Spontaneous Mesotheliomas in F344/N Rats Are Characterized by Dysregulation of Cellular Growth and Immune Function Pathways

Abstract

Aged male Fischer 344/N rats are prone to developing spontaneous peritoneal mesotheliomas that arise predominantly from the tunica vaginalis of the testes. A definitive cause for the predominance of this neoplasm in F344/N rats is unknown. Investigation of the molecular alterations that occur in spontaneous rat mesotheliomas may provide insight into their pathogenesis as well enable a better understanding regarding the mechanisms underlying chemically induced mesothelioma in rodents. Mesothelial cell function represents a complex interplay of pathways related to host defense mechanisms and maintenance of cellular homeostasis. Global gene expression profiles of spontaneous mesotheliomas from vehicle control male F344/N rats from 2-year National Toxicology Program carcinogenicity bioassays were analyzed to determine the molecular features of these tumors and elucidate tumor-specific gene expression profiles. The resulting gene expression pattern showed that spontaneous mesotheliomas are associated with upregulation of various growth factors, oncogenes, cytokines, pattern recognition response receptors, and pathogen-associated molecular patterns receptors, and the production of reactive oxygen and nitrogen species, as well as downregulation of apoptosis pathways. Alterations in these pathways in turn trigger molecular responses that stimulate cell proliferation and promote tumor survival and progression.

Keywords

mesothelioma F344/N rat National Toxicology Program microarray gene expression mesothelial cell Fred-PE cells

Introduction

Mesothelial cells form the lining of the heart, pleural and peritoneal cavities, and visceral surfaces of organs. The mesothelial lining of the peritoneum in males extends into the scrotum to form the tunica vaginalis that covers the testes and the epididymis (Hall et al. 1990). The mesothelial lining consists of a single layer of 2 morphologically distinct cell types, (1) flattened cells and (2) dome-shaped cuboidal to polygonal cells, overlying a very thin fibrovascular stroma. The flattened cells are held together by apical tight junctions, adherens junctions, zona occludens, and desmosomes (Antony 2003; Madison et al. 1979), and the dome-shaped cells surround pores that connect endothelial cells with the mesothelium, thus providing a direct role for the mesothelial cell in fluid and large molecule transport between the peritoneal cavity and lymphatic system (Hall et al. 1990). Both domed and flattened mesothelial cells bear microvilli that are involved in the absorption of fluid and maintenance of lubrication of peritoneal and pleural cavities. Mesothelial cells are a dynamic cell population, derived from embryonic mesoderm, and provide innate immune mechanisms and multiple cellular functions that aid in maintaining homeostasis for the pleural and peritoneal cavities. Mesothelial cells are capable of pinocytosis of small particles as well as phagocytosis of large particles. Their cytoplasm contains microfilaments, which are involved in cell movement and cytoskeletal rearrangement, as well as proper function of mitochondria, smooth and rough endoplasmic reticulum, and pinocytotic vesicles. Mesothelial cells have both epithelial and mesenchymal characteristics; they express a broad range of cytokeratins as well as vimentin (VIM), and when activated, they express desmin and smooth muscle actin (Afify et al. 2002; Kupryjanczyk and Karpinska 1998; Yung and Chan 2011). Additionally, they produce hyaluronic acid and express a variety of cell surface receptors including CD44 (Nasreen et al. 2002; Yung and Chan 2011), pathogen-associated molecular pattern (PAMP) receptors (e.g., CD14), and pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs), receptors for advanced glycation end products (RAGE; Jantz and Antony 2008), and mannose receptors.

Mesotheliomas occur rarely in rats and humans and are characterized by the neoplastic transformation of pleural or peritoneal mesothelial cells. In humans, these tumors have a long latency period (Hall et al. 1990) and an insidious nature that makes the diagnosis of mesotheliomas challenging, prognosis poor, and treatment options limited. Mesotheliomas occur at a low incidence in aged male F344/N rats (3.2%, all routes of vehicle exposure, all vehicles, National Toxicology Program [NTP 2011] historical controls), and arise most commonly from the tunica vaginalis (Hall et al. 1990). In females, mesotheliomas arise much less frequently (0.17%, all routes of vehicle exposure, all vehicles; NTP 2011), and typically arise from various locations within the peritoneal cavity, including adjacent to the ovary (Hall et al. 1990). There are three primary histologic phenotypes of mesothelioma in the rat: epitheliomatous, sarcomatous, and mixed. Most mesotheliomas in the rat are epitheliomatous, characterized by papillary projections overlying a fibrovascular stalk. Sarcomatous tumors are associated with spindyloid morphology, and mixed tumors have components of both epitheliomatous and sarcomatous phenotypes. All three histologic phenotypes that occur in the rat are also reported in humans (Blobel et al. 1985; Mackay, Tracy, and Craighead 1987), with the epitheliomatous subtype the most common (Remon et al. 2012). Both peritoneal and pleural mesotheliomas occur in humans, but by far, pleural mesotheliomas are the most common, occurring in up to 80% of all cases (Sekido 2013). Malignant pleural mesothelioma (MPM) is a rare disease in humans with approximately 2,000 to 3,000 new cases in the United States diagnosed annually (Kaufman and Pass 2008). The incidence of MPM has steadily increased over the last 20 years, with 70 to 80% of cases implicating asbestos exposure as the dominant risk factor (Kaufman and Pass 2008; Robinson and Lake 2005). Men have a higher incidence of pleural mesothelioma than women, possibly due to higher occupational exposures of asbestos (Yang, Testa, and Carbone 2008). The remaining 20 to 30% of cases are attributed to nonasbestos risk factors, such as germ line mutations in BRCA1-associated protein 1 (BAP1; Bott et al. 2011; Testa et al. 2011), SV40 viral infection (Carbone et al. 2003), and exposure to radiation or chemical agents (Carbone and Bedrossian 2006; Yang, Testa, and Carbone 2008).

Molecular alterations in human mesothelioma associated with asbestos exposure have been an area of intense interest, and a topic of several recent reviews and gene expression profile targeted publications; gene expression profiling and mutation analysis of these tumors in humans have revealed a variety of genetic alterations including mutations in tumor suppressor genes (TP53, WT1, NF2, and PTEN) or cell cycle genes (CDKN2A/2B and CDKN2C), and alterations in proteins associated with tumorigenesis (Collagen Type III [COL3], Chemokine (C-C motif) ligand 2 [CCL2], lectin, galactose binding, soluble 12 [LGALS]; Bott et al. 2011; Carbone and Bedrossian 2006; Gordon et al. 2005; Gueugnon et al. 2011; Romagnoli et al. 2009; Sekido 2013; Tomasetti et al. 2009; Tsujimura et al. 2012). These alterations in gene expression patterns appear to vary between different populations of people as well as between the various morphological subtypes of human mesothelioma.

