Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura: the emerging gene portrait of the mesothelioma phenotype

Abstract

Background: Malignant pleural mesothelioma is considered an almost incurable tumour with increasing incidence worldwide. It usually develops in the parietal pleura, from mesothelial lining or submesothelial cells, subsequently invading the visceral pleura. Chromosomal and genomic aberrations of mesothelioma are diverse and heterogenous. Genome-wide profiling of mesothelioma versus parietal and visceral normal pleural tissue could thus reveal novel genes and pathways explaining its aggressive phenotype.

Methodology and principal findings: Well-characterised tissue from five mesothelioma patients and normal parietal and visceral pleural samples from six non-cancer patients were profiled by Affymetrix oligoarray of 38 500 genes. The lists of differentially expressed genes tested for overrepresentation in KEGG PATHWAYS (Kyoto Encyclopedia of Genes and Genomes) and GO (gene ontology) terms revealed large differences of expression between visceral and parietal pleura, and both tissues differed from mesothelioma. Cell growth and intrinsic resistance in tumour versus parietal pleura was reflected in highly overexpressed cell cycle, mitosis, replication, DNA repair and anti-apoptosis genes. Several genes of the "salvage pathway" that recycle nucleobases were overexpressed, among them TYMS, encoding thymidylate synthase, the main target of the antifolate drug pemetrexed that is active in mesothelioma. Circadian rhythm genes were expressed in favour of tumour growth. The local invasive, non-metastatic phenotype of mesothelioma, could partly be due to overexpression of the known metastasis suppressors NME1 and NME2. Down-regulation of several tumour suppressor genes could contribute to mesothelioma progression. Genes involved in cell communication were down-regulated, indicating that mesothelioma may shield itself from the immune system. Similarly, in non-cancer parietal versus visceral pleura signal transduction, soluble transporter and adhesion genes were down-regulated. This could represent a genetical platform of the parietal pleura propensity to develop mesothelioma.

Conclusions: Genome-wide microarray approach using complex human tissue samples revealed novel expression patterns, reflecting some important features of mesothelioma biology that should be further explored.

Figure 1. Schematic presentation of mesothelioma, the parietal and visceral pleura.

Representative histology showing the most abundant cell types.

Figure 2. Venn diagram of significantly up- and down-regulated genes (n) in mesothelioma (T) versus normal parietal pleura (pp) and normal visceral pleura (pv) (P<0.05).

828 genes are overexpressed (red) and 1004 genes are down-regulated (green) in T versus pp. 341 genes are overexpressed (blue) and 52 genes downregulated (brown) in pv versus pp.

Figure 3. Selected pathways with distribution of differentially expressed genes (P<0.05).

This graph depicts the areas of differentially expressed genes in tumour (T), parietal pleura (PP) and visceral pleura (PV). Each dot represents a gene, where red represent genes overexpressed in tumour and green represent genes overexpressed in parietal pleura or visceral pleura. Gray represents all the genes of the chip and yellow represents the genes non-differentially expressed in each pathway. Genes associated to the cell cycle and the proteasome are uniformly overexpressed. More genes associated to apoptosis are downregulated than overexpressed and most genes involved in cytokine-cytokine receptor interaction are down-regulated. Important circadian rhythm genes are differentially regulated (see Fig. 8).

Figure 4. Protein expression of selected genes, AGGF1, TYMS and MSLN by immunohistochemistry.

A–C–E: normal parietal pleura. B–D–F: Biphasic mesothelioma with epithelial and sarcomarous components. A–B (x20): AGGF1(VG5Q) mRNA was overexpressed in mesothelioma, and clearly protein was expressed (brown) in both tumour components (arrows). Strong expression in normal mesothelium was seen (arrow) but the majority of endothelial and other pleural cells were negative. C–D (x40): TYMS (Thymidylate synthase) mRNA was overexpressed, also on the protein level (brown), mostly in the epithelial component (arrow) of tumour. Normal pleura was negative. E–F (x20): MSLN (Mesothelin) mRNA was not differentially expressed, that could be explained by the intense protein expression not only in epithelial tumour cells, but also in normal mesothelial and stromal cells.

