In normal liver, the JNK family is minimally or transiently activated, the latter usually physiological, whereas sustained activation is pathological. In addition to their central role in hepatic physiological responses (such as cell death, apoptosis, and cell cycle regulation) [27,28], JNK plays a carcinogenic role by promoting inflammation, proliferation, invasion, and angiogenesis, depending on the specific context and duration of the activation of the JNK signaling pathway [29,30]. Earlier studies have reported that the JNK signaling pathway plays a significant role in the development of HCC [31,32]. In particular, Jnk1 seems to be more important in malignant transformation and hepatocellular carcinoma (HCC) development [33,34]. However, during the progression of liver disease, the deletion of Jnk1 significantly exacerbated apoptosis, compensatory proliferation, and carcinogenesis in experimental chronic liver disease. In turn, Jnk2 was reported to modulate necrosis and inflammation [35]. Using Puma KO mice in diethylnitrosamine (DEN)-induced HCC, HCC formation was suppressed, a phenomenon associated with reduced cell death and compensatory proliferation of hepatocytes. However, such effects were inhibited by the administration of the JNK inhibitor SP600125 [36]. In human HCC cell lines, Jnk1 activation is associated with tumor size. By knocking down Jnk1 but not Jnk2, proliferation of human HCC occurs via upregulation of cmyc and downregulation of p21 [37].

The JNK signaling pathway has been recently implicated in CCA development. JNK plays an important role in regulating the interaction between different pro-apoptotic and anti-apoptotic proteins in response to both external and internal apoptotic stimuli [38]. In human CCA, a high expression of TNFA in cells near CCA lesions was found and phosphorylation of JNK in cholangiocytes (80% in CCA patients) as well as the accumulation of reactive oxygen species (ROS) around the peripheral hepatocytes occurred. Accumulation of ROS leads to caspase-3-dependent apoptosis via JNK. At the same time, Yuan et al. [39], showed that mitochondrial dysfunction and oxidative stress promoted cholangiocellular overgrowth and tumorigenesis. In this study, cholangiocellular proliferation and oncogenic transformation were promoted through the JNK signaling pathway, which was activated by Kupffer cells (KC), subsequently leading to liver damage, ROS, and the paracrine release of TNF. Therefore, KC-derived TNF might promote cholangiocellular cell proliferation, differentiation, and carcinogenesis, suggesting that the ROS/TNF/JNK axis plays a significant role in the development of CCA. Another study that tested the antioxidant activity of using guggulsterone, a steroid found in the resin of the guggul plant (Commiphora wightii) in both HuCC-T1 and RBE CCA cell lines, showed that guggulsterone could induce the apoptosis of human CCA cells via ROS-mediated activation of the JNK signaling pathway [40]. Feng et al. [41] reported that JNK exerted its carcinogenic effect in human CCA cells, partially because the mammalian target of the rapamycin (mTOR) pathway was regulated by the induction of glucose-regulated protein 78 (GRP78). They showed that eukaryotic initiation factor-α (eIF2a)/activated transcription factor 4 (ATF4) signaling promoted the accumulation of GRP78; whilst JNK was both promoted by the activation of mTOR and characterized by a high expression of GRP78. These results indicated that GRP78 contributed to the pro-tumorigenic function of JNK in human CCA cells, partly through promoting the eIF2α/ATF4/GRP78 pathway, and through JNK/mTOR signaling. As above-mentioned, the JNK/MAPKs signaling pathway may be a key target in the treatment of CCA; however, more studies are needed to test this possibility.

The p38 kinase plays a key role by promoting proliferation, invasion, inflammation, and angiogenesis in the occurrence and development of CCA [42,43,44,45], also affecting the growth of malignant human CCA cells, and maintaining the phenotype of transformed cells [45,46]. p38δ can be used for differential diagnosis of CCA, since it is not expressed in HCC cells [47]. IL-6 receptors and tyrosine kinases receptors such as Met (c-MET) and the EGFR family members ERBB2 and ERBB1 (EGFR) are key signaling pathways in CCA. Members of the EGFR family, particularly EGFR and ERBB2 (HER-2/neu), are involved in the pathogenesis of CCA [15,48,49]. Dai et al. [50] reported that both p38 and C-MET, the tyrosine kinase receptor for hepatocyte growth factor (HGF), promote the proliferation and invasion of human CCA cells, and p38 promotes CCA formation via sustained activation of C-MET. In addition, using ATO (arsenic trioxide) alone or in combination with metformin to treat CCA cell lines, Ling et al. [42] found that inactivation of p38 by the inhibitor SB203580 or specific siRNA could enhance the anticancer efficacy of single drug or combination of metformin and ATO, especially using ATO alone. Furthermore, the expression of P38δ was upregulated in CCA when compared to normal biliary tissue. Inhibition of P38δ expression by siRNA transfection significantly reduced CCA cell proliferation and invasion. Conversely, overexpression of P38δ in CCA cells can induce an increase in tumor invasiveness.

