Cholangiopathy: Genetics, Mechanism, and PathologyView this Special Issue
Review Article | Open Access
G. Fava, I. Lorenzini, "Molecular Pathogenesis of Cholangiocarcinoma", International Journal of Hepatology, vol. 2012, Article ID 630543, 7 pages, 2012. https://doi.org/10.1155/2012/630543
Molecular Pathogenesis of Cholangiocarcinoma
Epidemiological data from the last years show an increasing trend of incidence and mortality of cholangiocarcinoma (CC) worldwide. Many pathophysiologic aspects of this neoplasia are still unknown and need to be fully discovered. However, several progresses were recently made in order to establish the molecular mechanisms involved in the transformation and growth of malignant cholangiocytes. The principal concept that at least seems to be established is that cholangiocarcinogenesis is a multistep cellular process evolving from a normal condition of the epithelial biliary cells through a chronic inflammation status ending with malignant transformation. The bad prognosis related to CC justifies why a better identification of the molecular mechanisms involved in the growth and progression of this cancer is required for the development of effective preventive measures and valid treatment regimens. This Paper describes the scientific progresses made in the last years in defining the molecular pathways implicated in the generation of this devastating disease.
Cholangiocarcinoma is originated by a malignant transformation of cholangiocytes, the epithelial cells lining the biliary ducts . Since biliary cancers may arise from every portion of the biliary tree, they are anatomically classified as intrahepatic or extrahepatic . Epidemiological data show that intrahepatic cholangiocarcinoma is increasing in incidence, prevalence, and mortality worldwide [2, 3]. In particular, in the past three decades, a progressive increase of mortality for intrahepatic CC has been reported, while mortality for extrahepatic CC is stable or slightly decreasing .
The poor prognosis of this cancer is also explained from the fact that no useful tools for early diagnosis for this neoplasia are still available. Because of, the lack of specific symptoms coupled with high invasiveness and frequent involvement of critical anatomical organs [1, 4], the patient typically presents with an unresectable disease at the diagnostic approach. This aspect justifies why a surgical curative treatment is often impossible. Besides surgery, the other types of treatments for CC are chemotherapy and radiotherapy . However, CC cells do not respond or weakly respond to these approaches, which thus have often a palliative role. Recent therapeutic options include brachytherapy and photodynamic therapy (PDT), with promising results [1, 4].
CC develops from the accumulation of genetic and epigenetic alterations in regulatory genes in cholangiocytes that lead to the activation of oncogenes and the dysregulation of tumor suppressor genes (TSGs) [5–8]. The principal characteristics of malignant cholangiocytes can be summarized in (i) uncontrolled growth, (ii) high capacity of tissue invasiveness, and (iii) capacity to metastasize [5, 8]. In this paper, we have described in detail the principal molecular mechanisms involved in every passage of the multistep process of cholangiocarcinogenesis.
2. Molecular Mechanisms of Cholangiocarcinogenesis
The molecular mechanisms involved in the development of CC are incompletely defined in detail, although in the last years, several studies have contributed to codify them, at least in part . With the term “cholangiocarcinogenesis” are named all the complex mechanisms that lead to the malignant transformation of cholangiocytes. These mechanisms can be simply described as a multistep process (Figure 1). CC usually develops in an environment of chronic inflammation of bile ducts with consequent cholangiocyte damage associated with the obstruction of bile flow [1, 5]. This tumor, especially when originating in the perihilar bile ducts, can develop in normal liver [5, 9].
Primary sclerosing cholangitis (PSC) is the most recognized risk factor for CC development . Other risk factors for this cancer are specific parasites of endemic regions of Asia such as Opisthorchis viverrini, Clonorchis sinensis, and Schistosoma Japonica and bacteria such as Salmonella typhi [9, 10]. Hepatolithiasis, Caroli’s disease, congenital choledochal cysts, bilioenteric surgical drainage and anomalous pancreaticobiliary junction, age greater than 65 years, bile duct adenoma, papillomatosis, liver cirrhosis, smoking, diabetes mellitus, thorotrast, dioxin and vinyl chloride intoxication, and HIV and HCV infections [9, 10] are also risk conditions for CC. However, the role of most of these conditions as predisposing factors for biliary cancer is still debated. Independently from the presence of one of the mentioned factors, the malignant transformation of cholangiocyte arises in a background of chronic inflammation. The high amount of cytokines and factors secreted during chronic inflammatory processes triggers and maintains the process of cholangiocarcinogenesis [5, 8] (Figure 1). Molecules participating in chronic inflammation promote neoplastic process by damaging protooncogenes, DNA mismatch repair genes/proteins, and tumor suppressor genes involved in cell growth, apoptosis, invasiveness, and neoangiogenesis. The final result is the uncontrolled cell proliferation and invasion (Figure 1).
