Epigenetic mechanisms in COPD: implications for pathogenesis and drug discovery.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. The growing burden of COPD is due to continuous tobacco use, which is the most important risk factor of the disease, indoor fumes, occupational exposures and also aging of the world's population. Epigenetic mechanisms significantly contribute to COPD pathophysiology. AREAS COVERED: This review focuses on disease-relevant changes in DNA modification, histone modification and non-coding RNA expression in COPD, and provides insight into novel therapeutic approaches modulating epigenetic mechanisms. Recent findings revealed, among others, globally changed DNA methylation patterns, decreased levels of histone deacetylases and reduced microRNAs levels in COPD. The authors also discuss a potential role of the chromatin silencing Polycomb group of proteins in COPD. EXPERT OPINION: COPD is a highly complex disease and therapy development is complicated by the fact that many smokers develop both COPD and lung cancer. Of interest, combination therapies involving DNA methyltransferase inhibitors and anti-inflammatory drugs provide a promising approach, as they might be therapeutic for both COPD and cancer. Although the field of epigenetic research has virtually exploded over the last 10 years, particular efforts are required to enhance our knowledge of the COPD epigenome in order to successfully establish epigenetic-based therapies for this widespread disease.

Epithelial-mesenchymal transition in lung development and disease: does it exist and is it important?

Epithelial-mesenchymal transition (EMT) is a process when epithelial cells gradually transform into mesenchymal-like cells losing their epithelial functionality and characteristics. EMT is thought to be involved in the pathogenesis of numerous lung diseases ranging from developmental disorders, fibrotic tissue remodelling to lung cancer. The most important question--namely what is the importance and contribution of EMT in the pathogenesis of several chronic lung conditions (asthma, COPD, bronchiolitis obliterans syndrome and lung fibrosis)--is currently intensely debated. This review gives a brief insight into the mechanism and assessment methods of EMT in various pulmonary diseases and summarises the recent literature highlighting the controversial experimental data and conclusions.

Cigarette smoke-induced disruption of bronchial epithelial tight junctions is prevented by transforming growth factor-ß.

The airway epithelium constitutes an essential immunological and cytoprotective barrier to inhaled insults, such as cigarette smoke, environmental particles, or viruses. Although bronchial epithelial integrity is crucial for airway homeostasis, defective epithelial barrier function contributes to chronic obstructive pulmonary disease (COPD). Tight junctions at the apical side of epithelial cell-cell contacts determine epithelial permeability. Cigarette smoke exposure, the major risk factor for COPD, is suggested to impair tight junction integrity; however, detailed mechanisms thereof remain elusive. We investigated whether cigarette smoke extract (CSE) and transforming growth factor (TGF)-ß1 affected tight junction integrity. Exposure of human bronchial epithelial cells (16HBE14o(-)) and differentiated primary human bronchial epithelial cells (pHBECs) to CSE significantly disrupted tight junction integrity and barrier function. Specifically, CSE decreased transepithelial electrical resistance (TEER) and tight junction-associated protein levels. Zonula occludens (ZO)-1 and ZO-2 protein levels were significantly reduced and dislocated from the cell membrane, as observed by fractionation and immunofluorescence analysis. These findings were reproduced in isolated bronchi exposed to CSE ex vivo, as detected by real-time quantitative reverse-transcriptase PCR and immunohistochemistry. Combined treatment of 16HBE14o(-) cells or pHBECs with CSE and TGF-ß1 restored ZO-1 and ZO-2 levels. TGF-ß1 cotreatment restored membrane localization of ZO-1 and ZO-2 protein and prevented CSE-mediated TEER decrease. In conclusion, CSE led to the disruption of tight junctions of human bronchial epithelial cells, and TGF-ß1 counteracted this CSE-induced effect. Thus, TGF-ß1 may serve as a protective factor for bronchial epithelial cell homeostasis in diseases such as COPD.

Zyxin is a transforming growth factor-ß (TGF-ß)/Smad3 target gene that regulates lung cancer cell motility via integrin α5ß1.

