Ewa Nawrot

CTLA-4 Expression and Polymorphisms in Lung Tissue of Patients with Diagnosed Non-Small-Cell Lung Cancer

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a potent immunoregulatory molecule that downregulates T-cell activation and thus influences the antitumor immune response. CTLA-4 polymorphisms are associated with various cancers, and CTLA-4 mRNA/protein increased expression is found in several tumor types. However, most of the studies are based on peripheral blood mononuclear cells, and much less is known about the relationship between CTLA-4 expression, especially gene expression, and its polymorphic variants in cancer tissue. In our study we assessed the distribution of CTLA-4 two polymorphisms (+49A/G and −318C/T), using TaqMan probes (rs231775 and rs5742909, resp.), and CTLA-4 gene expression in real-time PCR assay in non-small-cell lung cancer (NSCLC) tissue samples. The increased CTLA-4 expression was observed in the majority of NSCLC patients, and it was significantly correlated with TT genotype (−318C/T) and with tumor size (T2 versus T3 + T4). The presence of G allele and GG genotype in cancer tissue (+49A/G) was significantly associated with the increased NSCLC risk. Additionally, we compared genotype distributions in the corresponding tumor and blood samples and found statistically significant differences. The shift from one genotype in the blood to another in the tumor may confirm the complexity of gene functionality in cancer tissue.

TGF-β and SMADs mRNA Expression in Pulmonary Sarcoidosis

Lung fibrosis is a complication of sarcoidosis, in which TGF-β/Smad pathway may play an important role. We evaluated gene expression of TGF-β1, SMAD2, 3 and 7 in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94) to better understand the mechanisms of sarcoid inflammation. The relative gene expression was analyzed by qPCR method. Selected clinical/radiological features and biochemical markers were taken into account in the analysis. We found that TGF-β1 and SMAD3 expressions in PB lymphocytes were significantly higher in sarcoidosis patients. Up-regulation of SMAD7 (inhibitory Smad) and down-regulation of SMAD3 in BAL cells in all subgroups were found. The expression of TGF-β1 in PB lymphocytes was the highest in patients with lung parenchymal involvement and in the insidious onset phenotype. The expression of TGF-β1 in BAL cells was higher in patients with abnormal spirometry (p=0.012), and TGF-β1 and SMAD3 in patients with restrictive pattern (p=0.034 and 0.031, respectively). Several statistically significant negative correlations were found between the expression levels of SMAD2 and 3 in BAL cells and various LFT parameters. We conclude that TGF-β/Smad pathway is involved in the pathogenesis of pulmonary sarcoidosis. These biomarkers (especially TGF-β1, SMAD2 and 3) are of a negative prognostic value.

Altered miRNA expression in pulmonary sarcoidosis

BackgroundmiRNAs control important cellular functions including angiogenesis/angiostasis or fibrosis and reveal altered expression during pathological processes in the lung.MethodsThe aim of the study was to investigate the expression of selected miRNAs (miR-let7f, miR-15b, miR-16, miR-20a, miR-27b, miR-128a, miR-130a, miR-192 miR-221, miR-222) in patients with pulmonary sarcoidosis (n = 94) and controls (n = 50). The expression was assessed by q-PCR in BALF cells and peripheral blood lymphocytes (PB lymphocytes). For statistical analysis, the Kruskal–Wallis test, Mann–Whitney U- test, Neuman–Keuls’ multiple comparison test, and Spearman’s rank correlation were used.ResultsIn BALF cells, significantly higher expression of miR-192 and miR-221 and lower expression of miR-15b were found in patients than controls. MiR-27b, miR-192 and miR-221 expression was significantly higher in patients without parenchymal involvement (stages I) than those at stages II-IV. Patients with acute disease demonstrated significantly higher miR-27b, miR-192 and miR-221 expression than those with insidious onset. For PB lymphocytes, patients demonstrated significantly greater miR-15b, miR-27b, miR-192, miR-221 and miR-222 expression, but lower miR-let7f and miR-130a expression, than controls. Stage I patients demonstrated significantly higher miR-16 and miR-15b expression than those in stages II-IV, and patients with the acute form demonstrated higher miR-130a and miR-15b expression. In BALF cells, miR-16 and miR-20a expression was significantly higher in patients with lung volume restriction, and miR-let7f was higher in the PB lymphocytes in patients with obturation. Several correlations were observed between the pattern of miRNA expression, lung function parameters and selected laboratory markers.ConclusionThe obtained results suggest that the studied miRNAs play a role in the pathogenesis of sarcoidosis, and that some of them might have negative prognostic value.

