Intrahepatic Cholangiocarcinoma: Continuing Challenges and Translational Advances.

Intrahepatic cholangiocarcinoma (iCCA) has over the last 10-20 years become the focus of increasing concern, largely due to its rising incidence and high mortality rates worldwide. The significant increase in mortality rates from this primary hepatobiliary cancer, particularly over the past decade, has coincided with a rapidly growing interest among clinicians, investigators, and patient advocates to seek greater mechanistic insights and more effective biomarker-driven targeted approaches for managing and/or preventing this challenging liver cancer. In addition to discussing challenges posed by this aggressive cancer, this review will emphasize recent epidemiological, basic, and translational research findings for iCCA. In particular, we will highlight emerging demographic changes and evolving risk factors, the critical role of the tumor microenvironment, extracellular vesicle biomarkers and therapeutics, intertumoral and intratumoral heterogeneity, and current and emerging targeted therapies regarding iCCA. Specifically, recent evidence linking non-bile duct medical conditions, such as nonalcoholic fatty liver disease and nonspecific cirrhosis, to intrahepatic cholangiocarcinogenesis together with geographic and ethnic variation will be assessed. Recent developments concerning the roles played by transforming growth factor-ß and platelet-derived growth factor D in driving the recruitment and expansion of cancer-associated myofibroblasts within cholangiocarcinoma (CCA) stroma as well as their therapeutic implications will also be discussed. In addition, the potential significance of extracellular vesicles as bile and serum biomarkers and therapeutic delivery systems for iCCA will be described. An integrated systems approach to classifying heterogeneous iCCA subtypes will be further highlighted, and recent clinical trials and emerging targeted therapies will be reviewed, along with recommendations for future translational research opportunities. Established international CCA networks are now facilitating collaborations aimed at advancing iCCA translational and clinical research.

Transforming Growth Factors α and ß Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model.

To gain insight into the cellular and molecular interactions mediating the desmoplastic reaction and aggressive malignancy of mass-forming intrahepatic cholangiocarcinoma (ICC), we modeled ICC desmoplasia and progression in vitro. A unique three-dimensional (3D) organotypic culture model was established; within a dilute collagen-type I hydrogel, a novel clonal strain of rat cancer-associated myofibroblasts (TDFSM) was co-cultured with a pure rat cholangiocarcinoma cell strain (TDECC) derived from the same ICC type as TDFSM. This 3D organotypic culture model reproduced key features of desmoplastic reaction that closely mimicked those of the in situ tumor, as well as promoted cholangiocarcinoma cell growth and progression. Our results supported a resident liver mesenchymal cell origin of the TDFSM cells, which were not neoplastically transformed. Notably, 3D co-culturing of TDECC cells with TDFSM cells provoked the formation of a dense fibrocollagenous stroma in vitro that was associated with significant increases in both proliferative TDFSM myofibroblastic cells and TDECC cholangiocarcinoma cells accumulating within the gel matrix. This dramatic desmoplastic ICC-like phenotype, which was not observed in the TDECC or TDFSM controls, was highly dependent on transforming growth factor (TGF)-ß, but not promoted by TGF-α. However, TGF-α was determined to be a key factor for promoting cholangiocarcinoma cell anaplasia, hyperproliferation, and higher malignant grading in this 3D culture model of desmoplastic ICC.

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. CONCLUSION: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).

Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2.

UNLABELLED: Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra- and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal-regulated kinase (ERK)1/2- and phosphatidylinositol-3-kinase/protein kinase B (AKT)-signaling pathways through sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes. In the current study, we demonstrate S1PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK1/2- and AKT-signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)-mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE-013, a chemical antagonist of S1PR2, or by lentiviral short hairpin RNA silencing of S1PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/"duct-like" structures, which was blocked by treatment with JTE-013. CONCLUSION: Our collective data support the hypothesis that CBAs promote CCA cell-invasive growth through S1PR2.

Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma.

