**2. Asbestos-induced carcinogenesis as an inflammation-driven process**

The association between exposure to asbestos fibers and development of lung cancer and mesothelioma is well established in both humans and animals models (Greillier & Astoul, 2008; Huang et al., 2011; Mossman & Churg, 1998; Yarborough, 2007). A variety of mediators, either generated directly from asbestos fibers or elaborated intracellularly or extracellularly by cells exposed to asbestos, are implicated in the initiation and promotion of mesothelial cell transformation.

The mechanisms underlying asbestos-induced carcinogenesis involve mutagenic and nonmutagenic pathways, the latter including inflammation, enhanced mitogenesis, cell signaling alterations, and cytotoxic apoptosis/necrosis. Neither of these two mechanisms alone fully accounts for the complex biological abnormalities produced by asbestos fibers, even though in MM asbestos appears to act as a complete carcinogen (Dong et al., 1994; Huang et al., 2011). Still, the chronic inflammatory response induced by asbestos inhalation seems to play a critical role in mesothelial cell transformation.

Asbestos exposure induces an inflammatory reaction with a large component of mononuclear phagocytes (Antony et al., 1993; Branchaud et al., 1993; Carbone et al., 2012; Choe et al., 1997). Upon differentiation into macrophages, these cells phagocytize asbestos fibers and, in response, release numerous cytokines and reactive oxygen species with mutagenic properties (Robledo & Mossman, 1999). Thus, many of the pathological consequences occurring in the lung following exposure to asbestos fibers are believed to arise from an inflammatory cascade involving both autocrine and paracrine events (Hillegass et al., 2010). Persistent pulmonary inflammation is observed in animal models of asbestosis that can be correlated with fibroproliferative responses (Mossman & Churg, 1998).

Experimental models, as well as *in vitro* studies, have shown that mesothelial cells are particularly susceptible to the cytotoxic effects of asbestos (Baldys et al., 2007; BéruBé et al., 1996; Broaddus et al., 1996). Asbestos does not induce transformation of primary human mesothelial cells *in vitro*, instead, it is very cytotoxic to this cell type, causing extensive cell death. This finding raised an apparent paradoxical issue of how asbestos causes MM if human mesothelial cells exposed to this mineral die (Liu et al., 2000). This apparent paradox is reconciled by the current hypothesis that the chronic inflammation induced by asbestos leads to the persistent activation of the nuclear factor kappa B (NF-κB) transcription factor, which in turn mediates the activation of prosurvival genes and prevents apoptosis of the damaged mesothelial cells (Mantovani et al., 2008; Micheau & Tschopp, 2003; Philip et al., 2004). This allows mesothelial cells with asbestos-induced DNA damage to survive and divide rather than die and, if sufficient genetic damage accumulates, to eventually develop into a MM (Miura et al., 2006; Nymark, 2007). In fact, apoptosis is an important mechanism by which cells with DNA damage are eliminated without eliciting an inflammatory response (Ullrich et al., 2008; Yoshida et al., 2010). However, failure of apoptosis in cells with unrepaired DNA and chromosomal damage after chronic exposure to asbestos may lead to permanent genetic alterations and trigger the development of a clone of cancerous cells (Roos & Kaina, 2006; Wu, 2006). Consistently, MM cells are found to be apoptosis-resistant as compared to primary cultured mesothelial cells (Fennel & Rudd, 2004; Villanova et al., 2008).

#### **2.1. Tumor Necrosis Factor-α and other pro-inflammatory cytokines**

50 Malignant Mesothelioma

mesothelial cell transformation.

microenvironment. They participate in serosal inflammation by secreting both pro- and antiinflammatory as well as immunomodulatory mediators. Besides, these cells can act as antigen presenting cells for T lymphocytes (Hausmann et al., 2000), regulate tissue repair, control fibrin deposition and breakdown, and modulate adhesion, growth and dissemination of tumor cells metastasizing to the serosal membranes (Mutsaers, 2002). In particular, in response to different types of stimuli, including cytokines and asbestos fibers, mesothelial cells have been reported to release prostaglandins, chemokines, reactive oxygen and nitrogen species and growth factors which represent key effectors in the modulation of inflammatory reactions that occur in response to pleural injury (Fleury-Feith et al., 2003; Mutsaers, 2002).