Immunoexpression of TGF-β/Smad and VEGF-A proteins in serum and BAL fluid of sarcoidosis patients

BackgroundThe chronic course of pulmonary sarcoidosis can lead to lung dysfunction due to fibrosis, in which the signalling pathways TGF-β/Smad and VEGF-A may play a key role.MethodsWe evaluated immunoexpression of TGF-β1, Smad2, 3, and 7, and VEGF-A in serum and bronchoalveolar lavage (BAL) fluid of patients (n = 57) classified according to the presence of lung parenchymal involvement (radiological stage I vs. II-III), acute vs. insidious onset, lung function test (LFT) results, calcium metabolism parameters, percentage of BAL lymphocytes (BAL-L%), BAL CD4+/CD8+ ratio, age, and gender. Immunoexpression analysis of proteins was performed by ELISA.ResultsThe immunoexpression of all studied proteins were higher in serum than in BAL fluid of patients (p >0.05). The serum levels of TGF-β1 (p = 0.03), Smad2 (p = 0.01), and VEGF-A (p = 0.0002) were significantly higher in sarcoidosis patients compared to healthy controls. There were no differences within the sarcoidosis group between patients with vs. without parenchymal involvement, acute vs. insidious onset, or patients with normal vs. abnormal spirometry results. In patients with abnormal spirometry results a negative correlation was found between forced vital capacity (FVC) % predicted value and TGF-β1 immunoexpression in BAL fluid, and positive correlations were observed between the intensity of lung parenchymal changes estimated by high-resolution computed tomography (HRCT scores) and Smad 2 level in serum.ConclusionsTGF-β/Smad signalling pathway and VEGF-A participate in the pathogenesis of sarcoidosis. BAL TGF-β1, and Smad 2 in serum seem to be promising biomarkers with negative prognostic value, but further studies are required to confirmed our observations.

Decreased FAM107A Expression in Patients with Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related death in the world. Early detection, based on molecular markers, could decrease mortality from this disease. Tumor development is often associated with inactivation or loss of tumor suppressor genes (TSGs). The aim of the present study was to analyze the expression level of FAM107A gene, a TSG located in 3p21.1, in lung cancer tumors and in tumor adjacent normal lung samples. Promoter methylation status of FAM107A was evaluated as the potential mechanism of its epigenetic silencing. The relationship between gene mRNA expression and tumor staging, metastasis status, and non-small cell lung cancer (NSCLC) histopathological subtypes in 60 patients was analyzed. Total RNA was isolated from tissue samples and gene expression was assessed in qPCR assay. Gene promoter methylation status was evaluated in MSP reactions, using bisulfite converted DNA and two pairs of primers: methylated and unmethylated. We found that the expression of the gene was dramatically decreased in all NSCLC samples and was significantly lower than in tumor adjacent normal lung tissue. Promoter methylation of FAM107A gene was confirmed only in the minority of NSCLCs. The results highlight the importance of FAM107A in lung carcinogenesis, although indicate other than promoter hypermethylation mechanism of the gene decreased expression.

Expression analysis of three miRNAs, miR-26a, miR-29b and miR-519d, in relation to MMP-2 expression level in non-small cell lung cancer patients: a pilot study

Lung cancer is the most common cause of death in men and second only to breast cancer in women. MicroRNAs (miRNAs) are involved in tumorigenesis and function as oncogenes or tumor suppressor genes. Among other genes, miRNAs regulate matrix metalloproteinases (MMPs), the proteolytic enzymes playing a significant role in the degradation of extracellular matrix, enhancing tumor invasion and metastasis. The aim of the study was to evaluate the expression levels of selected miRNAs: miR-26a, miR-29b and miR-519d, and their target gene, matrix metalloproteinase-2 (MMP-2) in patients with non-small cell lung cancer (NSCLC). The results were correlated with tumor staging, NSCLC histopathological subtypes and patients’ demographical features to assess the possible diagnostic/prognostic value of the studied miRNAs and MMP-2. Total RNA was isolated from 38 NSCLC tissue samples, and the expression analysis was performed using TaqMan® probes in qPCR assay. The results indicated underexpression of selected miRNAs and overexpression of MMP-2. The decrease in miRNA-29b expression was statistically significant and differentiated NSCLC histopathological subtypes. Additionally, statistically significant negative correlation was found between MMP-2 expression and its regulatory miR-26a. There are very few studies reporting miRNA-MMPs analysis on mRNA level in lung cancer, and no similar reports are available from Polish population. The results of our pilot study indicated the diagnostic potential of miR-29b and MMP-2, an inverse association between miR-26a and MMP-2, and proved the role of MMP-2 and the studied miRNAs in lung carcinogenesis. Further studies are needed to verify their potential usefulness for the treatment of lung cancer.

