Oncogene (2018) 37:2645–2659 https://doi.org/10.1038/s41388-018-0153-z ARTICLE How asbestos drives the tissue towards tumors: YAP activation, macrophage and mesothelial precursor recruitment, RNA editing, and somatic mutations 1 2 3 3 3 4 Hubert Rehrauer ● Licun Wu ● Walter Blum ● Lazslo Pecze ● Thomas Henzi ● Véronique Serre-Beinier ● 1 5 2 3 6 Catherine Aquino ● Bart Vrugt ● Marc de Perrot ● Beat Schwaller ● Emanuela Felley-Bosco Received: 1 September 2017 / Revised: 11 December 2017 / Accepted: 30 December 2017 / Published online: 6 March 2018 © The Author(s) 2018. This article is published with open access Abstract Chronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines, and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single-nucleotide variations, with an RNA-editing signature, which were 1234567890();,: even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naïve mice supporting the role of the environment in tumor progression. Introduction The association of exposure to asbestos with development of mesothelioma has been demonstrated in the seminal experimental work of Wagner in the 1960s [1]. In 1987, Kane and co-workers [2] observed that already a single dose These authors contributed equally: Hubert Rehrauer, Licun Wu. of asbestos fibers damages the mesothelium tissue and sti- Electronic supplementary material The online version of this article mulates regeneration with the additional recruitment of (https://doi.org/10.1038/s41388-018-0153-z) contains supplementary macrophages to the site of damage and were the first to material, which is available to authorized users. propose that persistent tissue injury leads to an inflamma- * Emanuela Felley-Bosco tory and regenerative response, which subsequently paves [email protected] the way to mesothelioma development. Tumor development greatly depends on the evasion from 1 Functional Genomics Center Zurich, ETH Zurich and University the immune surveillance. Tumor cells escaping the immune of Zurich, 8057 Zurich, Switzerland defense is enhanced by the induction of an immunosup- 2 Latner Thoracic Surgery Research Laboratories, Division of pressive tumor microenvironment [3]. For example, the Thoracic Surgery, Toronto General Hospital University Health Network, University of Toronto, Toronto, ON, Canada differentiation and activation of antigen-presenting dendritic cells, which are the key initiators of the adaptive immune 3 Department of Medicine, Unit of Anatomy, University of Fribourg, Route Albert-Gockel 1, CH-1700 Fribourg, Switzerland responses [4], are inhibited by signals, e.g., vascular endothelial growth factor (VEGF), present in the tumor 4 Department of Thoracic Surgery, University Hospitals of Geneva, Geneva, Switzerland microenvironment [5]. Tumor-associated macrophages polarized to a “M2” state [6] as well as a heterogeneous 5 Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland population of myeloid-derived suppressor cells, are potent suppressors of antitumor immunity [7]. Others and we have 6 Laboratory of Molecular Oncology, Lungen- und Thoraxonkologie Zentrum, University Hospital Zürich, previously observed that blockade of immunosuppressive Sternwartstrasse 14, 8091 Zurich, Switzerland signals enhances immunity against mesothelioma [8–12]. 2646 H. Rehrauer et al. This study assesses the loss of homeostasis in the after intraperitoneal exposure to asbestos has been widely mesothelial environment during tumor development after accepted as a bona fide surrogate to investigate exposure to asbestos. Since mesothelioma development mesothelium-dependent reaction, we used this model to Molecular changes during mesothelioma development 2647 Fig.1 Exposure to asbestos alters the profile of cell populations in In order to explore mechanisms putatively involved in peritoneal lavage. a Experimental scheme: 6–8-week-old C57Bl/6J the observed changes, the content of different inflammatory mice were exposed to crocidolite i.p. (400 µg/mouse) every 3 weeks cytokines (IL-6, IL-10, IFNγ), myeloid chemoattractant with in total eight treatments. Thirty-three weeks after initial exposure to crocidolite mice were sacrificed to collect peritoneal lavage, blood, chemokines (CCL-2, CCL5, CXCL1), and growth factors and tissues. Tumor-bearing mice were sacrificed to collect tumor tis- modulating the differentiation of myeloid cells (G-CSF, sue. b The lavage from crocidolite-exposed mice shows higher pro- GM-CSF) or vessel growth (VEGF), was measured in the portions of