BACKGROUND & AIMS: The Hedgehog signaling pathway contributes to cholangiocarcinoma biology. However, canonical Hedgehog signaling requires cilia, and cholangiocarcinoma cells often do not express cilia. To resolve this paradox, we examined non-canonical (G-protein coupled, pertussis toxin sensitive) Hedgehog signaling in cholangiocarcinoma cells. METHODS: Human [non-malignant (H69), malignant (HuCC-T1 and Mz-ChA-1)] and rat [non-malignant (BDE1 and NRC), and malignant (BDEneu)] cell lines were employed for this study. A BDE(ΔLoop2) cell line with the dominant-negative receptor Patched-1 was generated with the Sleeping Beauty transposon transfection system. RESULTS: Cilia expression was readily identified in non-malignant, but not in malignant cholangiocarcinoma cell lines. Although the canonical Hh signaling pathway was markedly attenuated in cholangiocarcinoma cells, they were chemotactic to purmorphamine, a small-molecule direct Smoothened agonist. Purmorphamine also induced remodeling of the actin cytoskeleton with formation of filopodia and lamellipodia-like protrusions. All these biological features of cell migration were pertussis toxin sensitive, a feature of G-protein coupled (Gis) receptors. To further test the role of Hedgehog signaling in vivo, we employed a syngeneic orthotopic rat model of cholangiocarcinoma. In vivo, genetic inhibition of the Hedgehog signaling pathway employing BDE(ΔLoop2) cells or pharmacological inhibition with a small-molecule antagonist of Smoothened, vismodegib, was tumor and metastasis suppressive. CONCLUSIONS: Cholangiocarcinoma cells exhibit non-canonical Hedgehog signaling with chemotaxis despite impaired cilia expression. This non-canonical Hedgehog signaling pathway appears to contribute to cholangiocarcinoma progression, thereby, supporting a role for Hedgehog pathway inhibition in human cholangiocarcinoma.

Polo-like kinase 2 is a mediator of hedgehog survival signaling in cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and thus rely on potent survival signals to circumvent cell death by TRAIL. Hedgehog (Hh) signaling is an important survival pathway in CCA. Herein, we further examine the mechanisms whereby Hh signaling mediates apoptosis resistance in CCA, revealing a pivotal role for the cell division regulating serine/threonine kinase polo-like kinase 2 (PLK2). We employed 50 human CCA samples (25 intrahepatic and 25 extrahepatic CCA) as well as human KMCH-1, Mz-CHA-1, and HUCCT-1 CCA cells for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. In human samples, polo-like kinase (PLK)1/2/3-immunoreactive cancer cells were present in the preponderance of intra- and extrahepatic CCA specimens. Inhibition of Hh signaling by cyclopamine reduced PLK2, but not PLK1 or PLK3, messenger RNA and protein expression in vehicle-treated and sonic Hh-treated CCA cells, confirming our previous microarray study. PLK2 regulation by Hh signaling appears to be direct, because the Hh transcription factors, glioma-associated oncogene 1 and 2, bind to the PLK2 promotor. Moreover, inhibition of PLK2 by the PLK inhibitor, BI 6727 (volasertib), or PLK2 knockdown was proapoptotic in CCA cells. BI 6727 administration or PLK2 knockdown decreased cellular protein levels of antiapoptotic myeloid cell leukemia 1 (Mcl-1), an effect reversed by the proteasome inhibitor, MG-132. Finally, BI 6727 administration reduced Mcl-1 protein expression in CCA cells, resulting in CCA cell apoptosis and tumor suppression in vivo. CONCLUSION: PLK2 appears to be an important mediator of Hh survival signaling. These results suggest PLK inhibitors to be of therapeutic value for treatment of human CCA.

Periostin in intrahepatic cholangiocarcinoma: pathobiological insights and clinical implications.