Expression of HIF-1A/VEGF/ING-4 Axis in Pulmonary Sarcoidosis

Angiogenesis/angiostasis regulated by hypoxia inducible factor-1A (HIF-1A)/vascular endothelial growth factor (VEGF)/inhibitor of growth protein 4 (ING-4) axis may be crucial for the course and outcome of sarcoidosis. Overexpression of angiogenic factors (activation of VEGF through HIF-1A) may predispose to chronic course and lung fibrosis, whereas immunoangiostasis (related to an overexpression of inhibitory ING-4) may be involved in granuloma formation in early sarcoid inflammation, or sustained or recurrent formation of granulomas. In this work we investigated gene expression of HIF-1A, VEGF and ING-4 in bronchoalveolar fluid (BALF) cells and in peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94), to better understand mechanisms of the disease and to search for its biomarkers. The relative gene expression level (RQ value) was analyzed by qPCR. The results were evaluated according to the presence of lung parenchymal involvement (radiological stage I vs. II-IV), acute vs. insidious onset, lung function tests, calcium metabolism parameters, percentage of lymphocytes (BALL%) and BAL CD4+/CD8+ in BALF, age, and gender. In BALF cells, the ING-4 and VEGF RQ values were increased, while HIF-1A expression was decreased. In PB lymphocytes all studied genes were overexpressed. Higher expression of HIF-1A in PB lymphocytes of patients with abnormal spirometry, and in BALF cells of patients with lung volume restriction was found. VEGF gene expression in BALF cells was also higher in patients with abnormal spirometry. These findings were in line with previous data on the role of HIF-1A/VEGF/ING-4 axis in the pathogenesis of sarcoidosis. Up-regulated HIF-1A and VEGF genes are linked to acknowledged negative prognostics.

RARβ Promoter Methylation as an Epigenetic Mechanism of Gene Silencing in Non-small Cell Lung Cancer.

The retinoid acid receptor-p (RARβ) gene is one of the tumor suppressor genes (TSGs), which is frequently deleted or epigenetically silenced at an early stage of tumor progression. In this study we investigated the promoter methylation and expression status of the RARβ gene in 60 surgically resected non-small cell lung cancer (NSCLC) tissue samples and 60 corresponding unchanged lung tissue samples, using methylation-specific PCR and real-time-polymerase chain reaction (qPCR) techniques. We correlated the results with the pathological features of tumors and clinical characteristics of patients. qPCR analysis detected a significantly lower RARβ expression in the patients with adenocarcinoma (AC) and large cell carcinoma (LCC) than in those with squamous cell carcinoma (SCC) (AC vs. SCC, p = 0.032; AC and LCC vs. SCC, p = 0.0 13). Additionally, significantly lower expression of the RARβ gene was revealed in the patients with non-squamous cell cancer with a history of smoking assessed as pack-years (PY < 40 vs. PY ≥ 40, p = 0.045). Regarding RARβ promoter methylation, we found significant differences in the methylation index in the SCC group when considering pTNM staging; with higher index values in T1a + T1b compared with T2a + T2b and T3 + T4 groups (p = 0.024). There was no correlation between the methylation status and expression level of the RARβ gene, which suggests that other molecular mechanisms influence the RARβ expression in NSCLC patients. In conclusion, different expression of the RARβ gene in SCC and NSCC makes the RARβ gene a valuable diagnostic marker for differentiating the NSCLC subtypes.

Small suitability of the DLEC1, MLH1 and TUSC4 mRNA expression analysis as potential prognostic or differentiating markers for NSCLC patients in the Polish population

According to the latest data, lung cancer is one of the most common cancer worldwide, men contributing nearly 21.2% and women 8.6% of all diagnosed cancers. Late detection of tumour drastically reduces the chance for a cure. Thus, it is important to search for candidate biomarkers for screening of early stage nonsmall cell lung carcinoma (NSCLC). Tumour suppressor genes, DLEC1, TUSC4 and MLH1, localized on 3p21 are recognized to play a role in NSCLC carcinogenesis. The aim of this study was to assess the relationship between the DLEC1, TUSC4 and MLH1 mRNA expression, and clinical features of NSCLC patients, tobacco addiction, and tumour histopathological characteristics. The DLEC1, TUSC4 and MLH1 expression was analysed in lung tumour tissue samples obtained from 69 patients diagnosed with NSCLC: squamous cell carcinoma (