L1CAM, INP10, P-cadherin, tPA and ITGB4 over-expression in malignant pleural mesotheliomas revealed by combined use of cDNA and tissue microarray
E. Kettunen1,
A.G. Nicholson2,
B. Nagy1,
H. Wikman1,3,
J.K. Seppänen4,
T. Stjernvall3,
T. Ollikainen3,
V. Kinnula5,
S. Nordling1,
J. Hollmén4,
S. Anttila1,3 and
S. Knuutila1,6
1 Department of Pathology and Department of Medical Genetics, Haartman Institute and HUSLAB, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland, 2 Department of Histopathology, Royal Brompton Hospital, London, UK, 3 Department of Occupational Medicine and Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland, 4 Laboratory of Computer and Information Science, Helsinki University of Technology, Espoo, Finland and 5 Department of Internal Medicine, Division of Pulmonary Diseases, Helsinki University Central Hospital, Helsinki, Finland
6 To whom correspondence should be addressed Email: sakari.knuutila{at}helsinki.fi
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Abstract
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Malignant pleural mesothelioma (MM) is a rare tumour with high mortality, which can exhibit various morphologies classified as epithelioid, biphasic and sarcomatoid subtypes. To investigate the molecular changes in these tumours, we studied gene expression patterns by combined use of cDNA arrays and tumour tissue microarrays (TMA). Deregulation of the expression of 588 cancer-related genes was screened in 16 MM comprising all three subtypes and compared with references, i.e. normal mesothelial cell lines and pleural mesothelium. Array data were analysed using three statistical methods; principal component analysis (PCA), permutation test and receiver operating characteristic (ROC) curves. Eleven genes were verified by real-time RTPCR. Genes encoding two adhesion molecules [COL1A2 and integrin ß4 (ITGB4)] and a chemokine (INP10) were up-regulated in MM compared with both the cell lines and pleural mesothelium. There was a type-specific up-regulation of semaphorin E, ITGB4 and P-cadherin in epithelioid MM, matrix metalloproteinase 9 (MMP9) and tissue-type plasminogen activator (tPA) in sarcomatoid MM and neural cell adhesion molecule L1 (L1CAM) and INP10 in biphasic MM. Immunohistochemistry on TMA containing 47 MM (26 epithelioid, 15 sarcomatoid and six biphasic) was performed for five proteins, ITGB4, P-cadherin, tPA, INP10 and L1CAM. INP10 expression was increased in MM in general compared with normal mesothelium, while increased expression of P-cadherin, L1CAM and ITGB4 was more specific in MMs exhibiting an epithelioid growth pattern. The over-expression of tPA was more frequent in epithelioid MM despite higher mRNA levels in sarcomatoid and biphasic MM. We conclude that several proteins, associated with cell adhesion either directly (ITGB4, L1CAM, P-cadherin) or as a regulatory factor (INP10), are differentially expressed in MM. In particular, INP10, ITGB4 and COL1A2 were up-regulated in MM compared with both reference sample types, suggesting a relationship with development of these tumours.
Abbreviations: IHC, immunohistochemistry; ITGB4, integrin ß4; L1CAM, neural cell adhesion molecule L1; MM, malignant mesothelioma; MMPs, matrix metalloproteinases; MMP9, matrix metalloproteinase 9; PCA, principal component analysis; ROC, receiver operating characteristic; SEMA3C, Semaphorin E; TIMPs, tissue inhibitor of metalloproteinases; TMA, tumour tissue microarray; tPA, tissue type plasminogen activator
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Introduction
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Malignant mesothelioma (MM) is a rare tumour of mesothelium lining the serosal cavities and occuring most often in the pleura. Histologically, MM exhibits various morphologies, which are classified as epithelioid, biphasic and sarcomatoid subtypes in the WHO/IASLC classification for lung and pleural tumours (1).
