pro-tumor inflammatory phenotypes while further reducing microbial diversity. Yet, how gut microbiome alterations impinge on LUAD development remains poorly understood. Here, we investigated the role of gut microbiome changes in LUAD development using fecal microbiota transfer and delineated a pathway by which gut microbiome alterations incurred by loss of Lcn2 fostered the proliferation of pro-inflammatory bacteria of the genus Alistipes, triggering gut inflammation. This inflammation propagated systemically, exerting immunosuppression within the tumor microenvironment, augmenting tumor growth through an IL-6-dependent mechanism and dampening response to immunotherapy. Corroborating our preclinical findings, we found that patients with LUAD with a higher relative abundance of Alistipes species in the gut showed diminished response to neoadjuvant immunotherapy. These insights reveal the role of microbiome-induced inflammation in LUAD and present new potential targets for interception and therapy.

Beyond bacteria: How the intratumor mycobiome modulates lung adenocarcinoma progression.

Increasing evidence suggests that tumors harbor diverse microbiomes, adding complexity to the tumor microenvironment. In this issue of Cancer Cell, Liu et al. highlight the role of the intratumor mycobiome, specifically Aspergillus sydowii, in promoting lung adenocarcinoma progression. A. sydowii enhances the recruitment and activation of myeloid-derived suppressor cells via IL-1ß signaling driven by the ß-glucan-mediated Dectin-1/CARD9 pathway.

Cell-by-Cell: Unlocking Lung Cancer Pathogenesis.

For lung cancers, cellular trajectories and fates are strongly pruned by cell intrinsic and extrinsic factors. Over the past couple of decades, the combination of comprehensive molecular and genomic approaches, as well as the use of relevant pre-clinical models, enhanced micro-dissection techniques, profiling of rare preneoplastic lesions and surrounding tissues, as well as multi-region tumor sequencing, have all provided in-depth insights into the early biology and evolution of lung cancers. The advent of single-cell sequencing technologies has revolutionized our ability to interrogate these same models, tissues, and cohorts at an unprecedented resolution. Single-cell tracking of lung cancer pathogenesis is now transforming our understanding of the roles and consequences of epithelial-microenvironmental cues and crosstalk during disease evolution. By focusing on non-small lung cancers, specifically lung adenocarcinoma subtype, this review aims to summarize our knowledge base of tumor cells-of-origin and tumor-immune dynamics that have been primarily fueled by single-cell analysis of lung adenocarcinoma specimens at various stages of disease pathogenesis and of relevant animal models. The review will provide an overview of how recent reports are rewriting the mechanistic details of lineage plasticity and intra-tumor heterogeneity at a magnified scale thanks to single-cell studies of early- to late-stage lung adenocarcinomas. Future advances in single-cell technologies, coupled with analysis of minute amounts of rare clinical tissues and novel animal models, are anticipated to help transform our understanding of how diverse micro-events elicit macro-scale consequences, and thus to significantly advance how basic genomic and molecular knowledge of lung cancer evolution can be translated into successful targets for early detection and prevention of this lethal disease.

Game of clones: Battles in the field of carcinogenesis.

Recent advances in bulk sequencing approaches as well as genomic decoding at the single-cell level have revealed surprisingly high somatic mutational burdens in normal tissues, as well as increased our understanding of the landscape of "field cancerization", that is, molecular and immune alterations in mutagen-exposed normal-appearing tissues that recapitulated those present in tumors. Charting the somatic mutational landscapes in normal tissues can have strong implications on our understanding of how tumors arise from mutagenized epithelium. Making sense of those mutations to understand the progression along the pathologic continuum of normal epithelia, preneoplasias, up to malignant tissues will help pave way for identification of ideal targets that can guide new strategies for preventing or eliminating cancers at their earliest stages of development. In this review, we will provide a brief history of field cancerization and its implications on understanding early stages of cancer pathogenesis and deviation from the pathologically "normal" state. The review will provide an overview of how mutations accumulating in normal tissues can lead to a patchwork of mutated cell clones that compete while maintaining an overall state of functional homeostasis. The review also explores the role of clonal competition in directing the fate of normal tissues and summarizes multiple mechanisms elicited in this phenomenon and which have been linked to cancer development. Finally, we highlight the importance of understanding mutations in normal tissues, as well as clonal competition dynamics (in both the epithelium and the microenvironment) and their significance in exploring new approaches to combatting cancer.

Chronic Exposure to Waterpipe Smoke Elicits Immunomodulatory and Carcinogenic Effects in the Lung.

Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using an experimental animal model that is prone to tobacco carcinogens and an exploratory observational analysis of human waterpipe smokers and nonsmokers. Mice exhibited elevated incidence of lung tumors following heavy WPS exposure (5 days/week for 20 weeks) compared to littermates with light WPS (once/week for 20 weeks) or control air. Lungs of mice exposed to heavy WPS showed augmented CD8+ and CD4+ T cell counts along with elevated protumor immune phenotypes including increased IL17A in T/B cells, PD-L1 on tumor and immune cells, and the proinflammatory cytokine IL1ß in myeloid cells. RNA-sequencing (RNA-seq) analysis showed reduced antitumor immune gene signatures in animals exposed to heavy WPS relative to control air. We also performed RNA-seq analysis of airway epithelia from bronchial brushings of cancer-free waterpipe smokers and nonsmokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe nonsmokers, harbored gene programs that were associated with poor clinical outcomes in patients with lung adenocarcinoma, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Our findings support the notion that WPS exhibits carcinogenic effects and constitutes a possible risk factor for lung cancer as well as warrant future studies that can guide evidence-based policies for mitigating waterpipe smoking. PREVENTION RELEVANCE: Potential carcinogenic effects of waterpipe smoking are very poorly understood despite its emergence as a socially acceptable form of smoking. Our work highlights carcinogenic effects of waterpipe smoking in the lung and, thus, accentuate the need for inclusion of individuals with exclusive waterpipe smoking in prevention and smoking cessation studies.

