| Abstract |
INTRODUCTION
Idiopathic pulmonary fibrosis (IPF) is a form of chronic progressive interstitial pneumonia that continues to be associated with high morbidity and mortality, despite the fact that new antifibrotic drugs have recently been added to our quiver[1].
The exact cause of the disease still remains inexplicable, even though it seems to be strongly related to aging and smoking, similarly with lung cancer. Interestingly, LC is a common co morbidity among patients with idiopathic pulmonary fibrosis with major impact in their survival. Increasing data support the hypothesis that lung cancer occurs secondarily on the ground of fibrosis rather than as a preceding finding. In the majority of patients, LC arises as nodular lesions in the peripheral area of fibrosis, mainly at the lower areas of the lungs with squamous cell and adenocarcinoma being the prevalent histological types [2, 3].
A deeper view at the pathophysiology of the two diseases reveals several similarities and common pathways involved. Crucial cellular mechanisms known to be implicated in proliferation and resistance to apoptosis are activated in both lung cancer and pulmonary fibrosis, such as Fas/Fas ligand pathway, PTEN (protein phosphatase and tensin homologue) , phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) and FAK (focal adhesion kinase). Epithelial–mesenchymal transition (EMT), is also a common event of cardinal importance both in the invasive feature of malignancies and in proliferation. Preneoplastic lesions (atypia, metaplasia, dysplasia), microsatellite instability and loss of heterozygosity in genes that underlie the tumorigenesis have been reported in lung fibrosis tissue also. Aging and the subsequent cellular senescense is a profound component of both diseases. Aggregating mutational burden, increased epigenetic gene silencing and telomere dysfunction frame the probable underline correlation[4, 5].
Common risk factors like aging, cigarette smoke and environmental effects seem to influence the activity of numerous genes by creating aberrant DNA methylation patterns and accelerating the aging of our epigenome. DNA methylation is carried out by three main DNA methyltransferases (DNMTs); DNMT1, which maintains pre-existing methylation patterns and DNMT3A and DNMT3B which yield novel methylation patterns. Genome wide hypomethylation and promoter hypermethylation affect genes involved in crucial cellular procedures in carcinogenesis. Similarly in IPF, the p53 pathway, Thy-1 and PTGER2 are suppressed by promoter hypermethylation, while global methylation profile in IPF was found different compared to controls and partially similar to cancer. [6, 7]
MicroRNAs have also been emerged as major regulators of gene expression through epigenetic and posttranscriptional mechanisms. MicroRNA profiling studies in lung cancer have currently proposed several microRNAs as potential epigenetic biomarkers in the diagnostic procedure. Similarly, in IPF several oncomirs have been consistently found deregulated, such as miR-21, let-7d and miR-29 family. Recently our team found miR185 and miR29a to be downregulated in IPF compared to controls and data supporting important pathophysiological extensions were shown as Mir185 downregulation activated AKT signaling pathway in macrophages. MiR-29a downregulation found to be correlated with the overexpression of the collagen gene COL1A1 suggesting that the miR-29a/COL1A1 pathway is also active in IPF BAL cells, as previously demonstrated in IPF tissues. [8] [9]
Aberrant DNA methylation patterns, miRNAs profiling studies and other epigenetic mediators consist the field of intense research for both LC and IPF, not only individually but also as face to face comparison, in an effort of revealing common underlying mechanisms and sharing therapeutic targets. Tissue specimens and cell lines are the predominant material of research. However the role of BALF as a minimally invasive tool to study alveolar macrophages and their implication in pathogenesis continually recovers ground. Alveolar macrophages in IPF, acquiring alternatively activated phenotype, are actively involved in the fibrotic process. The production of soluble mediators related with myofibroblasts’s survival, epithelial activation and EMT (such as CCL18, IL13, TGF-b, insulin-like growth factor) and the expression of profibrotic microRNAs are potential mechanisms through which alveolar macrophages promote fibrogenesis [10, 11]. On the other hand, there is plenty of evidence to support the altered alveolar macrophage function in patients with lung cancer. Those highly plastic cells display different phenotypes under the influence of tumor-associated polarizing events such as mediators and hypoxic tissue damage. [12, 13]
The aim of our study is to investigate possible common pathways between IPF and lung cancer at the level of key epigenetic regulators in bronchoalveolar lavage fluid. Similarities between IPF and LC were noticed at the level of miRNAs (miR-185 and miR-29a) with known implication in both diseases and their targets. However, DNMT1 and col1a1 seem to differ between the diseases, further reduced in the presence of malignant burden in BALF.
