RICERCA ITALIANA     

TOBACCO SMOKE, INFLAMMATION AND LUNG CANCER: BIOLOGICAL, MOLECULAR, CLINICAL AND PATHOLOGICAL FEATURES.

Università degli Studi di Modena e Reggio Emilia

Abstract

Tobacco smoke is the main risk factor both for chronic obstructive pulmonary disease and for lung cancer in industrialized countries and its impact on these disease is progressively growing in developing countries too. Moreover, it is not only the common etiologic factor for these disease but chronic lung inflammation due to tobacco smoking, especially when associated with airflow limitation, may represent "per se" a predisposing condition for the onset of the tumor. The aim of this multicenter, multidisciplinary research program is the study of biological and molecular modifications induced by tobacco smoke on pulmonary tissues and cells, and the "in vivo" evaluation of the corresponding changes at the clinical and pathological level.
Overall, the program involves seven Research Units. Some of them will conduct mainly experimental studies with cell biology, molecular biology or molecular genetics techniques, whose results may be subsequently used for interpreting the "in vivo" data obtained by the clinical Research Units. The potential associations between inflammation and lung cancer will be searched for and analysed within each project, when possible. Each single project has a clearly defined purpose which is intrinsically able to provide original results. However, the multidisciplinary structure of the research program, which represents its most original feature, among Units having the effects of tobacco smoke on human lung as the common target will be able to provide solid data which may be used also outside the specialty area. Finally, some of the research areas (e.g., gene expression after exposure to environmental matrices extract, role of decoy receptors in airways inflammation) are extremely new fields of research whose results will represents a further improvement to knowledge anyway. <<<

Principal Investigator

Leonardo FABBRI Università degli Studi di MODENA e REGGIO EMILIA

Research Objectives

Tobacco smoke is the main risk factor for chronic obstructive pulmonary disease (COPD) and lung cancer in Western countries, the incidence of morbidity and mortality for both progressively increasing. Moreover, if cigarette smoke is on one hand the common etiologic factor for these two diseases, chronic lung inflammation, especially when associated to airflow limitation, is a predisposing factor for lung cancer itself. The complex molecular and biological mechanisms which play a role in this pro-neoplastic action of lung inflammation are largely unknown, but they probably involve many different pathophysiologic pathways such as oxidative stress, tissue remodelling, cell proliferation and apoptosis, etc.
The aim of this interdisciplinary research program is the study of molecular and biological modifications induced by tobacco smoke on lung tissues and cells, both as favouring inflammation and carcinogenesis, and the "in vivo" evaluation of their clinical and pathological evidence. The program involves seven Research Units. In the part regarding "in vitro" studies the different Reseacrh Units will use corroborative analytical techniques (cell biology, molecular biology, molecular genetics) which allow the study of the role of chemokines and of chemokines decoy receptors in lung inflammatory disorders, the analysis of tissue expression of different isoforms of clusterin in lung inflammatory and neoplastic diseases, the gene expression profile of human bronchial epithelial cells after exposure to toxic (cigarette smoke, environmental matrices) or biological (virus) agents, and the study of regulatory T cells in lung tumors. On then other hand, the remaining research Units will perform the analysis of the expression of protein-kinases in smoke-related neuroendocrine proliferations, the definition of the clinical, functional, biological and radiological features of the predominant COPD phenotypes, and the study of the role of oxidative stress in the pathogenesis of COPD by the "in vivo" use of biological markers. Data obtained by the biological Units might be used to interpreting data obtained by the clinical Units. Each single project has a clear purpose and the potential to give original contributions. However, the multidisciplinary and corroborative structure is the main feature of the program as a whole and it may provide a broader and more solid basis for knowledge on this field of research. Finally, each project has new and original features which will be useful for the advancement of knowledge anyway. <<<

First Results

Definition of experimental protocols, generation of cell lines, accurate stting-up of the methodologies for preparation of study reagents.
Distribution of duties to different Units.
Definition of selection criteria (inclusion and exclusion)for study populations.In this second phase, the research Units will evaluate the expression of the activation molecules in study in plasmids, cell lines, biopsies and bronchoalveolar lavage samples.
Furthermore, we will analyze the responses of cell lines following stimulation by cigarette smoke, environmental preparation and extracts of viruses.
The role and the characterization of T lymphocytes involved in the pathogenesis of COPD and lung cancer will be analyzed in cell lines and biopsies.
The parameters of inflammatory cellular activation will be measured in patients selected based upon the criteria established during the previous phase.
The parameters for the discriminant analysis of patients with prevalent emphysema or prevalent bronchitis will be applied to a prospective cohort of patients.In this last phase of the project the data obtained in vitro will be collected and statistically analyzed.
These data will then be correlated with the clinical data of the patients from whom biopsies have been obtained. the results will be correlated mainly with severity of disease, survival and response to therapies.
The genetic data will be compared to those already available in public genetic databases. <<<