**2. Asbestos-induced carcinogenesis as an inflammation-driven process** 

The association between exposure to asbestos fibers and development of lung cancer and mesothelioma is well established in both humans and animals models (Greillier & Astoul, 2008; Huang et al., 2011; Mossman & Churg, 1998; Yarborough, 2007). A variety of mediators, either generated directly from asbestos fibers or elaborated intracellularly or extracellularly by cells exposed to asbestos, are implicated in the initiation and promotion of

The mechanisms underlying asbestos-induced carcinogenesis involve mutagenic and nonmutagenic pathways, the latter including inflammation, enhanced mitogenesis, cell signaling alterations, and cytotoxic apoptosis/necrosis. Neither of these two mechanisms alone fully accounts for the complex biological abnormalities produced by asbestos fibers, even though in MM asbestos appears to act as a complete carcinogen (Dong et al., 1994; Huang et al., 2011). Still, the chronic inflammatory response induced by asbestos inhalation

Asbestos exposure induces an inflammatory reaction with a large component of mononuclear phagocytes (Antony et al., 1993; Branchaud et al., 1993; Carbone et al., 2012; Choe et al., 1997). Upon differentiation into macrophages, these cells phagocytize asbestos fibers and, in response, release numerous cytokines and reactive oxygen species with mutagenic properties (Robledo & Mossman, 1999). Thus, many of the pathological consequences occurring in the lung following exposure to asbestos fibers are believed to arise from an inflammatory cascade involving both autocrine and paracrine events (Hillegass et al., 2010). Persistent pulmonary inflammation is observed in animal models of asbestosis that can

Experimental models, as well as *in vitro* studies, have shown that mesothelial cells are particularly susceptible to the cytotoxic effects of asbestos (Baldys et al., 2007; BéruBé et al., 1996; Broaddus et al., 1996). Asbestos does not induce transformation of primary human mesothelial cells *in vitro*, instead, it is very cytotoxic to this cell type, causing extensive cell death. This finding raised an apparent paradoxical issue of how asbestos causes MM if human mesothelial cells exposed to this mineral die (Liu et al., 2000). This apparent paradox is reconciled by the current hypothesis that the chronic inflammation induced by asbestos

seems to play a critical role in mesothelial cell transformation.

be correlated with fibroproliferative responses (Mossman & Churg, 1998).

Tumor Necrosis Factor-α (TNF-α) is probably the most studied candidate for initiating inflammatory and fibrotic events linked to lung diseases such as asbestosis. Asbestos fibers cause the accumulation of macrophages in the pleura and lung. When these macrophages encounter asbestos, they release TNF-α. At the same time, asbestos induces the secretion of TNF-α and the expression of TNF-α receptor I (TNF-RI) in mesothelial cells (Yang et al., 2006). Remarkably, treatment of mesothelial cells with TNF-α significantly reduced asbestos cytotoxicity. Indeed, TNF-α activates NF-κB, which in turn promotes mesothelial cell survival and resistance to the cytotoxic effects of asbestos. Thus, TNF-α signaling through NF-κBdependent mechanisms increases the percentage of mesothelial cells that survive asbestos exposure, thereby increasing the pool of asbestos-damaged cells susceptible to malignant transformation (Haegens et al., 2007; Janssen-Heininger et al., 1999; Yang et al., 2006).

It has been reported that rats receiving a single intratracheal instillation of fibrogenic chrysotile asbestos developed lung chronic inflammatory reactions characterized by the accumulation of alveolar macrophages producing elevated levels of both Interleukin (IL)-1 and IL-6 (Lemaire & Ouellet, 1996). An increased production and/or release of these cytokines triggers inflammatory cell recruitment, thus amplifying and sustaining local inflammation. It has also been demonstrated that crocidolite asbestos and TNF-α can stimulate a dose-dependent increase in IL-6 expression and secretion from cultured, transformed and normal, human alveolar type II epithelial cells that is dependent upon intracellular redox potential (Simeonova et al., 1997). Interestingly, although MM cells appear to express low levels of IL-6 receptor (IL-6R), IL-6 can act as a growth factor for these cells through a trans-signaling mechanism involving the interaction of macromolecular complexes of IL-6 and soluble IL-6R (sIL-6R) with the transmembrane glycoprotein gp130 expressed on the surface of MM cells (Adachi et al., 2006; Rose-John et al., 2007). High levels of both IL-6 and sIL-6R are typical of several chronic inflammatory conditions (Rose-John et al., 2007).

Thus, inflammatory cytokines such as TNF-α and IL-6 appear to play a dual role in MM pathogenesis: they induce and sustain pleural inflammation and at the same time can act as survival or mitogenic factors for normal and transformed mesothelial cells, respectively.

### **2.2. Reactive Oxygen and Nitrogen Species (ROS/RNS)**

The mechanisms of injury and disease development caused by asbestos fibers are presumed to be related to their greater fibrogenic and carcinogenic properties in comparison to other minerals. Asbestos–induced mutagenicity is mediated through both direct and indirect pathways. Asbestos fibers may induce mutagenicity and genotoxicity directly through physical interaction with the mitotic machinery after being phagocytized by the target cells, or indirectly as a result of DNA and chromosome damage caused by asbestos-induced reactive oxygen (ROS) and nitrogen species (RNS) (Kamp & Weitzman, 1999; Shukla et al., 2003a, 2003b). ROS and RNS can be generated primarily by asbestos fibers or secondarily through fiber-induced inflammation (Aust et al., 2011; Gulumian, 2005; Hoidal, 2001). Free radicals generated from asbestos fibers plus the direct damage induced by the fibers are linked to cell signaling, inflammation, and a plethora of other responses (mutagenesis, proliferation, etc.) associated with the pathogenesis of asbestos-associated diseases (Heinz et al., 2010; Manning et al., 2002; Shukla et al., 2003a, 2003b).

