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Chronic Cigarette Smoke-Induced Epigenomic Changes Precede Sensitization of Bronchial Epithelial Cells to Single-Step Transformation by KRAS Mutations
Abstract
We define how chronic cigarette smoke-induced time-dependent epigenetic alterations can sensitize human bronchial epithelial cells for transformation by a single oncogene. The smoke-induced chromatin changes include initial repressive polycomb marking of genes, later manifesting abnormal DNA methylation by 10 months. At this time, cells exhibit epithelial-to-mesenchymal changes, anchorage-independent growth, and upregulated RAS/MAPK signaling with silencing of hypermethylated genes, which normally inhibit these pathways and are associated with smoking-related non-small cell lung cancer. These cells, in the absence of any driver gene mutations, now transform by introducing a single KRAS mutation and form adenosquamous lung carcinomas in mice. Thus, epigenetic abnormalities may prime for changing oncogene senescence to addiction for a single key oncogene involved in lung cancer initiation.
... 42 Since both tumors and normal lung tissues are composed of multiple cell types, including epithelial cells, fibroblasts, hematopoietic cells, and endothelial cells, DNAme and gene expression profiles collected at the tissue level (''bulk'') may not accurately reflect alterations present in specific cell types. 43,44 Since the majority of DNAme alterations in lung cancers have been found to occur in epithelial cells, 45,46 we aimed at identifying DNAme alterations specific to the epithelial cell population. To resolve the confounding effects of other cell types, we leveraged previously validated computational methods to estimate the proportions of epithelial cells and infer epithelial-specific methylomes and transcriptomes ( Figure S3; STAR Methods). ...
... We then validated our results using an independent dataset of lung cancer epithelial cells. 46 Our results showed that the majority (86.6%-88.4%) of the differentially methylated CpGs identified from the independent dataset were also detected using our deconvoluted data for epithelial cells ( Figure S4). To further demonstrate the effectiveness of the deconvolution procedures, we compared the hypo-and hypermethylated CpGs identified using the bulk tissue data with those identified from the deconvoluted data. ...
... To validate the deconvolution results, we leveraged an independent dataset of human lung epithelial cells. 46 In this dataset, the DNAme data were collected from the HM450 array. We compared the DNAme profiles in KRAS-transformed lung epithelial cells (n = 4 replicates) to normal controls (n = 4 replicates). ...
DNA methylation (DNAme) is a major epigenetic factor influencing gene expression with alterations leading to cancer and immunological and cardiovascular diseases. Recent technological advances have enabled genome-wide profiling of DNAme in large human cohorts. There is a need for analytical methods that can more sensitively detect differential methylation profiles present in subsets of individuals from these heterogeneous, population-level datasets. We developed an end-to-end analytical framework named "EpiMix" for population-level analysis of DNAme and gene expression. Compared with existing methods, EpiMix showed higher sensitivity in detecting abnormal DNAme that was present in only small patient subsets. We extended the model-based analyses of EpiMix to cis-regulatory elements within protein-coding genes, distal enhancers, and genes encoding microRNAs and long non-coding RNAs (lncRNAs). Using cell-type-specific data from two separate studies, we discover epigenetic mechanisms underlying childhood food allergy and survival-associated, methylation-driven ncRNAs in non-small cell lung cancer.
... In addition, smoking might induce chronic inflammation and reactive oxygen species, leading to aberrant methylation followed by the recruitment of the silencing complex (DNMT1, DNMT3B, and members of Polycomb repressive complex 4) [165]. Furthermore, Vaz et al. demonstrated that chronic exposure to cigarette smoke induced abnormal methylation in human bronchial epithelial cells at genes enriched in stem cell differentiation and embryonic morphogenesis ontology [166]. They hypothesized that this exposure transitions cells to a more stem-cell-like nature via epigenetic changes, activating key signaling events and sensitizing cells to transformation by a single oncogenic event (e.g., KRAS mutations). ...
... The dynamic remodeling of DNAm is essential for lung development and cell fate decisions as stem cells exit pluripotency [195]. Many studies have shown that DNA hypermethylation at key developmental genes occupied by the Polycomb complex in embryonic stem cells is a common hallmark in many tumor types, including lung cancers [128,166,196]. The current model suggests that DNA hypermethylation could result in shifting the balance towards the silencing of these developmental genes. ...
... The knowledge would be essential to understand the underlying mechanisms driving tumorigenesis of different subtypes, identify biomarkers for accurate differential diagnosis, and develop effective personalized treatments. • While in lung cancers from smokers, tobacco-smoking carcinogens can provide a "fertile ground" for oncogenic mutations that drive tumor development [166] even in the early stages of lung carcinogenesis [199]. However, which exogenous/endogenous factors (e.g., environmental pollutants, inflammation, aging, chronic cellular stress) drive the epigenetic transformation of lung cancers in the absence of smoking carcinogens is unclear and warrants further investigation. ...
Lung cancer is the major leading cause of cancer-related mortality worldwide. Multiple epigenetic factors—in particular, DNA methylation—have been associated with the development of lung cancer. In this review, we summarize the current knowledge on DNA methylation alterations in lung tumorigenesis, as well as their associations with different histological subtypes, common cancer driver gene mutations (e.g., KRAS, EGFR, and TP53), and major epidemiological risk factors (e.g., sex, smoking status, race/ethnicity). Understanding the mechanisms of DNA methylation regulation and their associations with various risk factors can provide further insights into carcinogenesis, and create future avenues for prevention and personalized treatments. In addition, we also highlight outstanding questions regarding DNA methylation in lung cancer to be elucidated in future studies
... The condition in which demethylation and hypermethylation are present in various parts of the entire gene is called the aberrant DNA methylation. The progression of aberrant DNA methylation changes are key regulators of the aging process and contributors to the development of aging-associated diseases [207][208][209][210][211][212][213], including neoplastic growth [214][215][216][217][218] and senescence [219][220][221][222]. ...
... DNA methylation is susceptible to various lifestyle and living environments, such as environmental air pollution [223][224][225][226][227], smoking [218,[228][229][230][231][232], and excessive alcohol consumption [233][234][235][236]. It has been reported that the changes observed on DNA methylation induced by these environmental factors with negative health effects have similarities to those associated with aging-associated diseases, such as malignant transformation [218,232,237], CVDs [231,238], and the acceleration of senescence [229,230,239]. ...
... DNA methylation is susceptible to various lifestyle and living environments, such as environmental air pollution [223][224][225][226][227], smoking [218,[228][229][230][231][232], and excessive alcohol consumption [233][234][235][236]. It has been reported that the changes observed on DNA methylation induced by these environmental factors with negative health effects have similarities to those associated with aging-associated diseases, such as malignant transformation [218,232,237], CVDs [231,238], and the acceleration of senescence [229,230,239]. Contrarily, lifestyle habits considered having favorable consequences for health, such as exercise [240][241][242][243] and dietary restriction for obesity [244], also alter DNA methylation status, which is the opposite of the changes observed during aging. ...
Polyamines, spermidine and spermine, are synthesized in every living cell and are therefore contained in foods, especially in those that are thought to contribute to health and longevity. They have many physiological activities similar to those of antioxidant and anti-inflammatory substances such as polyphenols. These include antioxidant and anti-inflammatory properties, cell and gene protection, and autophagy activation. We have first reported that increased polyamine intake (spermidine much more so than spermine) over a long period increased blood spermine levels and inhibited aging-associated pathologies and pro-inflammatory status in humans and mice and extended life span of mice. However, it is unlikely that the life-extending effect of polyamines is exerted by the same bioactivity as polyphenols because most studies using polyphenols and antioxidants have failed to demonstrate their life-extending effects. Recent investigations revealed that aging-associated pathologies and lifespan are closely associated with DNA methylation, a regulatory mechanism of gene expression. There is a close relationship between polyamine metabolism and DNA methylation. We have shown that the changes in polyamine metabolism affect the concentrations of substances and enzyme activities involved in DNA methylation. I consider that the increased capability of regulation of DNA methylation by spermine is a key of healthy long life of humans.
... As expected, we observed significant enrichment for embryonic targets of the 309 PRC2 ( Figure 1C), including HOX clusters, GATA3, PAX6, and CIMP-high biomarker G0S2 310 ( Supp Fig 2A), mirroring other CIMP-high cancers. 311 312 PRC2 targets may gain DNA methylation in mammalian tissues with aging (14,15,37,38). To 313 determine if CIMP-high methylation reflects tissue origin, we profiled the DNA methylome of 314 fetal and adult adrenals ( Figure 1D). ...
... CIMP-high 576 DNA hypermethylation is uniform, with many targets possessing binary and complete 577 methylation (8). These data, consistent with literature examining etiology and emergence of 578 CIMP-high (14,15), suggest acquisition of this signature is an early selection event in 579 adrenocortical carcinogenesis. 580 ...
Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin–activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin–containing complexes, and the epigenome. On chromatin, β-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, β-catenin bound histone methyltransferase EZH2. SF1/β-catenin and EZH2/β-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/β-catenin from chromatin and favored EZH2/β-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities.
Significance
Oncogenic β-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for β-catenin–driven cancers.
... Whereas cluster 1 was mostly associated with cytoskeleton and cell adhesion, and cluster 2 with nucleic acid binding, clusters 3 and, especially, 4 revealed a significant association of the target proteins with histone modifications and chromatin remodeling ( Fig 1B). Although recent data validate the critical role of PP2A in regulating transcriptional elongation (Huang et al, 2020;Vervoort et al, 2021), and epigenetic gene regulation has an important role in RAS-mediated oncogenesis (Vaz et al, 2017), the role of PP2A and RAS in phosphorylation-dependent regulation of epigenetic complexes is very poorly understood. Based on these notions, we focused our downstream analysis on RAS and PP2A convergence on epigenetic gene regulation and transcription. ...
... Epigenetic gene regulation has an established role in cancer initiation and progression (Laugesen & Helin, 2014;Baylin & Jones, 2016;Cheng et al, 2019;Quagliano et al, 2020). Several loss-offunction studies across different species have also demonstrated an integral role of epigenetic gene regulation in signal transduction, development, and malignant progression downstream of RAS proteins (Vaz et al, 2017). However, it has been surprisingly poorly known how RAS impacts phosphorylation of epigenetic proteins, and whether RAS activity toward epigenetic gene regulation is modulated by PP2A-mediated protein dephosphorylation. ...
RAS-mediated human cell transformation requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A). However, the phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168, and TP53BP1. We validate RAS- and PP2A-elicited regulation of HDAC1/2 chromatin recruitment, of RNF168-TP53BP1 interaction, and of gene expression. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter derepression and activation of oncogenic transcription. Transcriptional derepression by PP2A inhibition was associated with an increase in euchromatin and a decrease in global DNA methylation. Collectively, the results indicate that epigenetic protein complexes constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Furthermore, the work provides an important resource for future studies focusing on phosphoregulation of epigenetic gene regulation in cancer and in other RAS/PP2A-regulated cellular processes.
... Tobacco carcinogens induce characteristic genomic as well as epigenomic alterations including copy number variations and point mutations as well as aberrant DNA methylation that collectively induce genomic instability and malignant transformation in airway epithelial cells (3)(4)(5)(6)(7)(8)(9). The fact that cigarette smoke induces timeand dose-dependent chromatin alterations in human respiratory epithelial cells (10,11), which sensitize these cells to transformation by a single oncogenic event (e.g., KRAS mutations) (12) attest to the significance of epigenetic perturbations during pulmonary carcinogenesis. ...
... Cigarette smoking is the major cause of lung cancer deaths (1,2), and mutational signatures associated with cigarette smoking are well established in human cancer (83). Cigarette smoking has been linked to aberrant DNA methylation in lung cancers (8,9,12). We previously showed that FILIP1L is down-regulated by promoter methylation in normal human respiratory epithelial cells following short-term exposure to tobacco condensates (13). ...
Lung adenocarcinoma (LUAD) is the major subtype in lung cancer, and cigarette smoking is essentially linked to its pathogenesis. We show that downregulation of Filamin A interacting protein 1-like (FILIP1L) is a driver of LUAD progression. Cigarette smoking causes its downregulation by promoter methylation in LUAD. Loss of FILIP1L increases xenograft growth, and, in lung-specific knockout mice, induces lung adenoma formation and mucin secretion. In syngeneic allograft tumors, reduction of FILIP1L and subsequent increase in its binding partner, prefoldin 1 (PFDN1) increases mucin secretion, proliferation, inflammation, and fibrosis. Importantly, from the RNA-sequencing analysis of these tumors, reduction of FILIP1L is associated with upregulated Wnt/β-catenin signaling, which has been implicated in proliferation of cancer cells as well as inflammation and fibrosis within the tumor microenvironment. Overall, these findings suggest that down-regulation of FILIP1L is clinically relevant in LUAD, and warrant further efforts to evaluate pharmacologic regimens that either directly or indirectly restore FILIP1L-mediated gene regulation for the treatment of these neoplasms.
Significance
This study identifies FILIP1L as a tumor suppressor in LUADs and demonstrates that downregulation of FILIP1L is a clinically relevant event in the pathogenesis and clinical course of these neoplasms.
... Next, a list of smoking-related DNA methylation probes was obtained from a previous study conducted by Vaz et al. Vaz et al performed two repeated experiments with respect to chronic-cigarette-smoking-induced hypermethylated probes [49].The union of all reported probes was extracted and their levels stratified by the mutant subsets were compared. Somatic mutation status of KEAP1/NFE2L2-mutant patients was analyzed to extract mutational signatures using the SignatureAnalyzer [50]. ...
