Chronic Obstructive Pulmonary Disease (COPD) and Its Association with Lung Cancer: Molecular Mechanism and Therapeutic Targets

    

    

    Figure 1: Linkage of lung cancer and COPD owing to their common exogenous and endogenous factors. Both share similar pathogenic mechanism which includes
NF-κB, oxidative stress, hypoxia and angiogenesis.
    

Genetic Predisposition in COPD and Lung Cancer

A large amount of genetic variation and environmental risk factors greatly influence the pathogenicity of COPD and lung cancer. Genome-wide Association Studies (GWAS), Whole Genome sequencing, transcriptome, and whole gene methylation are useful in studying the genetic variation of COPD and lung cancer [11-13].

SNP’s in COPD and Lung Cancer

The Single Nucleotide Polymorphism (SNPs) is the most common genetic variation which involves the substitution of a single nucleotide at a specific position. Pathogenic genes are believed to have high frequencies of SNP variations. SNP variations of genes like SNPsrs660652 and rs12910984 in the nicotinic Acetylcholine Receptors (nAChRs) family are related to COPD and lung cancer [14]. Variants in CHRNA5-HRNA3-CHRNB4 locus are strongly associated with smoking. Among SNP variations in nAChRs, rs16969968 was confirmed to be associated with lung cancer and COPD and hence proposed as the best potential target gene in COPD-Lung cancer transformation [15].

In addition to this SNP variation of inflammatory genes play a crucial role. rs1800469 in the promoter region of the TGFB1, rs139092247, and rs2568494 in IREB2, rs117916638 in EGLN2, position-308 of TNF-alpha were associated with cigarette smokers with COPD and lung cancer [16-19].

Differentially Expressed Genes in COPD and Lung Cancer

The copy number variation and expression of genes are equally important while exploring the association of COPD with lung cancer. CHRNA7 is up-regulated and proposed as a genetic biomarker of the co-existence of both diseases [20]. HER2 was overexpressed in patients with COPD and lung cancer but not in lung cancer patients. Hence it might be involved in the regulation of lung epithelial cell transformation from chronic inflammation to lung tumorigenesis [21]. The C-X-C motif chemokine ligand 14 (CXCL14) is involved in the transformation from smoking to lung cancer through upregulated expression. The expression of CXCL14 is high in the airway epithelium of healthy smokers, and it is furthermore in smokers with COPD, and most up-regulated in smokers with COPD and lung ADC [22]. The expression of a COPD-associated CXCL14-correlating cluster of genes was associated with a poor survival rate of patients with COPD and lung ADC. Reports by Wang et al., after analyzing gene expression profiles showed that some candidate genes are up or down-regulated in smokers with COPD or lung cancer and COPD [23].

Epigenetic Dysregulation in COPD and Lung Cancer

Epigenetics is defined as the study of heritable or acquired stable changes in gene expression that occur without variation in the DNA sequence. DNA methylation, post-translational histone modifications (including histone acetylation, methylation, phosphorylation, ubiquitination, and sumoylation) are the three main epigenetic marks. Evidence has increased that epigenetic mechanisms influence gene expression in chronic lung diseases like Chronic Obstructive Pulmonary Disease (COPD). Epigenetic silencing of genes through promoter methylation of cytosines and histone modifications is now recognized as a major and normal event affecting the expression of hundreds of genes during initiation and clonal development of lung cancer.

DNA Methylation and CpG Island in COPD & Lung Cancer

Epigenome-Wide Association Study (EWAS) is carried out to study the link between gene methylation in COPD and lung cancer. Repression of CCDC37 and MAP1B genes is significantly associated with both COPD and lung cancer [24] Immune-modulatory genes are hypomethylated to a greater extent in COPD and Lung cancer patients [25].

Clinical data from lung cancer patients have shown that DNA Methyltransferase 1 (DNMT1) is overexpressed which catalyzes methylation of DNA in CpG islands leads to p53 mutation and increased expression of specificity protein 1 (Sp1) [26-28]. Nicotinederived nitrosamine ketone (NNK) is also playing a major role in epigenetic alterations like hypermethylation of promoters of multiple tumor suppressor genes by induced activation of DNMT1 [29]. Identification of CpG methylation events in COPD as a precursor event in lung cancer will be of valuable clinical significance. Examples of such candidate genes that are hypermethylated in both diseases are IL-12Rβ2, Wif-1, P16, GATA4, KEAP1, and ECSOD [30].