Role of Inflammation and Angiogenic Growth Factors in Malignant Mesothelioma 53

Finally, in mesothelial and lung epithelial cells asbestos fibers, as opposed to nonpathogenic minerals, cause a persistent induction of the redox-sensitive transcription factors NF-κB and Activator Protein-1 (AP-1), which is accompanied by chronic alterations in gene expression (Heintz et al., 1993; Janssen et al., 1995). As mentioned above, the aberrant activation of the NF-κB pathway is regarded as a critical event for mesothelial cell transformation (Toyooka

NF-κB proteins are dimeric transcription factors composed of five different subunits, namely p65 (RelA), RelB, c-Rel, NF-κB1 p50 and NF-κB2 p52, which regulate gene expression events that impact on cell survival and differentiation. Moreover, since activation of NF-κB is critical in up-regulating the expression of many genes linked to proliferation, apoptosis resistance, and chemokine/cytokine production, this is undoubtedly a critical transcription factor in inflammatory responses occurring in target cells of asbestos-related

In unstimulated cells, the NF-κB transcription dimers are retained in the cytoplasm in an inactive state through the interaction with a family of inhibitors called IκBs (Inhibitors of κB) or with the p50 and p52 precursor proteins, p105 and p100, respectively (Hayden & Ghosh, 2008; Scheidereit, 2006). Indeed, p50 and p52 are translated as precursors proteins

Two different NF-κB-activation pathways exist: the classical and the alternative NF-κB pathway. The classical NF-κB pathway is initiated by signals elicited by diverse receptors, including TNF receptors type 1/2, Toll/IL-1 receptor, T-cell and B-cell receptors and EGF receptor, and also by cellular stresses and DNA damage (Hayden & Ghosh 2004; Le Page et al., 2005). These signals induce the activation of the IκB kinase (IKK) complex, which is composed by the catalytic subunits IKKα and IKKβ and by the regulatory subunit IKKγ/NEMO (Hayden & Ghosh, 2008; Scheidereit, 2006; Sun, 2011). The activated IKK complex phosphorylates IκB proteins, thereby triggering their proteasomal degradation. As a consequence, NF-κB dimers are released and can translocate into the nucleus. This pathway mainly leads to the activation of p50:RelA dimers (Sun, 2011). Conversely, the alternative NF-κB pathway predominantly targets activation of RelB:p52 complexes. This pathway relies on the inducible processing of p100 triggered by signaling from TNF receptor family members via the NF-κB-inducing kinase (NIK): NIK activates IKKα, which, in turn, phosphorylates p100 and triggers its processing to p52. This event results in the conversion of p100-inhibited NF-κB complexes into p52-containing NF-κB dimers, capable of translocating into the nucleus (Hayden & Ghosh, 2008; Scheidereit, 2006; Sun, 2011).

NF-κB-regulated genes have distinct requirements for NF-κB dimers. For instance, the NFκB binding site of the IL-2 gene has been reported to bind preferentially c-Rel homodimers

et al., 2008).

*2.3.1. NF-κB* 

**2.3. Transcription factors** 

diseases (Janssen et al., 1995, 1997).

containing an IκB-like C-terminal portion (Sun, 2011).

Several evidences indicate that a main factor in determining the surface and biological reactivity of different types of asbestos fibers is their ability to participate in redox reactions that generate free radicals (Kamp & Weitzman, 1999; Shukla et al., 2003a). Although the nature of the free radical-generating surface sites on asbestos fibers is not yet clear, asbestos fibers have an intrinsic redox activity and contain ferrous iron, which catalyzes reactions generating active oxygen intermediates on the fiber surface. Within the tissues several asbestos fiber types can produce reactive oxygen free radicals from hydrogen peroxide, a common product of intermediary tissue metabolism. Epidemiological studies have identified crocidolite as one of the most potent forms of asbestos associated with the induction of MM (Heintz et al., 2010). Crocidolite has a greater surface-area and a higher ferrous iron content compared to other fiber types such as chrysotile, and it is more biologically active in the generation of free radicals (Toyokuni, 2009). However, the ability of asbestos fibers to elicit these effects is not related to total iron content, suggesting the presence of specific iron active sites at the fibers' surface (Shukla et al., 2003a).