Such as in many other cancer types, K-ras, p53, p14ARF, p16INK4a, and β-catenin genes can be mutated during the development of CC .
Two other genes recently described as implicated in the development of CC are NKG2D and AID. The natural killer group 2, member D cell receptor, also known as NKG2D, is expressed by NK cells and T-lymphocytes and plays a critical role in tumor surveillance by cell-mediated cytotoxicity . Melum et al. recently showed that two single nucleotide polymorphisms (SNPs) of the NKG2D gene were associated with an increased risk of CC in PSC-affected patients . Contrarily, homozygous condition for the no-risk alleles is related with a low risk of CCs .
Activation-induced cytidine deaminase (AID) is a member of the DNA/RNA-editing enzyme family. Recently, it was shown that AID production was significantly increased in human biopsies of PSC and CC-affected patients compared with normal liver parenchyma . Aberrant expression of AID in biliary cells resulted in the generation of somatic mutations in tumor-related genes such as p53, c-myc and the promoter region of the INK4A/p16 sequences . The aberrant expression of AID gene induced by proinflammatory cytokines strengthens the link between chronic inflammation of the biliary tract and CC development .
3. Molecular Pathways Implicated in Cholangiocarcinogenesis
IL-6 has an important role in the pathogenesis and growth of CC . The mitogenic effect of IL-6 is suggested from the fact that the concentration of this molecule is increased during chronic inflammation of the biliary tract, a condition predisposing CC development . IL-6 acts by both an autocrine and a paracrine manner stimulating several intracellular pathways involved in survival and growth of malignant cholangiocytes . Among them, p44/p42 and p38 MAPKs have been largely studied . Tadlok et al. showed that activation of p38 MAPK by IL-6 decreases expression of p21(WAF1/CIP1), a cell cycle controller protein, and mediates growth independent of anchorage signals, whereas activation of p44/p42 MAPK mediates an anchorage signal-dependent growth pathway . IL-6 also influences the apoptotic process of malignant cholangiocytes. Several studies showed that IL-6 upregulates myeloid cell leukemia-1 or Mcl-1, a potent key antiapoptotic Bcl-2 family member protein. It has been recently shown that this effect of IL-6 is mediated by increased activation of STAT-3 (that is constitutively activated in malignant cholangiocytes), which regulates Mcl-1 transcription thus increasing resistance to apoptosis . In addition, Mcl-1 increases cancer cell resistance to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL)  and, therefore, this molecule appears to have a fundamental role in CC development . Conversely, inhibition of IL-6-induced iperexpression of Mcl-1 restores sensitivity to TRAIL .
The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is one of the key signaling mechanisms in CC cells, mediating their resistance to apoptosis . In a recent study, Blechacz et al. showed that the multikinase inhibitor sorafenib may also block JAK/STAT signaling with consequent inhibition of CC growth . The authors demonstrated that sorafenib induces STAT3 dephosphorylation by stimulating phosphatase SHP2 activity, sensitizes CC cells to TRAIL-mediated apoptosis, and is therapeutic in a syngeneic rat, orthotopic CC model that mimics human disease .
TGFβ is a cytokine implicated in several cell functions such as growth, differentiation, migration, apoptosis, adhesion, survival, and immunity. Several cell types of the liver secrete this cytokine . Cholangiocytes, for example, express TGFβ in course of cholestasis but not in normal conditions . It has been shown that TGFβ inhibits proliferation of human CC cells through modulation of the p21 cyclin dependent kinase inhibitor . However, the mutations of TGFβ receptor and the alterations of intracellular signaling mediators (e.g., Smad4) together with the intracellular overexpression of cyclin D1 [25, 26] in CC cells induce a resistance to the inhibitory effect of TGFβ cells . In addition, the lack of TGFβ signaling also stimulates the deposition of fibrotic tissue, abundantly expressed by biliary malignancies .
DCP4/Smad4 is a tumor suppressor gene and also a downstream of TGFβ signaling . Recently, it has been shown that Smad4 interacts with PTEN, another tumor suppressor gene, in order to regulate cellular cycle and escape the process of cholangiocarcinogenesis . To strength this data, the blockage of these tumor suppressor genes favors the development of CC [29, 30]. It was also demonstrated that pTNM stage of intrahepatic CC appears to be correlated with the degree of Smad4 loss .
The protooncogene-encoded receptor tyrosine kinase ErbB-2 is overexpressed in malignant cholangiocytes and plays an important role in the development and progression of biliary malignancies [31–33]. ErbB-2 acts in two different manners: first of all stimulates the proliferation of CC cells, then ErbB-2 stimulates the production of COX-2, which interacts with a subunit of the IL-6 receptor forming a complex . This effect suggests a close link between IL-6 and ErbB-2 signaling [1, 34]. The mitogenic effect of ErbB-2 is also suggested from the fact that when normal cholangiocytes are transfected with the neu (the rat homologue of ErbB-2) oncogene, they undergo a malignant transformation that resembles the molecular aspects of human CC .