MM is strongly resistant to all available therapies and its course is typically fatal. Sarcomatous MM has a particularly poor prognosis (2). Most patients with MM have been exposed to asbestos (3,4), although other molecular associations have also been suggested. These include genetic susceptibility caused by polymorphisms in critical metabolic genes, such as GSTM1 and NAT2 (5,6), and exposure to simian virus SV40 Tag (7,8). Also environmental factors and exposure to asbestos fibres cause variation in the expression of many genes, e.g. by activating the signal transduction pathways associated with reactive oxygen or nitrogen species (9) within the mesothelium.
A characteristic feature in the development of MM is its extremely long latency period (2040 years), which suggests that multiple different changes at the genomic level and/or involvement of several signalling pathways in mesothelial cell are needed for the malignant transformation. So far, certain alterations involving tumour-related genes, such as p16, NF2 and GPC3, have been recognized in MM (1013). However, the apparently complex genetic pattern leading to the tumorigenesis of MM remains largely unknown.
To bring insight into the deregulated genes related to primary MM, we performed a cDNA array screening and semi-quantitative real-time RTPCR validation on 16 MM tumours using normal mesothelial cell lines and pleural mesothelium as references. Morphologically different types of MM were compared with each other and the differences in the gene expression level between them were described. For five genes, the encoded protein levels were further assessed using immunohistochemistry (IHC) in tumour tissue microarray (TMA) consisting of 47 MM cases.
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Materials and methods
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Mesothelioma tumours in cDNA array experiments
Between 1997 and 2001 at the Royal Brompton Hospital, frozen tissue was collected from 29 patients with pleural MM. The diagnosis was confirmed by correlating the histological appearances with clinical and immunohistochemical data using three mesothelial [cytokeratin (K) 5/6, calretinin (CAL), thrombomodulin] and two epithelial (BerEP4 and CEA) markers, together with negative staining for neutral mucins using a diastase-periodic acid Schiff stain. The study protocol was approved by the Ethical Review Board of the Royal Brompton and Harefield Hospitals NHS Trust.
The frozen tissue samples were screened for gene expression patterns using cDNA array. Sections of tissues adjacent to those used for RNA extractions were microscopically examined by one pathologist (S.N.) and 16 representative tumours, out of 29 that produced high quality RNA, were included in the study. Tumours comprised eight epithelioid (MM-E), five mixed (MM-M) and three sarcomatoid (MM-S) MM. For each group one of the specimens was obtained from a female patient, others originating from male patients. The mean age for MM-E patients was 57 years (range 4975), for MM-M patients 61 (range 4176) and for MM-S patients 67 (range 5579). Five of the eight MM-E patients had been exposed to asbestos. Three of the MM-M patients and one MM-S patient had been exposed to asbestos whereas for one case of each type, the exposure data were not known.
Reference specimens in cDNA array experiments
Three primary normal mesothelial cultures were used as reference, two of them in cDNA array (Upl148 and Upl167) and two in RTPCR (Upl151 and Upl167). Cells were collected from pleural fluid of non-cancer patients and cultured as described previously (14). Normal mesothelial cells typically grow in culture for only three to four passages (14). The cultured cells possessed epithelioid morphology, and expressed cytokeratin mRNAs.
The fourth reference sample, used in cDNA array and RTPCR, was scraped from normal-looking pleural mesothelium obtained from a 65-year-old male patient with emphysematous bullae. The asbestos exposure status of the patient was unknown. The specimen was characterized histologically and, using K5/6 and CAL staining, was confirmed to comprise a normal mesothelial cell layer with some submesothelial connective tissue/stromal cells and a few leucocytes.