... Meanwhile, 8,981 hypermethylated probes were found in the C1 group, while 5,933 hypermethylated probes were found in the C2 group ( Figure 5B). Next, unique smoking-related probes were extracted according to Vaz et al [49]. Both P2 and C2 groups displayed a similar trend of hypermethylation compared with the P1 and C1 groups ( Figure 5C-D). ...
Backgrounds: Lung adenocarcinoma is one of the most common malignant tumors, in which KEAP1-NFE2L2 pathway is altered frequently. The biological features and intrinsic heterogeneities of KEAP1/NFE2L2-mutant lung adenocarcinoma remain unclear.
Methods: Multiplatform data from The Cancer Genome Atlas (TCGA) were acquired to identify two subtypes of lung adenocarcinoma harboring KEAP1/NFE2L2 mutations.
Bioinformatic analyses, including immune microenvironment, methylation level and mutational signature, were performed to characterize the intrinsic heterogeneities. Meanwhile, initial results were validated by using in silico assessment of common lung adenocarcinoma cell lines, which revealed consistent features of mutant subtypes. Furthermore, drug sensitivity screening was conducted based on public datasets.
Results: Two mutant subtypes (P1 and P2) of 89 patients were identified in TCGA. P2 patients had significantly higher levels of smoking and worse survival compared with P1 patients. The P2 subset was characterized by active immune microenvironment and more smoking-induced genomic alterations with respect to methylation and somatic mutations. Validations of the corresponding features in 20 mutant cell lines were achieved. Several compounds which were sensitive to mutant subtypes of lung adenocarcinoma were identified, such as inhibitors of PI3K/Akt and IGF1R signaling pathways.
Conclusions: KEAP1/NFE2L2-mutant lung adenocarcinoma showed potential heterogeneities. The intrinsic heterogeneities of KEAP1/NFE2L2 were associated with immune microenvironment and smoking-related genomic aberrations.
... The concept of epigenetic priming has been proposed in other cancers emerging from chronic health conditions or environmental factors, such as obesity in colon cancer or cigarette smoke in lung cancer [6,7]. In this model, precancerous cells assume a new, epigenetically defined identity, which sensitises them to oncogenic transformation. ...
... Our results reflect the recently proposed 'epigenetic priming' model, whereby epigenetic changes induced by chronic exposure to cigarette smoke were shown to sensitise cells to an oncogenic KRAS mutation by promoting EMT in lung cancer, or the epigenomic alterations driven by obesity were detectable in precancerous colonic epithelium [7,67]. Importantly, while many of the genes affected by epigenetic priming are not necessarily cancer drivers, in the case of hypomethylated/upregulated genes such as UBD and CREB5, these genes have been linked to prognosis and disease outcome [56,68,69]. ...
Hepatocellular carcinomas (HCCs) usually arise from chronic liver disease (CLD). Pre‐cancerous cells in chronically inflamed environments may be 'epigenetically primed', sensitising them to oncogenic transformation. We investigated whether epigenetic priming in CLD may affect HCC outcomes by influencing the genomic and transcriptomic landscapes of HCC. Analysis of DNA methylation arrays in ten paired CLD‐HCC identified 339 shared dysregulated CpG sites and 18 shared differentially methylated regions compared to healthy livers. These regions were associated with dysregulated expression of genes with relevance in HCC, including Ubiquitin D (UBD), Cytochrome P450 Family 2 Subfamily C Member 19 (CYP2C19) and O‐6‐Methylguanine‐DNA Methyltransferase (MGMT). Methylation changes were recapitulated in an independent cohort of nine paired CLD‐HCC. High CLD methylation score, defined using the 124 dysregulated CpGs in CLD and HCC in both cohorts, was associated with poor survival, increased somatic genetic alterations, and TP53 mutations in two independent HCC cohorts. Oncogenic transcriptional and methylation dysregulation is evident in CLD and compounded in HCC. Epigenetic priming in CLD sculpts the transcriptional landscape of HCC and creates an environment favouring the acquisition of genetic alterations, suggesting that the extent of epigenetic priming in CLD could influence disease outcome.
... Oncogenic mutations can further amplify this nongenetic heterogeneity, as seen in breast epithelial cell cultures where oncogenic mutations increase the rate of switching between nonstem and stem-like epithelial cells (12). An interplay between genetic and epigenetic alterations is likely to underlie complete malignant transformation (13). ...
... Beyond the role of mutations, epigenetic changes from age-related hypomethylation (68) likely contribute to the stability and rate of switching to an edge state with age. Though we do not find mutations underlying the edge cells in the pancreas, the edge state may represent a state primed for malignant transformation by oncogenic mutation (13) or other age-associated transcriptomic changes (17,69,70) Overall, our results support the notion of an edge transcriptomic state in healthy tissues that is premalignant. Pancreatic acinar cells likely switch between edge and non-edge states, although the time spent by cells in either state is unclear. ...
Pancreatic ductal adenocarcinoma (PDAC) tumors can originate either from acinar or ductal cells in the adult pancreas. We re-analyze multiple pancreas and PDAC single-cell RNA-seq datasets and find a subset of nonmalignant acinar cells, which we refer to as acinar edge (AE) cells, whose transcriptomes highly diverge from a typical acinar cell in each dataset. Genes upregulated among AE cells are enriched for transcriptomic signatures of pancreatic progenitors, acinar dedifferentiation, and several oncogenic programs. AE-upregulated genes are upregulated in human PDAC tumors, and consistently, their promoters are hypomethylated. High expression of these genes is associated with poor patient survival. The fraction of AE-like cells increases with age in healthy pancreatic tissue, which is not explained by clonal mutations, thus pointing to a nongenetic source of variation. The fraction of AE-like cells is also significantly higher in human pancreatitis samples. Finally, we find edge-like states in lung, liver, prostate, and colon tissues, suggesting that subpopulations of healthy cells across tissues can exist in pre-neoplastic states.
Significance
These findings show “edge” epithelial cell states with oncogenic transcriptional activity in human organs without oncogenic mutations. In the pancreas, the fraction of acinar cells increases with age.
... Further evaluation and validation allowed us to identify IGF1 as a potential immune-related critical gene for pulmonary vascular remodeling and fibrosis. Immunity is one of the critical factors involved in the pathogenesis of PH and PF besides genetic predisposition (25, 26), epigenetic regulation (27,28), metabolic derangement (26), and environment insults (28, 29). Both innate immunity and adaptive immunity contribute to all forms of PF and pulmonary artery vascular remodeling. ...
Pulmonary fibrosis (PF) and pulmonary hypertension (PH) have common pathophysiological features, such as the significant remodeling of pulmonary parenchyma and vascular wall. There is no effective specific drug in clinical treatment for these two diseases, resulting in a worse prognosis and higher mortality. This study aimed to screen the common key genes and immune characteristics of PF and PH by means of bioinformatics to find new common therapeutic targets. Expression profiles are downloaded from the Gene Expression Database. Weighted gene co-expression network analysis is used to identify the co-expression modules related to PF and PH. We used the ClueGO software to enrich and analyze the common genes in PF and PH and obtained the protein–protein interaction (PPI) network. Then, the differential genes were screened out in another cohort of PF and PH, and the shared genes were crossed. Finally, RT-PCR verification and immune infiltration analysis were performed on the intersection genes. In the result, the positive correlation module with the highest correlation between PF and PH was determined, and it was found that lymphocyte activation is a common feature of the pathophysiology of PF and PH. Eight common characteristic genes ( ACTR2, COL5A2, COL6A3, CYSLTR1, IGF1, RSPO3, SCARNA17 and SEL1L ) were gained. Immune infiltration showed that compared with the control group, resting CD4 memory T cells were upregulated in PF and PH. Combining the results of crossing characteristic genes in ImmPort database and RT-PCR, the important gene IGF1 was obtained. Knocking down IGF1 could significantly reduce the proliferation and apoptosis resistance in pulmonary microvascular endothelial cells, pulmonary smooth muscle cells, and fibroblasts induced by hypoxia, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β1 (TGF-β1), respectively. Our work identified the common biomarkers of PF and PH and provided a new candidate gene for the potential therapeutic targets of PF and PH in the future.
... Alteration of methylomes also bridges exogenous carcinogens and transformation by driver mutations; for example, chronic cigarette smoke replaces H3K4me3/H3K27me3 bivalent histone marks with 5mC at promoters of a set of low-expression genes and primes human bronchial epithelial cells for cancer initiation by a single KRAS mutation. 275 In addition to potentiating the oncogenic competence of driver mutations, methylation alteration plays a leading role in the initiation of some cancer types, which has been best demonstrated in some pediatric tumors with very few or no recurrent somatic mutations. For example, childhood ependymomas lack recurrent single nucleotide and focal copy number Fig. 4 Mutational cancer driver methylation modifier genes and the role of methylation dysregulation across the central dogma in tumor initiation and progression. ...
The proper transfer of genetic information from DNA to RNA to protein is essential for cell-fate control, development, and health. Methylation of DNA, RNAs, histones, and non-histone proteins is a reversible post-synthesis modification that finetunes gene expression and function in diverse physiological processes. Aberrant methylation caused by genetic mutations or environmental stimuli promotes various diseases and accelerates aging, necessitating the development of therapies to correct the disease-driver methylation imbalance. In this Review, we summarize the operating system of methylation across the central dogma, which includes writers, erasers, readers, and reader-independent outputs. We then discuss how dysregulation of the system contributes to neurological disorders, cancer, and aging. Current small-molecule compounds that target the modifiers show modest success in certain cancers. The methylome-wide action and lack of specificity lead to undesirable biological effects and cytotoxicity, limiting their therapeutic application, especially for diseases with a monogenic cause or different directions of methylation changes. Emerging tools capable of site-specific methylation manipulation hold great promise to solve this dilemma. With the refinement of delivery vehicles, these new tools are well positioned to advance the basic research and clinical translation of the methylation field.
... Since our NGEJ tissues were pathologically confirmed as inflammatory tissues but devoid of apparent BE, this result suggests that CGI hypermethylation may occur in inflamed GEJ. Interestingly, CGI hypermethylation has been observed in long-term-cultured colon organoids and cells upon prolonged exposure to cigarette smoke extract [29,30]. These data suggest that prolonged extrinsic pressure may result in DNA methylation changes at CGIs. ...
Background
As one of the most common malignancies, esophageal cancer has two subtypes, squamous cell carcinoma and adenocarcinoma, arising from distinct cells-of-origin. Distinguishing cell-type-specific molecular features from cancer-specific characteristics is challenging.
Results
We analyze whole-genome bisulfite sequencing data on 45 esophageal tumor and nonmalignant samples from both subtypes. We develop a novel sequence-aware method to identify large partially methylated domains (PMDs), revealing profound heterogeneity at both methylation level and genomic distribution of PMDs across tumor samples. We identify subtype-specific PMDs that are associated with repressive transcription, chromatin B compartments and high somatic mutation rate. While genomic locations of these PMDs are pre-established in normal cells, the degree of loss is significantly higher in tumors. We find that cell-type-specific deposition of H3K36me2 may underlie genomic distribution of PMDs. At a smaller genomic scale, both cell-type- and cancer-specific differentially methylated regions (DMRs) are identified for each subtype. Using binding motif analysis within these DMRs, we show that a cell-type-specific transcription factor HNF4A maintains the binding sites that it generates in normal cells, while establishing new binding sites cooperatively with novel partners such as FOSL1 in esophageal adenocarcinoma. Finally, leveraging pan-tissue single-cell and pan-cancer epigenomic datasets, we demonstrate that a substantial fraction of cell-type-specific PMDs and DMRs identified here in esophageal cancer are actually markers that co-occur in other cancers originating from related cell types.
Conclusions
These findings advance our understanding of DNA methylation dynamics at various genomic scales in normal and malignant states, providing novel mechanistic insights into cell-type- and cancer-specific epigenetic regulations.
... We performed cell growth assays in soft agar for the control cells, TET2 single KO, RYBP single KO, TET123 TKO and the RYBP/TET123 QKO cells (Fig. 5A-C). Confirming earlier data showing that HBEC3-KT cells are nontumorigenic (49,62), the parental control cells did not grow in soft agar and neither did TET2 and RYBP single knockouts cells (Fig. 5A-C). TET123 TKO cells showed few, very small colonies. ...
Hypermethylation of CpG islands (CGI) is a common feature of cancer cells and predominantly affects Polycomb-associated genomic regions. Elucidating the underlying mechanisms leading to DNA hypermethylation in human cancer could help identify chemoprevention strategies. Here, we evaluated the role of Polycomb complexes and 5-methylcytosine (5mC) oxidases in protecting CGIs from DNA methylation and observed that four genes coding for components of Polycomb repressive complex 1 (PRC1) are downregulated in tumors. Inactivation of RYBP, a key activator of variant PRC1 complexes, in combination with all three 5mC oxidases (TET proteins) in nontumorigenic bronchial epithelial cells led to widespread hypermethylation of Polycomb-marked CGIs affecting almost 4,000 target genes, which closely resembled the DNA hypermethylation landscape observed in human squamous cell lung tumors. The RYBP- and TET-deficient cells showed methylation-associated aberrant regulation of cancer-relevant pathways, including defects in the Hippo tumor suppressor network. Notably, the quadruple knockout cells acquired a transformed phenotype, including anchorage-independent growth and formation of squamous cell carcinomas in mice. This work provides a mechanism promoting hypermethylation of CGIs and shows that such hypermethylation can lead to cell transformation. The breakdown of a two-pronged protection mechanism can be a route towards genome-wide hypermethylation of CGIs in tumors.