The enzyme cyclooxygenase (COX) is responsible of the generation of prostaglandins, expressed in the course of the process of inflammation. COX exists in two specific isoforms: COX-1, normally expressed in many cell types and regulating the homeostatic functions of prostaglandins, and COX-2, the inducible isoform, which can be stimulated by a variety of molecules, such as cytokines and lipopolysaccharides . The expression of COX-2 is major during inflammation, condition predisposing the development of CC . Moreover, in rat CC cells, overexpression of COX-2 stimulates cell growth  while antisense depletion of COX-2 inhibits cell proliferation . Recent studies showed that COX-2 is activated in human CC cells in vitro  by oxysterols, derivatives from cholesterol, which are present in bile of patients in course of cholestasis and inflammatory processes of the biliary tract . The mitogenic effect of COX-2 towards CC cells justifies why the inhibition of COX-2-mediated pathway could represent a strategy to prevent CC development and growth. At this regard, recent data demonstrated that selective COX-2 inhibitors (e.g., celecoxib) reduce proliferation of CC cells by stimulating apoptosis [31, 37, 40, 41]. These studies also showed that the inhibitory effect towards CC cell growth by celecoxib was accompanied by an inhibition of PDK1 and PTEN, with a consequent reduction of Akt phosphorylation . Moreover, celecoxib inhibits CC cells proliferation through activation of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1, with consequent cell cycle arrest at G1/S phase . Sirica et al. recently demonstrated a link between the increase of COX-2 expression and CC development since the amount of this cytokine is high in cholangiocytes obtained from livers affected by PSC , a well known risk factor for CC . These promising in vitro data, however, do not correspond to a good outcome in clinical practice since not all COX-2 inhibitors were shown to have a benefit in reducing CC cell growth  and also because the use of high doses of COX-2 inhibitors could cause serious side effects .
Inducible nitric oxide (NO) synthase (iNOS) is an enzyme highly expressed during inflammatory and malignant processes of the biliary tract . The activation of iNOS in course of inflammation determines an increase of intracellular NO, which triggers the process of cholangiocarcinogenesis by different ways: (1) inhibits DNA repair system thus allowing an accumulation of DNA damage and mutations [8, 45]; (2) stimulates COX-2 expression .
The carcinogenic effect of NO is at least in part due to Notch-1 signaling . The role played by NO and Notch-1 in the development of biliary malignancies, as well as pancreatic cancer, is suggested by the evidence that Notch-1 is hyperexpressed both in cholangiocytes of PSC-affects patients and in CC cells where it colocalizes with iNOS . A recent study by Ishimura et al. showed that iNOS is able to stimulate COX-2 expression through NO generation. In particular, iNOS enhances COX-2 expression through activation of p38 MAPK and JNK1/2 . The link between these two proteins explains their important role in the development and growth of CC .
The process of apoptosis is fundamental to maintain the homeostasis of the biliary epithelium because it permits to remove the cells deeply damaged and with no reversible genomic mutations [47, 48]. After this premise, it is clear that a defect of apoptotic process favorites the survival of mutated cholangiocytes, which could go through a series of other mutations ending with the malignant transformation of the cell [47, 48].
Bcl-2 is a superfamily of antiapoptotic proteins. Bcl-2, which represents the prototype of this family  and is expressed in CC cells in a high amount. In these malignant cells, which possess a higher apoptotic threshold with respect to normal cholangiocytes , Bcl-2 exerts its antiapoptotic activity by reducing caspase 3 activation by preventing cytochrome-c release from the mitochondria .
Several other factors are implicated in the dysregulation of cholangiocyte apoptosis . Among them, NO inhibits apoptosis of biliary epithelial cells. At this regard, the transfection of CC cells with nitric-oxide-synthase- (NOS-) cDNA induces a resistance to etoposide-induced apoptosis, an event that happens by caspase 9 nitrosylation .
Furthermore, Notch-1 and COX-2 reduce TRAIL-mediated apoptosis  and high levels of COX-2 inhibit Fas-induced apoptosis in CC cells . To strengthened these data, a recent study demonstrated that celecoxib, a selective COX-2 inhibitor, induces cell death by apoptosis by the inhibition of the PI3-kinase signaling [37, 41].