Timescale

24 months

National and international background

Tobacco smoke represents the main risk factor for death, and causes a number of different diseases. In many developing countries the mortality due to diseases related to tobacco smoke is steadily increasing. It has been calculated that in year 2000 almost 5 million of premature deaths have been caused by cigarette smoke and of these almost 2 million were due to either chronic obstructive pulmonary disease (COPD) or lung cancer (1). Future perspectives for the forthcoming years consider a further increase in the incidence of these smoke-related events (2). Tobacco smoke can directly cause inflammation and tissue remodeling in the lungs of smokers (3). These effects are due, at least in part, to the oxidative stress induced by the smoke itself; the maintenance of the pulmonary oxidative processes is due both to the oxidants present in the cigarette smoke and to the elicited immune response (4). Cigarette smoke contains a large quantity of strong oxidants and increases the endogenous production of reactive oxygen species (ROS) by respiratory cells, in particular alveolar macrophages and neutrophils (5), the main sources of ROS. The final net result of these events is the generation of self-maintaining circuits, which can maintain and amplify the inflammatory processes associated with COPD, through paracrine and/or autocrine mechanisms.
Experimental evidence proves that cigarette smoke, the main risk factor for COPD, is one of the most important among the causes of the altered imbalance between oxidants and anti-oxidants in lung tissue. The overwhelming oxidative status in COPD patients has also been used for clinical purposes through the monitoring of different oxidative stress markers, among them 8-isoprostane and 4-hydroxi-2-nonenal (4-HNE). Interestingly, oxidative stress has been hypothesized to play a critical role also in the pathogenetic mechanisms of lung cancer (6).
The recruitment of inflammatory cells in airways is the critical event associated with the maintenance of the pathologic processes causing COPD (7). Both airway epithelial cells and alveolar macrophages produce several chemokines, particularly after exposure to cigarette smoke (8). A more detailed knowledge of chemokines and their receptors directly involved in the recruitment of leukocytes in the airways could therefore be crucial for the understanding of how this mechanism is modulated by cigarette smoke. Moreover, this could be the basis for the generation of chemokine inhibitors, potentially an useful therapeutic approach for the treatment of chronic inflammatory airway disorders (9).
In COPD patients, the normal inflammatory response to cigarette smoke is amplified (10), and the chronic inflammation caused by cigarette smoke is associated with an increased risk of lung cancer (11). The relationships between the chronic degenerative processes (inflammation) and those related to cellular proliferation (neoplastic) caused in the lung by tobacco smoke, is pertaining to many different pathologic processes.
Neuroendocrine (NE) cells can play a relevant role in determining if a smoker will develop during the lifetime a chronic airway obstruction or any other smoke-related disease, such as lung cancer (12). NE cells increase in number in the context of many different lung disorders characterized by both a restrictive (such as respiratory bronchiolitis and interstitial pneumonias) and an obstructive (such as pulmonary emphysema and chronic bronchitis) functional pattern. Neuroendocrine tumours are epidemiologically closely related to cigarette smoke.
Tobacco smoke can activate resident cells (macrophages, epithelial and dendritic cells), which can recruit from peripheral blood other cell types, among them neutrophils, monocytes and T lymphocytes. These cells produce inflammatory mediators, proteases and free radicals, relevant key players in the induction and the maintenance of the pathologic processes.
Oxidative stress can also produce apoptosis in epithelial and endothelial cells. Interestingly, apoptosis of type I pneumocytes can contribute to the induction of emphysema (13). Modifications of the cellular apoptotic activity can play an important role also in the development of cancer and in the determination of the level of response of cancer cells to anti-neoplastic drugs.
The complex process of apoptosis involves a huge number of events, involving several molecules. Among these molecules, clusterin (also known as APOJ or SGP-2) is an heterodimeric glycoprotein, present in the majority of tissues and organic liquids, secreted by different cell types. Clusterin expression is modulated in response to different mechanisms, among them inflammation, cellular neoplastic transformation and cancer progression in vivo (14). The heterogeneity of the results reported until now about clusterin can be explained by the existence of different isoforms of this molecule. One of these isoforms, glycosilated and secreted (70-80 kDa) has anti-apoptotic activity, conferring a protective action in respect to neoplastic growth. A different non-glycosilated isomeric form of clusterin (55 kDa) is localized within the nucleus, has anti-apoptotic activities and seems to be able to inhibit the proliferation of cancer cells (14). However, it is not known the exact role of this molecule in regard to inflammatory and neoplastic processes in the lung. Apoptosis can also be induced by the interaction among many different cellular types. As an example, in patients with pulmonary emphysema an association between CD8+ T lymphocytes and apoptosis of epithelial alveolar cells has been reported.