Histone Modification in COPD and Lung Cancer

Histone modification is a cellular process where histone tails are modified by nonhistone chromatin-associated proteins called chromatin-modifying enzymes. Various post-translational modifications in histones play a crucial role in genomic functions which in turn alter the structural property of chromatin [31]. Based on their function chromatin modification enzymes are classified, for example, acetylation by Histone Acetyltransferases (HATs), deacetylation by Histone Deacetylases (HDACs), methylation by Histone Methyltransferases (HMTs), and demethylation by Histone Demethylases (HDMs).

The resulting Post-translational modification will promote the activation or repression of chromatin-mediated gene expression for inflammatory mediators, apoptosis, senescence, growth factors, and tumor suppressor genes involved in COPD and lung cancer [32, 33]. The activities of these enzymes are greatly influenced by environmental cues like prolonged exposure to irritants or particulate matter most often involves cigarette smoke which occurs in patients with COPD and lung cancer [34].

Cigarette Smoke Mediated Epigenetic Modification in COPD & Lung Cancer

Cigarette smoke is one of the most important risk factor in the development of COPD emphysema and lung cancer. It contains more than 1014 oxidants and about 4700 reactive chemical compounds. Cigarette smoke challenges the lung through the generation and release of ROS and by activating various redox-sensitive transcription factors, which ultimately leads to increased expression of proinflammatory cytokines and chemokines. The next major impact of cigarette smoke is chromatin modification and epigenetic changes by causing post-translational modifications to HDAC2 and SIRT1 which ultimately results in chromatin remodelling [35].

HDAC2 is an important redox-sensitive protein and it prone to alteration by oxidants and free radicals which leads to decreased activity of this deacetylase enzyme. It has been reported that a significant reduction in the activity of HDAC2 in the lungs of smokers and patients with COPD compared to healthy non-smokers [36-38]. Several in vitro experiments showed that in response to cigarette smoke there is a decreased level of HDAC2 in human airway and bronchial epithelial cells and lungs of mice. This study helps to understand that HDAC2 could be modified post-translationally by aldehydes and protein nitration present in cigarette smoke [39]. HDAC2 deacetylase activity is decreased in monocytes, epithelial cells, and rodent lungs due to nitration of tyrosine residues on HDAC2 by nitric oxide (NO) [40]. Moreover, the level of nitrated HDAC2 is significantly increased in alveolar macrophages from patients with COPD [41]. The activity of HDAC2 has been drastically reduced by oxidative stress by proteasomal degradation. A basal level of phosphorylation is required for HDAC2 deacetylase activity [42]. Hyperphosphorylation of HDAC2 results in the dissociation of co-repressor complex and enhanced ubiquitination and finally degradation. Cigarette smoke induces increased phosphorylation of HDAC2 by a kinase-dependent mechanism [43, 44]. Regulation of HDAC2 activity by casein kinase 2 (CK2- downstream of phosphatidylinositol 3-kinase (PI3K)) is validated by Marwick and colleagues where they showed that stability and lack of phosphorylation of HDAC2 in phosphoinositide-3 kinase (PI3K) d knockout mice in response to cigarette smoke [45].

SIRT1 is another class of histone deacetylase enzyme that deacetylase both histone and non-histone proteins including transcription factors even other signaling molecules. Cigarette smoke exposure has drastically reduced the level of SIRT1 in the lungs of smokers and patients with COPD. SIRT1 undergo post-translational modification and there is increased nitration and carbonylation of SIRT1 were observed in the lungs of smokers on exposure to cigarette smoke and patients with COPD [46]. Disruption of SIRT1-RelA/p65 complex occurs once there is a degradation of SIRT1 after PTM which ultimately results in the release of high pro-inflammatory mediators [47] this high expression of pro-inflammatory genes leads to chronic inflammation which is the initial stage in the pathogenesis of COPD & lung cancer.

Thus Cigarette smoke greatly influences the development and progression of COPD and lung cancer by increasing the oxidative stress and the activation of various redox-sensitive transcription factors and chromatin remodeling by alteration in the redox- sensitive deacetylases.

Acetylation of histone H3 in macrophages and lungs of humans and rodents were observed in response to cigarette smoke, this work elucidates the role of histone acetylation in chromatin remodeling and subsequent activation of an inflammatory response in lungs of COPD patients [48-51].