Significance
Dysfunction of the Polycomb component RYBP in combination with loss of 5-methylcytosine oxidases promotes widespread hypermethylation of CpG islands in bronchial cells and induces tumorigenesis, resembling changes seen in human lung tumors.
... In rat lung, cigarette smoke leads to an enhanced expression of B1R (5x) and IL1β (30x), while no increase in levels of B2R or TNFα was observed . Nicotine also enhances EZH2 expression and EZH2 dependent gene silencing (Vaz et al., 2017;Kumari et al., 2018) which may contribute to the development of COPD (Anzalone et al., 2019). As discussed above, EZH2 is a negative regulator of ACE2 and a positive regulator of TMPRSS2. ...
Infection with SARS-CoV-2 coronavirus causes systemic, multi-faceted COVID-19 disease. However, knowledge connecting its intricate clinical manifestations with molecular mechanisms remains fragmented. Deciphering the molecular basis of COVID-19 at the whole-patient level is paramount to the development of effective therapeutic approaches. With this goal in mind, we followed an iterative, expert-driven process to compile data published prior to and during the early stages of the pandemic into a comprehensive COVID-19 knowledge model. Recent updates to this model have also validated multiple earlier predictions, suggesting the importance of such knowledge frameworks in hypothesis generation and testing. Overall, our findings suggest that SARS-CoV-2 perturbs several specific mechanisms, unleashing a pathogenesis spectrum, ranging from “a perfect storm” triggered by acute hyper-inflammation, to accelerated aging in protracted “long COVID-19” syndromes. In this work, we shortly report on these findings that we share with the community via 1) a synopsis of key evidence associating COVID-19 symptoms and plausible mechanisms, with details presented within 2) the accompanying “COVID-19 Explorer” webserver, developed specifically for this purpose (found at https://covid19.molecularhealth.com ). We anticipate that our model will continue to facilitate clinico-molecular insights across organ systems together with hypothesis generation for the testing of potential repurposing drug candidates, new pharmacological targets and clinically relevant biomarkers. Our work suggests that whole patient knowledge models of human disease can potentially expedite the development of new therapeutic strategies and support evidence-driven clinical hypothesis generation and decision making.
... Estos cambios afectan a 1) oncogenes, que son homólogos de los genes celulares normales y, cuando sufren mutaciones, determinan su activación y ganancia de la función; 2) genes supresores de tumores, que son genes del cáncer cuya pérdida de función por mutación elimina los sistemas de inhibición del control del crecimiento celular, y 3) factores de crecimiento. (11) Hasta un 19% de los pacientes con cáncer pulmonar no tienen inicialmente síntomas. ...
Introducción:
el cáncer de pulmón tiene elevada incidencia a nivel mundial. En cuanto a mortalidad, es la primera localización en ambos sexos, con una tasa de 58,0 por 100 000 y 32,5 por 100 000 en el año 2017 en hombre y mujeres respectivamente.
Objetivos:
identificar los factores relacionados con la supervivencia de los pacientes con cáncer de pulmón de células no pequeñas.
Métodos:
se realizó un estudio analítico y observacional de cohorte en pacientes atendidos con el diagnóstico de cáncer de pulmón de células no pequeñas en el servicio de Oncología de del hospital general universitario “Carlos Manuel de Céspedes” de Bayamo, Granma, en el período comprendido desde el 1º de enero del 2014 al 31 de diciembre de 2019.
Resultados:
el factor más relevante y de carácter independiente fue clasificar en estadio IV al incrementar el riesgo de morir a 1,304 veces (IC: 1,011-2,025; p: 0,000) seguido del estadio IIIB (HR: 1,070; IC: 1,004-2,113; p: 0,000). El modelo de regresión de Cox tiene una capacidad discriminativa adecuada para predecir los pacientes que fallecerán de los que no en pacientes con cáncer de pulmón de células no pequeñas (área: 0,727; IC: 0,516-0,737; p: 0,025).
Conclusiones:
el estadio del cáncer de pulmón de células no pequeñas tuvo una relación inversamente proporcional con la sobrevida de los pacientes; la edad avanzada incrementó el riesgo de morir. El modelo de regresión proporcional de Cox tuvo una capacidad discriminativa adecuada para precisar la menor supervivencia de los pacientes con el mencionado diagnóstico.
... Chronic cigarette smoke exposure (CSE) has been linked to inducing mutations involving C:G > A:T transversions 8 . Prolonged CSE of human bronchial epithelial cells (HBECs) has also been found to induce aberrant transcriptional control, promoting the transition to a mesenchymal phenotype [75][76][77] . These smoke-exposed HBECs acquire a mesenchymal phenotype, as indicated by elongation. ...
Tumor suppressor genes (TSGs) are often involved in maintaining homeostasis. Loss of tumor suppressor functions causes cellular plasticity that drives numerous types of cancer, including small-cell lung cancer (SCLC), an aggressive type of lung cancer. SCLC is largely driven by numerous loss-of-function mutations in TSGs, often in those encoding chromatin modifiers. These mutations present a therapeutic challenge because they are not directly actionable. Alternatively, understanding the resulting molecular changes may provide insight into tumor intervention strategies. We hypothesize that despite the heterogeneous genomic landscape in SCLC, the impacts of mutations in patient tumors are related to a few important pathways causing malignancy. Specifically, alterations in chromatin modifiers result in transcriptional dysregulation, driving mutant cells toward a highly plastic state that renders them immune evasive and highly metastatic. This review will highlight studies in which imbalance of chromatin modifiers with opposing functions led to loss of immune recognition markers, effectively masking tumor cells from the immune system. This review also discusses the role of chromatin modifiers in maintaining neuroendocrine characteristics and the role of aberrant transcriptional control in promoting epithelial-to-mesenchymal transition during tumor development and progression. While these pathways are thought to be disparate, we highlight that the pathways often share molecular drivers and mediators. Understanding the relationships among frequently altered chromatin modifiers will provide valuable insights into the molecular mechanisms of SCLC development and progression and therefore may reveal preventive and therapeutic vulnerabilities of SCLC and other cancers with similar mutations.
... Both histone deacetylation and DNA hypermethylation, which can change the structure of chromatin and lead to tumor suppressor genes being epigenetically silenced, have been related to nickel's carcinogenic effects (Law et al., 2019). Also, individuals' exposure to tobacco and secondhand smoke possesses many harmful effects due to chemicals found in smoke that damage DNA or lower the DNA repair mechanisms, bringing about genetic and epigenetic changes that promote the growth of cancer (Barrow et al., 2017;Vaz et al., 2017). ...
Cancer has been known since BC., with rare incidence. At that time, some scientists tried to treat cancer, and some tried to find out the reason(s) behind the occurrence of cancer, and they put up many hypotheses. Nowadays, with all progression in medical technology and diagnostic methods, the causes of cancer remain unclear. But the causes of cancer can be an interaction between a person's genetic factors and external carcinogens factors that can be divided into physical, chemical, and biological carcinogens. In this review, the risk factors of cancer were discussed.
... We suspect that this may be related to smoking, which needs to be further explored. 32,33 In terms of efficacy, the bevacizumab biosimilars have highly similar ORR (week 18), DCR (week 18), mDOR, mPFS, mOS and 1-year OS rate to the original drug. ORR is a direct measure of the efficacy of treatment, is approved by the FDA and the European Medicines Agency for comparing the antitumor activity of the putative biosimilar with that of the reference biologic, and has previously been used successfully for this purpose. ...
Introduction
The role of bevacizumab combined with paclitaxel and carboplatin in the first-line treatment of advanced non-squamous non-small-cell lung cancer (NSCLC) has been supported by a large number of data. However, whether bevacizumab biosimilars have the same efficacy and safety as the original drug is still controversial. This meta-analysis was designed to evaluate whether bevacizumab biosimilars have the same clinical efficacy and safety as the original drug in patients with advanced non-squamous NSCLC.
Methods
Electronic databases (PubMed, Embase, Cochrane, CNKI, Wanfang, and VIP) and the ClinicalTrail.gov website were extensively searched using relevant search criteria. We included phase III randomized controlled trials (RCTs) to compare the efficacy and safety of marketed biosimilars and Avastin in the first-line treatment of patients with advanced NSCLC. The risk of bias of the included studies was assessed using the RoB 2 assessment scale, and the RevMan 5.4 statistical software was used for meta-analysis.
Results
A total of 6360 patients were included in 11 RCTs. There was no statistical difference between the experimental group and the control group in terms of effectiveness [objective response rate (at week 18), disease control rate (at week 18), median duration of response, median progression-free survival, median overall survival (OS), and OS after 12 months]. In terms of safety [treatment-emergent adverse events (grade ⩾3) and treatment-related adverse events (grade ⩾3)], there was also no significant difference between biosimilars and Avastin.
Conclusions
The efficacy and safety of bevacizumab biosimilars in the treatment of advanced non-squamous NSCLC are highly similar to those of the original drug combined with paclitaxel and carboplatin, respectively.
... In this same line, more mechanistic studies have shown how spontaneous epigenetic lesions accumulated over time can facilitate oncogenic transformation, for example, in mouse colorectal organoids through the repression of key genes (Tao et al., 2019). Of course, time-associated alterations may also reflect the accumulation of lifestyle-related aggressions: for instance, long-term exposure to cigarette smoke condensate has been shown to produce epigenetic alterations leading to tumorigenesis in human lung cells (Belinsky et al., 2002;Vaz et al., 2017). With respect to the loss of DNAm, the commonalities between aging and cancer again appear to be related to cell division, and DNA hypomethylation has been observed at late-replicating, lamina-associated domains in both aging and cancer, with cancer once again manifesting stronger alterations (Dmitrijeva et al., 2018;Zhou et al., 2018). ...
Aging and cancer are clearly associated processes, at both the epidemiological and molecular level. Epigenetic mechanisms are good candidates to explain the molecular links between the two phenomena, but recent reports have also revealed considerable differences, particularly regarding the loss of DNA methylation in the two processes. The large‐scale generation and availability of genome‐wide epigenetic data now permits systematic studies to be undertaken which may help clarify the similarities and differences between aging and cancer epigenetic alterations. In addition, the development of epigenetic clocks provides a new dimension in which to investigate diseases at the molecular level. Here, we examine current and future questions about the roles of DNA methylation mechanisms as causal factors in the processes of aging and cancer so that we may better understand if and how aging‐associated epigenetic alterations lead to tumorigenesis. It seems certain that comprehending the molecular mechanisms underlying epigenetic clocks, especially with regard to somatic stem cell aging, combined with applying single‐cell epigenetic‐age profiling technologies to aging and cancer cohorts, and the integration of existing and upcoming epigenetic evidence within the genetic damage models of aging will prove to be crucial to improving understanding of these two interrelated phenomena. Aging and cancer are interrelated processes which share common epigenetic alterations. Nonetheless, there are differences in some of the DNA methylation changes which occur in both phenomena. The recent development of epigenetic clocks will help dissect the common and specific epigenetic characteristics of aging and cancer, although the mechanisms underlying epigenetic clocks are yet to be clarified, particularly in relation to somatic stem cell epigenetic aging.
... Lung cancer has the highest incidence among all cancer types and is the leading cause of cancer-related deaths (1); lung adenocarcinoma (LUAD) is its most common histological type. Several epidemiological investigations and experimental studies have attributed the onset and progression of LUAD primarily to environmental factors and genetic alterations (2)(3)(4). Given a large number of non-smokers with LUAD, previous theories based solely on environmental factors have been disproven and research attention has been re-focused on profound alterations in the genetic content. ...
The interaction between cancer-associated fibroblasts (CAFs) and the tumor microenvironment (TME) is a key factor for promoting tumor progression. In lung cancer, the crosstalk between CAFs and malignant and immune cells is expected to provide new directions for the development of immunotherapy. In this study, we have systematically analyzed a single-cell dataset and identified interacting genes between CAFs and other cells. Subsequently, a robust fibroblast-related score (FRS) was developed. Kaplan-Meier (KM) and ROC analyses showed its good predictive power for patient prognoses in the training set comprising of specimens from the cancer genome atlas (TCGA) and in three external validation sets from the Gene Expression Omnibus (GEO). Univariate and multivariate Cox regression analyses suggested that FRS was a significant prognostic factor independent of multiple clinical characteristics. Functional enrichment and ssGSEA analyses indicated that patients with a high FRS developed “cold” tumors with active tumor proliferation and immunosuppression capacities. In contrast, those with a low FRS developed “hot” tumors with active immune function and cell killing abilities. Genomic variation analysis showed that the patients with a high FRS possessed a higher somatic mutation burden and copy number alterations and were more sensitive to chemotherapy; patients with a low FRS were more sensitive to immunotherapy, particularly anti-PD1 therapy. Overall, these findings advance the understanding of CAFs in tumor progression and we generated a reliable FRS-based model to assess patient prognoses and guide clinical decision-making.
... Within the aforementioned percentage, 45% accounts for lung squamous cell carcinoma (LUSC) cases and 30% accounts for lung adenocarcinoma (LUAD) cases (2). Many epidemiological and experimental studies have attributed the occurrence and progression of LUAD mainly to environmental factors and genetic alterations (3,4). Recent multi-omics studies have shown that there are significant differences in the copy number variation (CNV) and methylation of LUAD subtypes with different prognoses (5,6), and the low expression of CNTN4 and RFTN1 predicts poorer clinical outcomes in LUAD patients (7). ...