cDNA array hybridizations
Total RNA was extracted from whole tissue sections as well as cell lines and treated with DNase I using Qiagen's RNeasy Kit and DNase I kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The integrity and the quality of RNA were verified by agarose gel electrophoresis. Clontech's Atlas Human Cancer Gene nylon filters, with 588 target genes, and cDNA array kit (Clontech, Palo Alto, CA) were used in the hybridization procedure. The list of the genes can be accessed at http://atlasinfo.clontech.com/atlasinfo. Samples of 1.32.5 µg of RNA were used in the cDNA synthesis, in which [
-33P]dATP (Amersham Pharmacia Biotech, Buckinghamshire, UK) was incorporated into the cDNA. The labelling and purification of the probe, overnight hybridizations and washing were done according to Clontech's recommended protocol. The filters were exposed onto the phosphoimaging plate (Fuji, Kanagawa, Japan) for 24 days and scanned using a Bio-Imaging Analyzer BAS-2500 (Fuji) to obtain 16-bit images.
Data acquisition and statistical analysis of cDNA array results
Filter images were imported to the AtlasImage 2.01 software (Clontech), which was used to obtain the adjusted gene intensity values for individual cDNA spots by subtracting the background value from each spot. The adjusted intensity values were then analysed using two methods, principal component analysis (PCA) (15) and another measure termed the g-score. Both have been applied in several previous cDNA array studies (1619). In brief, we use PCA as a projection method that finds the direction of the greatest variance in the expression space, and yields a coordinate along this direction for each of the genes. The second method (g-score) is a measure of separation that is less sensitive to absolute differences and more sensitive to relative differences, and it is validated by means of a permutation test. Both methods have been described previously in detail (16). PCA was used in all comparisons, and the g-score in comparisons of the subtypes of MM. An arbitrarily chosen cut-off level of 25 genes was used to define up- and down-regulated genes.
Because the contents of stromal cells varied in different MM specimens, the correlation between proportional tumour content and gene expression level of a tissue specimen was studied using receiver operating characteristic (ROC) curves (20,21). This method has been successfully used in our previous research (17). Specimens with a tumour volume of 60% or more (10 cases) were compared with those with a tumour volume <60% (six cases). We estimated the ROC curves with these two groups based on the gene expression and calculated the associated area under the ROC curve. The area has the following statistical interpretation: given a randomly chosen subject with high tumour content, and a randomly chosen subject with low tumour content, the probability that gene expression identifies which subject belongs to which group is equal to the normalized area. [It is thus equivalent to the non-parametric two-sample Wilcoxon statistic (20).] Using this method, we may establish a non-random association between the two groups. This process is repeated for each of the 588 genes. Moreover, we applied a permutation test (22) to verify that the findings were not due to random fluctuations in a finite data sample. We permutated (rearranged) the group labels and calculated the ROC curve and the associated area for 10 000 repetitions, and consequently drew samples from the null hypothesis of no association between expression and tumour content. Corresponding P-values were estimated for each gene from the empirical distribution.
We point out those genes whose expression level may be associated with the proportional tumour content in the sample. A probability threshold of 0.75 was used. As a P-value measuring the confidence of the finding, we used a conservative value of 0.1, which is more likely to include rather than exclude genes.
Semi-quantitative real-time RTPCR
cDNA was synthesized from 0.5 µg of DNase-treated total RNA. For reaction we used the First Strand cDNA Synthesis kit for RTPCR (Roche Diagnostics GmbH, Mannheim, Germany). Gene-specific primers were designed with LightCycler Probe Design Software Version 1.0 (Roche). Table I shows primer sequences for the 11 genes studied. PCR was performed in a LightCycler thermal cycler (Roche). Each reaction consisted of 1 µl of DNA Master SYBR Green I mix (LightCycler-FastStart DNA Master SYBR Green I kit, Roche), 1 µl of cDNA, from 1.625 to 2.25 mM MgCl2, water and 2.55.0 pmol of primers. The amplification programme included an initial denaturation for 7 min, 45 cycles with denaturation at 95°C for 10 s, annealing at 6065°C for 5 s and extension at 72°C for 10 s. Amplification was followed by melting curve analysis using one cycle at 95°C for 0 s, 65°C for 10 s and 95°C for 0 s at the acquisition step mode. Each sample was run in duplicate. Standard curves were obtained using serial dilutions of ß-globin gene (DNA Control kit, Roche). The relative mRNA concentrations were determined on the kinetic approach using the LightCycler software and normalized with PL2A concentration because its expression was steady among all samples and the expression level of PL2A was comparable with the expression of the genes of interest. The MM specimens were grouped according to histological types. The statistical significance of differences between each group and a group of references was evaluated with the help of permutation testing, with n = 10 000 repetitions.