The objective of this study was to investigate the gene expression profiles of spontaneous mesotheliomas in the Fischer 344/N rat. Previous studies have characterized the gene expression profiles or molecular features of some chemically induced mesotheliomas in F344/N rats or F344 crosses (Hu et al. 2010; Kim et al. 2006). However, global gene expression profiling of spontaneous mesotheliomas in rats has not been previously performed. Moreover, without a database of molecular changes or genetic signatures for spontaneously occurring mesotheliomas, separating changes due to chemical exposure from those inherent in mesothelioma pathogenesis is impossible. Characterization of spontaneous peritoneal mesotheliomas in rats may provide clues into the pathogenesis of chemically induced mesotheliomas and may also provide useful information as to the utility of the F344/N rat as a hazard identification model for nonasbestos-related mesothelioma in humans.

Materials and Methods

Sample Collection and Histopathology

Spontaneous mesotheliomas were collected from male vehicle control Fischer 344/N rats from three 2-year NTP carcinogenicity bioassay studies (Cobalt, Riddelliine, and Codeine). The incidence of malignant mesotheliomas in vehicle control male rats from these studies was 2/50 (4%), 4/50 (8%), and 1/50 (2%), for Cobalt, Riddelliine, and Codeine, respectively. Samples were sectioned in half: one half was fixed in 10% neutral buffered formalin (NBF) and the other half was flash frozen in liquid nitrogen. Five tumor samples were selected for molecular biology based on size from 3 animals (Riddelliine, peritoneum; Codeine, mesentery; Cobalt, tunica vaginalis, peritoneum, and mesentery; Table 1), and the morphology of each sample was reviewed histologically to assure minimal necrotic tissue (<20%) and maximum tumor to normal tissue (>80% tumor tissue) per section. The rarity of large spontaneous mesotheliomas available necessitated using multiple spontaneous tumors from 1 animal from the Cobalt study. Since isolation of normal mesothelium from rats would not yield adequate material for microarray, we used a nontransformed, immortalized mesothelial cell line (Fred-PE) which was grown and subcultured from the peritoneal cavity of untreated normal F344/N rats (a generous gift from Dr. Yongbaek Kim, North Carolina State University), which has been used as a mesothelium control in previous studies in F344/N rats (Crosby et al. 2000; Kim et al. 2006).

Table 1.

Spontaneous mesotheliomas from male F344/N rats used for global gene expression profiling.

Tumor sample	Study	Location	Tumors/animal	Morphology	EpCAM +	Vimentin +	CK18 +
SO2130	Riddelliine	Peritoneum	1	Epitheliomatous	Cytoplasm and nucleus	Cytoplasm	Cell membrane
MB194	Codeine	Mesentery	5	Epitheliomatous	Cytoplasm and nucleus	Cytoplasm	Cell membrane
BW4719	Cobalt	Peritoneum	6	Epitheliomatous	Cytoplasm and nucleus	Cytoplasm	Cell membrane
BW4721	Cobalt	Mesentery		Epitheliomatous	Cytoplasm and nucleus	Cytoplasm	Cell membrane
BW4723	Cobalt	Testes capsule		Epitheliomatous	Cytoplasm and nucleus	Cytoplasm	Cell membrane

Note: EpCAM = epithelial cell adhesion molecule; CK18 = cytokeratin 18.

Extraction and Quantification of RNA

Extraction of RNA was performed using the Invitrogen PureLink Mini Kit (Invitrogen cat no. 12183-081A). Frozen tissue samples were lyzed and homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) using a rotary-stator homogenizer. Isolation of RNA was performed according to the mini kit protocol. On-column DNase treatment was performed using the Invitrogen PureLink DNase kit (Invitrogen, Carlsbad, CA) to purify RNA samples. RNA quantification and RNA integrity number were measured on a bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples were aliquoted and stored at −80°C until used.

RNA Labeling, Microarray Hybridization, and Data Processing

Gene expression analysis was conducted using Affymetrix Rat Genome 230 2.0 GeneChip^® arrays (Affymetrix, Santa Clara, CA). One hundred nanograms of total RNA was amplified as directed in the Affymetrix 3′ IVT Express kit protocol. A total of 12.5 μg of amplified biotin-aRNAs were fragmented and 10 μg were hybridized to each array for 16 hr at 45°C in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed for antibody amplification according to the GeneChip Hybridization, Wash and Stain Kit, and user manual. Arrays were scanned in an Affymetrix Scanner 3000 and data were obtained using the GeneChip^® Command Console Software (AGCC; Version 1.1) using the MAS5 algorithm to generate .CHP files.

Probe intensity data from all arrays were entered into the R software environment (http://www.R-project.org) directly from .cel files using the R/affy package (Gautier, Bolstad, and Irizarry 2003). Data quality was assessed using image reconstruction, intensity histograms, and boxplots. Normalization was performed across all 11 samples (6 Fred-PE and 5 spontaneous mesothelioma samples) using the robust multiarray average (RMA) method to form 1 expression measure for each gene on each array (Irizarry et al. 2003). The RMA method adjusts the background of perfect match (PM) probes, applies a quantile normalization of the corrected PM values, and calculates final expression measures using the Tukey’s median polish algorithm. Treatment groups (cultured mesothelial cells [Fred-PE], spontaneous mesothelioma) were compared using a t-statistic whose null distribution was derived using 10,000 bootstrap samples by resampling the residuals (Efron and Tibshirani 1993). The bootstrap methodology was implemented in the ORIOGEN software package (Peddada et al. 2005). We applied Benjamini–Hochberg procedure (Benjamini and Hochberg 1995) for multiple testing with a nominal false discovery rate (FDR) of 0.01 to determine differential gene expression.

Core analysis of differentially expressed genes was conducted to determine biologic functions, canonical pathways, and transcription factor activation, and overrepresented classifications of genes were determined from statistical outcomes by testing for association with gene product relationships from a curated database of biological networks (Ingenuity Pathways Analysis™ [IPA] version 9.0; www.ingenuity.com). The Ingenuity Pathways Knowledge Base (IPKB) consists of data with known biological relationships between genes and gene products.

The significantly differentially expressed genes (p < .001) in the IPA core analysis were then grouped by pathways to account for upstream and downstream players as well as overlapping pathways. Upstream activation (formerly known as transcription factor activation in earlier versions of IPA) was based on IPA z scores > 2.0 with no bias.