The cytokine tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) selectively stimulates apoptosis only in malignant cells without having any toxicity in normal tissues [51, 52]. CC cells resist TRAIL-induced apoptosis because they express high levels of myeloid cell leukemia protein-1 (Mcl-1) . Thus, when specific small-interfering mRNA or stable transfection with Mcl-1 small hairpin RNA block Mcl-1 expression, CC cells become sensitive to TRAIL-induced apoptosis [19, 53]. The expression of Mcl-1 is also stimulated by bile acids, abundant in the course of cholestasis. Among them, deoxycholic acid, for example, increases Mcl-1 expression by blocking protein degradation through activation of an EGFR/Raf-1 pathway . Indeed, Raf-1 inhibitors block the increase of Mcl-1, rendering the cells much more sensitive to Fas-induced apoptosis . All these data suggest that TRAIL could be a target for novel drugs for the management of biliary tumors .
Biliary tumors proliferate surrounded by a rich vascular network, which provides an adequate support of oxygen and metabolites to malignant cholangiocytes in order to enhance tumor development and growth [1, 57]. The proliferation of blood vessels is favored by high levels of vascular endothelial growth factor (VEGF) [57, 58]. This protein is stimulated by TGF-β and β-catenin  and is expressed by the surrounding mesenchymal cells and, even if in a lesser extent, by the malignant cells themselves. This evidence suggests the existence of an autocrine/paracrine mechanism for the VEGF production by malignant cholangiocytes .
It is well known the mitogenic effect of estrogens for CC cells. At this regard, 17-β estradiol stimulates human CC cells growth  while tamoxifen, an estrogen antagonist, decreases the proliferation of these cells in vitro and in vivo  by stimulating apoptosis through the Fas/APO-1 (CD95) signaling pathway via a calmodulin-dependent mechanism . Alvaro et al. demonstrated that human intrahepatic CC cells express receptors for both estrogens and insulin-like growth factor 1 (IGF-1), which cooperate in the modulation of enhancing cell growth and reducing apoptosis . Furthermore, HuH-28 human intrahepatic CC cell line expresses VEGF-A, VEGF-C, and related receptors, which are enhanced by stimulation with estrogens , and the stimulatory effect of 17beta-estradiol is blocked by estrogen receptor or insulin-like growth factor-1 receptor antagonists . These data demonstrate that estrogens stimulate the proliferation of human CC by inducing the expression and secretion of vascular endothelial growth factor . The result of these studies could be applied to the management of biliary malignancies in clinical practice. In fact, measuring IGF-1 levels in bile could help distinguish extrahepatic CC from pancreatic cancer or benign biliary stenosis and blocking estrogen, IGF-1, and VEGF receptors could be crucial to arrest CC cell proliferation .
3.11. Neuropeptides and Hormones
In the last years, many studies described as a multitude of hormones and neuropeptides are able to interact and regulate CC cells growth and invasion. Among them, gastrin, endothelin, serotonin, secretin, histamine, the α-2-adrenoreceptor UK14, 304, NPY, GABA, leptin, and opioid receptor modulators regulate the proliferation and apoptosis of CC cells [66–75]. All these novel data could contribute to clarify the complex mechanisms governing the process of cholangiocarcinogenesis.
The increasing worldwide incidence of CC together with the lack of its effective therapeutic tools explains the growing general interest for the study of this cancer type. The bad prognosis of people affected by CC is given by the fact that this cancer is often diagnosed when already at an advanced stage. Unfortunately, at this point, only palliative approaches are possible. With these premises, in the recent years, many researchers have focused their studies on the investigation of the molecular mechanisms involved in the development and growth of CC. Several works demonstrated that the conditions of cholestasis and chronic inflammation induce a local release of a network of mitogenic factors that induce genomic damages thus triggering the malignant transformation of cholangiocytes. The complete codification of molecular pathways involved in the pathogenesis of CC is mandatory to discover novel tools for an early diagnosis and an efficacious specific therapy.
- K. N. Lazaridis and G. J. Gores, “Cholangiocarcinoma,” Gastroenterology, vol. 128, no. 6, pp. 1655–1667, 2005.
- T. Patel, “Worldwide trends in mortality from biliary tract malignancies,” BMC Cancer, vol. 2, p. 10, 2002.
- D. Alvaro, E. Crocetti, S. Ferretti, M. C. Bragazzi, and R. Capocaccia, “Descriptive epidemiology of cholangiocarcinoma in Italy,” Digestive and Liver Disease, vol. 42, no. 7, pp. 490–495, 2010.
- S. A. Khan, H. C. Thomas, B. R. Davidson, and S. D. Taylor-Robinson, “Cholangiocarcinoma,” The Lancet, vol. 366, no. 9493, pp. 1303–1314, 2005.
- G. Fava, M. Marzioni, A. Benedetti et al., “Molecular pathology of biliary tract cancers,” Cancer Letters, vol. 250, no. 2, pp. 155–167, 2007.
