Studies on epithelial mesenchymal transition (EMT) in 'smoking related' airway disease

Background: Chronic obstructive pulmonary disease (COPD) is a global health problem, killing approximately 3 million people annually and emerging as the third largest cause of human mortality worldwide. COPD is a poorly reversible chronic, slowly progressive airway obstructive respiratory disease. It is mainly smoking-related and primarily constitutes small airway fibrosis and destruction, more generalized “chronic bronchitis” and in some but not all later development of emphysema. Tobacco smoking is the main causative factor for development of COPD, both in developed and developing countries. Approximately 50% of smokers develop COPD eventually. COPD airways remodeling changes are mostly related to reduce airflow due to small airway fibrosis and ultimately obliteration. Various mechanisms lead to remodeling changes in the airways including perhaps most importantly, dysregulated epithelial basal stem cell function, one feature of which is active EMT. In EMT, epithelial cells transition into a mesenchymal phenotype with subsequent migration into the underlying lamina propria and may be associated with accumulation of myofibroblasts immediately adjacent in the lamina propria. EMT has been implicated in tissue fibrosis in the so called Type-2 form, and when associated with hyper vascularity, it is termed Type-3 which is strongly related to epithelial cancer development. EMT is a complex process which includes various intracellular pathways including Smad-dependent (TGFβ-Smad pathways) and non-Smad dependent pathways (e.g. the Wnt-βcatenin and hedgehog pathways), and various nuclear transcription factors (the β-catenin/twist/Snail clusters). It is notable that epithelial cancers make up approximately 90% of all human malignancies suggesting that epithelial cells are especially unstable and EMT- associated mechanisms may be the common factor. Local tissue angiogenesis is especially regarded as another key aspect of both the pre-malignant and malignant phases of epithelial cancer development. Moreover, COPD itself is a major independent risk factor for development of non-small cell (NSC) lung cancer with squamous cell tumors developing in central airways and adenocarcinoma more in the peripheral airway tree. Along with several genetic changes secondary to environmental insult (tobacco smoke) EMT, especially in association with hyper vascularity i.e. Type-3, also has a strong pro-cancerous influence Thus, the local airway microenvironment is very important in carcinogenesis and metastasis. Although the expression of EMT biomarkers has been investigated in smokers, the expression pattern of transcription factors and their pathways in COPD has not been reported until now. Aims: I have investigated the expression of EMT biomarkers in large and small airways, as well as involvement of transcriptional pathway and alterations in transcription factors cellular location. I have investigated smoker with normal spirometry, COPD subjects (both current smokers, CS, and ex-smokers, EX), and compared with normal control tissues. I have also investigated whether EMT biomarker expression in lung cancer is associated with tumor staging and differentiation and how this expression is related to that in the corresponding non-cancerous airway wall. Methodology: I evaluated the expression of EMT biomarkers (S100A4, Vimentin), an epithelial activation marker (EGFR), and Rbm vascularity (Type-IV collagen) cross-sectionally in both large airways (from resected lung tissue, RT) and small airways (also RT) from: 18 individuals with chronic airflow limitation [combination of smokers with SAD plus frank COPD] and 9 RT control tissues (NC). Differences between peripheral and central area in adeno and squamous cell carcinoma from these same subjects were also evaluated, using the same mesenchymal biomarkers and compared to their expression in large and small airways. I also evaluated the TGFβ-Smad pathway and the β-catenin/Snail/Twist transcription factor cluster in large airway bronchial biopsies (EBB) from 15 NLFS, 20 COPD-CS; 15 COPD-EX, and compared these with 15 NC. Anti-S100A4/Vimentin/Type-IV collagen/TGFβ-Smad 2/3/7 and anti-transcription factor (β-catenin/Snail/Twist) immunostaining on paraffin-embedded tissue was quantified using computer-aided image analysis. Results: Large and small airway epithelium showed markedly enhanced expression of S100A4 and vimentin in CAL (p<0.001) compared to normal controls (NC). A significant difference in term of increased Rbm vascularity (Type-IV collagen) was observed in large airway (Type-3 EMT) in comparison to small airway (Type-2 EMT) (p<0.001). Small airflow obstruction (FEF25-75%) was correlated significantly with small airway EMT biomarker expression (R= -0.52; p<0.03). In the large airway epithelium, TGFβ1 and pSmad2/3 overexpression was found along with pSmad7 under expression in all clinical groups compared with controls. TGFβ1 expression was especially high in COPD subjects throughout the airway wall (P<0.01), while pSmad 2/3 expression was most marked in the epithelial basal cells and Rbm cells and again especially in current smoking COPD (P<0.05). Expression of inhibitory pSmad-7 was also predominantly reduced in patients with COPD in contrast to smokers and controls (P<0.01). In addition, pSmad but not TGFβ1 expression, was related to airflow obstruction (both FEF25-75% and FER) [(R= -0.74; p<0.001) for pSmad2/3 and (R= 0.48; p<0.07) for psmad7] and the canonical EMT biomarker (S100 A4) expression (R= 0.48; p<0.05 for pSmad 2/3 and R= -0.59; p<0.02 for pSmad7). An overall increase in β-catenin/Snail/Twist expressing cells in the airway epithelium, Rbm and LP was uniformly found in all clinical groups compared to normal controls along with their significant cytoplasmic to nuclear relocation (p<0.05). However, such changes in Twist expression were not as marked as for the other two transcription factor and mainly significant for NLFS and COPD-Ex (p<0.05). In addition, transcription factors over-expression was significantly correlated not only with airflow obstruction (both FER and FEF25-75%) and EMT activity (S100A4), but also with the TGFβ-Smad pathway expression as well. Significantly increased EMT biomarker expression was found in the peripheral (leading edge) areas of the both adeno carcinoma (p<0.001) and squamous cell carcinoma (p<0.001), in comparison to central areas of these tumors. Moreover, peripheral tumor bio-marker expression was correlated significantly with TNM staging and differentiation score for the whole group of carcinomas, and also with airway epithelial EMT activity. Further, vascularity in the leading edge areas for peripheral squamous cell carcinoma was significantly correlated with large airway Rbm vascularity (R=0.68; p<0.05). Conclusions: The expression of EMT biomarkers (S100A4 and Vimentin) was upregulated in both small as well as large airways; with EMT categorized on the basis of Rbm vascularity as Type-2 in small airways (pro-fibrotic) and as Type-3 in large airways (pro-malignant as well as pro-fibrotic). In addition, transcription factors (β-catenin/Snail/Twist) and the transcriptional pathway (TGFβ-Smad) examined were upregulated in large airways in smokers, but in general, especially so in COPD. These data enhance the strength of evidence for EMT activity being a fundamental part of the pathogenic process of COPD in the airways, and the strong correlations seen between such activity and airway obstruction in especially compelling. Further, increased expression of EMT activity may also be crucial in the pathogenesis of malignancy related to COPD, with “imprinting into the cancer of EMT activity from the already abnormal airway epithelium.