Quantitative Real-time PCR (qPCR)

Quantitative gene expression levels were detected using real-time PCR with the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) and TaqMan MGB probes (FAM™ dye labeled). Primers and probes for all genes analyzed were purchased from Applied Biosystems Assays-on-Demand Gene Expression products. For amplification, diluted cDNA was combined with a reaction mixture containing TaqMan universal PCR Master Mix (Applied Biosystems, Catalog No. 4304437) according to manufacturer’s instructions. Samples were analyzed in duplicate, and a sample without reverse transcriptase (RT) was included with each plate to detect contamination by genomic DNA. Amplification was carried out as follows: (1) 50°C, 2 min (for uracil-N-glycosylase incubation); (2) 95°C, 10 min (denaturation); (3) 95°C, 15 sec, 60°C, 30 sec (denaturation/amplification) for 40 cycles. Fold increases or decreases in gene expression were determined by quantitation of cDNA from target samples relative to a calibrator sample (Fred PE passage 8). The 18S RNA gene was used as the endogenous control for normalization of initial RNA levels. To determine this normalized value, 2^−(ΔΔCt) values were compared between target and calibrator samples, where the changes in crossing threshold $(Δ {C t) = C t}_{T a r g e t g e n e} - {C t}_{18 S R N A}$ and $Δ Δ C t = Δ {C t}^{c o n t r o l} - Δ {C t}^{t a r g e t}$ .

Immunohistochemistry

Protein expression assayed by immunohistochemistry was used to characterize the immunophenotype of spontaneous rat mesotheliomas and to validate genes associated with mesothelioma development. Formalin-fixed, paraffin-embedded rat tissues were deparaffinized and rehydrated and counterstained with hematoxylin, dehydrated, cleared, and coverslipped in a similar manner for all 3 antibodies. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Antigen retrieval was performed with heat and pressure, using citrate buffer (Biocare Medical, Concord, CA). The sections were incubated with 10% normal donkey serum or normal horse serum (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 20 min based on the respective antibodies, followed by the Avidin–Biotin Blocking Kit (Vector Laboratories, Burlingame, CA). The sections were incubated with rabbit monoclonal VIM antibody (Clone EPR3776, Epitomics, Burlingame, CA) and rabbit IgG (negative control; Epitomics, Burlingame, CA) at 1:250 dilution or Keratin 18 (Clone C-04, Santa Cruz, CA) at a 1:100 dilution and a nonimmune mouse serum (negative control) diluted to match protein concentration of Keratin 18 antibody (Jackson ImmunoResearch, West Grove, PA) for 60 min. Sections were incubated with a donkey antirabbit secondary antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) or biotinylated horse antimouse (Vector Laboratories, Burlingame, CA) for 30 min at 1:500 dilution. Label incubation was also performed using Vector R.T.U. Vectastain Kit (Vector Laboratories, Burlingame, CA) for 30 min. Antigen–antibody complex was visualized using diaminobenzidine (DAB; Dako, Carpinteria, CA) for 6 min. Epithelial cell adhesion molecule (EpCAM) staining was performed on Discovery XT autostainer (Ventana Medical Systems, Tucson, AZ). Standard OmniMap Kit (Ventana Medical Systems, Tucson, AZ) reagents were used. Sections were incubated with rabbit monoclonal EpCAM antibody (Clone E144, Abcam, Inc, Cambridge, MA) at 1:350 dilution and normal rabbit serum (negative control; Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 32 min with no heat setting. The sections were incubated with Discovery XT polymer (Ventana Medical Systems, Tucson, AZ) for 16 min. Antigen–antibody complex was visualized using DAB (Dako, Carpinteria, CA) for 8 min.

Results

Histopathology of Spontaneous Mesotheliomas in F344 Rats

Spontaneously arising mesotheliomas from vehicle control animals (Table 1) were classified as epitheliomatous, characterized by papillary projections of 1 or more layers of atypical mesothelial cells overlying a fibrovascular stalk (Figure 1). Some tumors had a more solid phenotype, forming sheets of cells interspersed with variable amounts of fibrovascular stroma. Mesotheliomas frequently had mixed to mononuclear inflammatory infiltrates within the fibrovascular stroma. The mixed inflammatory component was variable from sample to sample with a prominence of eosinophils, mast cells, or neutrophils.

Figure 1.

Spontaneous mesothelioma, abdominal cavity, F344/N rat. Spontaneous mesotheliomas in F344/N rats exhibited an epitheliomatous phenotype, characterized by papillary projections of plump mesothelial cells overlying a fibrovascular stalk (A, H&E, 10×). Cells were polygonal to cuboidal, with scant to moderate faintly eosinophilic cytoplasm and a central to eccentric round to oval hyperchromatic nucleus and an occasional prominent nucleoli (B, H&E, 40×). H&E = Hematoxylin and eosin.

Transcriptomic Profiling Identifies Significant Differences in Global Gene Expression Profiles between Spontaneous Mesothelioma and Control (Fred-PE) Mesothelium

Of the 21,901 genes on the microarray, 15,714 genes were differentially expressed in rat spontaneous mesotheliomas compared to Fred-PE mesothelial cells. Of these genes, 10,294 were mapped in IPA and 5,420 were unrecognized by IPA and therefore designated as unmapped. Of the mapped genes, 7,953 were considered analysis ready according to IPA based on threshold of p < .001. Principal component analysis (PCA) of differentially expressed genes showed clear separation of spontaneous mesotheliomas from Fred-PE cells in terms of global gene expression (Figure 2). Spontaneous mesotheliomas sampled from different anatomic locations within the same animal (Cobalt) as well as from different anatomic locations and different carcinogenicity studies clustered closely together, indicating that there was little variation in gene expression profiles between these samples based on PCA. However, there was some variability in the gene expression profiles between cell lines based on PCA, which may be attributable to different cell passages or other in vitro cell culture effects. Regardless, hierarchical cluster analysis (HCA) showed distinct clustering of samples within groups based on statistically significant differentially upregulation and downregulation of genes across the genome (Figure 3). IPA core analysis was used to group the 7,953 significantly differentially expressed genes (p < .001) into relevant biological functions. Biological functions representative of the top upregulated and downregulated genes included inflammatory response, cell growth and proliferation, embryonic development, cell death, cancer, carbohydrate metabolism, cell cycle, and gene expression. Many of the significantly differentially expressed genes had overlapping biological functions in multiple categories. The microarray accession can be found in the GEO database (accession # GSE47581).

Figure 2.