Cells exposed to asbestos have also been reported to produce a higher amount of nitric oxide (NO). In this regard, it has been reported that in human mesothelial cells crocidolite increases the expression of the inducible NO synthase (NOS) isoform (iNOS), the activity of the constitutive endothelial NOS (eNOS), and the synthesis of NO via NF-κB and Akt activation (Riganti et al., 2007). Thus, the asbestos-induced upregulation of iNOS or NO in the lungs, as well as the induction of inflammation by fibers, may contribute along with ROS, to the pathogenesis of lung and pleural injury (Hussain et al., 2003; Tanaka et al., 1998). Indeed, ROS and RNS can cause breakage of DNA, lipid peroxidation, release of inflammatory cytokines such as TNF-α, and the modification of cellular proteins including phosphatases involved in cell signaling cascades (Gossart et al., 1996; Hussain et al., 2003), so that their increased synthesis by various cell types may have multiple roles in cellular events critical to the establishment of lung and pleural inflammation and uncontrolled cell proliferation.

Finally, in mesothelial and lung epithelial cells asbestos fibers, as opposed to nonpathogenic minerals, cause a persistent induction of the redox-sensitive transcription factors NF-κB and Activator Protein-1 (AP-1), which is accompanied by chronic alterations in gene expression (Heintz et al., 1993; Janssen et al., 1995). As mentioned above, the aberrant activation of the NF-κB pathway is regarded as a critical event for mesothelial cell transformation (Toyooka et al., 2008).

#### **2.3. Transcription factors**

#### *2.3.1. NF-κB*

52 Malignant Mesothelioma

proliferation.

**2.2. Reactive Oxygen and Nitrogen Species (ROS/RNS)** 

al., 2010; Manning et al., 2002; Shukla et al., 2003a, 2003b).

The mechanisms of injury and disease development caused by asbestos fibers are presumed to be related to their greater fibrogenic and carcinogenic properties in comparison to other minerals. Asbestos–induced mutagenicity is mediated through both direct and indirect pathways. Asbestos fibers may induce mutagenicity and genotoxicity directly through physical interaction with the mitotic machinery after being phagocytized by the target cells, or indirectly as a result of DNA and chromosome damage caused by asbestos-induced reactive oxygen (ROS) and nitrogen species (RNS) (Kamp & Weitzman, 1999; Shukla et al., 2003a, 2003b). ROS and RNS can be generated primarily by asbestos fibers or secondarily through fiber-induced inflammation (Aust et al., 2011; Gulumian, 2005; Hoidal, 2001). Free radicals generated from asbestos fibers plus the direct damage induced by the fibers are linked to cell signaling, inflammation, and a plethora of other responses (mutagenesis, proliferation, etc.) associated with the pathogenesis of asbestos-associated diseases (Heinz et

Several evidences indicate that a main factor in determining the surface and biological reactivity of different types of asbestos fibers is their ability to participate in redox reactions that generate free radicals (Kamp & Weitzman, 1999; Shukla et al., 2003a). Although the nature of the free radical-generating surface sites on asbestos fibers is not yet clear, asbestos fibers have an intrinsic redox activity and contain ferrous iron, which catalyzes reactions generating active oxygen intermediates on the fiber surface. Within the tissues several asbestos fiber types can produce reactive oxygen free radicals from hydrogen peroxide, a common product of intermediary tissue metabolism. Epidemiological studies have identified crocidolite as one of the most potent forms of asbestos associated with the induction of MM (Heintz et al., 2010). Crocidolite has a greater surface-area and a higher ferrous iron content compared to other fiber types such as chrysotile, and it is more biologically active in the generation of free radicals (Toyokuni, 2009). However, the ability of asbestos fibers to elicit these effects is not related to total iron content, suggesting the

presence of specific iron active sites at the fibers' surface (Shukla et al., 2003a).

Cells exposed to asbestos have also been reported to produce a higher amount of nitric oxide (NO). In this regard, it has been reported that in human mesothelial cells crocidolite increases the expression of the inducible NO synthase (NOS) isoform (iNOS), the activity of the constitutive endothelial NOS (eNOS), and the synthesis of NO via NF-κB and Akt activation (Riganti et al., 2007). Thus, the asbestos-induced upregulation of iNOS or NO in the lungs, as well as the induction of inflammation by fibers, may contribute along with ROS, to the pathogenesis of lung and pleural injury (Hussain et al., 2003; Tanaka et al., 1998). Indeed, ROS and RNS can cause breakage of DNA, lipid peroxidation, release of inflammatory cytokines such as TNF-α, and the modification of cellular proteins including phosphatases involved in cell signaling cascades (Gossart et al., 1996; Hussain et al., 2003), so that their increased synthesis by various cell types may have multiple roles in cellular events critical to the establishment of lung and pleural inflammation and uncontrolled cell NF-κB proteins are dimeric transcription factors composed of five different subunits, namely p65 (RelA), RelB, c-Rel, NF-κB1 p50 and NF-κB2 p52, which regulate gene expression events that impact on cell survival and differentiation. Moreover, since activation of NF-κB is critical in up-regulating the expression of many genes linked to proliferation, apoptosis resistance, and chemokine/cytokine production, this is undoubtedly a critical transcription factor in inflammatory responses occurring in target cells of asbestos-related diseases (Janssen et al., 1995, 1997).

