Malignant Pleural Mesothelioma is a rapidly fatal disease that is often difficult to diagnose pathologically and can be confused with other cancers, often, lung adenocarcinoma. Treatment of MPM relies on a rapid diagnosis and it would be advantageous to have specific biomarkers to differentiate it from other tumors 6
. MiRNA are being used clinically to aid in the diagnosis of this disease, although there is little experimental literature detailing the identification of differentially altered miRNA, or the biological pathways predicted to be affected by miRNA expression alterations. We identified a panel of miRNA that are specifically downregulated in MPM compared to lung adenocarcinoma. Expression of these miRNAs was not significantly different in the histological types of mesothelioma, suggesting that their downregulation is in fact, a general characteristic of mesothelioma, and distinguishes this disease from its main differential diagnosis, adenocarcinoma. Analysis of these miRNAs reveals that they can act as biomarkers which exceed the current guidelines for the diagnosis of mesothelioma (). In fact, miR-200c has a 100% Negative Predictive Value, indicating that detection of this miRNA is enough to rule out a diagnosis of mesothelioma. When pathological tests are inconclusive, measuring a combination of these miRNAs could lead to an accurate diagnosis which can greatly influence the treatment and outcome of the patient.
Understanding the normal, collective role of these miRNA can inform on the consequences of their loss in mesothelioma, and suggest novel targets for therapeutic intervention and disease screening, both of which are crucial to patient survival. Previous studies relied on cultured tumor cell lines to identify a panel of miRNAs which may be associated with disease 28
. Although most research on miRNAs done in cell lines can provide clues to human cancers, due to the inherent artificiality of in vitro
systems, conclusions drawn from experimental systems alone must be corroborated with data from human tumors. Experiments in our lab, as well as published reports have shown that cultured cell lines are inappropriate for miRNA biomarker identification andoften have no relation to the expression patterns in primary tumors 29
. Our approach using human tumors allowed us to identify a group of miRNAs whose downregulation in MPM are predictive of disease. Analysis of the overlapping targets of these miRNAs reveals the biological mechanisms through which they lead to mesothelial carcinogenesis. The gene clustering and familial pattern of these miRNAs indicated that they affect common pathways 13
and led us to predict aberrant wnt signaling as a mechanism for disease, providing potential new targets for treatment.
Experiments in simple organisms that show most miRNAs are not essential, and individually often have subtle effects on their downstream targets, provide evidence for the theory of miRNA redundancy 14, 15
. Five out of seven of our miRNAs are members of the same gene family (miR-200). This family is encoded in two clusters on chromosomes 1 (miR-200a/b/429) and 12 (miR-200c/141), and is evolutionarily conserved. Forty-two percent of human miRNAs are encoded in clusters less than 3000 nucleotides apart, suggesting that they are co-regulated and that their expression levels can be indicative of certain diseases and syndromes 13
. These miRNAs are grouped into a family due to homology in their seed sequences, and which messenger RNAs they will target. In fact, miR-200b/c/429 have identical seed sequences, indicating that they are redundant regulators of the same genes. The loss of expression of these closely related miRNAs by at least two separate events suggests they have redundant functions that are absent in the diseased state. By comparing overlapping targets of all of our classifying miRNAs, we were predict that absence of these miRNAs would result in the loss of multiple levels of post-transcriptional gene regulation of the wnt signaling pathways.
Our observation of the downregulation of seven potential miRNA wnt antagonists in mesothelioma suggests that dysregulation of this pathway, through the loss of miRNA expression, is an essential process in disease progression. The mechanism for this is not known but may occur in various ways. Two of the miRNAs are encoded individually; miR-203 on chromosome 14 and miR-205 on chromosome 1, and loss of their expression seems to be unrelated. Downregulation of the miR-200 gene family may be coordinate, however, as the genes for the miRNAs miR-200c/141 are clustered on chromosome 12, while the genes for miR-200a/b/429 are clustered on chromosome 1. This clustering, combined with evidence that distinct methylation alterations predict MPM, indicates epigenetic silencing as a possible mechanism of repression 30
. Indeed, the expression of miR-203 from a fragile region of chromosome 14 has been shown to be modulated by loss of heterozygocity and through hypermethylation in some acute lymphoblastic leukemias 31
. It is of interest to investigate the mechanisms for downregulation of these specific miRNAs in MPM in order to gain further insight into the genesis of this disease, as well as to develop novel modes of treatment.