Tumour tissue microarray
TMA was produced using formalin-fixed paraffin-embedded material of primary MM that were different from those used in the cDNA array. The tumours consisted of 26 MM-E (obtained from 21 males, 5 females), 6 MM-M (4 males, 2 females) and 15 MM-S (13 males, 2 females). The mean age for MM-E patients was 61 years (range 4982), for MM-M patients 69 (range 5177) and for MM-S patients 69 (range 5191). For MM-E, MM-M and MM-S, 14, 4 and 10 patients, respectively, had been exposed to asbestos. For 8, 1 and 5 patients, respectively, the exposure data were missing. Eleven other neoplasms were included in the TMA as controls (four pulmonary adenocarcinomas, five peritoneal serous carcinomas, a pleural pleomorphic liposarcoma and a malignant solitary fibrous tumour of the pleura). Most of the MM and control cases (43 and eight cases, respectively) were sent to the Finnish National Mesothelioma Panel for consultation.
TMA blocks were made using a manual TMA instrument (Beecher Instruments, Silver Springs, MD). Cylinders of either 0.6 or 1.0 mm in diameter were obtained from donor blocks and punched in four replicates (0.6 mm) or in duplicate (1.0 mm) to a TMA recipient block. As normal mesothelial controls we used five different paraffin-embedded specimens of peripheral lung covered by visceral pleura with a surface layer of mesothelial cells, in part reactive.
Immunohistochemistry
Sections 0.4-µm-thick were cut from TMA blocks, deparaffinized and rehydrated using a standard protocol. To retrieve antigens the slides were microwaved for 20 min either in Dako Target Retrieval Solution (DakoCytomation, Glostrup, Denmark) [antigens INP10, tissue type plasminogen activator (tPA) and neural cell adhesion molecule L1 (L1CAM)] or in TrisEDTA buffer, pH 9 (antigens ITGB4 and CDH3). IHC stainings were performed using a DAKO TechMateTM Horizon Instrument (Dako), incubating with primary antibody for 30 min at RT. For INP10, tPA and L1CAM, Dako ChemMateTM Detection Kit Peroxidase/AEC was used, whereas ITGB4 and CDH3 stainings were performed using Dako ChemMateTM EnVisionTM Detection Kit, Peroxidase/DAB as a chromogen; all counterstained with haematoxylin. The following antibodies and dilutions were used: rabbit polyclonal anti-human IP-10 at 1:15 (PeproTech EC, London, UK); rabbit polyclonal anti-L1CAM at 1:15 (Santa Cruz Biotehcnology, Santa Cruz, CA); rabbit polyclonal anti-ITGB4 at 1:150 (Santa Cruz Biotechnology); mouse monoclonal anti-CDH3 at 1:5 (NeoMarkers, Fremont, CA); and goat anti-tPA (Calbiochem, San Diego, CA) at 1:50. The immunoreactivity was evaluated by three authors independently and consensus was achieved afterwards in each case. The immunoreactivity for INP10, CDH3 and L1CAM was scored as negative, or weak/moderate or strong positive, and for ITGB4 and tPA as negative or positive. If the scores in different spots from the same tumour differed, the highest score was chosen. In order to detect differences in aberrant antigen reactivity levels between the MM types, we executed a hypothesis test of proportions with permutation testing, where the proportion of negative cases was compared with that of positive cases. For INP10, CDH3 and L1CAM, the weak/moderate and strong positive cases were grouped together for permutation testing. In regard to differentiating MM from other mesenchymal or epithelial neoplasms, each MM type was also compared with the group of 11 other neoplasms.