In unstimulated cells, the NF-κB transcription dimers are retained in the cytoplasm in an inactive state through the interaction with a family of inhibitors called IκBs (Inhibitors of κB) or with the p50 and p52 precursor proteins, p105 and p100, respectively (Hayden & Ghosh, 2008; Scheidereit, 2006). Indeed, p50 and p52 are translated as precursors proteins containing an IκB-like C-terminal portion (Sun, 2011).

Two different NF-κB-activation pathways exist: the classical and the alternative NF-κB pathway. The classical NF-κB pathway is initiated by signals elicited by diverse receptors, including TNF receptors type 1/2, Toll/IL-1 receptor, T-cell and B-cell receptors and EGF receptor, and also by cellular stresses and DNA damage (Hayden & Ghosh 2004; Le Page et al., 2005). These signals induce the activation of the IκB kinase (IKK) complex, which is composed by the catalytic subunits IKKα and IKKβ and by the regulatory subunit IKKγ/NEMO (Hayden & Ghosh, 2008; Scheidereit, 2006; Sun, 2011). The activated IKK complex phosphorylates IκB proteins, thereby triggering their proteasomal degradation. As a consequence, NF-κB dimers are released and can translocate into the nucleus. This pathway mainly leads to the activation of p50:RelA dimers (Sun, 2011). Conversely, the alternative NF-κB pathway predominantly targets activation of RelB:p52 complexes. This pathway relies on the inducible processing of p100 triggered by signaling from TNF receptor family members via the NF-κB-inducing kinase (NIK): NIK activates IKKα, which, in turn, phosphorylates p100 and triggers its processing to p52. This event results in the conversion of p100-inhibited NF-κB complexes into p52-containing NF-κB dimers, capable of translocating into the nucleus (Hayden & Ghosh, 2008; Scheidereit, 2006; Sun, 2011).

NF-κB-regulated genes have distinct requirements for NF-κB dimers. For instance, the NFκB binding site of the IL-2 gene has been reported to bind preferentially c-Rel homodimers

and p50:c-Rel, while that of the gene encoding IL-8 has been found to selectively bind Rel A (Hoffman et al., 2003, 2006). On the other hand, several genes are redundantly induced by more than one dimer (Hoffman et al., 2003, 2006; Saccani et al., 2003).

Role of Inflammation and Angiogenic Growth Factors in Malignant Mesothelioma 55

ERK, JNK and p38 MAPK (Chang & Karin, 2001). Indeed, the MAPK signal transduction pathway uses AP-1 as a converging point not only to regulate the expression of various

Several genes, which play very important roles in injury, repair, and differentiation, contain binding site(s) for AP-1 in their promoter and/or enhancer regions (Chang & Karin, 2001). These genes include extracellular matrix metalloproteinases (MMPs), antioxidant enzymes, growth factors and their receptors, differentiation markers, cytokines, chemokines and other

The Nuclear Factor of Activated T cells (NFAT) family of transcription factors consists of five proteins that are evolutionarily related to the Rel/NF-κB family. NFAT can be present in both the cytoplasm and the nucleus. In the cytoplasm NFAT is in a highly phosphorylated, inactive state. Cell stimuli leading to the elevation of intracellular Ca2+ levels induce the activation of the phosphatase PP2B/Calcineurin which dephosphorylates NFAT. This results in its nuclear relocalization and transcriptional activation. Interestingly, NFAT family members can act synergistically with AP-1 on composite DNA elements which contain adjacent NFAT and AP-1 binding sites (Macián et al., 2001). A functional cooperation has

Initially, NFAT was identified in lymphocytes and was reported to be expressed in activated but not resting T cells (Macián et al., 2005; Shaw et al., 1988). NFAT regulates not only T cell activation and differentiation but also the function of other immune cells, including dendritic cells (DCs), B cells and megakaryocytes. In addition, NFAT has crucial roles in

Dysregulation of NFAT signalling is now known to be associated with malignant transformation and the development of cancer (Mancini & Toker, 2009; Müller & Rao, 2010). The observation that NFAT can be activated by asbestos-induced oxidative stress suggests that this transcription factor may play multiple roles in asbestos-induced inflammation and carcinogenesis (Li et al., 2002). Indeed, NFAT mediates the expression of several inflammatory cytokines, including TNF-α, and is involved in cell transformation, proliferation, invasive migration, tumor cell survival and tumor angiogenesis (Mancini &