Principal component analysis (PCA) comparing global gene profiles of cultured Fred-PE mesothelial cells (meso-normal) and spontaneous mesotheliomas from control F344/N rats (CTL). PCA shows clear clustering of samples within groups, indicating significant similarities in global gene expression of samples within experimental groups, and clear separation of experimental groups in space, indicating significant differences between groups in terms of their global gene expression.

Figure 3.

Unsupervised hierarchal cluster analysis (HCA) comparing global gene expression profiles of Fred-PE mesothelial cells (meso-normal) and spontaneous mesotheliomas from control F344/N rats (CTL). HCA indicates significant differential expression of upregulated (red) and downregulated (green) genes across the genome between Fred-PE mesothelial cells and spontaneous F344/N rat mesotheliomas.

Global Gene Expression Profiling of Spontaneous Mesotheliomas in F344/N Rats Identifies Dysregulation of Genes Associated with Cell Proliferation and Tumorigenesis

Upregulation of genes (Table 2) associated with cell proliferation, cell motility and cell migration, cell invasion, and cell survival included growth factors (transforming growth factor, Tgfα, Tgfβ1), transporter molecules (Abcg1), solute carrier molecules (Slc7a9, Slc7a7, Slc28a2), adhesion molecules (EpCAM, Cdh22), bioactive lipids receptors (Sp1r1), oncogenes (v-Mafb, Lyn [v-yes1 homolog]), Allograft inflammatory factor 1 (Aif1), PRRs (Tlr8, Tlr7, Mannose receptor-1), C-type lectins (Clec4a3, Clec4A, Clec7A, Clec10A), and embryonic genes (Plac8, Plau, Wnt4, Gata5). In addition, there was upregulation of biomarkers for mesothelial cells and mesothelial cell hyperplasia (Ck18, Vim, Wt, Des, Thbd). Genes associated with up- and downstream regulation of transcription factor pathways with a Z score > 2 included interferon α and γ (Ifnα, Ifnγ), signal transducer and activator of transcription 1 (Stat1), and interleukin 10 (Il10). There was downregulation of critical human tumor suppressor genes (PTEN, Tp53, Rassf1, and CDKN2A/2B), cell cycle regulators (p21, p27), and genes associated with cell proliferation (Myc, Fos, Jun). In addition, there was upregulation of antiapoptotic factors such as Bcl2a1 and downregulation of proapoptotic factors such as Bax. These alterations in gene expression represent dysregulation of cell growth and proliferation pathways in mesothelial cells leading to transformation and oncogenesis in F344/N rat spontaneous mesotheliomas.

Table 2.

Select differentially expressed genes dysregulated in spontaneous mesotheliomas in F344/N rats.

Gene name	Gene symbol	Fold change
Transforming growth factor
Transforming growth factor, alpha	Tgfa	12.45
Transforming growth factor, beta 2	Tgfb2	−17.61
Transforming growth factor, beta receptor 1	Tgfbr1	2.14
Transforming growth factor, beta-induced, 68 kDa	Tgfbi	43.39
Platelet-derived growth factor
Platelet-derived growth factor D	Pdgfd	1.88
Platelet-derived growth factor C	Pdgfc	−1.75
Vascular endothelial growth factor
Vascular endothelial growth factor A	Vegfa	−2.03
Vascular endothelial growth factor B	Vegfb	−2.58
Vascular endothelial growth factor C	Vegfc	−7.65
Epidermal growth factor
Epidermal growth factor receptor	Egfr	1.53
Fibroblast growth factor
Fibroblast growth factor 13	Fgf13	−10.61
Fibroblast growth factor receptor 2	Fgfr2	−5.11
Insulin-like growth factor
Insulin-like growth factor 1 (somatomedin C)	Igf1	17.48
Insulin-like growth factor 2 binding protein 1	Igf2bp1	−2.73
Insulin-like growth factor 2 binding protein 2	Igf2bp2	−5.19
Insulin-like growth factor 2 binding protein 3	Igf2bp3	−9.54
Insulin-like growth factor binding protein 6	Igfbp6	10.20
Insulin-like growth factor binding protein 3	Igfbp3	32.46
Cell cycle
Cyclin-dependent kinase inhibitor 1A (p21, Cip1)	Cdkn1a	−4.53
Cyclin-dependent kinase inhibitor 1B (p27, Kip1)	Cdkn1b	−5.69
Cyclin D1	Ccnd1	−1.90
Oncogenes/Protooncogenes
v-Maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian)	Mafb	25.86
v-Myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)	Mycn	−29.75
FBJ (Finkel-Biskis-Jinkins) murine osteosarcoma viral oncogene homolog	Fos	−9.62
Jun B proto-oncogene	Junb	−4.69
v-yes-1 Yamaguchi sarcoma viral related oncogene homolog	LYN (v-yes)	5.12
RAS pathway
RAS, dexamethasone-induced 1	Rasd1	39.86
Rho family GTPase 1	Rnd1	−13.89
Rho family GTPase 3	Rnd3	−13.90
Tumor suppressor genes
Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)	Cdkn2b	−3.77
Tumor protein p53	Tp53	−2.25
Phosphatase and tensin homolog	Pten	−1.41
Wilms Tumor 1	Wt1	3.18
Ras association (RalGDS/AF-6) domain family member 1	Rassf1	−1.34
Adhesion molecule, integrins, catenins
Epithelial cell adhesion molecule	Epcam	22.65
Cadherin 22, type 2	Cd22	128.56
Catenin (cadherin-associated protein), beta 1, 88 kDa	Ctnnb1	−1.41
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)	Itgb2	22.11
Protein kinase
Protein kinase C, beta	Prkcb	73.34
Mitogen-activated protein kinase 12	Mapk12	5.33
Mitogen-activated protein kinase-activated protein kinase 3	Mapkapk3	15.96
Reactive oxygen species (ROS)
Dual oxidase 2	Duox2	104.54
Glutathione peroxidase 2 (gastrointestinal)	Gpx2	102.71
Collagen
Collagen, type VI, alpha 2	Col6a2	66.60
Collagen, type VI, alpha 1	Col6a1	64.48
Transporters and solute carriers
Solute carrier family 7, member 9	Slc7A9	12.70
Solute carrier family 7, member 7	Slc7A7	14.15
Solute carrier family 28, member 2	Slc28A2	14.08
ATP-binding cassette, subfamily A (ABC1), member 4	Abca4	33.23
Mesothelial cell marker
Cytokeratin 18	Krt18	162.94
Cytokeratin 19	Krt19	511.14
Thrombomodulin	Thbd	28.22
Desmin	Des	24.19
Embryonic/cell development (cell migration, differentiation)
Placenta-specific 8	Plac8	39.53
Wingless-type MMTV (mouse mammary tumor virus) integration site family, member 4	Wnt4	11.04
Plasminogen activator, urokinase	Plau	18.00
GATA-binding protein 5	Gata5	15.36
Hormone receptors
Progesterone receptor	Pgr	8.10
Estrogen receptor 1	Esr1	2.15
Estrogen receptor 2 (ER beta)	Esr2	−1.23
Androgen receptor	Ar	1.69
Growth arrest, apoptosis
Growth arrest and DNA-damage-inducible, beta	Gadd45b	−11.25
Growth arrest and DNA-damage-inducible, alpha	Gadd45a	−2.01
BCL2-associated X protein	Bax	−1.53
BCL2-related protein A1	Bcl2A1	12.93
Fas apoptotic inhibitory molecule 3	Faim3	9.96
Fas ligand (TNF superfamily, member 6)	Faslg	2.17

Note: BCL2 = B-cell lymphoma.