Insights Into Lung Cancer Immune-Based Biology, Prevention, and Treatment.

Lung cancer is the number one cause of cancer-related deaths. The malignancy is characterized by dismal prognosis and poor clinical outcome mostly due to advanced-stage at diagnosis, thereby inflicting a heavy burden on public health worldwide. Recent breakthroughs in immunotherapy have greatly benefited a subset of lung cancer patients, and more importantly, they are undauntedly bringing forth a paradigm shift in the drugs approved for cancer treatment, by introducing "tumor-type agnostic therapies". Yet, and to fulfill immunotherapy's potential of personalized cancer treatment, demarcating the immune and genomic landscape of cancers at their earliest possible stages will be crucial to identify ideal targets for early treatment and to predict how a particular patient will fare with immunotherapy. Recent genomic surveys of premalignant lung cancer have shed light on early alterations in the evolution of lung cancer. More recently, the advent of immunogenomic technologies has provided prodigious opportunities to study the multidimensional landscape of lung tumors as well as their microenvironment at the molecular, genomic, and cellular resolution. In this review, we will summarize the current state of immune-based therapies for cancer, with a focus on lung malignancy, and highlight learning outcomes from clinical and preclinical studies investigating the naïve immune biology of lung cancer. The review also collates immunogenomic-based evidence from seminal reports which collectively warrant future investigations of premalignancy, the tumor-adjacent normal-appearing lung tissue, pulmonary inflammatory conditions such as chronic obstructive pulmonary disease, as well as systemic microbiome imbalance. Such future directions enable novel insights into the evolution of lung cancers and, thus, can provide a low-hanging fruit of targets for early immune-based treatment of this fatal malignancy.

Cancer in Lebanon: A Review of Incidence Rates from 2008 to 2015 and Projections Till 2025.

Background and Objectives Cancer carries one of the heaviest burdens globally in terms of mortality. Lebanon is a middle-income Middle East country also plagued with cancer, as such a study and analysis of cancer trends and projections would serve a great benefit in the fight against the disease. Materials and Methods All data pertaining to cancers in Lebanon were extracted from the National Cancer Registry of Lebanon Web site. Data were analyzed to produce trends over the years of our study (2008-2015). Ten-year projections were further calculated for the top cancers by the primary site using logarithmic models. Results The top cancers in Lebanon are the breast, lung, colorectal, bladder, and prostate. The top cancers affecting females are the breast, lung, and colorectal. The top cancers affecting males are the prostate, lung, and bladder. Cancer cases are projected to increase in Lebanon over the next 10 years. Conclusion Lebanon had a steady incidence rate of cancer cases during the time of our study. A more complete understanding of cancer trends and their ultimate reduction will require further research into the origins of specific cancers and the means of prevention and control.

Genomics of adult and pediatric solid tumors.

Different types of cancers exhibit disparate spectra of genomic alterations (germline and/or somatic). These alterations can include single nucleotide variants (SNVs), copy number alterations (CNAs) or structural changes (e.g. gene fusions and chromosomal rearrangements). Identification of those genomic alterations has provided the opportune element to derive new strategies for molecular-based precision medicine of adult and pediatric cancers including risk assessment, non-invasive detection, molecular diagnosis and personalized therapy. Moreover, it is now becoming clear that the spectra of genomic-based alterations and mechanisms in pediatric malignancies are different from those predominantly occurring in adult cancer. Adult cancers on average exhibit substantially higher mutational burdens compared with the vast majority of childhood tumors. Accumulating evidence also suggests that the type of genomic alterations frequently encountered in adult cancers is different from those observed in pediatric malignancies. In this review, we discuss the state of knowledge on adult and pediatric cancer genomes (or "mutatomes"), specifically focusing on solid tumors. We present an overview of mutational signatures and processes in cancer as well as comprehensively compare and contrast the diverse spectra of genomic alterations (somatic and familial) among major adult and pediatric solid tumors. The review also discusses the role of genomics in molecular-based precision medicine of adult and pediatric solid malignancies as well as comprehending resistance mechanisms to various targeted therapies. In addition, we present a perspective that discusses upon emerging concepts in cancer genomics including intratumoral heterogeneity, the precancer (premalignant) genome as well as the interface between the host immune response and tumor genome - immunogenomics - as they relate to adult and pediatric tumors.

Smoking and Lung Cancer: A Geo-Regional Perspective.

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents the most frequently diagnosed subtype of this morbid malignancy. NSCLC is causally linked to tobacco consumption with more than 500 million smokers worldwide at high risk for this fatal malignancy. We are currently lagging in our knowledge of the early molecular (e.g., genomic) effects of smoking in NSCLC pathogenesis that would constitute ideal markers for early detection. This limitation is further amplified when considering the variable etiologic factors in NSCLC pathogenesis among different regions around the globe. In this review, we present our current knowledge of genomic alterations arising during early stages of smoking-induced lung cancer initiation and progression, including discussing the premalignant airway field of injury induced by smoking. The review also underscores the wider spectra and higher age-adjusted rates of tobacco (e.g., water-pipe smoke) consumption, along with elevated environmental carcinogenic exposures and relatively poorer socioeconomic status, in low-middle income countries (LMICs), with Lebanon as an exemplar. This "cocktail" of carcinogenic exposures warrants the pressing need to understand the complex etiology of lung malignancies developing in LMICs such as Lebanon.