Angiogenesis is a common feature of solid tumors. Indeed, the development of a clinically observable tumor requires the neoformation of a vascular network sufficient to sustain tumor growth (Ribatti et al., 2007). Tumor angiogenesis is stimulated by the secretion of angiogenic molecules which induce endothelial cells from nearby vessels to switch from a quiescent to an activated state. Further, upon the stimulation of angiogenic growth factors, activated endothelial cells disrupt the extracellular matrix, proliferate and migrate (Ribatti et

genes but also to autoregulate AP-1 gene transcription (Reuter et al., 2010).

also been reported to occur between NFAT and NF-κB (Jash et al., 2012).

**3. Multifaceted role of angiogenic growth factors in MM** 

transcription factors (Shaulian & Karin, 2001).

*2.3.3. Nuclear Factor of Activated T Cells (NFAT)* 

numerous developmental programs in vertebrates.

Toker, 2009).

A number of studies have shown that nuclear retention and DNA binding of NF-κB protein complexes are increased following exposure of various cell types to a variety of extracellular stimuli that include oxidative stress (Bowie & O'Neill, 2000), hypoxia (Jung et al., 2003; Royds et al., 1998) and inflammatory cytokines (Mantovani et al., 2008). These observations are consistent with the hypothesis that persistent activation of NF-κB can contribute to the induction of multiple genes that are critical to the pathogenesis of asbestos-associated diseases, since oxidants, local hypoxia and inflammatory cytokines are all components involved in the effects induced by asbestos exposure.

It is noteworthy that among various carcinogenic and non-carcinogenic fibers studied for their effect on nuclear translocation of NF-κB, only carcinogenic fibers were found to cause a dose-dependent translocation of this transcription factor to the nucleus, and this effect was reported to be oxidative stress-dependent (Brown et al., 1999). In lung macrophages, the asbestos-induced expression and secretion of TNF-α are mediated by iron-catalyzed ROS products (Simeonova & Luster, 1995) through a process that involves NF-κB activation (Cheng et al., 1999). In rat alveolar type 2 cells, the crocidolite-induced activation of NF-κB as well as the expression of the macrophage inflammatory protein-2 (MIP-2) gene have also been shown to be dependent on mitochondrial-derived oxidative stress (Driscoll et al., 1998).

### *2.3.2. AP-1*

AP-1 is a homo- or heterodimeric transcription factor composed by proteins encoded by the *fos* and *jun* early response proto-oncogenes. This family of proteins includes c-Fos, FosB, FosL1 (Fra-1), FosL2 (Fra-2), c-Jun, JunB and JunD (Milde-Langosch, 2005). Whereas Jun members are capable of forming homodimers able to bind DNA and regulate transcription, all Fos members must form heterodimers with Jun family members to bind DNA.

AP-1 is a redox-sensitive transcription factor typically associated with cell proliferation and tumor promotion (Eferl & Wagner, 2003). The first evidence showing that asbestos exerts regulatory effects linked to aberrant transcriptional responses, cell proliferation and cell transformation derives from studies in which asbestos fibers caused induction of *c*-*fos* and *cjun* proto-oncogene mRNAs in pleural mesothelial cells and tracheo-bronchial epithelial cells in a dose–response fashion (Heintz et al., 1993).

The persistent induction of AP-1 by asbestos suggests a model of asbestos-induced carcinogenesis involving chronic stimulation of cell proliferation through activation of early response genes (Schonthaler et al., 2011). Of note, early response genes are a set of genes whose transcription is rapidly induced in response to growth factors. Furthermore, AP-1 activity is induced by growth factors, pro-inflammatory cytokines and genotoxic stress (Jochum et al., 2001; Shaulian & Karin, 2002). These stimuli activate mitogen-activated protein kinase (MAPK) cascades through the phosphorylation of distinct substrates such as ERK, JNK and p38 MAPK (Chang & Karin, 2001). Indeed, the MAPK signal transduction pathway uses AP-1 as a converging point not only to regulate the expression of various genes but also to autoregulate AP-1 gene transcription (Reuter et al., 2010).

Several genes, which play very important roles in injury, repair, and differentiation, contain binding site(s) for AP-1 in their promoter and/or enhancer regions (Chang & Karin, 2001). These genes include extracellular matrix metalloproteinases (MMPs), antioxidant enzymes, growth factors and their receptors, differentiation markers, cytokines, chemokines and other transcription factors (Shaulian & Karin, 2001).

