## 3 Molecular Pathways in Cholangiocarcinoma  
A large spectrum of malignancies, such as CCA, originates in the background of chronic inflammation. In fact, inflammatory pathways are not only crucial in carcinogenesis but also in tumor invasion and migration, for example, through the promotion of oxidative stress [50]. Oxysterols from biliary cholesterol are oxidation products involved in tumor development and progression through the activation of the Hedgehog signaling pathway, which promotes the desmoplastic response and it is strictly related to CCA cell proliferation and migration [51]. Furthermore, inflammatory cytokinesactivate inducible nitric oxide synthase (iNOS) contributing to nitrosative stress and generating an excess of nitric oxide, which leads to the inhibition of DNA repair proteins and oxidative DNA lesions [52]. Moreover, different proinflammatory cytokines induce cyclooxygenase-2 (COX-2), which is implicated in the carcinogenesis of CCA [53]. In particular, interleukin-6 (IL-6) is produced by cholangiocarcinomaytes upon inflammatory stimuli and secreted by CCA cells [54]. IL-6 upregulates the antiapoptotic protein Bcl-2 and the myeloid cell leukemia sequence (Mcl-1) by an AKT-dependent mechanism. It also acts via a signal transducer and activator of transcription- (STAT) dependent mechanism to increase the presence of Mcl-1 [55]. Similarly, inhibition of the JAK kinases, which activates STAT3 downstream of IL-6 signaling, has been studied in models of CCA to confirm the important role of neutralizing antibodies for the use of IL-6 in the management of CCA (Figure 2). It is important to underline the key role of PI3K-AKT-mTOR signaling, where AKT activation leads to phosphorylation of the mammalian target of rapamycin (mTOR) pathway and its deregulation promotes tumor development, cell survival, and angiogenesis [56]. For this reason, several PI3K pathway inhibitors are under evaluation in a large group of human cancer. Likewise, Notch dysregulation, upregulated in PSC and CCA, is implicated in inflammation and carcinogenesis [57]. The inhibition of Notch signaling can occur via several mechanisms, such as the block of Notch receptor activation with \(\gamma\)-secretase inhibitors, the block of the ligand with monoclonal antibodies or the block of the transcriptional activity of Notch intracellular domain (NICD) by inhibiting peptides [58]. Together with the cytokines and the inflammatory process, various growth factor tyrosine kinases are involved in the carcinogenesis of CCA, such as the ERBB family of receptor tyrosine kinases and the hepatocyte growth factor (HGF) receptor, c-met. The first one leads to the downstream activation of mitogen-activated protein kinase (MAPK); in fact, the upregulation of ERBB2 is related to biliary epithelium tumor in mice [59]. In CCA patients, erlotinib, an EGFR inhibitor, has had limited success in human clinical trials, while lapatinib, a dual inhibitor of EGFR and HER2 (another ERBB receptor) is significantly effective in the inhibition of CCA cell lines compared to trastuzumab, that selectively blocks HER2 [60]. The second abovementioned growth  
Figure 2: Schematic illustration of IL-6/STAT3 signaling. This pathway can be activated in malignant cholangiocarcinomaytes and it consists in the binding of IL-6 to its receptor. This process promotes JAK with subsequent phosphorylation, activation, dimerization, and nuclear translocation of STAT3 in the form of STAT3 dimer. STAT3 upregulates Mcl-1 transcription, which play an important role in cellular growth. factor, HGF, activates MET in CCA, thus promoting tumor invasion, and protection from apoptosis and angiogenesis in association with the activation of ERBB family members, such as ERBB2 [61]. MET amplification has been reported in different malignancies, such as gastric, ovarian, lung cancer, and always correlated with a poor clinical outcome [62]. FGFR is also a tyrosine kinase receptor involved in cell transformation and angiogenesis.