- D. S. Sandhu, A. M. Shire, and L. R. Roberts, “Epigenetic DNA hypermethylation in cholangiocarcinoma: potential roles in pathogenesis, diagnosis and identification of treatment targets,” Liver International, vol. 28, no. 1, pp. 12–27, 2008.
- I. Tischoff, C. Wittekind, and A. Tannapfel, “Role of epigenetic alterations in cholangiocarcinoma,” Journal of Hepato-Biliary-Pancreatic Surgery, vol. 13, no. 4, pp. 274–279, 2006.
- K. Okuda, Y. Nakanuma, and M. Miyazaki, “Cholangiocarcinoma: recent progress. Part 2: molecular pathology and treatment,” Journal of Gastroenterology and Hepatology, vol. 17, no. 10, pp. 1056–1063, 2002.
- K. N. Lazaridis, “Cholangiocarcinoma: epidemiology, risk factors and molecular pathogenesis,” in Pathophysiologiy of the Intrahepatic Biliary Epithelium, S. De Morrow, S. Glaser, G. Alpini, M. Marzioni, and G. Fava, Eds., pp. 301–313, Transworld Research Network, Kerala, India, 2008.
- Y. H. Shaib, H. B. El-Serag, J. A. Davila, R. Morgan, and K. A. Mcglynn, “Risk factors of intrahepatic cholangiocarcinoma in the United States: a case-control study,” Gastroenterology, vol. 128, no. 3, pp. 620–626, 2005.
- J. D. Coudert and W. Held, “The role of the NKG2D receptor for tumor immunity,” Seminars in Cancer Biology, vol. 16, no. 5, pp. 333–343, 2006.
- E. Melum, T. H. Karlsen, E. Schrumpf et al., “Cholangiocarcinoma in primary sclerosing cholangitis is associated with NKG2D polymorphisms,” Hepatology, vol. 47, no. 1, pp. 90–96, 2008.
- J. Komori, H. Marusawa, T. Machimoto et al., “Activation-induced cytidine deaminase links bile duct inflammation to human cholangiocarcinoma,” Hepatology, vol. 47, no. 3, pp. 888–896, 2008.
- F. Meng, Y. Yamagiwa, Y. Ueno, and T. Patel, “Over-expression of interleukin-6 enhances cell survival and transformed cell growth in human malignant cholangiocytes,” Journal of Hepatology, vol. 44, no. 6, pp. 1055–1065, 2006.
- T. Akiyama, T. Hasegawa, T. Sejima et al., “Serum and bile interleukin 6 after percutaneous transhepatic cholangio-drainage,” Hepato-Gastroenterology, vol. 45, no. 21, pp. 665–671, 1998.
- K. Okada, Y. Shimizu, S. Nambu, K. Higuchi, and A. Watanabe, “Interleukin-6 functions as an autocrine growth factor in a cholangiocarcinoma cell line,” Journal of Gastroenterology and Hepatology, vol. 9, no. 5, pp. 462–467, 1994.
- L. Tadlock and T. Patel, “Involvement of p38 mitogen-activated protein kinase signaling in transformed growth of a cholangiocarcinoma cell line,” Hepatology, vol. 33, no. 1, pp. 43–51, 2001.
- H. Isomoto, S. Kobayashi, N. W. Werneburg et al., “Interleukin 6 upregulates myeloid cell leukemia-1 expression through a STAT3 pathway in cholangiocarcinoma cells,” Hepatology, vol. 42, no. 6, pp. 1329–1338, 2005.
- M. Taniai, A. Grambihler, H. Higuchi et al., “Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells,” Cancer Research, vol. 64, no. 10, pp. 3517–3524, 2004.
- H. Isomoto, J. L. Mott, S. Kobayashi et al., “Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing,” Gastroenterology, vol. 132, no. 1, pp. 384–396, 2007.
- S. Kobayashi, N. W. Werneburg, S. F. Bronk, S. H. Kaufmann, and G. J. Gores, “Interleukin-6 contributes to Mcl-1 up-regulation and TRAIL resistance via an Akt-signaling pathway in cholangiocarcinoma cells,” Gastroenterology, vol. 128, no. 7, pp. 2054–2065, 2005.
- B. R. A. Blechacz, R. L. Smoot, S. F. Bronk, N. W. Werneburg, A. E. Sirica, and G. J. Gores, “Sorafenib inhibits signal transducer and activator of transcription-3 signaling in cholangiocarcinoma cells by activating the phosphatase shatterproof 2,” Hepatology, vol. 50, no. 6, pp. 1861–1870, 2009.
- L. A. Saperstein, R. L. Jirtle, M. Farouk, H. J. Thompson, K. S. Chung, and W. C. Meyers, “Transforming growth factor-beta1 and mannose 6-phosphate/insulin-like growth factor-II receptor expression during intrahepatic bile duct hyperplasia and biliary fibrosis in the rat,” Hepatology, vol. 19, no. 2, pp. 412–417, 1994.