Although TGF-ß acts as a tumor suppressor in normal tissues and in early carcinogenesis, these tumor suppressor effects are lost in advanced malignancies. Single cell migration and epithelial-mesenchymal transition (EMT), both of which are regulated by TGF-ß, are critical steps in mediating cancer progression. Here, we sought to identify novel direct targets of TGF-ß signaling in lung cancer cells and have indentified the zyxin gene as a target of Smad3-mediated TGF-ß1 signaling. Zyxin concentrates at focal adhesions and along the actin cytoskeleton; as such, we hypothesized that cytoskeletal organization, motility, and EMT in response to TGF-ß1 might be regulated by zyxin expression. We show that TGF-ß1 treatment of lung cancer cells caused rapid phospho-Smad3-dependent expression of zyxin. Zyxin expression was critical for the formation and integrity of cell adherens junctions. Silencing of zyxin decreased expression of the focal adhesion protein vasodilator-activated phospho-protein (VASP), although the formation and morphology of focal adhesions remained unchanged. Zyxin-depleted cells displayed significantly increased integrin α5ß1 levels, accompanied by enhanced adhesion to fibronectin and acquisition of a mesenchymal phenotype in response to TGF-ß1. Zyxin silencing led to elevated integrin α5ß1-dependent single cell motility. Importantly, these features are mirrored in the K-ras-driven mouse model of lung cancer. Here, lung tumors revealed decreased levels of both zyxin and phospho-Smad3 when compared with normal tissues. Our data thus demonstrate that zyxin is a novel functional target and effector of TGF-ß signaling in lung cancer. By regulating cell-cell junctions, integrin α5ß1 expression, and cell-extracellular matrix adhesion, zyxin may regulate cancer cell motility and EMT during lung cancer development and progression.

Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2beta to the nucleus.

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2alpha and PI3K-C2beta act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pools of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2beta translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2beta resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme, firstly in the cytosol and secondly in the nuclei. Using detergent-treated nuclei, we showed that PI3K-C2beta co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically, and a phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2beta levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutations of PI3K-C2beta demonstrated that epidermal growth factor-driven translocation to the nucleus is dependent on a sequence of basic amino acid residues (KxKxK) that form a nuclear localization motif within the C-terminal C2 domain. Furthermore, when this sequence was expressed as an EGFP (enhanced green fluorescent protein) fusion protein, it translocated fluorescence into nuclei with an efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2beta in nuclei. Further, this effect is dependent on a nuclear localization signal present within the C-terminal C2 domain, indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2beta into the nucleus.

Autonomous growth and hepatocarcinogenesis in transgenic mice expressing the p53 family inhibitor DNp73.

p53 family proteins carry on a wide spectrum of biological functions from differentiation, cell cycle arrest, apoptosis and chemosensitivity of tumors. Conversely, N-terminally truncated p73 (DNp73) functions as a potent inhibitor of all these tumor suppressor properties, implicating its participation in malignant transformation and oncogenesis. Several reports indicated considerable up-regulation of DNp73 in hepatocellular carcinoma (HCC) that correlates with reduced survival of patients, but little is known about the functional significance of DNp73 to tumorigenesis in vivo due to the lack of an appropriate model. To address this, we generated transgenic mice in which DNp73 expression is directed to the liver by the albumin promoter. Gene expression was tested by mRNA and protein analyses. Transgenic mice exhibited prominent hepatic histological abnormalities including increased hepatocyte proliferation resulting in preneoplastic lesions (liver cell adenomas) at 3-4 months. Among 12- to 20-month-old mice, 83% of animals developed hepatic carcinoma. HCC displayed a significant increase of hyperphosphorylated inactive retinoblastoma, whereas p53-regulated inhibitors of cell cycle progression were down-regulated in the tumors. Our data firmly establish the unique oncogenic capability of DNp73 to drive hepatocarcinogenesis in vivo, supporting its significance as a marker for disease severity in patients and as target for cancer prevention. This model offers new opportunities to further delineate DNp73-mediated liver oncogenesis but may also enable the development of more effective cancer therapies.

Proteomic analysis of the E2F1 response in p53-negative cancer cells: new aspects in the regulation of cell survival and death.