Histone Methyl Transferases will affect global methylation and are deregulated in several types of cancers. Gene activation and repression depend on the methylation level of histones. For example, when H3K4, H3K36, and H3K79 are methylated it linked to gene activation, whereas methylation at H3K9, H3K27, and H3K20 leads to gene repression [52] Histone Demethylases (HDMs) are classified into two types, such as lysine-specific demethylase 1 (LSD1) and Jumonji C (JmjC) domain family proteins which are involved in the regulation of gene expression [53]. HDM expression is dysregulated during tumor initiation and progression. It has been observed that there is an alteration in the expression level of JMJD1A, JMJD2B & JARID1A during hypoxic conditions in the lungs of patients with COPD [54-56].

Role of Inflammation in COPD & Lung Cancer

Inflammation is a biological process in which our cells recruit a diverse set of immune cells to fight against pathogens. The small airways of COPD patients are inflamed and there is an alteration in inflammatory cytokine level. Chronic inflammation is due to the activation and recruitment of macrophages, CD4+, and CD8+ T cells, dendritic cells, B cells, and neutrophils. Some studies showed that the degree of inflammation directly correlates with COPD severity and both COPD & Lung cancer are associated with an inflammatory environment.

Reports showed that upregulation of Tumor necrosis factor a (TNF-a) in COPD induces an immune suppressive effect within the tumor microenvironment thereby promoting lung tumorigenesis [57]. A high level of TNF- a is observed in tumor samples from lung cancer patients [58]. Bioinformatic analysis also highlighted that innate defense, lymphocyte trafficking, and dendritic cell pathways are enriched in COPD associated lung cancer. Some of the most important inflammatory mediators and intracellular signaling pathways involved in the pathogenesis of lung cancer and COPD are as follows.

Oxidative Stress and Mitochondrial Damage in COPD and Lung Cancer

Several mechanisms have been proposed to account for the association between COPD and lung cancer. Among these, oxidative stress and chronic inflammation are playing a crucial role in the process of carcinogenesis. Mitochondrial dysfunction is the key player of these two conditions which promotes tumorigenesis in COPD patients. Excessive Reactive Oxygen Species (ROS) exposure to airways leads to an increase in oxidative stress among COPD patients compared with non-COPD smokers. Mitochondrial ROS is increased in the airways of patients with COPD which is the effect of mitochondrial damage [59]. Impairment of antioxidant NRF2 (nuclear factor erythroid 2-related factor 2) and various pathways involved in oxidant and antioxidant responses are differentially expressed in COPD patients [60, 61]. Oxidative stress-related proteins like catalase and thioredoxin are differentially expressed in both COPD and COPD-associated NSCLC compared with nonsmokers [62]. Induction of cellular senescence is mediated by increased oxidative stress which aids in the process of carcinogenesis. Mitochondria are the main source of ROS, from the oxidative phosphorylation system. Also, it acts as a sensor for inflammatory damage signals and it is the site of initiation of inflammatory response. Mitochondrial Antiviral Signaling protein (MAVS) initiates the NLRP3 inflammasome, a multimolecular complex .Change in the conformation of this inflammasome results in the activation of caspase-1 and hence induces the expression of pro-inflammatory cytokines, like Interleukin (IL)-1β and IL-18 [63].

Cigarette smoke affects the structure and function of mitochondria thus leads to increased expression of the mitochondrial fusion protein, Mfn-2, in alveolar epithelial cells [64]. Work done by Lou et al., shows that the expression of Mfn-2 is high in lung adenocarcinoma when compared to adjacent normal lung tissue [65]. Damaged mitochondria undergo uncoupling and release of increased ROS. Mitophagy is activated to clear the damaged mitochondria and if mitophagy is impaired then there will be mitochondrial DNA accumulation in the cytoplasm.