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Results
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cDNA array results
ROC curve analysis. The expression profiles were analysed with ROC to ensure that the gene expression pattern of a tissue specimen was not biased by varying proportions of tumour tissue in the specimens. Only IGFBP6 and CAV2 out of the 588 genes showed significant P-values for lack of confidence (data not shown).
MM versus references. The expression profiles of 588 genes, associated with cancer, were studied in 16 primary MM. The results were analysed using PCA (all cDNA array data score values are available on request). For MM, two types of references were used: (i) two mesothelial cell lines and (ii) a specimen comprising pleural mesothelium. For evaluation of the references, the expression patterns of cytokeratins were examined. Cytokeratins K2E, K8, K10, K18 and K19 were over-expressed in MM compared with pleural mesothelium (Table II).
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Table II. cDNA array data from cytokeratin-encoding genes that were differentially expressed in MM versus references
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The top 25 genes that were over- or under-expressed in MM are shown in Table III. They include genes deregulated in either MM versus mesothelial cell lines (genes possibly related to cell reactivity) or also versus pleural mesothelium (genes possibly related to malignancy). A third group of genes was under-expressed in MM versus normal mesothelial cell lines but over-expressed versus pleural mesothelium, or vice versa (Table III). In addition, some genes were deregulated only in MM compared with pleural mesothelium (PCA score values not shown). This category included 25 deregulated genes that encode growth factors, cytokines and chemokines, and proteins involved in cell adhesion, motility, invasion regulation and cellcell interactions. For example, procollagen 2
1 (COL2A1) and 3
1 (COL3A1) and tissue inhibitor of metalloproteinases 1 (TIMP1) were over-expressed in MM, while CD9 (also shown in RTPCR), metalloproteinases MMP2 and MMP11, and TIMP3 were under-expressed. Also, five genes involved in apoptosis, cell fate and development, and angiogenesis regulation (such as PIG7 and PDGFRA) as well as three genes encoding proto-oncogenes (FOS, KIT and C-fgr) were under-expressed in MM, while a cell cycle regulator (Cyclin B1, CCNB1) and four genes encoding intermediate filaments (see Table II) were over-expressed. In addition, two house-keeping genes, liver glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and a 23-kDa highly basic protein were up-regulated in MM compared with pleural mesothelium.
Specific expression patterns in MM types. The gene intensity levels in different histological types of MM, i.e. epithelioid (MM-E), sarcomatoid (MM-S) and biphasic/mixed (MM-M), were compared separately with the references and with each other using two different statistical methods, PCA and g-score. In the 18 comparisons, altogether 213 genes were among the top 25 over- or under-expressed genes (data not shown).
The expression patterns of genes over-expressed in either MM-E or MM-S or MM-M versus both references and versus the counterpart(s) are shown in Figure 1. Semaphorin E (SEMA3C), integrin ß4 (ITGB4), P-cadherin/CDH3 and COL6A1, had the highest over-expression in MM-E. L1CAM, K14 and INP10 were up-regulated mainly in MM-M, whereas matrix metalloproteinase 9 (MMP9) and plexin 3 (PLXN3) were expressed especially in MM-S, and tPA both in MM-M and MM-S.

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Fig. 1. Expression patterns of 10 genes that were expressed in a type-specific manner in MM. To achieve the adjusted gene intensity values, the value of each gene was normalized by subtracting from the original intensity value (i) the background value, (ii) the averaged intensity value of a given gene in normal mesothelial cell lines and (iii) the averaged gene intensity value of a given array filter. MM-E, epithelial MM; MM-M, mixed MM; MM-S, sarcomatoid MM.