E2F1 is an essential transcription factor that regulates cell-cycle progression and apoptosis. Overexpression of E2F1 sensitizes neoplastic cells to apoptosis and leads to tumor growth suppression, making it an interesting target for anticancer therapy. Use of E2F1 as a therapeutic, however, requires a detailed knowledge of the mechanisms by which it controls cellular proliferation and apoptosis, and of other potential E2F1 activities. In this study, a differential proteome analysis was performed to identify proteins associated with E2F1 activity in inducible p53-deficient Saos-2ERE2F1 osteosarcoma cells. 2-DE revealed a distinct protein profile at 32 h after E2F1 activation. Thirty-three proteins were reproducibly identified as either up-regulated or down-regulated. Proteins were identified by MALDI-MS. They included hitherto unknown E2F1 target proteins of cytoskeletal origin, chaperones, enzymes, proteasomal proteins, and several heterogeneous nuclear ribonucleoproteins, suggesting its role in the ER-stress response, protein degradation, and modulation of pre-mRNA splicing. Protein analysis-derived results were verified by Western blot using representative protein candidates. Thirteen identified proteins were the products of genes known to be cancer related. Thus, proteome analysis provides new information about the complexity of E2F1 activities in human cancer cells that may be considered when using E2F1 as a drug.

C-terminal p73 isoforms repress transcriptional activity of the human telomerase reverse transcriptase (hTERT) promoter.

Activation of telomerase is linked to tumorigenesis and has been observed in a variety of human tumors. Previous reports demonstrated that p53 represses human telomerase reverse transcriptase (hTERT), a key component for telomerase activity. The p73 protein displays a tumor suppressor activity similar to p53. In the present study, we examined the effect of transactivation competent p73 isoforms on hTERT expression in p53-negative human H1299 cells. Overexpression of C-terminal p73 isoforms (alpha, beta, gamma, delta) resulted in a clear down-regulation of hTERT promoter activity. The strongest inhibitory effect, comparable with p53, was observed for p73beta. Moreover, suppression of hTERT expression was also mediated by endogenous p73 after activation of E2F1 in H1299ER-E2F1 cells. Mutations in the Sp1 transcription factor-binding sites of the proximal core promoter region significantly abolished p73-induced repression, suggesting that the effect is mediated by Sp1. Finally, we demonstrate that p73 directly interacts with Sp1, suggesting that formation of a p73-Sp1 complex is the underlying mechanism for p73-triggered inhibition of hTERT expression. Our findings provide additional evidence that p73 mimics p53 in many aspects in cells lacking functional p53, thereby contributing to tumor surveillance.

Role of MEN2A-derived RET in maintenance and proliferation of medullary thyroid carcinoma.

BACKGROUND: Dominant-activating mutations in the RET protooncogene, a receptor tyrosine kinase, have been identified as a cause of medullary thyroid carcinoma. Such oncogenic RET mutations induce its ligand-independent constitutive trans-autophosphorylation. We investigated the role of endogenous oncogenic RET autophosphorylation in maintaining the neoplastic phenotype in medullary thyroid carcinoma cells and orthotopic medullary thyroid carcinomas in RET transgenic mice. METHODS: We constructed adenoviral vectors expressing a dominant-negative truncated form of RET, termed RET(DeltaTK), and analyzed its effect on cell viability, apoptosis, and proliferation of TT medullary thyroid carcinoma cells. We investigated the effect of RET(DeltaTK) on downsteam signaling by assessing alterations in phosphorylation or in gene expression. The effect of RET(DeltaTK) in primary medullary thyroid carcinomas in transgenic mice was assessed by monitoring tumor growth. All statistical tests were two-sided. RESULTS: Cell viability was reduced. Phosphorylation of Akt and extracellular signal-regulated kinase (ERK), components of downstream signal transduction pathways, was abolished, and cell cycle progression was reduced. Expression of cell cycle regulator cyclin D1 was decreased, and expression of cell cyle regulators p21(CIP1/WAF1) and p27(KIP1) was increased. Apoptosis was stimulated and concurrently the expression of BCL-2 was decreased. All in vitro experiments compared TT cells expressing RET(DeltaTK) with untreated control cells or control vector-treated cells. Furthermore, 2 weeks after injecting adenovirus-carrying RET(DeltaTK) into thyroid glands of transgenic mice with orthotopic medullary thyroid carcinoma, tumors were statistically significantly smaller than their initial size in mice treated with RET(DeltaTK) (43.6%, 95% confidence interval [CI] = 30.7% to 56.5%; P =.010; two-sided unpaired Student's t test), whereas tumors in mice treated with a control vector were larger than their initial size (139.8%, 95% CI = 120.3% to 159.3%; P<.001). CONCLUSION: Selective disruption of oncogenic RET signaling in medullary thyroid carcinoma in vitro and in vivo is associated with loss of the neoplastic phenotype of medullary thyroid carcinoma and should be investigated further as the basis for new therapeutic approaches for this disease.