Alterations in Inflammatory and Immune Cell Function Are Overrepresented in Global Gene Expression Profiles of Spontaneous F344/N Rat Mesotheliomas

The top upregulation and down upregulated genes from the 7,953 analyzed were associated with inflammatory/immune response (Table 3), and included cytokines (Il18, Il10, Il24) and chemokines (Ccl11, Cxcl13, Ccl5), inflammatory pathways/products of inflammatory cells (arachidonic acid metabolism, Mcpt8, Lyz1/2, Chi3l1), major histocompatibility complexes (Hla-dra, Hla-dqa1, Hla-dqb1), the complement pathway (Cfh, C1qb, Serping1), integrins and adhesion molecules (Cdh22, Itgb2), and oxidative stress/response to reactive oxygen species (ROS; Duox2, Gpx2). Many of the genes with greater than 5-fold change in expression were inflammatory mediators associated with macrophage activation and maturation, lymphocyte (T and B cell), leukocyte, and natural killer (NK) cell activation and recruitment, and immune evasion (Il18, Il24, Il10, Cd40, Col6a1, Cd68, Cd163, Cd53, Cd74, Stab1). Select differentially expressed genes were validated by qPCR (Table 4). Genes were selected based on known involvement with cancer progression, embryonic development, cell survival/decreased apoptosis, mesothelial cell function, immune response, or for their role as a tumor biomarker. In all cases, RNA expression of selected genes matched the same directional change observed in the microarray.

Table 3.

Select differentially expressed genes associated with immune response in spontaneous mesotheliomas in F344/N rats.

Gene name	Gene symbol	Fold change
Chemokines
Chemokine (C-C motif) ligand 11	Ccl11	875.96
Chemokine (C-X-C motif) ligand 10	Cxcl10	9.45
Chemokine (C-C motif) ligand 5	Ccl5	20.40
Chemokine (C-C motif) ligand 6	Ccl6	79.68
Chemokine (C-X-C motif) ligand 11	Cxcl11	7.27
Chemokine (C-X-C motif) ligand 13	Cxcl13	167.52
Chemokine (C-X-C motif) ligand 2	Cxcl2	−133.25
Cytokines and cytokine receptors
Interleukin 1, beta	Il1b	3.21
Interleukin 4 receptor	Il4r	20.95
Interleukin 18 (interferon-gamma-inducing factor)	IL18	41.81
Interleukin 34	Il34	8.49
Interleukin 6 receptor	Il6r	7.20
Interleukin 7 receptor	Il7r	8.49
Tumor necrosis factor receptor superfamily, member 11a, NFKB activator	Tnfrsf11a	1.90
Tumor necrosis factor receptor superfamily, member 11b	Tnfrsf11b	45.94
Interleukin 24	Il24	27.76
Interleukin 10	Il10	24.21
Interleukin 1 receptor antagonist	Il1rn	10.35
Interleukin 23A	Il23a	−5.99
CD40 molecule, TNF receptor superfamily member 5	Cd40	10.29
Jak/Stat pathway
Signal transducer and activator of transcription 1, 91 kDa	Stat1	7.03
Signal transducer and activator of transcription 2, 113 kDa	Stat2	4.31
Signal transducer and activator of transcription 3 (acute-phase response factor)	Stat3	1.77
Janus kinase 2	Jak2	6.86
Janus kinase 1	JAK1	1.60
Histocompatibility markers
Major histocompatibility complex, class II, DR alpha	Hla-dra	425.51
Major histocompatibility complex, class II, DQ alpha 1	Hla-dqa1	172.26
Major histocompatibility complex, class II, DQ beta 1	Hla-dqb1	166.25
CD74 molecule, major histocompatibility complex, class II invariant chain	Cd74	564.33
Complement
Complement factor H	Cfh	306.57
Complement component 1, q subcomponent, B chain	C1qb	115.07
Complement component 1, q subcomponent, A chain	C1qa	132.21
Serpin peptidase inhibitor, clade G (C1 inhibitor), member 1	Serping1	303.39
Pattern recognition receptors
Toll-like receptor 1	Tlr1	3.04
Toll-like receptor 2	Tlr2	8.58
Toll-like receptor 8	Tlr8	23.83
Toll-like receptor 7	Tlr7	10.98
Mannose receptor, C type 1	Mrc1	73.69
Interferon pathway
Interferon, alpha 16	Ifna16	−1.28
Interferon gamma receptor 1	Ifngr1	2.33
Interferon regulatory factor 8	Irf8	11.31
Interferon regulatory factor 7	Irf7	7.42
Interferon regulatory factor 9	Irf9	4.02
Interferon regulatory factor 5	Irf5	2.27
Interferon regulatory factor 3	Irf3	1.43
Interferon induced transmembrane protein 1	Ifitm1	21.44
Inflammatory mediators/enzymes/miscellaneous
Allograft inflammatory factor 1	AIF1	189.47
Prostaglandin D2 synthase 21 kDa	Ptgds	288.68
Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)	Ptgs1	27.33
Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)	Ptgs2	−101.38
Lysozyme 2	Lyz1/lyz2	269.89
Mast cell protease 8	Mcpt8	249.84
Hemoglobin, alpha 1	Hba1/hba2	241.31
Tryptophan 2,3-dioxygenase	Tdo2	212.85
Ubiquitin D	Ubd	191.62
Carbonic anhydrase IV	Ca4	159.31
DEAD (Asp-Glu-Ala-Asp) box polypeptide 60	Ddx60	149.65
Cytochrome b-245, beta polypeptide	Cybb	134.29
Phospholipase A2, group IIA	Pla2g2a	96.46
Lymphatic vessel endothelial hyaluronan receptor 1	Lyve1	81.91
Activated macrophage products
Chitinase 3-like 1	Chi3l1	149.52
SPARC-like 1 (hevin)	Sparcl1	71.34
Complement component 1, q subcomponent, B chain	C1qb	115.07
Complement component 1, q subcomponent, A chain	C1qa	132.21
Cell surface markers/receptors
Sphingosine-1-phosphate receptor 1	S1pr1	24.39
Fc fragment of IgG, low affinity IIb, receptor (CD32)	Fcgr2b	198.66
Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide	Fcer1a	38.51
Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide	Fcer1g	117.78
Fc fragment of IgG, low affinity IIIa, receptor (CD16a)	Fcgr3a	57.05
Stabilin 1	Stab1	60.71
Fc fragment of IgG, high affinity Ia, receptor (CD64)	Fcgr1a	30.53
Fc fragment of IgG, low affinity IIa, receptor (CD32)	Fcgr2a	51.26
CD 163 molecule	Cd163	49.51
CD 68 molecule	Cd68	32.09
CD 53 molecule	Cd53	130.44
C-type lectin domain family 4, member a3	Clec4a3	123.54
C-type lectin domain family 10, member A	Clec10a	46.75
C-type lectin domain family 4, member A	Clec4a	36.02
C-type lectin domain family 7, member A	Clec7a	28.97
CD36 molecule (thrombospondin receptor)	Cd36	148.28