Periostin is a modular glycoprotein frequently observed to be a major constituent of the extracellular milieu of mass-forming intrahepatic cholangiocarcinoma and other desmoplastic malignant tumors. In intrahepatic cholangiocarcinoma, as well as in desmoplastic pancreatic ductal adenocarcinoma, periostin is overexpressed and hypersecreted in large part, if not exclusively, by cancer-associated fibroblasts within the tumor stroma. Through its interaction with specific components of the extracellular tumor matrix, particularly collagen type I and tenascin-C, and with cell surface receptors, notably integrins leading to activation of the Akt and FAK signaling pathways, this TGF-ß family-inducible matricellular protein appears to be functioning as a key extracellular matrix molecule regulating such critically important and diverse malignant tumor behaviors as tumor fibrogenesis and desmoplasia, invasive malignant cell growth, chemoresistance, and metastatic colonization. This review will discuss current evidence and basic molecular mechanisms implicating periostin as a mediator of intrahepatic cholangiocarcinoma invasive growth. In addition, its significance as a potential prognostic biomarker for intrahepatic cholangiocarcinoma patients, as well as future possibilities and challenges as a molecular target for cholangiocarcinoma therapy and/or prevention, will be critically evaluated.

Immunization with aspartate-ß-hydroxylase-loaded dendritic cells produces antitumor effects in a rat model of intrahepatic cholangiocarcinoma.

UNLABELLED: Dendritic cells (DCs) capture and process proteins and present peptides on the cell surface in the context of major histocompatibility complex I and II molecules to induce antigen-specific T cell immune responses. The aims of this study were to (1) employ an expanded and purified DC population and load them with aspartate-ß-hydroxylase (ASPH), a highly expressed tumor-associated cell surface protein, and (2) to determine if immunization induced antitumor effects in an orthotopic rat model of intrahepatic cholangiocarcinoma. Splenocytes were incubated with ASPH-coated beads and passed through a magnetic field to yield an 80% pure DC OX62+ population. This DC subset was stimulated with granulocyte-macrophage colony-stimulating factor, interleukin-4, CD40L, and interferon-γ, resulting in a 40-fold increase in interleukin-12A messenger RNA expression to subsequently generate a T helper 1-type immune response. After incubation with the cytokine cocktail, DCs were found to have matured, as demonstrated by increased expression of CD40, CD80, and CD86 costimulatory molecules. Immunization with ASPH-loaded DCs induced antigen-specific immunity. A clone of the parental tumorigenic rat BDEneu cholangiocyte cell line, designated BDEneu-CL24, was found to have the highest number of cells expressing this surface protein (97%); it maintained the same phenotypic characteristics of the parental cell line and was used to produce intrahepatic tumors in immunocompetent syngeneic Fisher-344 rats. Immunization with ASPH-loaded DCs generated cytotoxicity against cholangiocarcinoma cells in vitro and significantly suppressed intrahepatic tumor growth and metastasis, and was associated with increased CD3+ lymphocyte infiltration into the tumors. CONCLUSION: These findings suggest that immunization with ASPH-loaded DCs may constitute a novel therapeutic approach for intrahepatic cholangiocarcinoma, because this protein also appears to be highly conserved and expressed on human hepatobiliary tumors.

Myofibroblast-derived PDGF-BB promotes Hedgehog survival signaling in cholangiocarcinoma cells.

UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and, therefore, are dependent upon potent survival signals to circumvent TRAIL cytotoxicity. CCAs are also highly desmoplastic cancers with a tumor microenvironment rich in myofibroblasts (MFBs). Herein, we examine a role for MFB-derived CCA survival signals. We employed human KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1 CCA cells, as well as human primary hepatic stellate and myofibroblastic LX-2 cells, for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. Coculturing CCA cells with myofibroblastic human primary hepatic stellate cells or LX-2 cells significantly decreased TRAIL-induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing platelet-derived growth factor (PDGF)-BB antiserum. Cytoprotection by PDGF-BB was dependent upon Hedgehog (Hh) signaling, because it was abolished by the smoothened (SMO; the transducer of Hh signaling) inhibitor, cyclopamine. PDGF-BB induced cyclic adenosine monophosphate-dependent protein kinase-dependent trafficking of SMO to the plasma membrane, resulting in glioma-associated oncogene (GLI)2 nuclear translocation and activation of a consensus GLI reporter gene-based luciferase assay. A genome-wide messenger RNA expression analysis identified 67 target genes to be commonly up- (50 genes) or down-regulated (17 genes) by both Sonic hedgehog and PDGF-BB in a cyclopamine-dependent manner in CCA cells. Finally, in a rodent CCA in vivo model, cyclopamine administration increased apoptosis in CCA cells, resulting in tumor suppression. CONCLUSIONS: MFB-derived PDGF-BB protects CCA cells from TRAIL cytotoxicity by a Hh-signaling-dependent process. These results have therapeutical implications for the treatment of human CCA.

