br Materials and methods br Results br

Materials and methods
Results
Discussion Several major signaling and developmental pathways (e.g. EGFR, ALK, KRAS, TGFβ, Notch and Wnt/β-catenin) have been shown to be involved in lung carcinogenesis and they are being exploited as targets of the non-small cell lung cancer therapy (for reviews see (Eser and Janne, 2018; Pancewicz-Wojtkiewicz, 2016; Rotow and Bivona, 2017; Tomasini et al., 2016; Yang et al., 2016)). The complex crosstalk of AhR pathway with lung cancer-associated pathways mentioned above has been already observed in multiple tissues (Schneider et al., 2014; Ye et al., 2017). Given these reports and the fact that test PAHs present in cigarette smoke (BaP and BkF) are potent AhR agonists, we attempted to identify biological processes/pathways directly deregulated by AhR activation or inhibition. We identified interferon gamma (IFNɣ), growth factors receptor (e.g. EGFR, PDGFR), insulin/insulin-like growth factor (IGF), small GTPase, Wnt/β-catenin and p38/MAPK to be the most confidently and significantly deregulated signaling pathways in response TCDD and/or CH223191. The same analyses focusing on biological processes suggested Cell cycle/proliferation, Death receptor signaling/Apoptosis, Metabolism of xenobiotics and of Oxidative and Endoplasmic reticulum (ER) stress to be the most affected processes in TCDD-exposed cells. In addition, biological processes involved in mechanisms, which control Development, Endocrine system homeostasis and Lipid metabolism (e.g. Sphingosine-1-phosphate- S1P pathway) were predicted as significantly altered as well. Indeed, numerous reports have already indicated that AhR plays a major role in biological processes including xenobiotic metabolism, Phosphoramidon Disodium Salt regulation, apoptosis, oxidative stress (Bock, 2013; Marlowe and Puga, 2005), or that interplay of AhR signaling with Wnt/β-catenin, p38/MAPK or EGF signaling is an integral part of its cellular effects (Esser and Rannug, 2015; Haarmann-Stemmann et al., 2009; Puga et al., 2009; Schneider et al., 2014). In contrast, very little is known about AhR signaling − ER stress relationship during e.g. lung cancer progression, although both cellular events have been shown to be tightly connected in e.g. cytoprotection of liver cells or in inhibition of gastric tumor growth (Joshi et al., 2015; Lai et al., 2014). In addition, cigarette smoke has been already documented to induce ER stress in malignant lung cancer cells (Jorgensen et al., 2008). Similarly, although IFNγ, S1P and PDGF-BB signaling may be implicated in the development of various lung diseases e.g. lung metastasis spreading, little is known about their mutual crosstalk with AhR signaling within lung cancer context (van der Weyden et al., 2017; Ni et al., 2017; Hosaka et al., 2013; Jaguin et al., 2015; Lee et al., 2017; McMillan et al., 2007; Wang et al., 2016). Fig. 4 represents a graphical compilation of the proposed AhR signaling-associated cellular candidates as predicted in TCDD-exposed human model of lung carcinoma cells (Fig. 4). Considering CH223191 to be an inhibitor of basal AhR signaling, results from global gene profiling performed in A549 cells acutely exposed to CH223191 provide us with an opportunity to speculate about the implications of AhR activation status on character of lung cancer cell disposition/behavior. Jagged-1 (JAG1), gene upregulated in both CH223191-treated (see Results chapter 3.1) and siAhR-treated cells (Salisbury et al., 2014), has been reported to be a potential metastasis enhancer in lung cancer and it has been found to be induced in lung cells exposed to DEP (diesel exhaust particle) (Chang et al., 2016; Xia et al., 2015). The observed upregulation of JAG1 expression, together with suppression of genes necessary for reduction of oxidative stress (e.g. CAT, CYP1B1, HMOX1; Fig. 2), may thus provide lung cancer cells pro-survival and pro-invasive signals. These data support the hypothesis that AhR may exhibit tumor suppressor activity during lung cancer progression, based on the observation that AhR signaling reduces lung cancer cell invasion both in vivo and in vitro, and depletion of its cytosolic form may contribute to epithelial-to-mesenchymal transition (EMT) and cancer cell dissemination (Li et al., 2017). Therefore, AhR itself and its documented overexpression in adenocarcinomas might represent a protective mechanism, which helps to restrain lung cancer cell from spreading (Fan et al., 2010; Lin et al., 2003). In contrast, a sustained activation of AhR induced by its toxic environmental ligands may provide a switch to pro-carcinogenic transcriptional program(s).