Out of the seven miRNAs that we describe as under-expressed in MPM compared to lung adenocarcinoma, two (miR-203 and miR-205), have previously been shown to be upregulated in lung adenocarcinoma compared to matched, noncancerous tissue 17
. Even higher expression of miR-205 has been suggested as a biomarker for squamous cell carcinoma, as its upregulation can distinguish this tumor from adenocarcinoma 32
. The miRNAs miR-203 and miR-429 have recently been shown to be downregulated in MPM 33
. We found a panel of miRNAs (miR-200c/141/200b/200a*/429/203/205) downregulated in MPM that we propose as biomarkers for disease and predict that these miRNAs play overlapping redundant roles in modulation of proteins associated with wnt signaling. This is significant, as it has been shown that activation of the wnt pathway, specifically the nuclear translocation of β-catenin, occurs in mesothelioma cell lines 34
. In fact, we identified multiple wnt-pathway gene products that have been shown to be upregulated in MPM, including Jun, Myc, EGR1 and Wnt5B, which are predicted to modulated but our panel of microRNAs 35
The importance of wnt signaling has long been studied in development. In the canonical wnt pathway, wnt binding to its receptor leads to the stabilization of β-catenin. β-catenin can then translocate to the nucleus and interact with the transcription factors LEF/TCF (lymphoid enhancer-binding factor/T cell factor) to activate target genes 36
. In Drosophila
, the members of the miR-8 gene family have been shown to antagonize this signaling pathway in multiple ways, including through the inhibition of TCF translation, a positive regulator of wnt signaling, as well as by targeting effectors of the pathway 25
. In human cancers, reduction in expression of the orthologous miR-200 gene family and its target E-cadherin, as well as lower miR-205 expression, is associated with metastases 37, 38
. Specifically, it has been demonstrated that miR-200c can block the translation of the zinc finger transcription factor ZEB1 (TCF8/dEF1) and result in an increase in E-cadherin 39
. Other evidence indicates that decreased expression of the miR-200 gene family results in the up-regulation of the E-cadherin repressors ZEB1 and ZEB2, promoting the epithelial-to-mesenchymal transition (EMT) that is indicative of malignant tumor progression 40
. This pathway was revealed to be part of a regulatory loop when it was shown that ZEB1 can directly repress miR-200c and miR-141 41
. The effect of the loss of cadherins on the wnt signaling pathway is still being investigated but it is known that cadherin can tether β-catenin to the cellular membrane. In conjunction with a disruption in the β-catenin degradation pathway, the loss of this adhesion molecule may elevate wnt signaling by allowing translocation of β-catenin to the nucleus 42
Interestingly, dysregulation of non-miRNA regulators of the wnt signaling pathway has been reported in the metastases lung adenocarcinoma. In this case, disruption of the WNT/TCF signaling occurs through the transcription factors LEF1 and HOXB9 and promotes invasion and colonization of multiple organ types, and is not associated with primary lung adenocarcinoma 43
. Target prediction software indicates that the miRNAs downregulated in MPM have conserved and redundant roles in the regulation of the wnt signaling pathway. Our data, combined with previously published observations provide evidence that wnt signaling dysregulation in MPM occurs through the miRNAs, but aberrant wnt signaling in lung adenocarcinoma occurs through other mechanisms.
In summary, our data shown that MPM can be distinguished from lung adenocarcinoma through the downregulation of miRNAs: miR-141, miR-200a*, miR-200b, miR-200c, miR-203, miR-205 and miR-429. The downregulation of these miRNAs are characteristic of mesothelioma regardless of histological subtype. We propose that these miRNAs can be used as biomarkers to develop an assay for detection of MPM and aid in distinguishing it from lung adenocarcinoma. In addition, analysis of the cumulative loss of these miRNAs identified aberrant regulation of the wnt signaling pathway, effected through the loss of its miRNA antagonists, as a key component of MPM. Further study of this phenomenon should lead to a better understanding of the mechanism of MPM, as well as provide new strategies for the treatment of this disease.