- M. Miyazaki, R. Ohashi, T. Tsuji, K. Mihara, E. Gohda, and M. Namba, “Transforming growth factor-beta 1 stimulates or inhibits cell growth via down- or up-regulation of p21/Waf1,” Biochemical and Biophysical Research Communications, vol. 246, no. 3, pp. 873–880, 1998.
- Y. Yamagiwa and T. Patel, “Cytokine regulation of cholangiocyte growth,” in The Pathophysiology of Biliary Epithelia, G. Alpini, D. Alvaro, M. Marzioni, G. LeSage, and N. F. LaRusso, Eds., pp. 227–234, Landes Bioscience, Georgetown, Tex, USA, 2004.
- Y. Zen, K. Harada, M. Sasaki et al., “Intrahepatic cholangiocarcinoma escapes from growth inhibitory effect of transforming growth factor-beta1 by overexpression of cyclin D1,” Laboratory Investigation, vol. 85, no. 4, pp. 572–581, 2005.
- S. Yazumi, K. Ko, N. Watanabe et al., “Disrupted transforming growth factor-beta signaling and deregulated growth in human biliary tract cancer cells,” International Journal of Cancer, vol. 86, no. 6, pp. 782–789, 2000.
- S. C. Chuang, K. T. Lee, K. B. Tsai et al., “Immunohistochemical study of DPC4 and p53 proteins in gallbladder and bile duct cancers,” World Journal of Surgery, vol. 28, no. 10, pp. 995–1000, 2004.
- X. Xu, S. Kobayashi, W. Qiao et al., “Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice,” Journal of Clinical Investigation, vol. 116, no. 7, pp. 1843–1852, 2006.
- Y. K. Kang, W. H. Kim, and J. J. Jang, “Expression of G1-S modulators (p53, p16, p27, cyclin D1, Rb) and Smad4/Dpc4 in intrahepatic cholangiocarcinoma,” Human Pathology, vol. 33, no. 9, pp. 877–883, 2002.
- A. E. Sirica, G. H. Lai, K. Endo, Z. Zhang, and B. I. Yoon, “Cyclooxygenase-2 and ERBB-2 in cholangiocarcinoma: potential therapeutic targets,” Seminars in Liver Disease, vol. 22, no. 3, pp. 303–313, 2002.
- K. Endo, B. I. Yoon, C. Pairojkul, A. J. Demetris, and A. E. Sirica, “ERBB-2 overexpression and cyclooxygenase-2 up-regulation in human cholangiocarcinoma and risk conditions,” Hepatology, vol. 36, no. 2, pp. 439–450, 2002.
- S. I. Aishima, K. I. Taguchi, K. Sugimachi, M. Shimada, K. Sugimachi, and M. Tsuneyoshi, “c-erbB-2 and c-Met expression relates to cholangiocarcinogenesis and progression of intrahepatic cholangiocarcinoma,” Histopathology, vol. 40, no. 3, pp. 269–278, 2002.
- Y. Qiu, L. Ravi, and H. J. Kung, “Requirement of ErbB2 for signalling by interleukin-6 in prostate carcinoma cells,” Nature, vol. 393, no. 6680, pp. 83–85, 1998.
- G. H. Lai, Z. Zhang, X. N. Shen et al., “erbB-2/neu transformed rat cholangiocytes recapitulate key cellular and molecular features of human bile duct cancer,” Gastroenterology, vol. 129, no. 6, pp. 2047–2057, 2005.
- J. R. Brown and R. N. DuBois, “COX-2: a molecular target for colorectal cancer prevention,” Journal of Clinical Oncology, vol. 23, no. 12, pp. 2840–2855, 2005.
- Z. Zhang, G. H. Lai, and A. E. Sirica, “Celecoxib-induced apoptosis in rat cholangiocarcinoma cells mediated by Akt inactivation and Bax translocation,” Hepatology, vol. 39, no. 4, pp. 1028–1037, 2004.
- J. H. Yoon, A. E. Canbay, N. W. Werneburg, S. P. Lee, and G. J. Gores, “Oxysterols induce cyclooxygenase-2 expression in cholangiocytes: implications for biliary tract carcinogenesis,” Hepatology, vol. 39, no. 3, pp. 732–738, 2004.
- W. G. Haigh and S. P. Lee, “Identification of oxysterols in human bile and pigment gallstones,” Gastroenterology, vol. 121, no. 1, pp. 118–123, 2001.
- T. Wu, J. Leng, C. Han, and A. J. Demetris, “The cyclooxygenase-2 inhibitor celecoxib blocks phosphorylation of Akt and induces apoptosis in human cholangiocarcinoma cells,” Molecular Cancer Therapeutics, vol. 3, no. 3, pp. 299–307, 2004.