Targeting PDGFR-ß in Cholangiocarcinoma.

BACKGROUND: Cholangiocarcinomas (CCAs) are highly desmoplastic neoplasms with a tumour microenvironment plentiful in myofibroblasts (MFBs). MFB-derived PDGF-BB survival signalling is a mediator of CCA cell resistance to apoptotic stimuli. This raises the concept that targeting PDGFR-ß, a cognate receptor of PDGF-BB, represents a potential strategy for the treatment of human CCA. AIMS: Herein, we examine a role for inhibiting PDGFR-ß in restoring CCA cell sensitivity to apoptotic stimuli in vitro and in vivo. METHODS: We employed human CCA samples from 41 patients (19 intrahepatic and 22 extrahepatic CCA samples), the human CCA cell lines KMCH-1 and HUCCT-1 as well as shPDGFR-ß-KMCH-1 and human myofibroblastic LX-2 cells for these studies. In vivo-experiments were conducted using a syngeneic rat orthotopic CCA model. RESULTS: Of several MFB-derived growth factors profiled, PDGF-BB and CTGF were most abundantly expressed; however, only PDGF-BB attenuated tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Co-culturing CCA cells with PDGF-BB-secreting MFBs significantly decreased TRAIL-induced CCA cell apoptosis when compared with monoculture conditions; this cytoprotective effect was abrogated in the presence of the tyrosine kinase inhibitors imatinib mesylate or linifanib, which inhibit PDGFR-ß. Consistent with these findings, MFB-imparted cytoprotection also was abolished when PDGFR-ß was knocked down as demonstrated in shPDGFR-ß-KMCH-1 cells. Finally, administration of imatinib mesylate increased CCA cell apoptosis and reduced tumour growth in a rodent in vivo-CCA model that mimics the human disease. CONCLUSIONS: Targeting PDGFR-ß sensitizes CCA cells to apoptotic stimuli and appears to be therapeutic in vivo.

Matricellular proteins in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is typically characterized by a prominent desmoplastic stroma that is often the most dominant feature of the tumor. This tumor reactive stroma is comprised of a dense fibro-collagenous-enriched extracellular matrix (ECM) surrounding the cancer cells, together with other ECM proteins/peptides, specifically secreted matricellular glycoproteins and proteolytic enzymes, growth factors, and cytokines. Moreover, as enjoined by cholangiocarcinoma cells, this enriched tumor microenvironment is populated by various stromal cell types, most prominently, cancer-associated myofibroblasts (CAFs), along with variable numbers of tumor-associated macrophages (TAMs), inflammatory and vascular cell types. While it is now well appreciated that the interplay between cholangiocarcinoma cells, CAFs, and TAMs in particular play a critical role in promoting cholangiocarcinoma progression, therapeutic resistance, and immune evasion, it is also becoming increasingly evident that over-expression and secretion into the tumor microenvironment of functionally overlapping matricellular glycoproteins, including periostin, osteopontin, tenascin-C, thrombospondin-1, mesothelin and others have an important role to play in regulating or modulating a variety of pro-oncogenic cellular functions, including cholangiocarcinoma cell proliferation, invasion, and metastasis, epithelial-mesenchymal transition, ECM remodeling, and immune evasion. Matricellular proteins have also shown promise as potential prognostic factors for iCCA and may provide unique therapeutic opportunities particularly in relation to targeting iCCA pre-metastatic and metastatic niches, tumor cell dormancy, and immune evasion. This review will highlight timely research and its translational implications for salient matricellular proteins in terms of their structure-function relationships, as modulators of intrahepatic cholangiocarcinoma microenvironment and progression, and potential clinical value for iCCA prognosis and therapy.