Table 4.

Quantitative PCR (qPCR) validation of select upregulated and downregulated genes in spontaneous mesotheliomas in F344/N rats.

Entrez gene name	Gene symbol	qRT-PCR	Microarray
GATA-binding protein 5	Gata5	604.09	14.2
Interleukin-10	Il10	697.23	24.21
Interleukin-18	Il18	159.52	41.81
Glutathione peroxidase 2	Gpx2	484.44	102.71
Cytokeratin 18	Krt18	1,079.19	162.94
Tumor necrosis factor receptor 11b	Tnfr11b	729.76	45.94
Plasminogen activator, urokinase	Plau	278.72	17.9
Wingless-type MMTV integration site family, member 4	Wnt4	7.84	11.19
CD40 molecule, TNF receptor superfamily member 5	Cd40	843.49	10.18
Epithelial adhesion molecule	EpCAM	481.96	22.65
Protein kinase C, beta	Prkcb	274.06	73.34
Transforming growth factor alpha	Tgf-a	12.38	12.45
Cyclin-dependent kinase 1a (p21)	Cdkn1a	−2.88	−4.53
Fatty acid binding protein 4	Fabp4	2,705.77	68.98
Placenta-specific 8	Plac8	16,744.15	39.53
RAS, dexamethasone-induced 1	Rasd1	86.8	39.86
Tumor protein p53	Tp53	−1.11	−2.25

Downstream Protein Expression of Spontaneous F344/N Rat Mesotheliomas Confirm a Biphasic Phenotype and Share Protein Markers with Human Mesothelioma

Spontaneous mesotheliomas in the current study were evaluated immunohistochemically with antibodies to cytokeratin 18 (CK18) and VIM (Figure 4). Rat spontaneous mesotheliomas strongly expressed both VIM and CK18 (Table 1), and protein expression correlated positively with directional fold change in gene expression on the microarray. Expression of CK18 was restricted to the cell membrane in mesotheliomas, similar to the expression in positive controls (rat salivary gland) compared to negative controls. VIM was coexpressed in the cytoplasm of mesothelioma cells, and expression was similar to positive controls (rat kidney). Mesotheliomas expressed EpCAM (Table 1), a protein involved in the maintenance of tight junctions that is overexpressed in a variety of human cancers including mesothelioma. However, while expression of EpCAM was restricted to the cell membrane in positive controls (rat gastrointestinal tract mucosa), in spontaneous rat mesotheliomas, there was translocation of protein to the cytoplasm and nucleus, suggesting dysregulation of this protein (Figure 4).

Figure 4.

Immunohistochemical validation of mesothelioma markers in spontaneous mesotheliomas from control F344/N rats. Neoplastic cells in spontaneous mesotheliomas (A, C, E, 10×; B, D, F, 40×) showed strong membrane immunoreactivity with anticytokeratin 18 antibody (A–B), strong cytoplasmic immunoreactivity with antivimentin antibody (C–D), and strong cytoplasmic and nuclear immunoreactivity for antibodies to epithelial cell adhesion molecule (E–F; hematoxylin counterstain).

Discussion

Our study has characterized the genomic profile of spontaneously occurring peritoneal mesotheliomas in male Fisher 344/N rats. In their roles as a primary barrier to pathogens and maintenance of peritoneal homeostasis, mesothelial cells have a wide range of initiation and response mechanisms associated with cellular growth, cytokine and growth factor release, regulation of PRRs, and PAMPs (Izzi et al. 2012; Wu et al. 2010; Yung and Chan 2007). Carcinogenesis is a very complex process involving dysregulation of multiple cell growth and proliferation pathways, metabolic perturbations, inflammation and immune dysfunction, and hormonal factors, and consistent with this, we have shown that the genomic profiles observed in spontaneous F344/N rat mesotheliomas are similarly complex, involving dysregulation of numerous intersecting and interrelated pathways related to, cell growth and proliferation, contributions by inflammatory mediators, and interactions with ROS and free radicals, all well-known and contributory mechanisms in carcinogenesis.

Oncogenes, Tumor Suppressor Genes, and Apoptosis Mediators

Alterations in tumor suppressor genes and oncogenes are key genetic factors related to tumor development and progression. Spontaneous F344/N rat mesotheliomas showed downregulation of several tumor suppressor genes (Tp53, Pten, Gadd45A, Cdkn2b, Cdkn1a, Cdkn1b) and overexpression of oncogenes (v-MafB, v-Yes). MAFB (V-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is overexpressed in human mesothelioma (Gordon et al. 2005), and c-YES is a mediator of cell proliferation in human malignant mesothelioma cells (Sato et al. 2012). In addition, a variety of tumor suppressor genes (CKDN2A, TP53, PTEN, NF2, BAP1) and their downstream targets are similarly affected in human mesothelioma (Bueno et al. 2010; Gordon et al. 2005; Gueugnon et al. 2011; Tsujimura et al. 2012). While promoters of cell cycle progression and cellular proliferation such as FOS, MYC, JUN, and CCND1 are typically upregulated in asbestos-associated human mesothelioma, these genes were downregulated in spontaneous rat mesothelioma in this study. Finally, spontaneous rat mesotheliomas were associated with an antiapoptotic phenotype characterized by the overexpression of antiapoptotic Bcl mediators (Bcl2a1), downregulation of proapoptotic Bcl members (Bax) and Fadd and Fas mediators, and upregulation of Fas apoptotic inhibitory molecule 3 (Faim3). Alterations in tumor suppressor genes, oncogenes, and apoptosis mediators have a profound effect on the expression of downstream cellular growth factors and the inflammatory response, two other significantly represented categories of altered gene expression in this study.