Immune Defects in COPD and Lung Cancer

Reports are there to show that adaptive immune response also involves in the pathogenesis of COPD because mature lymphoid follicles with a germinal center and separated T and B cells zone occur in the lungs of COPD patients and it associates with the severity of COPD [66]. There is also marked alteration of the innate immune system in COPD. COPD patients have intense immune dysregulation which leads to enhanced activity of regulatory T cells, CD4+, PD-1+ exhausted effector T cells, and myeloid-derived suppressor cells. Mast cell activation is also aberrant in the lungs of COPD and lung cancer patients [67]. In addition to that, natural killer T cells, a cellular interface between innate and adaptive immunity also deregulated in COPD [68]. There are some specific mediators like Serum Amyloid A (SAA) implicate the links between COPD and Lung cancer. SAA is highly expressed in COPD and it helps the production of tumors [69]. In response to cigarette smoke, alveolar macrophages (AM) in COPD patients have an impaired phagocytic capacity which is associated with the deregulated release of extracellular sphingosine-1-phosphate (S1P) released from airway epithelial cells [70]. Mitochondrial dysfunction occurs in COPD and lung cancer results in impairment of the HMG-CoA reductase pathway which is involved in the production of cholesterol, and all steroid hormones. The defective HMG-CoA pathway affects innate immune response which might link COPD with an increased incidence of lung cancer [71].

NF-κB Pathway in COPD & Lung Cancer

The most important intracellular signaling pathway involves in the pathogenesis of COPD and lung cancer is NF-κB. NF-κB is a critical pro-inflammatory transcription factor and it is well known that both canonical and non-canonical NF-κB pathways induce increased expression of pro-inflammatory mediators leading to chronic inflammation in the lung. Several genes including cytokines, adhesion molecules, angiogenic factors, anti-apoptotic factors, and Matrix Metalloproteinases (MMPs) that all have shown to be associated with tumor progression and metastases are regulated by NF- κB. STAT 3 and NF- κB activation are key players in the development of lung cancer from COPD. NF- κB suppresses the expression of p53 thereby stimulates proliferation and inhibit cell death [72]. The pro-inflammatory environment which consists of activated macrophages and regulatory T cells depends on NF-κB and links COPD with lung cancer [73].

Gene Editing as a New Therapeutic Target for Lung Cancer & COPD

Respiratory diseases ranging from relatively mild to severe conditions such as COPD, Asthma, and lung cancer are influenced by environmental conditions as well as genetic or epigenetic changes as their root cause. Hence there is a need for novel tools that manipulate the function of endogenous genes. Gene Editing technology offers tools that allow us to investigate the role of a particular gene in the physiology and pathophysiology of the respiratory system. Epigenetic editing helps to rewrite the epigenetic marks at the defined locus of our gene of interest. Zinc Finger Nucleases (ZFN) and Transcription Activator-Like Effector Nucleases (TALEN) are widely used DNA targeting system until the introduction of the much more straightforward editing platform CRISPR/Cas9. A system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins, is now taking the research field to new heights by providing new insights about the underlying mechanism of the diseases.

Application of CRISPR/Cas9 in Lung Cancer Modelling, Tumorigenesis

CRISPR/Cas 9 mediated gene editing has accelerated the progress of lung cancer research in any direction by assisting in the identification of target genes, drug-resistance genes, and aids in constructing animal tumor models. One example of this is the deletion of gene33 which leads to the reduction of cell proliferation and also increases cell migration. Due to this deletion, there is an upregulation of UCHL1 (deubiquitination enzyme) which is associated with the early stages of lung epithelial cell transformation and tumorigenesis [74]. Manguso et al. showed that deletion of PTPN2 improves the efficiency of immunotherapy by enhancing interferon mediated effect on growth suppression and antigen presentation [75]. Inhibition of miR584-ROCK1 pathway by CRISPR Cas9 FLI1 gene knockout results in cessation of tumor progression [76]. Identification of drug resistance genes is done by Wang et al., They have shown that CRISPR Cas9 based knockout of the receptor uPAR (Urokinase plasminogen activator receptor) results in a decrease in resistance of lung cancer cells towards drugs like 5-FU, Doxorubicin, cisplatin, and docetaxel. This knockout also resulted in inhibition of cancer cell proliferation, invasion, and migration [77].

Clinical Trials using CRISPR/Cas9 Genome Editing

Even though being a potential genome editing tool, very few CRISPR/Cas9 projects got approved to conduct human clinical trials. One study involves direct administration of the CRISPR/Cas9 system into the human body after editing the immune checkpoint genes to improve the efficacy of T cell therapy in advanced NSCLC patients. It mainly aims to evaluate the safety of PD-1 knockout engineered T cells and the results from a first-in-human phase I clinical trial (ClinicalTrials.gov NCT02793856) showed that it is safe and feasible [78].