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Semi-quantitative real-time RTPCR results
The over-expression of MMP9, tPA, SARP1, SEMA3C, CDH11, CDH3, ephrin A5 (eph) and IGFBP4 and the under-expression of BAX, K7 and CD9 in 16 MM were verified using semi-quantitative real-time RTPCR, two mesothelial cell lines (Upl151 and Upl167) and the pleural mesothelium as references (Figure 2). Differences of average relative expression levels between the MM types and the reference samples and statistical significance of the differences are shown in Table IV.

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Fig. 2. Averaged relative mRNA concentrations obtained using real-time RTPCR in different types of MM and in references (cell lines and tissue). The error bars represent the standard deviation (SD). The relative concentration of a given gene was determined using the amplification product of ß-globin gene as a standard. The concentration was normalized using PL2A concentration of each specimen. For illustration the mRNA concentration values for CDH3 were multiplied by 10 and the values for CDH11 and CK7 were divided by 10, and for IGFBP4 by 100. (A) Relative expression levels of the genes that were up-regulated in MM as compared with the references. Cell lines expressed undetectable level of SARP1 mRNA. (B) Relative expression levels of the genes that were down-regulated in MM as compared with the references.
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Immunohistochemistry
One to four spots per MM were evaluated for ITGB4, CDH3, tPA and L1CAM antigens. The expression of INP10 was less homogeneous than that of the other antigens. Therefore, a minimum of two spots per tumour were evaluated for INP10. The staining results are shown in Table V and the patterns of staining and cellular localization of the antigens in Figure 3.
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Table V. Immunohistochemically studied expression of five antigens in MM types (epithelioid, MM-E; mixed, MM-M; sarcomatoid, MM-S) and in other neoplasms
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Fig. 3. IHC staining patterns in epithelioid (E) or sarcomatoid (S) malignant mesothelioma (MM). Staining for ITGB4 antigen in MM-E (A) and MM-S (B), L1CAM in MM-E (C) and MM-S (D), INP10 in MM-E (E) and MM-S (F), tPA in MM-E (G) and MM-S (H) and CDH3/P-cadherin in MM-E (I) and in MM-S (J).
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In contrast to normal mesothelium, which was negative, reactive pleura showed weak immunoreactivity for ITGB4, INP10 and tPA. Altogether 4378% of the other epithelial and mesenchymal neoplasms were positive for these antigens, indicating that none of the protein expression patterns were specific for MM in general. However, when each MM type was compared with other neoplasms using permutation testing, that is comparing the proportion of negative cases with that of positive cases, INP10 showed significant P-values (<0.0001) in all MM types. Nevertheless, analysed sample sizes (22 MM versus five other neoplasms) were small due to the heterogeneous staining pattern of INP10. In addition to INP10, MM-E showed significant P-values for L1CAM (0.0320), tPA (0.0333) and ITGB4 (<0.0001). In contrast, less antigens were significantly over-expressed in MM-M (INP10 and tPA, P-value <0.0001) and in MM-S (INP10, P-value <0.0001) in comparison with other neoplasms. Moreover, significant P-values for ITGB4 (0.0012), tPA (0.0250), L1CAM (0.0486) and CDH3 (<0.0001) were shown when antigen expression levels of MM-E were compared with MM-S, and for CDH3 (0.0373) when MM-E was compared with MM-M, using permutation testing.
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Discussion
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Analysis using a cDNA array technique, with 588 target genes, revealed COL1A2, SEMA3C, ITGB4, CDH3, COL6A1, L1CAM, K14, INP10, tPA, MMP9 and PLXN3 as the most differentially expressed genes in 16 primary MM and in specific MM types when compared with the references. Over-expression of INP10, ITGB4, CDH3, L1CAM and tPA was also demonstrated using IHC in 47 separate MM.