YAP1 activation and Hippo pathway signaling in the pathogenesis and treatment of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA), the second most common primary liver cancer, is a highly lethal epithelial cell malignancy exhibiting features of cholangiocyte differentiation. iCCAs can potentially develop from multiple cell types of origin within liver, including immature or mature cholangiocytes, hepatic stem cells/progenitor cells, and from transdifferentiation of hepatocytes. Understanding the molecular mechanisms and genetic drivers that diversely drive specific cell lineage pathways leading to iCCA has important biological and clinical implications. In this context, activation of the YAP1-TEAD dependent transcription, driven by Hippo-dependent or -independent diverse mechanisms that lead to the stabilization of YAP1 is crucially important to biliary fate commitment in hepatobiliary cancer. In preclinical models, YAP1 activation in hepatocytes or cholangiocytes is sufficient to drive their malignant transformation into iCCA. Moreover, nuclear YAP1/TAZ is highly prevalent in human iCCA irrespective of the varied etiology, and significantly correlates with poor prognosis in iCCA patients. Based on the ubiquitous expression and diverse physiologic roles for YAP1/TAZ in the liver, recent studies have further revealed distinct functions of active YAP1/TAZ in regulating tumor metabolism, as well as the tumor immune microenvironment. In the current review, we discuss our current understanding of the various roles of the Hippo-YAP1 signaling in iCCA pathogenesis, with a specific focus on the roles played by the Hippo-YAP1 pathway in modulating biliary commitment and oncogenicity, iCCA metabolism, and immune microenvironment. We also discuss the therapeutic potential of targeting the YAP1/TAZ-TEAD transcriptional machinery in iCCA, its current limitations, and what future studies are needed to facilitate clinical translation.

Preclinical assessment of simultaneous targeting of epidermal growth factor receptor (ErbB1) and ErbB2 as a strategy for cholangiocarcinoma therapy.

UNLABELLED: Overexpression of epidermal growth factor receptor (ErbB1) and/or ErbB2 has been implicated in the pathogenesis of cholangiocarcinoma, suggesting that combined ErbB1/ErbB2 targeting might serve as a target-based therapeutic strategy for this highly lethal cancer. To test this strategy, we investigated targeting with the ErbB1 inhibitor tryphostin AG1517 and the ErbB2 inhibitor tryphostin AG879, in combination and alone, as well as with the dual ErbB1/ErbB2 inhibitor lapatinib, to assess the effectiveness of simultaneous targeting of ErbB1 and ErbB2 signaling over single inhibitor treatments in suppressing cholangiocarcinoma cell growth in vitro and the therapeutic efficacy of lapatinib in vivo. Our in vitro studies were carried out using rat (BDEneu and C611B) and human (HuCCT1 and TFK1) cholangiocarcinoma cell lines. The efficacy of lapatinib to significantly suppress liver tumor growth was tested in an orthotopic, syngeneic rat model of intrahepatic cholangiocarcinoma progression. Our results demonstrated that simultaneous targeting of ErbB1 and ErbB2 signaling was significantly more effective in suppressing the in vitro growth of both rat and human cholangiocarcinoma cells than individual receptor targeting. Lapatinib was an even more potent inhibitor of cholangiocarcinoma cell growth and inducer of apoptosis than either tryphostin when tested in vitro against these respective cholangiocarcinoma cell lines, regardless of differences in their levels of ErbB1 or ErbB2 protein expression and/or mechanism of activation. Lapatinib treatment also produced a significant suppression of intrahepatic cholangiocarcinoma growth when administered early to rats, but was without effect in inhibiting liver tumor growth in rats with more advanced tumors. CONCLUSION: Our findings suggest that simultaneous targeting of ErbB1 and ErbB2 could be a potentially selective strategy for cholangiocarcinoma therapy, but is likely to be ineffective by itself against advanced cancer.