Lung adenocarcinoma (LUAD) is one of the most common histological subtypes of lung cancer. The aim of this study was to construct consensus clusters based on multi-omics data and multiple algorithms. In order to identify specific molecular characteristics and facilitate the use of precision medicine on patients we used gene expression, DNA methylation, gene mutations, copy number variation data, and clinical data of LUAD patients for clustering. Consensus clusters were obtained using a consensus ensemble of five multi-omics integrative algorithms. Four molecular subtypes were identified. The CS1 and CS2 subtypes had better prognosis. Based on the immune and drug sensitivity predictions, we inferred that CS1 may be less responsive to immunotherapy and less sensitive to chemotherapeutic drugs. The high immune infiltration of CS2 cells may respond well to immunotherapy. Additionally, the CS2 subtype may also respond to EGFR molecular targeted therapy. The CS3 and CS4 subtypes were associated with poor prognosis. These two subtypes had more mutations, especially TP53 ones, as well as higher sensitivity to chemotherapeutics for lung cancer. However, CS3 was enriched in immune-related pathways and may respond to anti-PD1 immunotherapy. In addition, CS1 and CS4 were less sensitive to ferroptosis inhibitors. We performed a comprehensive analysis of the five types of omics data using five clustering algorithms to reveal the molecular characteristics of LUAD patients. These findings provide new insights into LUAD subtypes and potential clinical treatment strategies to guide personalized management and treatment.
... The function of EMT in small-airway fibrosis of COPD remains to be investigated. Cigarette smoke condensate induces EMT in cultured human airway epithelial cells in vitro [23][24][25][26][27][28] . Active EMT has been detected in the small and large airways of smokers and COPD patients 25,27,29-31 . ...
Small airway fibrosis is a common pathology of chronic obstructive pulmonary disease (COPD) and contributes to airflow obstruction. However, the underlying fibrogenic mechanism is poorly understood. Epithelial-mesenchymal transition (EMT) has been proposed as a driver of fibrosis. EMT occurs in the airways of COPD patients and smokers, but it remains elusive whether EMT may contribute to airway fibrosis. We previously reported that FBXO11 is a critical suppressor of EMT and Fbxo11 deficiency in mice causes neonatal lethality and EMT in epidermis. Here, we found that Fbxo11-deficient mouse embryonic lungs showed impaired epithelial differentiation, excess fibroblast cells surrounding the airways, and thickened interstitial mesenchyme. We further generated conditional mutant mice to ablate Fbxo11 selectively in the club airway epithelial cells in adult mice, which induced partial EMT in the airways. To determine the effect of EMT on airway fibrosis, Fbxo11 conditional mutant mice were exposed to cigarette smoke. Airway-specific loss of Fbxo11 markedly enhanced smoking-induced airway fibrotic remodeling and collagen deposition. Taken together, our study suggests that EMT in the airway epithelium exacerbates cigarette smoke-induced airway fibrosis.
... In particular, as previously discussed, widespread DNA hypermethylation targeting CpG islands (CIMP-high) is a feature of COC3 ACC and a marker of aggressive disease, frequently associated with constitutive cell-cycle activation, differentiation, and activation of Wnt/β-catenin signaling (4,23). However, the role of CIMP-high in transcriptional dysregulation has not been characterized, and it is possible that this is a bystander phenomenon secondary to rapid proliferation, widespread genomic instability (212), or an abnormal metabolic state (213). DNA hypermethylation is written by the DNA methyltransferases DNMT1 and DNMT3A/B, which are also regulated in a cell cycle-dependent manner by transcription factors of the E2F family-explaining the link with a constitutively active cell cycle observed in ACC (214,215). ...
The adrenal glands are paired endocrine organs that produce steroid hormones and catecholamines required for life. Adrenocortical carcinoma (ACC) is a rare and often fatal cancer of the peripheral domain of the gland, the adrenal cortex. Recent research in adrenal development, homeostasis, and disease have refined our understanding of the cellular and molecular programs controlling cortical growth and renewal, uncovering crucial clues into how physiologic programs are hijacked in early and late stages of malignant neoplasia. Alongside these studies, genome-wide approaches to examine adrenocortical tumors have transformed our understanding of ACC biology, and revealed that ACC is comprised of distinct molecular subtypes associated with favorable, intermediate, and dismal clinical outcomes. The homogeneous transcriptional and epigenetic programs prevailing each ACC subtype suggest likely susceptibility to any of a plethora of existing and novel targeted agents, with the caveat that therapeutic response may ultimately be limited by cancer cell plasticity. Despite enormous biomedical research advances in the last decade, the only potentially curative therapy for ACC to date is primary surgical resection, and up to 75% of patients will develop metastatic disease refractory to standard-of-care adjuvant mitotane and cytotoxic chemotherapy. A comprehensive, integrated and current bench-to-bedside understanding of our field's investigations into adrenocortical physiology and neoplasia is crucial to developing novel clinical tools and approaches to equip the one-in-a-million patient fighting this devastating disease.
... Previous studies have shown that smoking contributes to lung remodeling prior to COPD, where CSE induces bronchial epithelial damage by increasing the susceptibility to respiratory infections (47)(48)(49). During this process, cells tend to undergo EMT and anchor-independent growth (50). In the present study, 16-HBE cells exposed to CSE exhibited EMT characteristics in a dose-dependent manner, with the highest N-cadherin, slug and α-SMA protein expression levels and the lowest E-cadherin protein expression levels being observed following 20% CSE exposure. ...
Vitamin D3 supplementation has been previously reported to inhibit the occurrence and development of chronic obstructive pulmonary disease (COPD). However, the underlying mechanism remains unclear. Epithelial-mesenchymal transition (EMT) and fibrogenesis have been associated with the development of COPD. The aim of the present study was to investigate the potential effects and mechanism of vitamin D3 in an in vitro model of cigarette smoke (CS)-induced EMT and fibrosis, with specific focus on the role of club cell protein 16 (CC16). CS extract (CSE) at different concentrations (5, 10 and 20%) was used to treat 16-HBE cells to induce EMT and fibrogenesis following which they were treated with vitamin D3. Subsequently, the 20% CSE group was selected for further experiments, where 16-HBE cells were divided into the following five groups: The control group; the CSE group; the low-dose vitamin D3 group (250 nM); the medium-dose vitamin D3 group (500 nM); and the high-dose vitamin D3 group (1,000 nM). Western blot analysis was used to detect the protein expression levels of the EMT-related proteins E-cadherin, N-cadherin, Slug and α-SMA, fibrogenesis-related proteins collagen Ⅳ and fibronectin 1, proteins involved in the TGF-β1/SMAD3 signaling pathway and CC16. Immunofluorescence was used to measure the protein expression levels of E-cadherin, N-cadherin and collagen Ⅳ. Specific CC16 knockdown was performed using short hairpin RNA transfection to investigate the role of CC16. The results of the present study found that vitamin D3 could increase the protein expression level of CC16 to inhibit the activation of the TGF-β1/SMAD3 signaling pathway; thereby reducing the 20% increase in CSE-induced EMT- and fibrogenesis-related protein expression levels. Following CC16 knockdown, the inhibitory effects of vitamin D3 on EMT- and fibrogenesis-related protein expression were partially reversed. To conclude, these results suggest that vitamin D3 can inhibit the protein expression levels of EMT- and fibrogenesis-related proteins induced by CSE, at least partially through the function of CC16. These findings are expected to provide novel theoretical foundations and ideas for the pathogenesis and treatment of COPD.
... Our knowledge on epigenetic marks in IPF and their cell type-specific contribution to disease pathogenesis and progression is still very limited. However, it is well-known that IPF risk factors like cigarette smoke or particulate matter, for instance, induce epigenetic alterations in bronchial epithelial cells [146][147][148], indicating that such changes may be frequent in IPF. Furthermore, increased expression and activity of histone deacetylases in IPF has been localized to myofibroblasts, but also to aberrant basal cells in IPF [145]. ...
Idiopathic pulmonary fibrosis (IPF) is a fatal disease with incompletely understood aetiology and limited treatment options. Traditionally, IPF was believed to be mainly caused by repetitive injuries to the alveolar epithelium. Several recent lines of evidence, however, suggest that IPF equally involves an aberrant airway epithelial response, which contributes significantly to disease development and progression. In this review, based on recent clinical, high-resolution imaging, genetic, and single-cell RNA sequencing data, we summarize alterations in airway structure, function, and cell type composition in IPF. We furthermore give a comprehensive overview on the genetic and mechanistic evidence pointing towards an essential role of airway epithelial cells in IPF pathogenesis and describe potentially implicated aberrant epithelial signalling pathways and regulation mechanisms in this context. The collected evidence argues for the investigation of possible therapeutic avenues targeting these processes, which thus represent important future directions of research.
... As a consequence, Wnt signaling is augmented, driving the malignant progression of LUAD. Consistent with our data, oncogenic Kras mutations alone are insufficient to endow lung epithelial cells with aggressive tumorigenic properties for rapid LUAD development, but augmented Wnt signaling can cooperate to accelerate lung tumorigenesis (60)(61)(62)(63)(64). Therapeutically, our findings provide a rationale for targeting Wnt signaling pathway as a therapeutic option in lung cancers harboring TET mutations, and point to epigenetic interventions to correct aberrant methylation as one modality for lung cancer treatment. ...
Significance
Previous studies have identified the tumor-suppressive function of TET enzymes in hematological cancers. Given the differential mutational incidence and lacking functional validation, how TET contributes to carcinogenesis in solid tumors remains largely undefined. Here, we report that TET mutations co-occur with KRAS mutations, and such co-occurrence predicts poor survival in human LUAD. Using genetically engineered mouse models (GEMMs), we show that inactivation of TET cooperates with oncogenic KRAS to potentiate LUAD development, and that this effect is preferentially induced by augmented Wnt signaling as a consequence of impaired expression of Wnt-associated antagonists due to DNA hypermethylation. Our work reveals the tissue-specific and context-dependent roles of TET during carcinogenesis and implicates Wnt signaling as a therapeutic modality for TET -mutant lung tumors.
... Nevertheless, the single somatic mutation and copy number variation analysis in this article clearly signifies the importance of these EMT TFs during the development and progression of cancer in specific cell/tissue types. Significantly, besides facilitating the metastatic dissemination of cancer cells, EMT has also been observed during early stages of tumorigenesis (244,245), therefore suggesting a wider role for these EMT TFs during cancer development. it is quite evident that EMT is involved during various stages of carcinogenesis and is responsible for acquisition of different characteristics such as stem cell properties, chemoresistance, as well as resistance to immunotherapy by the cancer cells. ...
Epithelial-mesenchymal transition or EMT is an extremely dynamic process involved in conversion of epithelial cells into mesenchymal cells, stimulated by an ensemble of signaling pathways, leading to change in cellular morphology, suppression of epithelial characters and acquisition of properties such as enhanced cell motility and invasiveness, reduced cell death by apoptosis, resistance to chemotherapeutic drugs etc. Significantly, EMT has been found to play a crucial role during embryonic development, tissue fibrosis and would healing, as well as during cancer metastasis. Over the years, work from various laboratories have identified a rather large number of transcription factors including the master regulators of EMT, with the ability to regulate the EMT process directly. In this review, we put together these EMT transcription factors and discussed their role in the process. We have also tried to focus on their mechanism of action, their inter-dependency, and the large regulatory network they form. Subsequently, it has become clear that the composition and structure of the transcriptional regulatory network behind EMT probably varies based upon various physiological and pathological contexts, or even in a cell/tissue type dependent manner.
... This approach is advantageous in recapitulating pathophysiologic endogenous carcinogenic events and in unravelling key events taking place during early tumour initiation, knowledge which can prove valuable for the development of LUAD early detection of chemoprevention strategies. Along these lines, we recently showed that as early as two weeks following treatment of lineage-marked mice with urethane (ethyl carbamate, a chemical carcinogen contained in tobacco smoke) [76], Kras Q61R driver mutations accumulate specifically in club and not in ATII cells [20], in line with evidence from human airway epithelial cells found to be sensitised by tobacco smoke to a single-hit KRAS mutation [77] and from a massive parallel sequencing approach [78]. These results are also in accord with earlier studies that dictate that only club cells possess the cytochrome CYP2E1 [79,80] that is required to convert the tobacco pre-carcinogen urethane (ethyl carbamate) to carcinogenic derivatives vinyl carbamate and its epoxide [81,82], which in turn have a half-life of a few femtoseconds and can thus only injure the DNA of the same cell that metabolically activates them [83,84]. ...
The cellular origin of lung adenocarcinoma remains a focus of intense research efforts. The marked cellular heterogeneity and plasticity of the lungs, as well as the vast variety of molecular subtypes of lung adenocarcinomas perplex the field and account for the extensive variability of experimental results. While most experts would agree on the cellular origins of other types of thoracic tumours, great controversy exists on the tumour-initiating cells of lung adenocarcinoma, since this histologic subtype of lung cancer arises in the distal pulmonary regions where airways and alveoli converge, occurs in smokers as well as nonsmokers, is likely caused by various environmental agents, and is marked by vast molecular and pathologic heterogeneity. Alveolar type II, club, and their variant cells have all been implicated in lung adenocarcinoma progeny and the lineage hierarchies in the distal lung remain disputed. Here we review the relevant literature in this rapidly expanding field, including results from mouse models and human studies. In addition, we present a case for club cells as cells of origin of lung adenocarcinomas that arise in smokers.
... Ma et al. [28] found that smoking carcinogenesis was closely related to DNA methylation in the study of the epigenetic mechanism of tobacco carcinogenesis. In a long-term study, Vaz et al. [29] found that long-term exposure of human bronchial epithelial cells to cigarette smoke would cause high methylation of gene promoters and activate key signaling pathways that drive tumorigenesis. They also found that these methylation abnormalities were highly consistent with the methylation abnormalities in non-small cell lung cancer (NSCLC). ...