#### *2.3.3. Nuclear Factor of Activated T Cells (NFAT)*

54 Malignant Mesothelioma

*2.3.2. AP-1* 

and p50:c-Rel, while that of the gene encoding IL-8 has been found to selectively bind Rel A (Hoffman et al., 2003, 2006). On the other hand, several genes are redundantly induced by

A number of studies have shown that nuclear retention and DNA binding of NF-κB protein complexes are increased following exposure of various cell types to a variety of extracellular stimuli that include oxidative stress (Bowie & O'Neill, 2000), hypoxia (Jung et al., 2003; Royds et al., 1998) and inflammatory cytokines (Mantovani et al., 2008). These observations are consistent with the hypothesis that persistent activation of NF-κB can contribute to the induction of multiple genes that are critical to the pathogenesis of asbestos-associated diseases, since oxidants, local hypoxia and inflammatory cytokines are all components

It is noteworthy that among various carcinogenic and non-carcinogenic fibers studied for their effect on nuclear translocation of NF-κB, only carcinogenic fibers were found to cause a dose-dependent translocation of this transcription factor to the nucleus, and this effect was reported to be oxidative stress-dependent (Brown et al., 1999). In lung macrophages, the asbestos-induced expression and secretion of TNF-α are mediated by iron-catalyzed ROS products (Simeonova & Luster, 1995) through a process that involves NF-κB activation (Cheng et al., 1999). In rat alveolar type 2 cells, the crocidolite-induced activation of NF-κB as well as the expression of the macrophage inflammatory protein-2 (MIP-2) gene have also been shown

AP-1 is a homo- or heterodimeric transcription factor composed by proteins encoded by the *fos* and *jun* early response proto-oncogenes. This family of proteins includes c-Fos, FosB, FosL1 (Fra-1), FosL2 (Fra-2), c-Jun, JunB and JunD (Milde-Langosch, 2005). Whereas Jun members are capable of forming homodimers able to bind DNA and regulate transcription,

AP-1 is a redox-sensitive transcription factor typically associated with cell proliferation and tumor promotion (Eferl & Wagner, 2003). The first evidence showing that asbestos exerts regulatory effects linked to aberrant transcriptional responses, cell proliferation and cell transformation derives from studies in which asbestos fibers caused induction of *c*-*fos* and *cjun* proto-oncogene mRNAs in pleural mesothelial cells and tracheo-bronchial epithelial

The persistent induction of AP-1 by asbestos suggests a model of asbestos-induced carcinogenesis involving chronic stimulation of cell proliferation through activation of early response genes (Schonthaler et al., 2011). Of note, early response genes are a set of genes whose transcription is rapidly induced in response to growth factors. Furthermore, AP-1 activity is induced by growth factors, pro-inflammatory cytokines and genotoxic stress (Jochum et al., 2001; Shaulian & Karin, 2002). These stimuli activate mitogen-activated protein kinase (MAPK) cascades through the phosphorylation of distinct substrates such as

to be dependent on mitochondrial-derived oxidative stress (Driscoll et al., 1998).

all Fos members must form heterodimers with Jun family members to bind DNA.

more than one dimer (Hoffman et al., 2003, 2006; Saccani et al., 2003).

involved in the effects induced by asbestos exposure.

cells in a dose–response fashion (Heintz et al., 1993).

The Nuclear Factor of Activated T cells (NFAT) family of transcription factors consists of five proteins that are evolutionarily related to the Rel/NF-κB family. NFAT can be present in both the cytoplasm and the nucleus. In the cytoplasm NFAT is in a highly phosphorylated, inactive state. Cell stimuli leading to the elevation of intracellular Ca2+ levels induce the activation of the phosphatase PP2B/Calcineurin which dephosphorylates NFAT. This results in its nuclear relocalization and transcriptional activation. Interestingly, NFAT family members can act synergistically with AP-1 on composite DNA elements which contain adjacent NFAT and AP-1 binding sites (Macián et al., 2001). A functional cooperation has also been reported to occur between NFAT and NF-κB (Jash et al., 2012).

Initially, NFAT was identified in lymphocytes and was reported to be expressed in activated but not resting T cells (Macián et al., 2005; Shaw et al., 1988). NFAT regulates not only T cell activation and differentiation but also the function of other immune cells, including dendritic cells (DCs), B cells and megakaryocytes. In addition, NFAT has crucial roles in numerous developmental programs in vertebrates.

Dysregulation of NFAT signalling is now known to be associated with malignant transformation and the development of cancer (Mancini & Toker, 2009; Müller & Rao, 2010). The observation that NFAT can be activated by asbestos-induced oxidative stress suggests that this transcription factor may play multiple roles in asbestos-induced inflammation and carcinogenesis (Li et al., 2002). Indeed, NFAT mediates the expression of several inflammatory cytokines, including TNF-α, and is involved in cell transformation, proliferation, invasive migration, tumor cell survival and tumor angiogenesis (Mancini & Toker, 2009).