A smac mimetic reduces TNF related apoptosis inducing ligand (TRAIL)-induced invasion and metastasis of cholangiocarcinoma cells.

UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand that, failing to kill CCA cells, instead promotes their tumorigenicity and especially the metastatic behaviors of cell migration and invasion. Second mitochondria-derived activator of caspase (smac) mimetics are promising cancer therapeutic agents that enhance proapoptotic death receptor signaling by causing cellular degradation of inhibitor of apoptosis (IAP) proteins. Our aim was to examine the in vitro and in vivo effects of the smac mimetic JP1584 in CCA. Despite JP1584-mediated loss of cellular inhibitor of apoptosis-1 (cIAP-1) and cIAP-2, TRAIL failed to induce apoptosis in KMCH-1, TFK-1, and BDEneu CCA cells; a finding consistent with a downstream block in death signaling. Because cIAP-1 and cIAP-2 also promote nuclear factor kappa B (NF-kappaB) activation by the canonical pathway, the effect of JP1584 on this signaling pathway was examined. Treatment with JP1584 inhibited TRAIL-induced NF-kappaB activation as well as TRAIL-mediated up-regulation of the NF-kappaB target gene, matrix metalloproteinase 7 (MMP7). JP1584 also reduced TRAIL-mediated CCA cell migration and invasion in vitro. Finally, in a syngeneic rat orthotopic CCA model, JP1584 administration reduced MMP7 messenger RNA levels and extrahepatic metastases. CONCLUSION: : Although the smac mimetic JP1584 does not sensitize cells to apoptosis, it reduces TRAIL-induced CCA cell metastatic behavior. These data support the emerging concept that IAPs are prometastatic and represent targets for antimetastatic therapies.

Cancer-associated fibroblasts in intrahepatic cholangiocarcinoma.

PURPOSE OF REVIEW: The aim of this brief review is to provide an up-to-date view of the role played by α-smooth muscle actin-positive cancer-associated fibroblastic cells in promoting intrahepatic cholangiocarcinoma progression. RECENT FINDINGS: An increase in α-smooth muscle actin-positive cancer-associated fibroblastic cells in the stroma of intrahepatic cholangiocarcinoma has recently been demonstrated to accelerate cholangiocarcinoma progression. However, our understanding of the evolving cellular and molecular interactions between these stromal cells and cholangiocarcinoma cells in relation to promoting intrahepatic cholangiocarcinoma progression is only just beginning to be elucidated. Imbalances in multifactorial growth factor/cytokine signaling, activation of Hedgehog-GLI signaling and of proteases involved in extracellular matrix remodeling, and matricellular protein-protein and protein-cholangiocarcinoma cell interactions, as well as hypoxia, all appear to factor into the complex and dynamic interactive mechanisms through which cancer-associated fibroblastic cells crosstalk with cholangiocarcinoma cells to promote intrahepatic cholangiocarcinoma progression. Novel three-dimensional organotypic co-culture models are being developed to facilitate relevant studies of cancer-associated fibroblastic cell/cholangiocarcinoma cell interactions that may more accurately mimic physiologically pertinent features of the tumor. SUMMARY: Increasing our understanding of critical interactive pathways by which cancer-associated fibroblastic cells crosstalk with cholangiocarcinoma cells to promote tumor progression can lead to the development of novel multitargeting strategies for intrahepatic cholangiocarcinoma therapy.

Intrahepatic cholangiocarcinoma: Morpho-molecular pathology, tumor reactive microenvironment, and malignant progression.