Growth Factor Pathways

Dysregulation of cellular growth is an integral part of carcinogenesis. In spontaneous F344/N rat mesotheliomas, there was upregulation of mediators in a number of growth factor pathways, including the Tgfα/β, insulin-like growth factor (Igf), p38 mitogen-associated protein kinase (p38 Mapk), and nuclear factor kappa-B (Nfκb) pathways. TGFβ was originally discovered as a mediator involved in tumor cell survival (Abbas and Lichtman 2003), regulates IGF1 and inhibits cell adhesion and enhances cell–collagen interactions associated with invasion and metastasis. TGFα is associated with positive regulation of cell proliferation, differentiation, and development, and negatively regulates apoptosis. Upregulation of TGFα/β is associated with a variety of cancers including breast, liver, kidney, lung, pancreatic, and hematologic cancers. In some cancers, TGFβ plays conflicting roles in transformation and tumor progression; for example, it has been shown to act as a tumor suppressor in early stages of breast cancer development, but acts to promote invasion and metastasis late in the course of disease (Akhurst and Derynck 2001; Tang et al. 2003). TGFα and β are also involved in a large number of physiologic processes, and interface with other growth factor pathways such as the MAP-kinase (MAPK) and NFκB signaling pathways. In addition, TGFβ1 is produced by and regulates cells of the immune system (Letterio and Roberts 1998; Yang, Pang, and Moses 2010), representing another overlapping component of the interlacing pathways associated with mesotheliomas in this study. Several members of the Igf pathway were upregulated in spontaneous mesotheliomas (Igf1, Igfbp1, Igfbp2-7), similar to a variety of human cancers including mesothelioma, where they are associated with high-grade or poorly differentiated tumors (Gullu, Karabulut, and Akkiprik 2012; Jean et al. 2012; Whitson and Kratzke 2006). The p38 Mapk pathway is regulated by a wide variety of molecules and conditions including growth factors, cytokines, cytosolic proteins, and environmental stresses (Cuadrado and Nebreda 2010). Several mediators within the p38 Mapk pathway (p38 Mapkα/β/γ, Stat1, Tgfβr1, Tgfβ1, Tnfrs11b, Tnfrs14, Tnfrs25) were upregulated in spontaneous mesotheliomas. Mediators in this pathway are involved in cell survival and proliferation, and there is moderate crosstalk between other growth pathways such as the NFkB (nuclear factor kappa-light-chain-enhancer of B cells), WNT (wingless type MMTV integration site family), JNK (janus kinase), and AKT (v-akt murine thymoma viral oncogene homolog/protein kinase B) pathways (Cuadrado and Nebreda 2010). Moreover, p38 Mapk is activated in rat mesothelial cells exposed to asbestos (Swain, O’Byrne, and Faux 2004) and regulates cell proliferation in rat pleural mesothelioma cell lines (Zhong et al. 2011). The various p38 Mapk isoforms act across many pathways and may be activated by environmental stress (oxidative stress, UV irradiation, hypoxia, ischemia) and inflammatory cytokines such as IL1 and tumor necrosis factor (TNFα; Cargnello and Roux 2011).

EpCAM is a transmembrane p-glycoprotein reported to have multiple roles including cell signaling, migration, proliferation, and differentiation (Patriarca et al. 2012). It is a well-known epithelial tumor marker that is overexpressed in breast, bladder, colorectal, pancreas, prostate and ovarian cancer, and epitheliomatous mesotheliomas in humans (Ryan et al. 1997; Shield, Callan, and Devine 1994). Alterations in EpCAM interfere with cadherin-mediated cell to cell adhesion (Litvinov et al. 1997), and cytoplasmic and nuclear translocation of its intercellular domain (EpICD/EpCAM intercellular domain) is associated with stimulation of cell proliferation (Maetzel et al. 2009). In addition to upregulation of EpCAM at the gene expression level, there was cytoplasmic and nuclear translocation of EpCAM protein in spontaneous rat mesotheliomas in this study (Figure 4); the EpCAM antibody used in this study recognizes the C terminus on EpICD (Patriarca et al. 2012), confirming the nuclear translocation of the intercellular domain and implicating EpCAM as mediator of cell proliferation and mesotheliomagenesis in both F344/N rats and humans. It is well known that mesothelial cells have a biphasic nature, expressing a broad spectrum of low and high molecular weight epithelial keratins and VIMs (Mullink et al. 1986; Whitaker, Papadimitriou, and Walters 1980). As such, spontaneous mesotheliomas in this study expressed both VIM and CK18 by immunohistochemistry.

Inflammatory Pathways and Immune Dysfunction

Inflammation and immune dysregulation are mechanisms central to many neoplastic processes, including the development of mesotheliomas, and are considered the seventh hallmark of cancer (Colotta et al. 2009; Hanahan and Weinberg 2011). In fact, a key component of early stage asbestos-related mesothelioma in humans is a nonspecific inflammatory response (Boutin and Rey 1993). Given the nature of the disease in humans, a chronic inflammatory response is not surprising. What is interesting, however, is the predominance of genomic changes associated with altered immune function and inflammation in spontaneous rat mesotheliomas, which arise in the absence of any initiating chemical or physical agent. This response could be attributed to chronic inflammation, cancer-related inflammation, dysregulation of mesothelial cell function, and/or intrinsic genetic events. Mesothelial cell proliferation may be stimulated through the induction of proinflammatory pathways by activated macrophages or mesothelial cells, with the subsequent production of proinflammatory cytokines (TNF, IL-1) and growth factors (IL6 [interleukin 6], GM-CSF [granulocyte/monocyte-colony stimulating factor]), upregulation of prostaglandins, nitrous oxide (NO), angiogenic factors, and adhesion molecules. Research on the etiology of mesotheliomas in humans (asbestos) and animal models (multiwalled carbon nanotubes, MWCNT) suggest that a major pathophysiologic factor in the development of these tumors is frustrated phagocytosis leading to biopersistence of the initiating agent, which results in chronic oxidative stress and inflammation (Walker, Everitt, and Barrett 1992; Yang, Testa, and Carbone 2008). Similarly, rat spontaneous mesotheliomas in this study were associated with upregulation of a variety of cytokines and cytokine receptors associated with chronic inflammation (Il6r, Tnfrs11b, Tnfrs14, Tnfrs25, and Il1b), including Il10, which promotes cell survival and abrogation of immune surveillance. It is known that asbestos in fact can cause alterations in normal cells that result in decreased tumor immunity (Matsuzaki et al. 2012). Whether these are contributing factors in the induction and pathogenesis of this tumor in rats or merely secondary to the presence of the neoplasm and the tissue damage associated with its invasion is uncertain. Certainly, the chronic tissue damage and inflammation associated with asbestos exposure in human mesotheliomagenesis is due to the persistence of the asbestos fiber and its interaction with cellular components within the lung parenchyma, ultimately leading to generation of ROS and secondary DNA damage (Matsuzaki et al. 2012). In terms of nonasbestos etiologies in human mesothelioma, many of these are also associated with inflammatory processes in the lung (SV40 viral infection), exposure to radiation or chemical agents (Carbone and Bedrossian 2006; Yang, Testa, and Carbone 2008); whereas others, such as germ line mutations in BAP1 (Bott et al. 2011; Testa et al. 2011), arise without such associated tissue damage and inflammation. Similarly, mesothelioma in vehicle control F344/N rats arises spontaneously without an inciting cause related to tissue damage or inflammation and may represent an inherent genetic susceptibility that could be influenced by chemical exposure.