Results
Demographic data and PFTs are summarised in Table 1. IPF patients were older than the patients in the LC group by 4.3(±4.6) years, while LC patients were heavier smokers as could be expected. The majority of LC was NSCLC (29/32) with the rest being SCLC. The group of LC patients was subdivided according to the endobronchial findings as described in materials and methods. 14 LC patients were assigned to the group “same side” (SS) and 9 to “opposite side” (OS) according to the site of broncoscopy. 14 LC patients had no malignant cell detected in their BAL and were assigned to “negative cytology” (NC) group and 7 to the “positive cytology” (PC). 19 patients had obvious “endobronchial lesion” (OEL) and 7 were assigned to “no endobronchial lesion” (NOEL).
MiR-185 and Mir-29a levels are similar between IPF and LC.
MicroRNA expression levels in BAL cells were measured by qRT-PCR and normalized using small nucleolar RNA RNU19. We have previously shown that both microRNAs were significantly downregulated in IPF relative to controls [8]. The downregulation of both micro-RNAs was also noted for LC since the expression of miR-29a and miR-185 did not differ between IPF and LC patients (Figure 4Α and Β). miR-29a and miR-185 expression levels were remarkably correlated within the IPF group (Spearman’s R 0.81, p=6e-14) and the LC group (Spearman’s R 0.71, p=3.6e-5).
DNMTs/AKT/ mir-29a/mir-185 axis in IPF and LC
DNMT1 and DNMT3b are common targets of miR-185 and mir-29 in IPF and LC. mRNA expression levels in BAL cells were measured by qRT-PCR and normalized using GAPDH. DNMT1 levels previously measured in IPF relative to controls showed no differences. Interestingly, DNMT1 levels were significantly lower in LC patients compared to IPF patients. (Figure 5Α Table 3). Similar transcript levels of DNMT3b, AKT1 or AKT2 were found in IPF and LC (Table 3).
Effect of malignant burden on DNMT1 levels in BAL cells compared to IPF.
Comparing IPF patients with the LC group in detail, further reduced levels of DNMT1 were detected in the samples where the BAL procedure was performed at the side of the lesion (SS: same side) as opposed to the lesion free side (OS: opposite side). Moreover, LC patients with positive BAL cytology results (PC: positive cytology) had a more pronounced reduction in DNMT1 levels than those without the presence cells with malignant features (NC: negative cytology), when compared with IPF (Figure 5Β). Regarding the histological type of LC, the more obvious reduction in DNMT1 mRNA levels compared with IPF was found in NSLC type (Table 2).
COL1a1/mir-29a/mir-185 axis in IPF and LC.
Next we analysed miR29a specific target Col1a1 by qRT-PCR and normalized using GAPDH. Our previous result showed that IPF BAL cells express significantly higher levels of Col1a1 mRNA than controls. In this study we observe that the levels of Col1a1 mRNA were significantly lower in LC patients compared with IPF patients. (Figure 6Α Table 3).
Effect of malignant burden on Col1a1 levels in BAL cells compared to IPF .
The mRNA levels of COL1a1 were quite reduced in LC patients compared with IPF overall as it was noted previously. Assorting LC patients depending on the side the BAL procedure was performed and comparing them with IPF, further reduced levels of COL1a1 was detected in LC patients when the BAL procedure was done ipsilaterally of the lesion than contralaterally. The presence of an endobronchial lesion during the bronchoscope signified more clearly reduced levels of COL1a1 (compared with IPF) than in the case of no endobronchial lesion. (Figure 6Β Table 3). Patients with the histological type of SCLC had COL1a1 levels similar with IPF levels, opposing the reduced levels in the NSCLC patients subgroup.
Discussion
This is a study of expression of major epigenetic molecules with known implication both in IPF and LC in BAL cells, such as key microRNAs miR29a and miR185, previously known to be downregulated in both diseases and their common targets DNMTs (DNMT1 and DNMT3b), the enzymes been actively involved in DNA methylation procedure. The relationship between those lethal diseases which often coexist is an active field of research as common pathogenic pathways emerge, with major therapeutic implications. Our cardinal findings were: a) both miR-185 and miR-29a were comparably expressed in IPF and LC BAL samples. b) no direct correlation with miR-29a or miR-185 and their targets was observed, albeit DNMT1 downregulation was characteristic of LC and collagen 1a upregulation was representative of IPF BAL samples. c) In LC the malignant burden affected both DNMT1 and collagen 1a expression.