Figure 5. Schematic presentation of the results of differential expression of the purine and pyrimidine pathways in tumour versus parietal pleura (P<0.05).

Genes encoding proteins responsible for DNA and RNA synthesis and recycling of purines and pyrimidines are overexpressed (red), while genes having the opposite or regulating role (green) are down-regulated. Genes encoding de novo synthesis of adenosine, guanosine, thymidine, cytidine and uracil were not differentially expressed (not shown). This pattern may represent salvage pathways facilitating tumour growth. Up: CTPS = CTP synthase, DTYMK = deoxythymidylate kinase, TYMS = thymidylate synthase, UCK2 = uridine-cytidine kinase, UMPS = uridine monophosphate synthase, POLR1A = polymerase (RNA) I polypeptide A, POLR3B; polymerase (RNA) III (DNA directed) polypeptide B, NME = non-metastatic cells 1, NME2 = non-metastatic cells 2, PKM2 = pyruvate kinase, muscle, PRIM2A = primase, DNA, polypeptide 2, PNPT1 = polyribonucleotide nucleotidyltransferase 1, PCNA = proliferating cell nuclear antigen, RRM1 = ribonucleotide reductase M1. Down: ADCY4 = adenylate cyclase 4, GUCY1A3 = guanylate cyclase 1, soluble, alpha 3, PDE2A = phosphodiesterase 2A, cGMP-stimulated, PDE4A = phosphodiesterase 4A, cAMP-specific, PDE5A = phosphodiesterase 5A, cGMP-specific, ENTPD3 = ectonucleoside triphosphate diphosphohydrolase 3.

Figure 6. Schematic presentation of some of the overexpressed genes related to their activity in the various phases of the cell cycle (P<0.05).

The M-phase genes are overrepresented.

Figure 7. Differentially overexpressed genes in tumour (red boxes) depicted in the Cell Cycle map from KEGG PATHWAYS (Kanehisa et al., 2008) (P<0.05).

21 of 21 cell cycle genes were overexpressed in mesothelioma versus normal parietal pleura tissue. Potential targets for anti-tumour treatment described in the litterature are marked (see text). Abbreviations: CDK7 = cyclin-dependent kinase 7, CHEK1 = checkpoint homolog, E2F2 = E2F transcription factor 2, ORC6L = origin recognition complex, subunit 6 like, MCM2-3-4-6 = minichromosome maintenance complex component 2-3-4-6, PCNA = proliferating cell nuclear antigen, RB1 = retinoblastoma, BUB1 = budding uninhibited by benzimidazoles 1 homolog, BUB1B = BUB1 beta, CDC7 = cell division cycle 7 homolog, APC/C = CDC23, cell division cycle 23 homolog, anaphase-promoting complex subunit 8, CCNB1 = cyclin B1, CCNB2 = cyclin B2, ESPL1 = extra spindle pole bodies homolog 1, CDC2/CDK1 = cell division cycle 2, G1 to S and G2 to M, CDC6 = cell division cycle 6 homolog, CDC20 = cell division cycle 20 homolog, CDC25A = cell division cycle 25 homolog A.

Figure 8. Circadian rhythm genes differentially expressed in tumour shown with KEGG PATHWAYS (modified from Kanehisa et al., 2008) (P<0.05).

CRY2, PER1, PER3 and NR1D1/Rev-Erb alpha that function as negative regulators of transcription are down-regulated (green) whereas both genes encoding the active transcriptional heterodimeric complex Bmal1(ARNTL):Npas2 (NPAS2) are overexpressed in mesothelioma versus normal parietal pleura. Damaged circadian rhythms may be a key to the continuous replicative force in tumour cells, and thus possible treatment targets.