A key question in cDNA array studies is to find the most suitable reference for each tumour entity. In MM the progenitor cell is believed to be a mesothelial cell of mesenchymal origin. Due to biphasic differentiation potential mesothelial cells differentiate in culture towards an epithelial or a fibromatous phenotype (23) and accordingly MM manifests in morphologically different histologic subtypes. Thus, the optimal reference needs to be nominated for each subtype. Our approach was to use short-term cultured normal mesothelial cells of epithelioid morphology collected from pleural fluid of non-cancer patients and, in addition, a pleural specimen that contained stromal cells and a small pool of leucocytes in addition to the mesothelium. In this way we were able to distinguish those gene expression patterns that were either unique to tumour cells or related to some other phenomenon, such as cell reactivity or mesothelial cell characteristics. Secondly, by comparing different MM types with each other, we could reveal gene expression patterns characteristic to each MM type.
Three genes, encoding procollagen 1
2 subunit precursor (COL1A2), interferon
-induced protein precursor (INP10) and ITGB4, were over-expressed in MM compared with both references. Up-regulation of COL1A2 in MM has been reported recently (24).
INP10, not reported before as deregulated in MM, encodes a chemokine with pleiotropic effects, e.g. chemotactic and mitogenic activity, as well as the capability to modulate the expression of adhesion molecules. It has been suggested that INP10 inhibits angiogenesis and tumour growth in vivo (25). This human homologue of the RAS target gene Mob-1 is over-expressed in a majority of colorectal cancers (26). Signal transduction through a novel INP10-specific receptor has been described in several epithelial cell types and it has also been suggested that this receptor may have a role in metastasis (27). In normal peritoneal mesothelial cells the expression of INP10 can be induced by a chemical stimulus or physical injury, suggesting its role in a defence mechanism (28,29). Over-expressed INP10 may have a similar role in MM. INP10 antigen was over-expressed in all MM types and in some other neoplasms although the expression pattern differentiated significantly in MM compared with other neoplasms. Thus, a larger sample size is needed to draw any conclusions about the diagnostical value of INP10 in MM.
The product of ITGB4 forms a heterodimer with integrin
6, which functions as a laminin receptor. Positive immunoreactivity for anti-ITGB4 has been shown in MM previously (3032). In some studies no positive ITGB4 staining could be found in MM-S (30,32), but in our study 46% of the MM-S stained positively for ITGB4, similar to data reported by Giuffrida et al. (31), as well as all MM-E. ITGB4 functions not only as an adhesion molecule but also as a component of the metastasis/scattering machinery associated with the HGF-receptor Met (33).
Semaphorins are also implicated in invasive growth, and we found up-regulation of semaphorin E (i.e. SEMA3C) in MM-E. As MM-E usually has a better prognosis than MM-S (2), it is interesting that the expression level of SEMA3C has been shown to be higher in good-outcome versus poor-outcome MM (34). The over-expression of SEMA3C in recurrent lung carcinomas and in a cisplatin-resistant MM cell line associates with drug resistance not related to MDR1 (35). As plexins are receptors for semaphorins, it is also notable that the expression of PLXN3 was considerably lower in MM-E than in MM-S.
Whereas comprehensive studies have been published on N-cadherin and E-cadherin in MM (3638), there seem to be no reports of P-cadherin in MM. Our study showed in MM-E an increase in P-cadherin, which was statistically significant at the protein level. Basal and parabasal layers of stratified epithelia selectively express P-cadherin, which also appears to be the predominant cadherin type in peritoneum (39,40). The Wnt pathway has recently been shown to be activated (41) in MM through an over-expression of Dishevelled and downstream signalling through ß-catenin, one of the proteins with which cadherins are interacting (42) and it has been suggested that P-cadherin/ß-catenin complexes associate with ovarian tumour progression (40). Our data support a role for this protein in the development of MM.
We show here for the first time in MM the up-regulation of L1CAM (neural cell adhesion molecule L1). Besides cell adhesion, L1CAM has recently been implicated in cell proliferation and using genetic suppressor cDNA elements, L1CAM has been identified as a potential new target for anticancer drugs (43).