#### **3. Multifaceted role of angiogenic growth factors in MM**

Angiogenesis is a common feature of solid tumors. Indeed, the development of a clinically observable tumor requires the neoformation of a vascular network sufficient to sustain tumor growth (Ribatti et al., 2007). Tumor angiogenesis is stimulated by the secretion of angiogenic molecules which induce endothelial cells from nearby vessels to switch from a quiescent to an activated state. Further, upon the stimulation of angiogenic growth factors, activated endothelial cells disrupt the extracellular matrix, proliferate and migrate (Ribatti et al., 2007). Angiogenic growth factors include, among the others, Vascular Endothelial Growth Factor (VEGF), Placenta Growth Factor (PlGF), Platelet-Derived Growth Factor (PDGF) and acidic and basic Fibroblast Growth Factors (FGF-1 and -2, respectively). VEGF is regarded as the most important player in angiogenesis (Ono, 2008).

Role of Inflammation and Angiogenic Growth Factors in Malignant Mesothelioma 57

expression of the antiapoptotic factors bcl-2 (Harmey & Bouchier-Hayes, 2002) and survivin (Kanwar et al., 2011). In this context, MM cells have been shown to express high amounts of VEGF, VEGF receptors and co-receptors both *in vitro* and *in vivo*, and VEGF has been demonstrated to act as an autocrine growth factor for this tumor cell type (Albonici et al.,

VEGF-R1 participates in cell migration; it has an important role in monocyte chemotaxis and promotes recruitment of circulating endothelial precursor cells from bone marrow (Hattori et al., 2002). Its expression is increased in various tumors, correlates with disease progression and can predict poor prognosis, metastasis and recurrent disease in humans (Dawson et al., 2009; Fischer et al., 2008; Kerber et al., 2008). This receptor is also expressed by MM cells *in vitro* and *in vivo*, where it appears to mediate proliferative and cell survival

VEGF-R2 is the main mediator of VEGF-stimulated endothelial cell migration, proliferation, survival and enhanced vascular permeability (Olsson et al., 2006; Shibuya, 2006). VEGF-R2 expression is induced in conjunction with active angiogenesis, such as during the reparative process, and in pathological conditions associated with neovascularization, such as cancer (Plate et al., 1993). VEGF-R2 is overexpressed in MM cells and specimens, and VEGF-R2 silencing by small intefering RNA has been shown to induce cell death in MM or immortalized mesotelial cells *in vitro* (Albonici et al., 2009; Catalano et al., 2009; Pompeo et al., 2009; Strizzi et al., 2001a). Interestingly, it has been reported that in MM cells this receptor can be activated also via the semaphorin-6D receptor Plexin-A1, triggering a prosurvival program that promotes anchorage-independent growth through a NF-κBdependent pathway (Catalano et al., 2009). Remarkably, the expression of plexin-A1 is induced by asbestos fibers and overexpression of plexin-A1 in non-malignant mesothelial cells inhibits cell death after asbestos exposure, thus suggesting a role for this receptor not only in MM promotion and progression but also in asbestos-induced mesothelial

*In vitro* studies have shown that transfection of normal mesothelial cells with SV40 Tag potently increases VEGF protein and mRNA levels (Cacciotti et al., 2002) as well as mesothelial cell proliferation (Catalano et al., 2002). These data indicate that VEGF regulation by SV40 transforming proteins can also represent a key event in MM onset and

PlGF, originally identified in the placenta during the early embryonic development (Khaliq et al., 1996; Maglione et al., 1991), is expressed in several other organs including the heart, lung, thyroid, skeletal muscle and adipose tissue (Persico et al., 1999) but not normal

Although the role exerted by PlGF in tumor growth is controversial yet, PlGF can stimulate vessel growth and maturation directly by affecting endothelial and mural cells, as well as indirectly by recruiting pro-angiogenic cell types (Barillari et al., 1998; Carmeliet, 2003). It

2009; Ohta et al., 1999; Pompeo et al., 2009; Strizzi et al., 2001a).

responses (Albonici et al., 2009; Strizzi et al., 2001a).

carcinogenesis (Catalano et al., 2009).

mesothelium (Albonici et al., 2009).

progression.

*3.1.2. PlGF* 

The link between angiogenesis and tumor progression is provided by the negative prognostic value of intratumoral microvascular density (IMD) (Folkman, 2006; Kerbel, 2008). In MM the IMD has an independent prognostic value (Kumar-Singh et al., 1997). MM demonstrates a higher IMD than colon and breast tumors and, consistently, presents with minimal central necrosis despite its huge size (Gasparini & Harris, 1995; Kumar-Singh et al., 1997).

On the other hand, the involvement of angiogenic growth factors in MM goes beyond the stimulation of angiogenesis. Indeed, as discussed below, MM cells express receptors for several angiogenic factors which, accordingly, can directly modulate MM cell behavior.