Many of the receptors associated with innate immunity that is constitutively expressed in mesothelial cells were upregulated in spontaneous mesotheliomas in this study. Pleural and peritoneal mesothelial cells express a wide range of PRRs, such as the TLRs, which are triggered by PAMPs, including components of bacteria, fungi, or viruses, and are therefore used in defense systems employed by the mesothelium (Takeuchi and Akira 2010). TLRs are PRRs that trigger activation of the NFkB pathway and/or MAPK activation, and increased production of proinflammatory cytokines and interferons (Takeuchi and Akira 2010). The genomic profile of spontaneous rat mesotheliomas is highlighted by the prominent upregulation of PRRs (TLR1, 2, 7, 8, Mrc1) that trigger activation of the TLR-NFkB and interferon pathways, with subsequent release of inflammatory cytokines and other inflammatory mediators. Endogenous TLR ligands also have an increasing role in tumorigenesis through promotion of cancer cell survival and proliferation, immune system evasion, and enhancement of metastasis and chemoresistance (Yu, Wang, and Chen 2012). Endogenous molecules that are released from mesothelial cells following tissue injury can act as danger-associated molecular pattern receptors (DAMPs) and activate TLRs (Yu, Wang, and Chen 2012). Several mediators that act as DAMPs (Mrc1, S100a8/9, hyaluronan, Lyve1, Stab1) were upregulated in spontaneous rat mesotheliomas; these mediators can promote tumorigenesis and invasion through the activation of various TLRs (Turovskaya et al. 2008; Voelcker et al. 2008; Yu, Wang, and Chen 2012). Spontaneous rat mesotheliomas were also associated with upregulation of interferon pathway genes associated with immunomodulation (If135, Psmb8, Ifitm1, Tap1, Aif1, Irf1/7/9). Overexpression of some of these immunomodulatory genes are also associated with cell proliferation, inhibition of apoptosis, and tumor growth (Liu et al. 2008), and upregulation of the interferon receptor pathway results in stimulation of a variety of other cell growth signaling networks including the Jak/Stat pathway, glucocorticoid pathway, and immune function and cytokine expression. This suggests a significant inflammatory component to the process of spontaneous mesothelioma development in these animals.

Evidence supporting the hypothesis that the immune system plays an important role in modulating cancer has been increasing over the last decade. Indeed, in addition to their role as activators of the immune response, protumorigenic activity has been described for upregulated TLRs in cancer cells or tumor-associated macrophages (TAMs; Yu, Wang, and Chen 2012). Moreover, TLR stimulation has been associated with tumor promotion and invasion in rodents and humans (Ochi et al. 2012; Xie et al. 2010). Mediators expressed by activated and/or TAMs have been shown to have tumor-promoting qualities (Traves, Luque, and Hortelano 2012). TAMs are present in variety of cancer types and are associated with tumor progression, tumor cell invasion, metastasis, and angiogenesis (Pollard 2004). Cell surface markers associated with TAMs in rat spontaneous mesotheliomas in the current study included Cd163, Cd68, Cd53, and C-type lectins (Cd93), many of which play roles in cell–cell and cell–matrix interactions (Bos et al. 2012), cell adhesion, migration, and inflammation (Greenlee-Wacker, Galvan, and Bohlson 2012), prosurvival signaling through the AKT/PIK3 (phosphatidylinositol 3 kinase) pathway (Yunta and Lazo 2003), or are expressed by tumor cells and TAMs in a variety of human cancers (Maniecki et al. 2012; Shabo and Svanvik 2011;Ch’ng et al. 2013; Feng et al. 2012; Jia et al. 2008). The upregulation of these cell surface markers in spontaneous rat mesotheliomas may be due to overexpression by mesothelioma cell themselves or due to the presence of activated or TAMs within these tumors.

In this study, a complex interaction of dysregulated mesothelial cell processes including growth, differentiation, and immune system function was observed. It is clear that mesothelioma in humans (Yung and Chan 2007) and rats (Xu et al. 2012) is associated with risk factors of chronic inflammation; however, in spontaneous mesothelioma, the prominent inflammatory phenotype is unexpected, and it is unclear as to whether the prominent immune cell response was a contributing factor for the development of mesothelioma, or merely a response to the presence of the neoplasms in these animals.

Conclusion

This study shows that there is dysregulation of numerous genes in overlapping pathways including those associated with cellular proliferation and survival, inflammation and immune system dysregulation, inhibition of apoptosis, and dysregulation of oncogenes, tumor suppressor and embryonic genes in the process of spontaneous mesotheliomagenesis. Importantly, this data set now provides a point of reference to better understand the events that occur in chemically induced mesothelioma in the F344/N rat, and how these events may be relevant to human hazard characterization.

Footnotes

Abbreviations

Acknowledgments

We acknowledge Norris Flagler, Eli Ney, and Beth Mahler for their imaging expertise and assistance with figures. We would like to thank the CMPB Histology and Immunohistochemistry Core Laboratories, and the NIEHS Microarray Core for their technical expertise.

Authors’ Note

This article may be the work product of an employee or group of employees of the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH); however, the statements, opinions, or conclusions contained therein do not necessarily represent the statements, opinions, or conclusions of the NIEHS, NIH, or the U.S. government.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Institutes of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), and The Division of the National Toxicology Program (DNTP).

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