- G. H. Lai, Z. Zhang, and A. E. Sirica, “Celecoxib acts in a cyclooxygenase-2-independent manner and in synergy with emodin to suppress rat cholangiocarcinoma growth in vitro through a mechanism involving enhanced Akt inactivation and increased activation of caspases-9 and -3,” Molecular Cancer Therapeutics, vol. 2, no. 3, pp. 265–271, 2003.
- J. R. Testa and A. Bellacosa, “AKT plays a central role in tumorigenesis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 20, pp. 10983–10985, 2001.
- C. Han, J. Leng, A. J. Demetris, and T. Wu, “Cyclooxygenase-2 promotes human cholangiocarcinoma growth: evidence for cyclooxygenase-2-independent mechanism in celecoxib-mediated induction of p21waf1/cip1 and p27kip1 and cell cycle arrest,” Cancer Research, vol. 64, no. 4, pp. 1369–1376, 2004.
- N. Ishimura, S. F. Bronk, and G. J. Gores, “Inducible nitric oxide synthase upregulates cyclooxygenase-2 in mouse cholangiocytes promoting cell growth,” AJP—Gastrointestinal and Liver Physiology, vol. 287, no. 1, pp. G88–G95, 2004.
- M. Jaiswal, N. F. LaRusso, and G. J. Gores, “Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis,” AJP—Gastrointestinal and Liver Physiology, vol. 281, no. 3, pp. G626–G634, 2001.
- N. Ishimura, S. F. Bronk, and G. J. Gores, “Inducible nitric oxide synthase up-regulates Notch-1 in mouse cholangiocytes: implications for carcinogenesis,” Gastroenterology, vol. 128, no. 5, pp. 1354–1368, 2005.
- G. Alpini, J. M. McGill, and N. F. LaRusso, “The pathobiology of biliary epithelia,” Hepatology, vol. 35, no. 5, pp. 1256–1268, 2002.
- N. J. Torok and G. J. Gores, “Apoptosis of biliary epithelial cells,” in The Pathophysiology of Biliary Epithelia, G. Alpini, D. Alvaro, M. Marzioni, G. LeSage, and N. F. LaRusso, Eds., pp. 219–226, Landes Bioscience, Georgetown, Tex, USA, 2004.
- D. M. Harnois, F. G. Que, A. Celli, N. F. LaRusso, and G. J. Gores, “Bcl-2 is overexpressed and alters the threshold for apoptosis in a cholangiocarcinoma cell line,” Hepatology, vol. 26, no. 4, pp. 884–890, 1997.
- N. J. Torok, H. Higuchi, S. Bronk, and G. J. Gores, “Nitric oxide inhibits apoptosis downstream of cytochrome c release by nitrosylating caspase 9,” Cancer Research, vol. 62, no. 6, pp. 1648–1653, 2002.
- H. Walczak, R. E. Miller, K. Ariail et al., “Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo,” Nature Medicine, vol. 5, no. 2, pp. 157–163, 1999.
- T. Yamanaka, K. Shiraki, K. Sugimoto et al., “Chemotherapeutic agents augment TRAIL-induced apoptosis in human hepatocellular carcinoma cell lines,” Hepatology, vol. 32, no. 3, pp. 482–490, 2000.
- J. L. Mott, S. Kobayashi, S. F. Bronk, and G. J. Gores, “mir-29 regulates Mcl-1 protein expression and apoptosis,” Oncogene, vol. 26, no. 42, pp. 6133–6140, 2007.
- K. Cheng and J. P. Raufman, “Bile acid-induced proliferation of a human colon cancer cell line is mediated by transactivation of epidermal growth factor receptors,” Biochemical Pharmacology, vol. 70, no. 7, pp. 1035–1047, 2005.
- U. C. Nzeako, M. E. Guicciardi, J. H. Yoon, S. F. Bronk, and G. J. Gores, “COX-2 inhibits Fas-mediated apoptosis in cholangiocarcinoma cells,” Hepatology, vol. 35, no. 3, pp. 552–559, 2002.
- S. Tanaka, K. Sugimachi, K. Shirabe, M. Shimada, and J. R. Wands, “Expression and antitumor effects of TRAIL in human cholangiocarcinoma,” Hepatology, vol. 32, no. 3, pp. 523–527, 2000.
- E. Gaudio, P. Onori, and A. Franchitto, “Vascularization of the intrahepatic biliary tree and its role in the regulation of cholangiocyte growth,” in The Pathophysiology of Biliary Epithelia, G. Alpini, D. Alvaro, M. Marzioni, G. LeSage, and N. F. LaRusso, Eds., Landes Bioscience, Georgetown, Tex, USA, 2004.
