Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling

Abstract

Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing approximately 22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n = 40), normal lung specimens (n = 4), normal pleura specimens (n = 5), and MPM and SV40-immortalized mesothelial cell lines (n = 5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma that correlated loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (ie, software) to create and explore complex physiological pathways. Combined, all of these data may advance our understanding of mesothelioma tumorigenesis, pathobiology, or both.

Figure 1

Defining MPM subclasses using unsupervised hierarchical clustering. Two-dimensional unsupervised hierarchical clustering was performed using the 1405 most variable transcripts across MPM tumor samples (n = 40, black letters), normal lung samples (n = 4, green letters), normal pleura samples (n = 5, red letters), and cell lines (n = 5, blue letters). The dendrogram specifying the arrangement of samples is shown across the top with major nontumor tissue types (ie, normal or cell line) and both major tumor subclasses (ie, C1 and C2) identified using yellow bars found immediately below. Individual gene expression levels (in rows) for each sample (in columns) were normalized and expressed relative to the median value for each gene in all samples according to the scale at the bottom left. Bars to the right of the figure refer to regions of differentially expressed genes shown in greater detail in Figures 2 and 3 (for genes overexpressed in subclasses C1 and C2, respectively) and Supplemental Figures 2 to 6 at http://ajp.amjpathol.org (for numbered bars 1 to 5, respectively).

Figure 2

Genes with elevated expression levels in C1 MPM tumors. Genes whose expression levels are elevated in C1 MPM tumors (from Figure 1) are shown in greater detail and annotated with gene symbol (ie, LocusLink identifier) and gene name (ie, Unigene title). Individual sample identifiers have been removed for the sake of clarity. Relative gene expression levels are given by the scale at the bottom left.

Figure 3

Genes with elevated expression levels in C2 MPM tumors. Genes whose expression levels are elevated in C2 MPM tumors (from Figure 1) are shown in greater detail and annotated with gene symbol (ie, LocusLink identifier) and gene name (ie, Unigene title). Individual sample identifiers have been removed for the sake of clarity. Relative gene expression levels are given by the scale at the bottom left.

Figure 4

Validation of select MPM molecular markers. We examined, in normal and tumor cell lines with and without EGF in the culture medium, the mRNA levels of three genes that were found to be significantly differentially expressed between MPM tumors and normal tissues, as described in the text. We performed quantitative RT-PCR using four MPM cell lines (MS589, MS428, MS924, and JMN1B) and one normal mesothelial cell line (HM3). Individual mRNA levels were normalized to GAPDH and expressed relative to those in HM3 cells (−EGF) for CRI1 (A), NME2 (B), and PDGFC (C). Expression levels for tumor markers were typically at least twofold higher in tumor cell lines relative to HM3 (−EGF) cells for all three genes. Black bars, −EGF; white bars, +EGF.

Figure 5

Protein analysis of candidate MPM tumor-related genes in vitro. Western blot analysis was performed for CRI1 and NME2 (A) and GSN (B) in nontumorigenic SV40-immortalized mesothelial cells (Met-5A) and six human MPM cell lines (98-483, MS589, MS428, JMN1B, 211-H, H-2052) as described in the Materials and Methods using β-actin as a loading control and pooled normal pleura as a control tissue.

Figure 6

Protein analysis of candidate MPM tumor-related genes in human MPM tissues. We performed indirect immunohistochemical analysis of MPM tissue arrays consisting of 66 MPM tumors, 2 lung adenocarcinoma tumors, and 4 normal pleura tissues using antibodies to NME2 (A and B) and CRI1 (C). Expression of NME2 antigen varied widely in intensity in a diverse percentage of tumor cells (see Table 1), while CRI1 antigen was consistently expressed at moderate levels in all tumors examined. Neither protein was detected in normal pleura tissue or stromal elements of tumor tissues. Original magnifications: ×4 (A); ×10 (B and C).

Figure 7

Pathway analysis of candidate MPM tumor-related genes. Genes found to be statistically significantly up-regulated in MPM were used to create a combined network of physiological pathways as described in the Materials and Methods. A portion of the resulting pathway is shown here. The intensity of the node color (red) indicates the experimentally determined degree of up-regulation of MPM tumor-associated genes expressed as a fold-increase in average expression levels relative to normal tissues. Other node colors are white (genes that are not user specified but are incorporated in networks through relationships with other genes) and yellow (genes that are shared by two or more networks in a merged diagram). Nodes are displayed using various shapes that represent the functional class of the gene product (see legend). Edges are displayed with various labels and shapes that describe the nature of the relationship between the nodes (eg, B for binding, T for transcriptional regulation) and links to experimental results and gene summaries supporting such a relationship. The edge labels and shapes have been removed from the diagram for the sake of clarity but can be found in Supplemental Figure 7 at http://ajp.amjpathol.org. The length of an edge reflects the evidence supporting that node-to-node relationship. Edges supported by more articles from the literature are shorter. For simplicity, genes are referred to by their LocusLink symbol.