In invasion and metastasis the processing state of extracellular matrix and basement membrane is unbalanced (44). This is determined by members of several different protein families, e.g. matrix metalloproteinases (MMPs), TIMPs and activators and inhibitors of fibrinolysis. Several MMPs and TIMPs have been implicated in MM (4547), although their roles are poorly understood. In our analysis MMP9 and a serine protease tPA were over-expressed in MM-S and MM-M compared with both references. Also, RTPCR verified these mRNA levels, which were higher than in MM-E. IHC showed, however, that the tPA antigen level was increased in MM-E more than in MM-S. (MMP9 antigen was not studied here.) It has been suggested that the counteraction of the increased plasminogen activator activity by PA inhibitor 1 may reflect the metastasis pattern typical for MM (48). In our study, the PAI1 mRNA was under-expressed in MM compared with mesothelial cell lines but over-expressed compared with mesothelium. Nevertheless, the expression pattern of PAI1 was not specific to any certain type. The antibody used reacts with both free tPA and tPA inhibitor complexes.
A comparison of the present results on gene expression patterns in MM to our earlier published data in MM cell lines (49) reveals that only one-third of the deregulated genes were the same. However, the distribution of growth patterns in the specimens differ between this and the previous study, plus the references were only partially the same. This may explain the apparent discrepancy between the studies. Genes that were similarly deregulated in MM cell lines and in all primary MM could be classified as reactivity related genes. In general, there were no discrepant results between the MM cell lines and the primary MM. It can therefore be suggested that there is a close relationship between the gene expression pattern in cell lines and primary tumours of a similar origin, as has also been indicated recently (50).
Few other cDNA array studies have been performed on primary MM (24,34,5052). The comparison of the results is difficult due to variable array platforms and study designs used, and sometimes very limited material has been available. However, some conclusions can be drawn. Similar to our study, genes especially involved in cytoskeletal remodelling pathways, adhesion and molecular recognition, such as ITGB4, CDH11 and COL1A2, were shown to be markedly up-regulated in MM (24,50). Also, activation of glucose metabolism (GAPDH) and protein translational pathways (not implicated in our study) (24) and cell protection and resistance (e.g.
-2-M) (50) have been recognized as of great importance in MM. Gordon et al. presented a set of genes with prime criteria of having diagnostic value for MM compared with adenocarcinoma of the lung (51). None of those genes were in our gene lists. However, other clinical applications of cDNA array technique that were used to identify gene ratios or expression profiles as outcome predictors, revealed some genes with similar expression patterns than in our study, as discussed above in relation to SEMA3C. (34). The over-expression of IGFBP5 and integrin
6, a gene encoding a dimerization partner for ITGB4, was likewise associated with better survival in MM (52). However, none of the previous studies have presented expression patterns specific to MM subtypes.
In conclusion, using cDNA array screening with complementary statistical methods and TMA we have shown that novel proteins associated with cell adhesion either directly (ITGB4, L1CAM, P-cadherin) or as a regulatory factor (INP10) are expressed in MM. In particular, INP10, ITGB4 and COL1A2 were up-regulated in MM compared with both references, suggesting a relationship with development of these tumours. Moreover, INP10, ITGB4, SEMA3C, L1CAM and P-cadherin have been implicated in metastasis or progression of some other tumour types, which may further suggest their potential role in MM.
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Acknowledgments
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We are grateful to Professor Heikki Mannila for advice on bioinformatics. We also wish to thank Messrs Peter Goldstraw, George Ladas and Michael Dusmet, Department of Thoracic Surgery, Royal Brompton Hospital and Dr Dia Kamel, Department of Histopathology, Royal Brompton Hospital for their help in this study. This work was supported by grants from K.Albin Johanssons Stiftelse, Helsinki University Central Hospital Research Fund, Jalmari and Rauha Ahokas Foundation, and the Finnish Cancer Society.
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Received December 17, 2003;
revised January 26, 2004;
accepted August 27, 2004.