Intrahepatic cholangiocarcinoma (iCCA) is a relatively rare, but highly lethal and biologically complex primary biliary epithelial cancer arising within liver. After hepatocellular carcinoma, iCCA is the second most common primary liver cancer, accounting for approximately 10-20% of all primary hepatic malignancies. Over the last 10-20 years, iCCA has become the focus of increasing concern largely due to its rising incidence and high mortality rates in various parts of the world, including the United States. The challenges posed by iCCA are daunting and despite recent progress in the standard of care and management options for iCCA, the prognosis for this cancer continues to be dismal. In an effort to provide a framework for advancing our understanding of iCCA malignant aggressiveness and therapy resistance, this review will highlight key etiological, biological, molecular, and microenvironmental factors hindering more effective management of this hepatobiliary cancer. Particular focus will be on critically reviewing the cell origins and morpho-molecular heterogeneity of iCCAs, providing mechanistic insights into high risk fibroinflammatory cholangiopathies associated with iCCA development, and notably discussing the deleterious role played by the tumor reactive desmoplastic stroma in regulating iCCA malignant progression, lymphangiogenesis, and tumor immunobiology.

Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly lethal adenocarcinoma of the hepatobiliary system, which can be classified as intrahepatic, perihilar and distal. Each anatomic subtype has distinct genetic aberrations, clinical presentations and therapeutic approaches. In endemic regions, liver fluke infection is associated with CCA, owing to the oncogenic effect of the associated chronic biliary tract inflammation. In other regions, CCA can be associated with chronic biliary tract inflammation owing to choledocholithiasis, cholelithiasis, or primary sclerosing cholangitis, but most CCAs have no identifiable cause. Administration of the anthelmintic drug praziquantel decreases the risk of CCA from liver flukes, but reinfection is common and future vaccination strategies may be more effective. Some patients with CCA are eligible for potentially curative surgical options, such as resection or liver transplantation. Genetic studies have provided new insights into the pathogenesis of CCA, and two aberrations that drive the pathogenesis of non-fluke-associated intrahepatic CCA, fibroblast growth factor receptor 2 fusions and isocitrate dehydrogenase gain-of-function mutations, can be therapeutically targeted. CCA is a highly desmoplastic cancer and targeting the tumour immune microenvironment might be a promising therapeutic approach. CCA remains a highly lethal disease and further scientific and clinical insights are needed to improve patient outcomes.

Sorafenib inhibits signal transducer and activator of transcription-3 signaling in cholangiocarcinoma cells by activating the phosphatase shatterproof 2.

UNLABELLED: The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is one of the key signaling cascades in cholangiocarcinoma (CCA) cells, mediating their resistance to apoptosis. Our aim was to ascertain if sorafenib, a multikinase inhibitor, may also inhibit JAK/STAT signaling and, therefore, be efficacious for CCA. Sorafenib treatment of three human CCA cell lines resulted in Tyr(705) phospho-STAT3 dephosphorylation. Similar results were obtained with the Raf-kinase inhibitor ZM336372, suggesting sorafenib promotes Tyr(705) phospho-STAT3 dephosphorylation by inhibiting Raf-kinase activity. Sorafenib treatment enhanced an activating phosphorylation of the phosphatase SHP2. Consistent with this observation, small interfering RNA-mediated knockdown of phosphatase shatterproof 2 (SHP2) inhibited sorafenib-induced Tyr(705) phospho-STAT3 dephosphorylation. Sorafenib treatment also decreased the expression of Mcl-1 messenger RNA and protein, a STAT3 transcriptional target, as well as sensitizing CCA cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. In an orthotopic, syngeneic CCA model in rats, sorafenib displayed significant tumor suppression resulting in a survival benefit for treated animals. In this in vivo model, sorafenib also decreased tumor Tyr(705) STAT3 phosphorylation and increased tumor cell apoptosis. CONCLUSION: Sorafenib accelerates STAT3 dephosphorylation by stimulating phosphatase SHP2 activity, sensitizes CCA cells to TRAIL-mediated apoptosis, and is therapeutic in a syngeneic rat, orthotopic CCA model that mimics human disease.