The crucial role of CD8+ T lymphocytes in the pathogenesis of COPD has already been established (15). Interestingly a similar role could be hypothesized also in lung cancer induction. In this context, among the inhibitory activities of CD8+ T lymphocytes, 3 different populations can be identified, all of them capable of significantly inhibitory activity for antigen-specific T cell clones (16). The first 2 populations are lacking the phenotypic expression of the CD28 receptor (CD8+CD28-), while no information is available in this regard on the phenotypic characteristics of the third population. Type 1 suppressor T cells inhibit the expression of co-stimulatory molecules on antigen presenting cells. Type 2 T cells produce 2 main cytokines; gamma interferon and interleukin 6 and therefore do not require a direct inter-cellular contact to exert their suppressor effect. This suggest a potential pathogenetic role of these effector T cells in the progression of cancer.
Cigarette smoke is the main risk factor for both COPD and lung cancer; however, these 2 diseases are caused by the interaction between endogenous (genetic) and environmental (cigarette smoke, pollution) factors (17, 18). In individuals genetically susceptible, the level of exposure to the single environmental factors or their combination induce the type and the intensity of the tissue response, responsible for the disease. In COPD, in addition to the effects of cigarette smoke, environmental pollution and respiratory infections, in particular those caused by viruses and during the infancy, can play a causal or con-causal pathogenetic role. It has been shown that particles of diesel exhaustions, main constituents of environmental pollution, induce an inflammatory response in bronchial epithelial human cells and an increased expression of the P4501A1 cytochrome, activating aromatic polycyclic hydrocarbures (19). The induction of enzymes activating cancerogenic molecules of cigarette smoke, induced by the smoke itself in human lung is markedly present among patients affected by lung cancer (20), thus suggesting the existence of a genetic susceptibility for this type of cancer. Viruses organotropic for the lung (adenovirus, respiratory syncithial virus and rhinovirus) might play a pathogenetic role and can be often cause exacerbations of COPD, in particular in elderly patients. Viral infection induces a marked inflammatory response of bronchial human cells, partly due to the mechanisms of the oxidative stress. Furthermore, it is possible that many exogenous factors interact in determining the noxious effect at the level of single individual. In this context, it has been reported that the exposure to particles of diesel exhausts increases the inflammatory response of bronchial cells following viral infections (21).
Alpha-1 anti-trypsin deficiency is a well know risk factor for the development of emphysema in smokers; on the same line of evidence, a role for the products of several genes has been demonstrated in the pathogenesis of COPD: proteases, activating or detoxifying enzymes, antioxidants, pro-inflammatory mediators, modifiers of the muco-ciliary clearance (22). Moreover, the tissue gene expression in non neoplastic lung tissue is different between non smokers with adenocarcinoma of the lung and smokers with the same type of lung cancer, thus suggesting the existence of different pathways for cellular transformation (23).
A subgroup of smokers appears to be more susceptible to the negative effects of cigarette smoke and this different susceptibility could be depend upon the relative role of different pathways of cellular transformation (e.g. neuroendocrine cells). In neuroendocrine lung cancer, the main genetic defects reported are relative to the p53/Rb gene; however, many other autocrine/paracrine pathways, characterized by the hyper expression of protein-kinase receptors and ligands, seem to be relevant for the regulation of growth, differentiation and survival of cancer cells (24). The pathogenetic mechanisms of COPD and of lung cancer caused by cigarette smoke are therefore quite complex and involve many different molecular aspects, partly not even recognized. The relevance of the characterization of these complex mechanisms resides not only in the field of basic knowledge of cancer and inflammation but also in the potential clinical application of these notions.
Functional abnormalities and many complications of COPD can be related to the exaggerated inflammatory response to smoke, and the phenotypic expression of this abnormal inflammatory response may vary greatly among different patients. Chronic airflow limitation, the main functional characteristic of COPD patients, is caused by the combination of modifications of both airways and pulmonary parenchyma (25). Chronic airway inflammation causes fibrosis and distortion of the airway wall, mucous impacts and contraction of smooth airway muscles, and reduction of airways calibre. Chronic inflammation of the lung parenchyma induces the destruction of the alveolar walls and the loose of the alveolar junctions, causing a reduction of the pulmonary elastic recoil. These modifications reduce the diameter of the airways. All of these effects do not cause a single homogeneous clinical pattern. As an example, patients with COPD and a prevalence of pulmonary emphysema have a more reduced FEV1, FEV1/FVC ratio and alveolar diffusing capacity, compared to patients with COPD and a prevalence of chronic bronchitis. In these same subjects, the radiologic score for emphysema was greater in the first group of patients. These clinical data do reflect biological markers; the number of lymphocytes in sputum is increased in patients with emphysema (26) and this finding goes on the same line with the notion that CD8+ lymphocytes can cause cytolysis and apoptosis of alveolar epithelial cells, through the production of many mediators, such as perforins, granzyme-B and TNF-alpha. <<<