DNA methylation is a work of adding a methyl group to the 5th carbon atom of cytosine in DNA sequence under the catalysis of DNA methyltransferase (DNMT) to produce 5-methyl cytosine. Some current studies have elucidated the mechanism of lung cancer occurrence and causes of lung cancer progression and metastasis from the perspective of DNA methylation. Moreover, many studies have shown that smoking can change the methylation status of some gene loci, leading to the occurrence of lung cancer, especially central lung cancer. This review mainly introduces the role of DNA methylation in the pathogenesis, early diagnosis and screening, progression and metastasis, treatment, and prognosis of lung cancer, as well as the latest progress. We point out that methylation markers, sample tests, and methylation detection limit the clinical application of DNA methylation. If the liquid biopsy is to become the main force in lung cancer diagnosis, it must make efficient use of limited samples and improve the sensitivity and specificity of the tests. In addition, we also put forward our views on the future development direction of DNA methylation.
... Interestingly, in a model wherein hematopoietic progenitors are proposed to be the cells of origin in MM, an aberrant epigenetic program persisting through normal cell differentiation is implicated in tumor initiation [80]. Further analysis of this aberrant DNA methylation program revealed strong enrichment of biological functions associated with developmental regulation (Additional file 3: Figure S29), suggesting that a disruption in developmental pathways does not prevent differentiation into plasma cells but could play a key role in the initiation of MM and increase susceptibility to oncogene transformation in response to environmental changes [81]. Moreover, considering the link between age and cancer predisposition demonstrated by Tao et al. [79], we can assume that disturbance of developmental genes in MM is partially age-related. ...
Background
Cancer evolution depends on epigenetic and genetic diversity. Historically, in multiple myeloma (MM), subclonal diversity and tumor evolution have been investigated mostly from a genetic perspective.
Methods
Here, we performed an analysis of 42 MM samples from 21 patients by using enhanced reduced representation bisulfite sequencing (eRRBS). We combined several metrics of epigenetic heterogeneity to analyze DNA methylation heterogeneity in MM patients.
Results
We show that MM is characterized by the continuous accumulation of stochastic methylation at the promoters of development-related genes. High combinatorial entropy change is associated with poor outcomes in our pilot study and depends predominantly on partially methylated domains (PMDs). These PMDs, which represent the major source of inter- and intrapatient DNA methylation heterogeneity in MM, are linked to other key epigenetic aberrations, such as CpG island (CGI)/transcription start site (TSS) hypermethylation and H3K27me3 redistribution as well as 3D organization alterations. In addition, transcriptome analysis revealed that intratumor methylation heterogeneity was associated with low-level expression and high variability.
Conclusions
We propose that disrupted DNA methylation in MM is responsible for high epigenetic and transcriptomic instability allowing tumor cells to adapt to environmental changes by tapping into a pool of evolutionary trajectories.
... Indeed, the environmental risks might result in epigenomic modifications, with the alteration in key genes regulation contributing to the pathogenesis of IPF. To this purpose, exposure of cigarette smoke (CS) triggers DNA damagerelated chromatin binding changes, and alterations in DNA methylation, influencing gene transcription and the downstream response of cells to injury (Vaz et al., 2017). Overall, however, the mechanisms and the correlation between environmental risk factors, genetic predisposition, aging and IPF pathogenesis need to be further elucidated. ...
Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of idiopathic interstitial pneumonias, characterized by chronic and progressive fibrosis subverting the lung’s architecture, pulmonary functional decline, progressive respiratory failure, and high mortality (median survival 3 years after diagnosis). Among the mechanisms associated with disease onset and progression, it has been hypothesized that IPF lungs might be affected either by a regenerative deficit of the alveolar epithelium or by a dysregulation of repair mechanisms in response to alveolar and vascular damage. This latter might be related to the progressive dysfunction and exhaustion of the resident stem cells together with a process of cellular and tissue senescence. The role of endogenous mesenchymal stromal/stem cells (MSCs) resident in the lung in the homeostasis of these mechanisms is still a matter of debate. Although endogenous MSCs may play a critical role in lung repair, they are also involved in cellular senescence and tissue ageing processes with loss of lung regenerative potential. In addition, MSCs have immunomodulatory properties and can secrete anti-fibrotic factors. Thus, MSCs obtained from other sources administered systemically or directly into the lung have been investigated for lung epithelial repair and have been explored as a potential therapy for the treatment of lung diseases including IPF. Given these multiple potential roles of MSCs, this review aims both at elucidating the role of resident lung MSCs in IPF pathogenesis and the role of administered MSCs from other sources for potential IPF therapies.
... Overall, EMT is defined as a process for developmental processes such as gastrulation and neural crest migration that can be hijacked by cancer cells for enhancing their invasion ability [17,18]. Noteworthy, it has been reported that EMT is considered as a principal mechanism for carcinogenesis [19,20]. As EMT possesses various roles from physiological to pathological conditions, different kinds of EMT including type I (involved in development), type II (involved in wound healing) and type III (involved in cancer migration) [21]. ...
Epithelial-to-mesenchymal transition (EMT) mechanism is responsible for metastasis and migration of cancer cells to neighboring cells and tissues. Morphologically, epithelial cells are transformed to mesenchymal cells, and at molecular level, E-cadherin undergoes down-regulation, while an increase occurs in N-cadherin and vimentin levels. Increasing evidence demonstrates role of EMT in mediating drug resistance of cancer cells. On the other hand, paclitaxel (PTX) and docetaxel (DTX) are two chemotherapeutic agents belonging to taxene family, capable of inducing cell cycle arrest in cancer cells via preventing microtubule depolymerization. Aggressive behavior of cancer cells resulted from EMT-mediated metastasis can lead to PTX and DTX resistance. Upstream mediators of EMT such as ZEB1/2, TGF-β, microRNAs, and so on are involved in regulating response of cancer cells to PTX and DTX. Tumor-suppressing factors inhibit EMT to promote PTX and DTX sensitivity of cancer cells. Furthermore, three different strategies including using anti-tumor compounds, gene therapy and delivery.
... The advent of precision medicine has led to the identification of actionable driver mutations regardless of tumor site or origin [2]. The presence of this Darwinian dynamic drives cancer evolution in the process of natural selection of "driver" somatic mutations that give rise to persisting epigenetic changes responsible for proliferation and tumorigenesis [3,4]. The identification and the classification of these mutational drivers has unlocked the potential to therapeutically inhibit this process of perpetual autonomous expansion through targeted therapy [5][6][7][8][9]. ...
Metastasis continues to be the primary cause of all cancer-related deaths despite the recent advancements in cancer treatments. To evaluate the role of mutations in overall survival (OS) and treatment outcomes, we analyzed 957 metastatic patients with seven major cancer types who had available molecular testing results with a FoundationOne CDx® panel. The most prevalent genes with somatic mutations were TP53, KRAS, APC, and LRP1B. In this analysis, these genes had mutation frequencies higher than in publicly available datasets. We identified that the somatic mutations were seven mutually exclusive gene pairs and an additional fifty-two co-occurring gene pairs. Mutations in the mutually exclusive gene pair APC and CDKN2A showed an opposite effect on the overall survival. However, patients with CDKN2A mutations showed significantly shorter OS (HR: 1.72, 95% CI: 1.34–2.21, p < 0.001) after adjusting for cancer type, age at diagnosis, and sex. Five-year post metastatic diagnosis survival analysis showed a significant improvement in OS (median survival 28 and 43 months in pre-2015 and post-2015 metastatic diagnosis, respectively, p = 0.00021) based on the year of metastatic diagnosis. Although the use of targeted therapies after metastatic diagnosis prolonged OS, the benefit was not statistically significant. However, longer five-year progression-free survival (PFS) was significantly associated with targeted therapy use (median 10.9 months (CI: 9.7–11.9 months) compared to 9.1 months (CI: 8.1–10.1 months) for non-targeted therapy, respectively, p = 0.0029). Our results provide a clinically relevant overview of the complex molecular landscape and survival mechanisms in metastatic solid cancers.
... Human carcinogens in different content elicit detrimental effects to human bodies in genotoxic or non-genotoxic ways [145]. Evidence has shown that cigarette smoke causes oncogenic mutations and epigenetic changes [146,147]. Tobacco smoking can alter miRNA encoding genes [148][149][150]. Genes with aberrant levels further participate in a myriad of pathological processes, including tumorigenesis and tumor progression [151]. ...
As one of the most studied ribonucleic acid (RNA) modifications in eukaryotes, N6‐methyladenosine (m6A) has been shown to play a predominant role in controlling gene expression and influence physiological and pathological processes such as oncogenesis and tumor progression. Writer and eraser proteins, acting opposite to deposit and remove m6A epigenetic marks, respectively, shape the cellular m6A landscape, while reader proteins preferentially recognize m6A modifications and mediate fate decision of the methylated RNAs, including RNA synthesis, splicing, exportation, translation, and stability. Therefore, RNA metabolism in cells is greatly influenced by these three classes of m6A regulators. Aberrant expression of m6A regulators has been widely reported in various types of cancer, leading to cancer initiation, progression, and drug resistance. The close links between m6A and cancer shed light on the potential use of m6A methylation and its regulators as prognostic biomarkers and drug targets for cancer therapy. Given the notable effects of m6A in reversing chemoresistance and enhancing immune therapy, it is a promising target for combined therapy. Herein, we summarize the recent discoveries on m6A and its regulators, emphasizing their influences on RNA metabolism, their dysregulation and impacts in diverse malignancies, and discuss the clinical implications of m6A modification in cancer. As one of the best studied ribonucleic acid (RNA) modifications in eukaryotes, N6‐methyladenosine (m6A) plays a predominant role in controlling gene expression and further influences physiological and pathological processes, including oncogenesis and tumor progression. Here we review the functions of m6A and its regulators in RNA metabolism control, their oncogenic or tumor suppressive roles in diverse malignancies, as well as the application of m6A methylation in cancer diagnosis and therapeutics.
... 8 Transient effects such as tolerance and persistence have been suggested to provide initial resistance, while the addition of drugs then induces epigenetic reprogramming until secondary mutations convert the progression to relapse, and the cells become genetically hardwired. 5,56,57 The present study further suggested that this "rewiring" involving epigenetic chromatin modifications not only conferred initial fitness, but also facilitated the hardwiring process giving rise to inheritable resistance. ...
Drug resistance is a significant obstacle to effective cancer treatment. Drug resistance develops from initially reversible drug‐tolerant cancer cells, which offers therapeutic opportunities to impede cancer relapse. The mechanisms of resistance to proteasome inhibitor (PI) therapy have been investigated intensively; however, the ways by which drug‐tolerant cancer cells orchestrate their adaptive responses to drug challenges remain largely unknown. Here, we demonstrated that cyclin A1 suppression elicited the development of transient PI tolerance in mixed‐lineage leukemia (MLL) cells. This adaptive process involved reversible down‐regulation of cyclin A1, which promoted PI resistance through cell cycle arrest. PI‐tolerant MLL cells acquired cyclin A1 dependency, regulated directly by MLL protein. Loss of cyclin A1 function resulted in the emergence of drug tolerance, which was associated with patient relapse and reduced survival. Combination treatment with PI and deubiquitinating enzyme (DUB) inhibitors overcame this drug resistance by restoring cyclin A1 expression through chromatin crosstalk between histone H2B monoubiquitination and MLL‐mediated histone H3 lysine 4 methylation. These results reveal the importance of cyclin A1‐engaged cell cycle regulation in PI resistance in MLL cells, and suggest that cell cycle re‐entry by DUB inhibitors may represent a promising epigenetic therapeutic strategy to prevent acquired drug resistance.
... The risk of lung cancer in smokers was 30 times higher than that of nonsmokers. Tobacco smoke can lead to gene mutations, DNA damage, abnormal DNA methylation, DNA adduct formation, lung in ammation, oxidative stress, abnormal proliferation, abnormal differentiation, epithelial-mesenchymal transition (EMT) and so on [22][23][24][25][26]. ...
Background: With the popularization of high-resolution computed tomography (HRCT), the detection rate of synchronous multiple primary lung cancer (SMPLC) is increasing. We retrospectively analyzed the surgical results of SMPLC patients in our hospital to determine the best treatment, surgical prognosis and survival analysis.
Methods: A total of 78 SMPLC patients met the diagnostic criteria underwent complete resection and lymph node dissection or sampling without any preoperative induction therapy in the Department of Thoracic Surgery, Qilu Hospital, Cheeloo College of Medicine, Shandong University. We analyzed the postoperative survival rate, and further studied the relationship between survival rates and sex, age, preoperative symptoms, tumor location, tumor number, tumor size, lymph node metastasis, TNM stage, surgical type, surgical frequency, histopathologic types, vascular infiltration, visceral pleural invasion and postoperative therapy.
Results: Among 78 patients, the 1-,2-,3-,4- and 5-year disease free survival (DFS) rates were 93.42%, 86.84%, 77.78%, 62.96%, and 60.00%, respectively, while the 1-,2-,3-,4- and 5-year overall survival (OS) rates were 94.73%, 92.11%, 82.22%, 77.78%, and 65.00%, respectively. TNM stage of the largest tumor (II:HR=7.40,III:9.01,p=0.002) was an independent risk factor for DFS. Smoking history (HR=4.34,p=0.039) and TNM stage of the largest tumor (II:HR=9.38,III:9.42,p=0.003) were independent risk factors for overall survival.