Previous studies have reported an upregulation of the RAS-ERK1/2 signaling pathway in CCA [51]. TGF-β transmits signals through SMAD2/3 phosphorylation, but can also transduce signals via RAS-ERK1/2, PI3K, p38, and Rho pathways [52,53,54]. Rac1, a member of the Rho family of small GTPases, plays a pivotal role in the development of CCA [55,56]. Li et al. [57] demonstrated that integrin β6 promoted tumor cell malignant behavior by activating Rac1. In biliary dysplasia, hyperplasia and CCA, TGF-β is overexpressed, a mechanism associated with CCA initiation and formation [51,58,59]. Moreover, ERK1/2 activation has also been linked with TGF-β-induced epithelial to mesenchymal transition (EMT) and cell invasiveness. One study using CCA cell lines indicated that TGF-β enhanced cell invasiveness and mesenchymal features in CCA cell lines [58]. Recently, a study using CCA cell lines (KKU-M213 and HuCCA-1) showed that TGF-β activates ERK1/2 via the SMAD2/3 pathway, enhancing TGF-β activity to promote tumor growth [60]. As above-mentioned, TGF-β can also transduce signals via the RAS-ERK/PI3K pathways [52,53,54]. The PI3K/AKT/PTEN signaling pathway is significantly overexpressed in human CCA tissue [61], therefore it is another important pathway involved in the development of CCA associated with RAS/ERK/MAPKs. PI3K is one of the most important factors in RAS activation and can be involved in the regulation of various functions including cell growth, cell cycle entry, cell survival, cytoskeleton reorganization, and apoptosis [15,62,63] (Figure 1). Loss of phosphatase function of PTEN will result in the constitutive activation of (PI3K/protein kinase B) AKT signaling pathway in CCA. Inhibition of PI3K/AKT signaling effectively blocked the proliferation and invasive behavior of CCA [61]. At the same time, Hyunho et al. [64] using CCA cell lines (SCK and Choi-CK) with inactivation of AKT, showed decreased expression of BCL2, and enhanced expression of BAX, thereby inducing the apoptosis of resistant cells; whilst the inhibition of ERK1/2 activation did not induce apoptosis, but decreased tumor cell growth. These results indicated that the AKT/ERK1/2 signaling transduction pathway might mediate apoptosis in CCA cell lines. Furthermore, in CCA, the expression of the HGF receptor (HGFR) encoded by the MET gene, also known as cMET, is increased (12–58%). Activation of MET promotes cell invasion and triggers metastasis by directly participating in tumor angiogenesis [65]. Experiments with CCA cell lines indicate that HGFR-dependent CCA cell invasiveness occurs via the AKT/ERK signaling pathway [66,67,68]. In summary, abundant evidence suggests that the ERK signaling pathway is intimately related to cholangiocarcinogenesis. In the future, the ERK signaling pathway may be an effective target for the diagnosis and treatment of CCA; however, more research is needed to confirm this.

Kinases such as transforming growth factor-β-activated kinase 1 (TAK1) belong to the MAP3Ks family. TAK1 is activated in response to cytokines such as TNF, LPS, and TGF-β in multiple cellular systems [69]. Activation of the TGF-β receptor triggers downstream signaling mediated by SMAD family of proteins, which promotes EMT progression in CCA (discussed below). However, phosphorylated TAK1 activates IKK (IκB kinase) and MKK4/7, causing the activation of NF-κB and JNK. Deletion of TAK1 in liver parenchymal cells (both hepatocytes and cholangiocytes) promotes hepatocyte death, inflammation, fibrosis, and hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis [70,71,72]. ASK1 is another member of the MAPK family, known as MAP3K5 (mitogen-activated protein kinase kinase kinase 5), which can activate the P38 and JNK pathways in response to both oxidative and ER (endoplasmic reticulum) stress as well as stress-induced via inflammatory cytokines (such as TNFα) [73,74,75].

2. Biomarkers and Diagnosis