Application of CRISPR/Cas9 in COPD

Till now very limited reports are available regarding the application of gene editing tools in COPD. Hypersecretion of airway mucus is one of the main causes of the pathogenesis of COPD. SPDEF (SAMpointed domain-containing Ets-like factor) is a master regulator of mucus production. Song et al. showed that targeted silencing of SPDEF using ZFPs and CRISPR/Cas9 has successfully reduced the mucus-related gene expression which might be a new therapeutic approach for patients with excessive mucus secretion [79].

Epigenetic editing of gene expression in the lung is complicated due to the difficulties in delivering the epigenetic editor. Several barriers like Physical and immune barriers of lung hamper successful delivery to the lung. Excessive mucus production and inflammation will further worsen the situation. To tackle these problems, several gene transfer systems were developed which employ viral vectors like Retroviral, Adenoviral. Thus Gene editing based technologies like CRISPR systems shows promising therapeutic possibilities for lung and respiratory tract diseases.

Therapeutic Targets in COPD and Lung Cancer

COPD and lung cancer are debilitating lung disorders whose underlying molecular and cellular mechanisms are not completely understood. Many patients fail to respond to broad-spectrum antiinflammatory molecules like corticosteroids. Bronchodilators have been advised to the patient as a part of the management of the disease; however, it fails to target chronic inflammatory response which is a key player in both disorders. This implies the need for finding a novel therapeutic target for COPD and lung cancer.

Several studies support the role of chemokines and their receptors including chemokine CXCR2 and CXCR3 with smallmolecule receptor antagonist (SB332235, NBI74330) [89]. This antiinflammatory therapy will reduce the inflammatory profile in COPD, which is unaffected by existing pharmacotherapy.

Inhibitors that target the core component of apoptotic signaling are also at an early stage of development. Selective inhibitors of caspases-3, 7, and 9 are carried out by IAPS- Inhibitors of apoptosis proteins. Enabling apoptosis to take place could be a potential treatment of malignancy and thus several agents that target IAPs are gaining more interest [90].

Antioxidant therapy is one of the emerging tools which target oxidative stress with antioxidants or boosting the endogenous level of antioxidants. Thiol molecules (glutathione and mucolytic drugs, such as N-acetyl-L-cysteine and N-acystelyn), dietary polyphenols (curcumin, resveratrol, green tea, catechins/quercetin), erdosteine, which have been reported to control oxidative stress by inhibiting NF-κB activation and maintaining the balanced redox state of the cell. Thus administration of multiple antioxidants will be effective in the treatment of lung diseases [91].

Proteomic profiling studies help to identify the dysregulated pathways in small cell lung cancer which leads to the development of therapies that specifically target those pathways. There is emerging evidence shows that the MAPK pathway contributes to several COPD-associated phenotypes. The classical MAP kinase can be inhibited at several points. Inhibition at the membrane blocks receptor dimerization. Inhibitors of Receptor Tyrosine Kinase exist, such as EGFR inhibitors (AG1478, ZD1839). ZD1839 has been tried in a mouse model and it has been shown to block lung injury. These drugs have shown promising results in clinical trials but the adverse effects are an issue. One of the promising compounds is CI-1040. CI- 1040 (PD 184352), a specific small-molecule inhibitor of MEK1/2 is well tolerated in humans and has shown efficacy as an anti-tumor agent [92].

miRNA as a Feature Therapeutic Target

MicroRNAs (miRNAs) are small, endogenous non-coding RNAs that regulate gene expression. miRNA bind to its target messenger RNAs in a sequence-specific manner, which leads to direct inhibition of protein synthesis or degradation of the transcript. Several miRNAs are known to regulate genes involving in the process of the developmental and physiological process including cellular proliferation, apoptosis, and differentiation, innate and adaptive immune responses (Figure 2). For instance, miR-146a-5p has been found to be a regulator of inflammation [80]. miR-146a-5p expression is impaired in fibroblast of patients with COPD, which release the break on the IL-1R/NF- κB pathway and contribute to abnormal inflammation. Treating fibroblast with miR-146a-5pmimic results in a reduced level of IL-8.This confirms the anti-inflammatory role of miR-146a-5p [81]. In vitro and in vivo studies have shown the role of miRNA in the pathogenesis of COPD. Recent in vivo and gene set enrichment analysis explained the role of miR-218-5P in cigarette smoke-induced inflammatory response as it was downregulated in the epithelium of COPD patients [82].

    

    

    Figure 2: MicroRNA involved in the pathogenesis of Lung cancer and COPD. Dysregulation of these miRNA aids in the development of both disorders.