- E. Gaudio, B. Barbaro, D. Alvaro et al., “Vascular endothelial growth factor stimulates rat cholangiocyte proliferation via an autocrine mechanism,” Gastroenterology, vol. 130, no. 4, pp. 1270–1282, 2006.
- K. Sugimachi, K.-I. Taguchi, S.-I. Aishima et al., “Altered expression of beta-catenin without genetic mutation in intrahepatic cholangiocarcinoma,” Modern Pathology, vol. 14, no. 9, pp. 900–905, 2001.
- C. Benckert, S. Jonas, T. Cramer et al., “Transforming growth factor beta 1 stimulates vascular endothelial growth factor gene transcription in human cholangiocellular carcinoma cells,” Cancer Research, vol. 63, no. 5, pp. 1083–1092, 2003.
- L. K. Sampson, S. M. Vickers, W. Ying, and J. O. Phillips, “Tamoxifen-mediated growth inhibition of human cholangiocarcinoma,” Cancer Research, vol. 57, no. 9, pp. 1743–1749, 1997.
- G. Pan, S. M. Vickers, A. Pickens et al., “Apoptosis and tumorigenesis in human cholangiocarcinoma cells: involvement of Fas/APO-1 (CD95) and calmodulin,” American Journal of Pathology, vol. 155, no. 1, pp. 193–203, 1999.
- D. Alvaro, B. Barbaro, A. Franchitto et al., “Estrogens and insulin-like growth factor 1 modulate neoplastic cell growth in human cholangiocarcinoma,” American Journal of Pathology, vol. 169, no. 3, pp. 877–888, 2006.
- A. Mancino, M. G. Mancino, S. S. Glaser et al., “Estrogens stimulate the proliferation of human cholangiocarcinoma by inducing the expression and secretion of vascular endothelial growth factor,” Digestive and Liver Disease, vol. 41, no. 2, pp. 156–163, 2009.
- D. Alvaro, G. Macarri, M. G. Mancino et al., “Serum and biliary insulin-like growth factor I and vascular endothelial growth factor in determining the cause of obstructive cholestasis,” Annals of Internal Medicine, vol. 147, no. 7, pp. 451–459, 2007.
- N. Kanno, S. Glaser, U. Chowdhury et al., “Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+-dependent protein kinase C-alpha,” Journal of Hepatology, vol. 34, no. 2, pp. 284–291, 2001.
- N. Kanno, G. Lesage, J. L. Phinizy, S. Glaser, H. Francis, and G. Alpini, “Stimulation of alpha2-adrenergic receptor inhibits cholangiocarcinoma growth through modulation of Raf-1 and B-Raf activities,” Hepatology, vol. 35, no. 6, pp. 1329–1340, 2002.
- G. Fava, L. Marucci, S. Glaser et al., “Gamma-aminobutyric acid inhibits cholangiocarcinoma growth by cyclic AMP-dependent regulation of the protein kinase A/extracellular signal-regulated kinase 1/2 pathway,” Cancer Research, vol. 65, no. 24, pp. 11437–11446, 2005.
- G. Fava, S. De Morrow, E. Gaudio et al., “Endothelin inhibits cholangiocarcinoma growth by a decrease in the vascular endothelial growth factor expression,” Liver International, vol. 29, no. 7, pp. 1031–1042, 2009.
- G. Alpini, P. Invernizzi, E. Gaudio et al., “Serotonin metabolism is dysregulated in cholangiocarcinoma, which has implications for tumor growth,” Cancer Research, vol. 68, no. 22, pp. 9184–9193, 2008.
- S. DeMorrow, P. Onori, J. Venter et al., “Neuropeptide Y inhibits cholangiocarcinoma cell growth and invasion,” American Journal of Physiology—Cell Physiology, vol. 300, no. 5, pp. C1078–C1089, 2011.
- G. Fava, G. Alpini, C. Rychlicki et al., “Leptin enhances cholangiocarcinoma cell growth,” Cancer Research, vol. 68, no. 16, pp. 6752–6761, 2008.
- M. Marzioni, P. Invernizzi, C. Candelaresi et al., “Human cholangiocarcinoma development is associated with dysregulation of opioidergic modulation of cholangiocyte growth,” Digestive and Liver Disease, vol. 41, no. 7, pp. 523–533, 2009.
- P. Onori, C. Wise, E. Gaudio et al., “Secretin inhibits cholangiocarcinoma growth via dysregulation of the cAMP-dependent signaling mechanisms of secretin receptor,” International Journal of Cancer, vol. 127, no. 1, pp. 43–54, 2010.
- H. Francis, P. Onori, E. Gaudio et al., “H3 histamine receptor-mediated activation of protein kinase Calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo,” Molecular Cancer Research, vol. 7, no. 10, pp. 1704–1713, 2009.
Copyright © 2012 G. Fava and I. Lorenzini. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.