macrophages and mesothelial precursor populations as peritoneal lavage (Fig. 2). Levels were increased in compared to sham-exposed mice. Flow-cytometry analysis of samples fi is shown in the left panel. A significant decrease in the total number of crocidolite-exposed mice paralleling the pro le change T and B cells was observed in the peritoneal lavage 12 weeks after last observed for the CD68/F480 and mesothelial precursor cell exposure to crocidolite. c The proportion of T cells was significantly populations. A strong correlation was observed between increased in the spleen of asbestos-exposed mice. No difference for B CCL2 and IL-6 levels (r = 0.7, p < 0.0001) consistent with cells was observed in the spleen. N = 6–8 mice. Mean ± SE. *p < 0.05, **p < 0.01, Mann–Whitney test the known reciprocal regulation of these two cytokines [20]. RNA-seq transcriptome profiling investigate in-depth how perturbation of homeostatic con- trol leads to tumor development. Nf2+/− mice on a C57Bl/ Tumors showing spindled morphology and benign meso- 6J genetic background were used for several reasons thelial proliferations cells that had grown in asbestos- including the observation that tumor that develop in Nf2 exposed mice stained positive for podoplanin, WT-1, +/− mice exposed to asbestos show common genomic mesothelin, vimentin, and cytokeratin (Supplementary alterations with human mesothelioma and loss of NF2 Figure 1). function has a driver role in mesothelioma development We collected scraped mesothelium (Fig. 3a) and per- [13–16], and C57Bl/6J mice being widely used in func- formed RNA-seq to identify gene expression changes dur- tional studies. ing mesotheliomagenesis. We analyzed three groups by RNA-seq: sham, age-matched crocidolite-exposed, and age- matched crocidolite-exposed with observable tumors. Results Among those groups we performed differential expression analysis between crocidolite-exposed and sham, and Exposure to asbestos alters the profile of cell detected 5976 differentially expressed genes (p < 0.01, populations and signaling molecules in the false-discovery-rate (FDR) < 0.022). Additionally we peritoneal cavity in non-tumor bearing mice assessed the expression differences between crocidolite exposed with tumors and crocidolite exposed, and identified C57Bl/6J Nf2+/− mice were exposed eight times to cro- 8416 genes (p < 0.01, FDR < 0.017). In Fig. 3b, we show a cidolite (blue asbestos) every 3 weeks then were sacrificed heatmap of the significant genes where we applied an 33 weeks after first crocidolite exposure in order to have the additional fold-change threshold of higher than two-fold possibility to investigate pre-cancer and cancer stages (Fig. (2316 genes up, 84 genes down when comparing 1a). In mesothelioma-free mice, the population of CD68/ crocidolite-exposed and sham). The crocidolite response is F480 macrophages in the peritoneal lavage was five-fold dominated by an upregulation of genes. It is interesting to higher compared to sham-exposed mice (Fig. 1b). Both, the note that among the 2316 genes with a positive response, numbers of T and B cells (Fig. 1b) in the peritoneal lavage 1989 have an even higher average expression in the of crocidolite-treated mice were significantly decreased. crocidolite-with-tumor samples as compared to the croci- Besides macrophage recruitment after tissue damage, dolite samples and only 327 genes have a lower average free-floating mesothelial-like cells were shown to be expression. However, this asymmetry is not generally true, incorporated into peritoneal wound surfaces and to con- when looking at all genes significantly changed between tribute to the regeneration of the damaged mesothelium crocidolite with tumors and crocidolite without tumors. We [17]. This population is characterized as mesothelin+ (Msln find 3014 genes with more than two-fold upregulation and +)-bone marrow-derived progenitor cells [18]. During 3008 genes with more than two-fold downregulation. A embryonic development, a mesothelial precursor population total of 1234 genes of these two upregulated gene sets were has been identified as Msln+ CD105low, CD90high, CD44low, overlapping, as shown in the Venn diagram (Fig. 3c). The and CD34high [19]. Accordingly, we observed that CD90 commonly upregulated 1234 genes represent 53% of the +CD34+Msln+ cells paralleled the CD68/F480 cell profile upregulated genes in the crocidolite-exposed tissue vs. (Fig. 1b). In the spleen a significant increase of T cells, both sham-exposed