Conclusions: First, SMPLC is different from intrapulmonary metastasis and its clinical stage is also different from the 8th (2015) edition TNM classification for lung cancer. Second, when pulmonary function permits, surgery (complete resection and lymph node dissection) is a significantly beneficial treatment for patients with SMPLC. Third, TNM stage of the largest tumor (II:HR=7.40,III:9.01,p=0.002) was an independent risk factor for DFS. Smoking history (HR=4.34,p=0.039) and TNM stage of the largest tumor (II:HR=9.38,III:9.42,p=0.003) were independent risk factors for overall survival.
Fusobacterium nucleatum (Fn) has been frequently detected in colorectal cancer (CRC). A high load of Fn has been associated with sub-types of CRCs, located in the proximal colon, exhibiting microsatellite instability-high (MSI-H), MLH1 promoter hypermethylation, the CpG island hypermethylation phenotype-high (CIMP-H), or BRAF mutation in some studies. Although these features characterize the sessile serrated pathway (SSP) of colon cancers, other studies have shown that Fn infection is associated with KRAS mutations mainly characteristic of non-serrated neoplasia. It is also not clear at what point the association of Fn infection with these genomic alterations is established during colorectal carcinogenesis. Here we show that MSI-H, MLH1 hypermethylation, BRAF mutation or KRAS mutations were independently associated with Fn infection in CRC. On the other hand, increasing Fn copy number in tissues was associated with increased probability to exhibit MSI-H, MLH1 hypermethylation or BRAF mutations but not KRAS mutations in CRC. We also show that Fn load was significantly less than that of CRC and no association was detected between BRAF/KRAS mutations or MLH1 hypermethylation and Fn infection in adenomas. Our combined data suggest that increasing loads of Fn during and/or after adenoma-carcinoma transition might promote SSP but not KRAS-driven colorectal carcinogenesis. Alternatively, Fn preferentially colonizes CRCs with SSP and KRAS mutations but can expand more in CRCs with SSP.
Altered DNA methylation (DNAm) might be a biological intermediary in the pathway from smoking to cancer. In this study, we investigated the contribution of differential blood DNAm to explain the association between smoking and lung cancer incidence. Blood DNAm was measured in 2321 Strong Heart Study (SHS) participants. Incident lung cancer was assessed as time to event diagnoses. We conducted mediation analysis, including validation using DNAm and paired gene expression data from the Framingham Heart Study (FHS). In the SHS, current versus never smoking and pack-years single-mediator models showed, respectively, 29 and 21 differentially methylated positions (DMPs) for lung cancer (14 of 20 available, and five of 14 available, replicated, respectively, in FHS) with statistically significant mediated effects. In FHS, replicated DMPs showed gene expression downregulation largely in trans, and were related to biological pathways in cancer. The multimediator model identified that DMPs annotated to the genes AHRR and IER3 jointly explained a substantial proportion of lung cancer. Thus, the association of smoking with lung cancer was partly explained by differences in baseline blood DNAm at few relevant sites. Experimental studies are needed to confirm the biological role of identified eQTMs and to evaluate potential implications for early detection and control of lung cancer.
Objectives:
E-cigarettes are the most commonly used nicotine containing products among youth. In vitro studies support the potential for e-cigarettes to cause cellular stress in vivo; however, there have been no studies to address whether exposure to e-liquid aerosols can induce cell transformation, a process strongly associated with pre-malignancy. We examined whether weekly exposure of human bronchial epithelial cell (HBEC) lines to e-cigarette aerosols would induce transformation and concomitant changes in gene expression and promoter hypermethylation.
Materials and methods:
An aerosol delivery system exposed three HBEC lines to unflavored e-liquid with 1.2% nicotine, 3 flavored products with nicotine, or the Kentucky reference cigarette once a week for 12 weeks. Colony formation in soft agar, RNA-sequencing, and the EPIC Beadchip were used to evaluate transformation, genome-wide expression and methylation changes.
Results:
Jamestown e-liquid aerosol induced transformation of HBEC2 and HBEC26, while unflavored and Blue Pucker transformed HBEC26. Cigarette smoke aerosol transformed HBEC4 and HBEC26 at efficiencies up to 3-fold greater than e-liquids. Transformed clones exhibited extensive reprogramming of the transcriptome with common and distinct gene expression changes observed between the cigarette and e-liquids. Transformation by e-liquids induced alterations in canonical pathways implicated in lung cancer that included axonal guidance and NRF2. Gene methylation, while prominent in cigarette-induced transformed clones, also affected hundreds of genes in HBEC2 transformed by Jamestown. Many genes with altered expression or epigenetic-mediated silencing were also affected in lung tumors from smokers.
Conclusions:
These studies show that exposure to e-liquid aerosols can induce a pre-malignant phenotype in lung epithelial cells. While the Food and Drug Administration banned the sale of flavored cartridge-based electric cigarettes, consumers switched to using flavored products through other devices. Our findings clearly support expanding studies to evaluate transformation potency for the major categories of e-liquid flavors to better inform risk from these complex mixtures.
Chronic cigarette smoke condensate (CSC) exposure is one of the preventable risk factors in the CS-induced lung cancer. However, understanding the mechanism of cellular transformation induced by CS in the lung remains limited. We investigated the effect of long term exposure of CSC in human normal lung epithelial Beas-2b cells, and chemopreventive mechanism of organosulphur garlic compounds, diallyl sulphide (DAS) and diallyl disulphide (DADS) using Next Generation Sequencing (NGS) transcriptomic analysis. CSC regulated 1077 genes and of these 36 genes are modulated by DAS while 101 genes by DADS. DAS modulated genes like IL1RL1 (interleukin-1 receptor like-1), HSPA-6 (heat shock protein family A, member 6) while DADS demonstrating ADTRP (Androgen-Dependent TFPI Regulating Protein), ANGPT4 (Angiopoietin 4), GFI1 (Growth Factor-Independent 1 Transcriptional Repressor), TBX2 (T-Box Transcription Factor 2), with some common genes like NEURL-1 (Neuralized E3-Ubiquitin Protein Ligase 1), suggesting differential effects between these two garlic compounds. They regulate genes by influencing pathways including HIF-1alpha, STAT-3 and matrix metalloproteases, contributing to the chemoprotective ability of organosulfur garlic compounds against CSC-induced cellular transformation. Taken together, we demonstrated CSC induced global gene expression changes pertaining to cellular transformation which potentially can be delayed with dietary chemopreventive phytochemicals like DS and DADS influencing alterations at the transcriptomic level.
Epithelial to mesenchymal transition (EMT) is a developmental cellular program driving metastasis and chemo-resistance in cancer, but its pharmacological treatment has been so far challenging. Targeting deregulated metabolic processes in cancer is emerging as a realistic therapeutic strategy. Here, we used an EMT-focussed integrative functional genomic approach and identified negative association of the short-chain fatty acids propionate and butanoate with EMT in non-small cell lung cancer (NSCLC) patients. Strikingly, in vitro treatment of lung cancer cell lines with propionate reinforced the epithelial transcriptional program promoting cell adhesion and reverting the aggressive and chemoresistant EMT phenotype. Propionate treatment reduced cells metastatic ability in nude mice and limited lymph nodal spread in a genetic NSCLC mouse model. Further analyses indicated chromatin remodeling via H3K27 acetylation (p300-mediated) as the mechanism shifting the EMT balance towards epithelial state upon propionate. Propionate administration could be tested in the clinic for reducing NSCLC aggressiveness.
Highlights
An EMT-centric investigation of metabolic processes in a comprehensive lung cancer transcriptome profiles identified negative associations between EMT and SCFAs (propionate and butyrate)
Propionate enhances the epithelial features both at the molecular and cellular levels
Pre-treatment of cells with propionate inhibits EMT associated processes including migration and sensitizes the cells to chemotherapeutic drug cisplatin
Oral administration of propionate inhibits EMT-mediated lung colonization ability of NSCLC cells, and lymph node metastasis in a genetic mouse NSCLC model
Molecular mechanistic investigation of propionate revealed chromatin remodelling through p300-mediated histone acetylation in E-cadherin gene regulation along with epithelial features reinforcement
BackgroundKRAS is commonly mutated in non-small cell lung cancer (NSCLC); however, the prognostic and predictive impact of each G12 substitution has not been fully elucidated. The approval of specific G12C inhibitors has modified the idea of KRAS “undruggability”, and although the first-line standard consists of immune checkpoint inhibitors (ICIs) with or without chemotherapy, as suggested at ASCO 2022, the outcome in KRAS-mutated population is still controversial.Methods
We retrospectively described the clinical and pathological characteristics of a homogeneous G12 mutated cohort of 219 patients treated in four Italian oncologic units. We evaluated the outcome (PFS at 18 months and OS at 30 months) of those who underwent standard first-line treatment according to PD-L1 status, focusing on differences across single mutations.ResultsIn the study population, 47.9% of patients harbor the KRAS G12C mutation; 20.5%, G12V; 17.4%, G12D; and 8.2%, G12A. Smoking was a common behavior of patients harboring transversions and transition mutations. PD-L1 expression does not show particular distribution in the case series, although we recorded a prevalence of PD-L1 <1% in G12V (51.4%) compared to G12A (26.7%). ICIs alone was the clinician’s choice in 32.7% of patients, and the chemo-immune combination in 17.3% of patients. We described the independent prognostic role of young age (p = 0.007), female gender (p = 0.016), and an ICI-based regimen (p = 0.034) regardless of mutations. Overall, our data confirm the worst prognostic value of G12V mutation apart from treatment choice unlike the other major mutations (C, D, and A) that showed a favorable trend in PFS.ConclusionsKRAS G12 mutations are confirmed to have different characteristics, and the outcome is influenced by ICI first-line regimen. This study provides valuable information for further analysis in the future.
The output of the genome is controlled by the interaction of transcription factors with the epigenome. Epigenetic processes such as DNA methylation, histone modification, histone variants, noncoding RNAs, and nucleosomal remodeling machines interact with each other to ensure stable states of gene expression. These processes can become dysregulated during aging, exposure to environmental stressors, and the development of cancer and other diseases. DNA methylation patterns can be relatively easily read by high throughput techniques and provide information reflecting the influence of the environment and aging on the functionality of the epigenome. Analysis of DNA methylation patterns, therefore, provides an exciting new route to understanding how the environment interacts with the epigenome to cause disease. Despite the promise of DNA methylation patterns for epidemiologic studies, caution in interpreting data from surrogate tissues is necessary and cellular heterogeneity can also complicate interpretation of the data. In addition, DNA methylation within the body of genes can influence the response of the genome to the environment. Hypomethylation of repetitive elements can lead to genomic instability and ectopic gene expression. Methylation of coding regions can directly increase the rate of spontaneous hydrolytic mutations and increase the mutational frequency induced by carcinogens and radiation. Epigenetic processes can therefore contribute in multiple ways to the development of human diseases particularly cancer.KeywordsHistone variantCytosine residueEpigenetic processNormal human bronchial epithelial cellSurrogate tissue
The presence and role of microbes in human cancers has come full circle in the last century. Tumors are no longer considered aseptic, but implications for cancer biology and oncology remain underappreciated. Opportunities to identify and build translational diagnostics, prognostics, and therapeutics that exploit cancer's second genome—the metagenome—are manifold, but require careful consideration of microbial experimental idiosyncrasies that are distinct from host‐centric methods. Furthermore, the discoveries of intracellular and intra‐metastatic cancer bacteria necessitate fundamental changes in describing clonal evolution and selection, reflecting bidirectional interactions with non‐human residents. Reconsidering cancer clonality as a multispecies process similarly holds key implications for understanding metastasis and prognosing therapeutic resistance while providing rational guidance for the next generation of bacterial cancer therapies. Guided by these new findings and challenges, this Review describes opportunities to exploit cancer's metagenome in oncology and proposes an evolutionary framework as a first step towards modeling multispecies cancer clonality. Gut and intratumoral microbiomes comprise cancer's “second” genome and influence cancer biology. Cancer type and stage‐specific tissue and blood‐derived microbes enable microbially‐informed cancer diagnostics and prognostics. Critically, intratumoral and intracellular microbes shape cancer cell fitness and clonality while having distinct selection pressures, motivating redefining clonal evolution as a multispecies process.
The pathogenesis of idiopathic pulmonary fibrosis (IPF) involves a complex interplay of cell types and signaling pathways. Recurrent alveolar epithelial cell (AEC) injury may occur in the context of predisposing factors (e.g., genetic, environmental, epigenetic, immunologic, and gerontologic), leading to metabolic dysfunction, senescence, aberrant epithelial cell activation, and dysregulated epithelial repair. The dysregulated epithelial cell interacts with mesenchymal, immune, and endothelial cells via multiple signaling mechanisms to trigger fibroblast and myofibroblast activation. Recent single-cell RNA sequencing studies of IPF lungs support the epithelial injury model. These studies have uncovered a novel type of AEC with characteristics of an aberrant basal cell, which may disrupt normal epithelial repair and propagate a profibrotic phenotype. Here, we review the pathogenesis of IPF in the context of novel bioinformatics tools as strategies to discover pathways of disease, cell-specific mechanisms, and cell-cell interactions that propagate the profibrotic niche.
Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 17 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Epigallocatechin‑3‑gallate (EGCG) has been demonstrated to exhibit anticancer effects; however, the mechanisms behind these are not yet clear. The objective of the present study was to assess the effect of EGCG on smoking‑induced, precancerous, bronchial epithelial cell lesions and determine a potential protective mechanism. Human bronchial epithelial (HBE) cells were treated with cigarette smoke extract (CSE). Benzopyrene‑DNA adducts were detected by immunofluorescence cytochemistry. Changes to microRNA (miRNA) expression levels were detected via microarray. The effects of EGCG on smoke‑induced benzopyrene‑DNA adduct formation and the subsequent change in miRNA expression were analyzed. Subsequently, the protective effect of EGCG on smoke inhalation‑induced precancerous lesions was investigated. The expression levels of miRNA target genes were also analyzed. After CSE treatment, benzopyrene‑DNA adducts appeared in HBE cells, along with a resultant change in miRNA expression. EGCG inhibited the effects of CSE exposure; benzopyrene‑DNA adduct formation was reduced and miRNA expression changes were suppressed. In vivo, EGCG significantly reduced benzopyrene‑DNA adduct formation and the subsequent development of precancerous lesions in rat lungs induced by cigarette smoke inhalation. Moreover, EGCG downregulated CYP1A1 overexpression, a target gene of multiple smoking‑induced miRNAs, in rat lungs. EGCG may reduce the risk of lung cancer by downregulating the expression of the key gene CYP1A1, preventing the formation of smoking‑induced benzopyrene‑DNA adducts and alleviating smoking‑induced bronchial epithelial dysplasia and heterogeneity.
Purpose:
Chronic obstructive pulmonary disease (COPD), a progressive and irreversible respiratory disease, becomes the third leading cause of death and results in enormous economic burden on healthcare costs and productivity loss worldwide by 2020. Thus, it is urgent to develop effective anti-COPD drugs.
Materials and methods:
In the present study, two published GEO profiles were used to re-analyze and ascertain the relationships between circulating miRNAs and bronchial epithelial cells (BECs) mRNAs in COPD. The microRNA levels of miR-361-5p and miR-196-5p in plasma of COPD patients and healthy volunteers were detected by qRT-PCR. Next, the effects of γ-sitosterol (GS) on the expression of miR-361-5p and miR-196-5p and cell proliferation were investigated in BEC and H292 cell lines. Finally, whether specific miRNA-mRNA pathways involved in the effect of GS on BECs was assayed using Western Blot, real-time PCR and immunofluorescence.
Results:
miR-196-5p and miR-361-5p were, respectively, up- and down-regulated in COPD patients compared with healthy controls. Luciferase assays demonstrated that miR-361-5p and miR-196-5p were, respectively, targeting abca1 and arhgef12 3'UTR in BEAS-2B cells. GS significantly suppressed miR-196-5p and promoted miR-361-5p levels in BEAS-2B cells and inhibited BECs proliferation in vitro. GS promoted miR-361-5p expression, which inhibited BCAT1 mRNA and protein levels and weaken mTOR-pS6K pathway, resulted in anti-proliferation in BEAS-2B cells. In addition, RhoA was activated by ARHGEF12 due to the inhibitory effect of miR-196-5p on arhgef12-3'UTR which was partially abolished by GS suppressing miR-196-5p expression. Activated RhoA further activated ROCK1-PTEN pathway and finally inhibited mTOR pathway, resulting in induced BECs proliferation. The anti-proliferation effect of GS was not observed in H292 cells.
Conclusion:
These findings indicate that miR-361-5p/abca1 and miR-196-5p/arhgef12 axis mediated GS inducing dual anti-proliferation effects on BECs.
N⁶-methyladenosine (m⁶A) has recently emerged as an important regulatory mechanism for gene expression and aberrant m⁶A modification plays an important role in tumor progression. Emerging evidence has shown that aberrant m⁶A modification induced by cigarette smoking is involved in carcinogenesis, but whether cigarette smoking affects m⁶A modification and thus deteriorates to NSCLC is still unclear. Here, we identified a tumor suppressor gene-DAPK2 which was significantly associated with poor prognosis of NSCLC patients, especially in patients with a smoking history. Low levels of DAPK2 were detected in smokers and in NSCLC tissues. Cigarette smoking induced aberrant N⁶-methyladenosine modification of DAPK2, which resulted in decreased DAPK2 mRNA stability and expression of its mRNA and protein. This modification was mediated by the m⁶A “writer” METTL3 and the m⁶A “reader” YTHDF2. Mechanistically, we further demonstrated that DAPK2 functions as a tumor suppressor and downregulation of DAPK2 substantially enhances the proliferation and migration abilities in vitro and in vivo by activating NF-κB signaling pathway. Notably, the BAY 11–7085, a NF-κB signaling selective inhibitor, was shown to efficiently suppressed downregulation of DAPK2-induced oncogenic phenotypes of NSCLC cells. Our study reveals that cigarette smoking induces aberrant N⁶-methyladenosine of DAPK2 to promote NSCLC progression, which provides new insight into the mechanisms of NSCLC progression and a specific therapeutic target for NSCLC patients with a smoking history.
Purpose
Epigenetic age acceleration (EAA) is robustly linked with mortality and morbidity. This study examined risk factors of EAA and its association with overall survival (OS), progression-free survival (PFS), and quality of life (QOL) in patients with head and neck cancer (HNC) receiving radiotherapy.
Methods and Materials
Patients without distant metastasis were enrolled and followed before and end of radiotherapy, and 6-months and 12-months post-radiotherapy. EAA was calculated with DNAmPhenoAge at all four times. Risk factors included demographics, lifestyle, clinical characteristics, treatment-related symptoms, and blood biomarkers. Survival data were collected until August 2020; QOL was measured using Functional Assessment of Cancer Therapy–HNC.
Results
Increased comorbidity, HPV-unrelated, and severer treatment-related symptoms were associated with higher EAA (p=0.03 to <0.001). A non-linear association (quadratic) between body mass index (BMI) and EAA was observed: decreased BMI (when BMI<35,p=0.04) or increased BMI (when BMI≥35,p=0.01), was linked to higher EAA. Increased EAA (per year) was associated with worse OS (hazard ratio (HR)=1.11,95% CI=[1.03,1.18],p=0.004; HR=1.10,95% CI=[1.01,1.19], p=0.02, for EAA at 6-months and 12-months post-treatment, respectively), PFS (HR=1.10, 95% CI=[1.02,1.19], p=0.02; HR=1.14, 95% CI=[1.06,1.23], p<0.001; HR=1.08,95% CI=[1.02,1.14], p=0.01, for EAA before, end, and 6-months post-radiotherapy, respectively), and QOL over time (β=-0.61,p=0.001). An average of 3.25-3.33 years of age acceleration across time, which was responsible for 33% to 44% higher HRs of OS and PFS, was observed in those who died or developed recurrences compared to those who did not (all p<0.001).
Conclusion
Compared to demographic and lifestyle factors, clinical characteristics were more likely to contribute to faster biological aging in patients with HNC. Acceleration in epigenetic age resulted in more aggressive adverse events including OS and PFS. EAA could be considered as a marker for cancer outcomes, and decelerating aging could improve survival and QOL.
Purpose:
Exome and whole-genome sequencing of muscle-invasive bladder cancer (BC) has revealed important insights into the molecular landscape; however, there are few studies of non-muscle invasive BC with detailed risk factor information.
Experimental design:
We examined the relationship between smoking and other BC risk factors and somatic mutations and mutational signatures in bladder tumors. Targeted sequencing of frequently mutated genes in BC was conducted in 322 formalin-fixed paraffin-embedded bladder tumors from a population-based case-control study. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), evaluating mutations and risk factors. We used SignatureEstimation to extract four known single base substitution mutational signatures and Poisson regression to calculate risk ratios (RRs) and 95%CIs, evaluating signatures and risk factors. Results:Non-silent KDM6A mutations were more common in females than males (OR=1.83,95%CI:1.05-3.19). There was striking heterogeneity in the relationship between smoking status and established single base substitution signatures: current smoking status was associated with greater ERCC2-Signature mutations compared to former (p-value=0.024) and never smoking (RR=1.40, 95% CI: 1.09-1.80, p-value=0.008); former smoking was associated with greater APOBEC-Signature13 mutations (p-value=0.05); and never smoking was associated with greater APOBEC-Signature2 mutations (RR=1.54, 95% CI: 1.17-2.01, p-value=0.002). There was evidence that smoking duration (the component most strongly associated with BC risk) was associated with ERCC2-Signature mutations and APOBEC-Signature13 mutations among current (p-trend=0.005) and former smokers (p=0.0004), respectively.
Conclusions:
These data quantify the contribution of BC risk factors to mutational burden and suggest different signature enrichments among never, former, and current smokers.
DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.
Reciprocal interactions between cancer cells and the tumor microenvironment drive multiple clinically significant behaviors including dormancy, invasion, and metastasis as well as therapy resistance. These microenvironment-dependent phenotypes share typical characteristics with cancer stem cells (CSC). However, it is poorly understood how metabolic stress in the confined tumor microenvironment contributes to the emergence and maintenance of CSC-like phenotypes. Here, we demonstrate that chronic metabolic stress (CMS) in a long-term nutrient deprivation induces a Wnt-dependent phenoconversion of non-stem cancer cells toward stem-like state and this is reflected in the transcriptome analysis. Addition of Wnt3a as well as transfection of dominant-negative Tcf4 establishes an obligatory role for the Wnt pathway in the acquisition of CSC-like characteristics in response to metabolic stress. Furthermore, systematic characterization for multiple single cell-derived clones and negative enrichment of CD44+/ESA+ stem-like cancer cells, all of which recapitulate stem-like cancer characteristics, suggest stochastic adaptation rather than selection of pre-existing subclones. Finally, CMS in the tumor microenvironment can drive a CSC-like phenoconversion of non-stem cancer cells through stochastic state transition dependent on the Wnt pathway. These findings contribute to an understanding of the metabolic stress-driven dynamic transition of non-stem cancer cells to a stem-like state in the tumor metabolic microenvironment.
Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK a
The Myelodysplastic syndrome (MDS) is a clonal hematologic disorder that frequently evolves to acute myeloid leukemia (AML). Its pathogenesis remains unclear, but mutations in epigenetic modifiers are common and the disease often responds to DNA methylation inhibitors. We analyzed DNA methylation in the bone marrow and spleen in two mouse models of MDS/AML, the NUP98-HOXD13 (NHD13) mouse and the RUNX1 mutant mouse model. Methylation array analysis showed an average of 512/3445 (14.9%) genes hypermethylated in NHD13 MDS and 331 (9.6%) genes hypermethylated in RUNX1 MDS. 32% of genes in common between the two models (2/3 NHD13 mice, and 2/3 RUNX1 mice) were also hypermethylated in at least 2 out of 19 human MDS samples. Detailed analysis of 41 genes in mice showed progressively drift in DNA methylation from young to old normal bone marrow and spleen; to MDS, where we detected accelerated age-related methylation; and finally to AML, which markedly extends DNA methylation abnormalities. Most of these genes showed similar patterns in human MDS and AML. Repeat element hypomethylation was rare in MDS but marked the transition to AML in some cases. Our data show consistency in patterns of aberrant DNA methylation in human and mouse MDS and suggest that epigenetically, MDS displays an accelerated aging phenotype.
Lung cancer in never smokers (NS) shows striking demographic, clinicopathological and molecular distinctions from the disease
in smokers (S). Studies on selected genetic and epigenetic alterations in lung cancer identified that the frequency and profile
of some abnormalities significantly differ by smoking status. This study compared the transcriptome of lung adenocarcinoma
cell lines derived from S (n = 3) and NS (n = 3) each treated with vehicle (control), histone deacetylation inhibitor (trichostatin A) or DNA methylation inhibitor (5-aza-2′-deoxycytidine).
Among 122 genes reexpressed following 5-aza-2′-deoxycytidine but not trichostatin A treatment in two or more cell lines (including
32 genes in S-only and 12 NS-only), methylation was validated for 80% (98/122 genes). After methylation analysis of 20 normal
tissue samples and 14 additional non–small cell lung cancer cell lines (total 20), 39 genes frequently methylated in normal
(>20%, 4/20) and 21 genes rarely methylated in non–small cell lung cancer (≤10%, 2/20) were excluded. The prevalence for methylation
of the remaining 38 genes in lung adenocarcinomas from S (n = 97) and NS (n = 75) ranged from 8–89% and significantly differs between S and NS for CPEB1, CST6, EMILIN2, LAYN and MARVELD3 (P < 0.05). Furthermore, methylation of EMILIN2, ROBO3 and IGDCC4 was more prevalent in advanced (Stage II–IV, n = 61) than early (Stage I, n = 110) tumors. Knockdown of MARVELD3, one of the novel epigenetically silenced genes, by small interfering RNA significantly reduced anchorage-independent growth
of lung cancer cells (P < 0.001). Collectively, this study has identified multiple, novel, epigenetically silenced genes in lung cancer and provides
invaluable resources for the development of diagnostic and prognostic biomarkers.
Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states, notably cancer.