Similar levels of miR-29a and miR-185 were detected between IPF and lung cancer in BAL cells while our previous study showed that both miR-29a and miR-185 were downregulated in IPF relative to controls [8]. The downregulation of miR-29a and miR-185 in lung cancer tissue specimens has been previously established however, this is the first study in lung cancer BAL cells. Our results suggest that miR-185 is a novel common microRNA deregulation in IPF and LC next to previously identified microRNAs such as miR-29a. MiR-29a and miR-185 expression showed remarkable association in both IPF and LC BAL samples. A possible common regulation of the expression of the two microRNAs may be related to the increased levels of TGFb in both IPF and LC BAL [14, 15]. Activation of TGF-β signaling and excessive accumulation of ECM proteins are observed in IPF and lung cancer, highlighting a common molecular mechanism in both diseases that is directly linked to both microRNAs[16] [17]. Interestingly, miRNA based-therapeutic strategies are already under evaluation for their use in several malignancies[18] and that imposes the need for in depth study of similarities and differences between IPD and LC, focusing on key molecules involved in the multifarious function of alveolar macrophages.
For this reason, we examined the expression of a common target of miR-29a and miR-185 induced by TGFb and central fibrosis mediator collagen 1a in LC in comparison to IPF. Our previous results supported that the downregulation of miR-29a in IPF is associated with the overexpression of col1a1 gene in BAL cells, confirming the active role of the miR-29a/col1a1 pathway also in AMs alike lung tissue while the expression profile of collagen 1a in LC has not been yet clarified. Collagen 1a could be involved in carcinogenesis as aberrant expression levels were revealed in several malignancies including hepatocellular carcinoma [19], NSCLC tissue [20] and in malignant gastric tissue also [21]. Our findings however showed significantly increased levels of collagen 1a in IPF relative to LC in BAL cells that establishes the characteristic fibrotic profile of BAL cells in IPF that appears to be lacking in LC BAL cells. The further reduced levels of col1a1 accordingly with increased malignant burden cannot so far be interpreted and further functional analysis of AMs in LC is needed.
A second pathway/axis affected by both miR-29a and miR-185 is the expression of DNA methyltransferases (DNMTs). An important concept currently put forward for the pathogenesis of IPF as in cancer is that common risk factors like aging, cigarette smoke and environmental effects induce errors in the maintenance of the methylation marks of the genome creating aberrant DNA methylation patterns and accelerating the aging of our epigenome [22, 23]. Global methylation profile in IPF lung tissue was found different compared to controls and partially similar to cancer. [6, 7] Furthermore, increased expression of the DNMTs was observed in both cancer and IPF lung tissues studies [7] [24], leading to site-specific hypermethylation and gene silencing.
We have previously reported that DNMT1 mRNA levels in the BAL cells of IPF patients were similar to controls[8]. In the current study, we observed a significant reduction in the mRNA levels of DNMT1 in LC BAL cells while, DNMT3b levels were similar in IPF and LC. DNMT1, in contrast to DNMT3’s, appears to function in cooperation with DNA damage repair pathways in order to maintain genomic stability and ablation or reduction of DNMT1 promotes mutagenic events [25], microsatellite instability and chromosomal translocations[26]. Interestingly, the samples obtained near the malignant lesion or with positive malignant cell cytology results showed a more pronounced reduction of DNMT1 expression suggesting that reduced DNMT1 levels were associated with increased malignant burden.
LC is a common and prognostically determinant comorbidity among IPF patients. BAL procedure is a less invasive and harmless tool for revealing new, disease specific biomarkers regarding LC in IPF patients, for screening, risk stratification and diagnostic purposes, verging the promising concept of cancer liquid biopsy. It would be of great interest to study he expression profile of those miRNAs and their targets also in patients who simultaneously suffer from IPF and LC. MicroRNA based-therapeutic strategies are already under evaluation for their use in several malignancies[18] and IPF[27] and would greatly benefit from in depth study of similarities and differences between IPF and LC, focusing on key molecules involved in the multifarious function of alveolar macrophages.
Tissue specimens and cell lines are the predominant material of research, however the role of BAL as a minimally invasive tool to study alveolar macrophages and their implication in pathogenesis continually recovers ground. In IPF alveolar macrophages deriving from monocytes recruited to the injured lungs are actively involved in the fibrotic process[28]. Altered alveolar macrophage function in patients with lung cancer has been recorded under the influence of tumor-associated polarizing events such as mediators and hypoxic tissue damage [12]. Our study provides some insight with respect to common alveolar macrophage function in the two groups as demonstrated by the commonly reduced expression of miR-29a and miR-185. Further research is needed in order to identify BAL biomarkers for high risk patients and more targeted therapies.
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