We evaluated the contribution of three genetic alterations (p53 knockdown, K-RASV12 ,a ndmutant EGFR )t o lung tumorigenesis using human bronchial epithelial cells (HBEC) immortalized with telomerase and Cdk4-mediated p16 bypass. RNA interference p53 knockdown or oncogenic K-RASV12 resulted in enhanced anchorage-independent growth and increased saturation density of HBECs. The combination of p53 knockdown and K-RASV12 further enhanced the tumori- genic phenotype with increased growth in soft agar and an invasive phenotype in three-dimensional organotypic cultures but failed to cause HBECs to form tumors in nude mice. Growth of HBECs was highly dependent on epidermal growth factor (EGF) and completely inhibited by EGF receptor (EGFR) tyrosine kinase inhibitors, which induced G1 arrest. Introduc- tion of EGFR mutations E746-A750 del and L858R progressed HBECs toward malignancy as measured by soft agar growth, including EGF-independent growth, but failed to induce tumor formation. Mutant EGFRs were associated with higher levels of phospho-Akt, phospho-signal transducers and activators of transcription 3 (but not phospho-extracellular signal-regulated kinase (ERK) 1/2), and increased expression of DUSP6/MKP-3 phosphatase (an inhibitor of phospho- ERK1/2). These results indicate that (a) the HBEC model system is a powerful new approach to assess the contribution of individual and combinations of genetic alterations to lung cancer pathogenesis; (b) a combination of four genetic alterations, including human telomerase reverse transcriptase overexpression, bypass of p16/RB and p53 pathways, and mutant K-RASV12 or mutant EGFR, is still not sufficient for HBECs to completely transform to cancer; and (c) EGFR tyrosine kinase inhibitors inhibit the growth of preneoplastic HBEC cells, suggesting their potential for chemoprevention. (Cancer Res 2006; 66(4): 2116-28)
Owing to novel therapy strategies in epidermal growth factor receptor (EGFR)-mutated patients, molecular analysis of the EGFR and KRAS genome has become crucial for routine diagnostics. Till date these data have been derived mostly from clinical trials, and thus collected in pre-selected populations. We therefore screened 'allcomers' with a newly diagnosed non-small cell lung carcinoma (NSCLC) for the frequencies of these mutations.
A cohort study.
Lung cancer centre in a tertiary care hospital.
Within 15 months, a total of 552 cases with NSCLC were eligible for analysis. PRIMARY AND SECONDARY OUTCOME MEASURES: Frequency of scrutinising exons 18, 19 and 21 for the presence of activating EGFR mutation and secondary codon 12 and 13 for activating KRAS mutations.
Of the 552 patients, 27 (4.9%) showed a mutation of EGFR. 19 of these patients (70%) had deletion E746-A750 in codon 19 or deletion L858R in codon 21. Adenocarcinoma (ACA) was the most frequent histology among patients with EGFR mutations (ACA, 22/254 (8.7%) vs non-ACA, 5/298 (1.7%); p<0.001). Regarding only ACA, the percentage of EGFR mutations was higher in women (16/116 (14%) women vs 6/138 (4.3%) men; p=0.008). Tumours with an activating EGFR mutation were more likely to be from non-smokers (18/27; 67%) rather than smoker (9/27; 33%). KRAS mutation was present in 85 (15%) of all cases. In 73 patients (86%), the mutation was found in exon 12 and in 12 cases (14%) in exon 13. Similarly, ACA had a higher frequency of KRAS mutations than non-ACA (67/254 (26%) vs 18/298 (6.0%); p<0.001).
We found a lower frequency for EGFR and KRAS mutations in an unselected Caucasian patient cohort as previously published. Taking our results into account, clinical trials may overestimate the mutation frequency for EGFR and KRAS in NSCLC due to important selection biases.
Background
Abnormal epigenetic marking is well documented in gene promoters of cancer cells, but the study of distal regulatory siteshas lagged behind.We performed a systematic analysis of DNA methylation sites connected with gene expression profilesacross normal and cancerous human genomes.
Results
Utilizing methylation and expression data in 58 cell types, we developed a model for methylation-expression relationships in gene promoters and extrapolated it to the genome. We mapped numerous sites at which DNA methylation was associated with expression of distal genes. These sites bind transcription factors in a methylation-dependent manner, and carry the chromatin marks of a particular class of transcriptional enhancers. In contrast to the traditional model of one enhancer site per cell type, we found that single enhancer sites may define gradients of expression levels across many different cell types. Strikingly, the identified sites were drastically altered in cancers: hypomethylated enhancer sites associated with upregulation of cancer-related genes and hypermethylated sites with downregulation. Moreover, the association between enhancer methylation and gene deregulation in cancerwas significantly stronger than the association of promoter methylationwith gene deregulation.
Conclusions
Methylation of distal regulatory sites is closely related to gene expression levels across the genome. Single enhancers may modulate ranges of cell-specific transcription levels, from constantlyopen promoters. In contrast to the remote relationships between promoter methylation and gene dysregulation in cancer, altered methylation of enhancer sites is closely related to gene expression profiles of transformed cells.
We used CDK4/hTERT–immortalized normal human bronchial epithelial cells (HBEC) from several individuals to study lung cancer pathogenesis by introducing combinations of common lung cancer oncogenic changes (p53, KRAS, and MYC) and followed the stepwise transformation of HBECs to full malignancy. This model showed that: (i) the combination of five genetic alterations (CDK4, hTERT, sh-p53, KRASV12, and c-MYC) is sufficient for full tumorigenic conversion of HBECs; (ii) genetically identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation; (iii) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; (iv) high levels of KRASV12 are required for full malignant transformation of HBECs, however, prior loss of p53 function is required to prevent oncogene-induced senescence; (v) overexpression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; (vi) growth of parental HBECs in serum-containing medium induces differentiation, whereas growth of oncogenically manipulated HBECs in serum increases in vivo tumorigenicity, decreases tumor latency, produces more undifferentiated tumors, and induces epithelial-to-mesenchymal transition (EMT); (vii) oncogenic transformation of HBECs leads to increased sensitivity to standard chemotherapy doublets; (viii) an mRNA signature derived by comparing tumorigenic versus nontumorigenic clones was predictive of outcome in patients with lung cancer. Collectively, our findings show that this HBEC model system can be used to study the effect of oncogenic mutations, their expression levels, and serum-derived environmental effects in malignant transformation, while also providing clinically translatable applications such as development of prognostic signatures and drug response phenotypes.
Visual Overview: http://mcr.aacrjournals.org/content/11/6/638/F1.large.jpg.
Mol Cancer Res; 11(6); 638–50. ©2013 AACR.
Neuroblastomas are tumors of peripheral sympathetic neurons and are the most common solid tumor in children. To determine the genetic basis for neuroblastoma, we performed whole-genome sequencing (6 cases), exome sequencing (16 cases), genome-wide rearrangement analyses (32 cases) and targeted analyses of specific genomic loci (40 cases) using massively parallel sequencing. On average, each tumor had 19 somatic alterations in coding genes (range of 3-70). Among genes not previously known to be involved in neuroblastoma, chromosomal deletions and sequence alterations of the chromatin-remodeling genes ARID1A and ARID1B were identified in 8 of 71 tumors (11%) and were associated with early treatment failure and decreased survival. Using tumor-specific structural alterations, we developed an approach to identify rearranged DNA fragments in sera, providing personalized biomarkers for minimal residual disease detection and monitoring. These results highlight the dysregulation of chromatin remodeling in pediatric tumorigenesis and provide new approaches for the management of patients with neuroblastoma.
Many DNA-hypermethylated cancer genes are occupied by the Polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that ~75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESCs and adult stem/progenitor cells. A large number of these genes are key developmental regulators, and a subset, which we call the "DNA hypermethylation module," comprises a portion of the PcG target genes that are down-regulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find that these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions.
While DNA methyltransferase1 (DNMT1) is classically known for its functions as a maintenance methyltransferase enzyme, additional
roles for DNMT1 in gene expression are not as clearly understood. Several groups have shown that deletion of the catalytic
domain from DNMT1 does not abolish repressive activity of the protein against a reporter gene. In our studies, we examine
the repressor function of catalytically inactive DNMT1 at endogenous genes. First, potential DNMT1 target genes were identified
by searching for genes up-regulated in HCT116 colon cancer cells genetically disrupted for DNMT1 (DNMT1−/− hypomorph cells). Next, the requirement for DNMT1 activity for repression of these genes was assessed by stably restoring
expression of wild-type or catalytically inactive DNMT1. Both wild-type and mutant proteins are able to occupy the promoters and repress the expression of a set of target genes,
and induce, at these promoters, both the depletion of active histone marks and the recruitment of a H3K4 demethylase, KDM1A/LSD1.
Together, our findings show that there are genes for which DNMT1 acts as a transcriptional repressor independent from its
methyltransferase function and that this repressive function may invoke a role for a scaffolding function of the protein at
target genes.
While the adult murine lung utilizes multiple compartmentally restricted progenitor cells during homeostasis and repair, much less is known about the progenitor cells from the human lung. Translating the murine stem cell model to humans is hindered by anatomical differences between species. Here we show that human bronchial epithelial cells (HBECs) display characteristics of multipotent stem cells of the lung. These HBECs express markers indicative of several epithelial types of the adult lung when experimentally tested in cell culture. When cultured in three different three-dimensional (3D) systems, subtle changes in the microenvironment result in unique responses including the ability of HBECs to differentiate into multiple central and peripheral lung cell types. These new findings indicate that the adult human lung contains a multipotent progenitor cell whose differentiation potential is primarily dictated by the microenvironment. The HBEC system is not only important in understanding mechanisms for specific cell lineage differentiation, but also for examining changes that correlate with human lung diseases including lung cancer.
Aberrant Kras signaling is observed in a high percentage of human lung cancers while activating mutations in the Wnt/b-catenin signaling pathway are only rarely found. Our recent work has shown that the combined activation of both Kras and Wnt/b-catenin signaling leads to a dramatic increase in both tumor incidence and size. Moreover, lung tumors generated by the combined activation of both of these pathways exhibit a distinct phenotype similar to embryonic progenitors found in the developing lung. Thus, combinatorial activation of Kras and Wnt/b-catenin pathways leads to a significant increase in lung tumor formation characterized by a more progenitor like phenotype.
Although mutations in Kras are present in 21% of lung tumors, there is a high level of heterogeneity in phenotype and outcome among patients with lung cancer bearing similar mutations, suggesting that other pathways are important. Wnt/β-catenin signaling is a known oncogenic pathway that plays a well-defined role in colon and skin cancer; however, its role in lung cancer is unclear. We have shown here that activation of Wnt/β-catenin in the bronchiolar epithelium of the adult mouse lung does not itself promote tumor development. However, concurrent activation of Wnt/β-catenin signaling and expression of a constitutively active Kras mutant (KrasG12D) led to a dramatic increase in both overall tumor number and size compared with KrasG12D alone. Activation of Wnt/β-catenin signaling altered the KrasG12D tumor phenotype, resulting in a phenotypic switch from bronchiolar epithelium to the highly proliferative distal progenitors found in the embryonic lung. This was associated with decreased E-cadherin expression at the cell surface, which may underlie the increased metastasis of tumors with active Wnt/β-catenin signaling. Together, these data suggest that activation of Wnt/β-catenin signaling can combine with other oncogenic pathways in lung epithelium to produce a more aggressive tumor phenotype by imposing an embryonic distal progenitor phenotype and by decreasing E-cadherin expression.
DNA methylation is an epigenetic mark critical for regulating transcription, chromatin structure and genome stability. Although many studies have shed light on how methylation impacts transcription and interfaces with the histone code, far less is known about how it regulates genome stability. We and others have shown that DNA methyltransferase 1 (DNMT1), the maintenance methyltransferase, contributes to the cellular response to DNA damage, yet DNMT1's exact role in this process remains unclear. DNA damage, particularly in the form of double-strand breaks (DSBs), poses a major threat to genome integrity. Cells therefore possess a potent system to respond to and repair DSBs, or to initiate cell death. In the current study, we used a near-infrared laser microirradiation system to directly study the link between DNMT1 and DSBs. Our results demonstrate that DNMT1 is rapidly but transiently recruited to DSBs. DNMT1 recruitment is dependent on its ability to interact with both PCNA and the ATR effector kinase CHK1, but is independent of its catalytic activity. In addition, we show for the first time that DNMT1 interacts with the 9-1-1 PCNA-like sliding clamp and that this interaction also contributes to DNMT1 localization to DNA DSBs. Finally, we demonstrate that DNMT1 modulates the rate of DSB repair and is essential for suppressing abnormal activation of the DNA damage response in the absence of exogenous damage. Taken together, our studies provide compelling additional evidence for DNMT1 acting as a regulator of genome integrity and as an early responder to DNA DSBs.
The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.
Functional analysis of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics
scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment analysis is a promising high-throughput
strategy that increases the likelihood for investigators to identify biological processes most pertinent to their study. Approximately
68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely
categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections,
unique tool classifications and associated questions/issues will provide a more comprehensive and up-to-date view regarding
the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the
survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details
of particular tool categories/tools, enabling them to make the best choices for their particular research interests.
A better understanding of key molecular changes during transformation of lung epithelial cells could affect strategies to reduce mortality from lung cancer. This study uses an in vitro model to identify key molecular changes that drive cell transformation and the likely clonal outgrowth of preneoplastic lung epithelial cells that occurs in the chronic smoker. Here, we show differences in transformation efficiency associated with DNA repair capacity for two hTERT/cyclin-dependent kinase 4, immortalized bronchial epithelial cell lines after low-dose treatment with the carcinogens methylnitrosourea, benzo(a)pyrene-diolepoxide 1, or both for 12 weeks. Levels of cytosine-DNA methyltransferase 1 (DNMT1) protein increased significantly during carcinogen exposure and were associated with the detection of promoter hypermethylation of 5 to 10 genes in each transformed cell line. Multiple members of the cadherin gene family were commonly methylated during transformation. Stable knockdown of DNMT1 reversed transformation and gene silencing. Moreover, stable knockdown of DNMT1 protein before carcinogen treatment prevented transformation and methylation of cadherin genes. These studies provide a mechanistic link between increased DNMT1 protein, de novo methylation of tumor suppressor genes, and reduced DNA repair capacity that together seem causal for transformation of lung epithelial cells. This finding supports the development of demethylation strategies for primary prevention of lung cancer in smokers.