Combined Inhibition of Smoothened and the DNA Damage Checkpoint WEE1 Exerts Antitumor Activity in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aberrant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2-M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical development of novel therapeutic approaches in iCCA.

Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

DNA Damage Response Inhibitors in Cholangiocarcinoma: Current Progress and Perspectives.

Cholangiocarcinoma (CCA) is a poorly treatable type of cancer and its incidence is dramatically increasing. The lack of understanding of the biology of this tumor has slowed down the identification of novel targets and the development of effective treatments. Based on next generation sequencing profiling, alterations in DNA damage response (DDR)-related genes are paving the way for DDR-targeting strategies in CCA. Based on the notion of synthetic lethality, several DDR-inhibitors (DDRi) have been developed with the aim of accumulating enough DNA damage to induce cell death in tumor cells. Observing that DDRi alone could be insufficient for clinical use in CCA patients, the combination of DNA-damaging regimens with targeted approaches has started to be considered, as evidenced by many emerging clinical trials. Hence, novel therapeutic strategies combining DDRi with patient-specific targeted drugs could be the next level for treating cholangiocarcinoma.

Metabolic reprogramming in cholangiocarcinoma.

Metabolic reprogramming is a hallmark of cancer and allows tumour cells to meet the increased energy demands required for rapid proliferation, invasion, and metastasis. Indeed, many tumour cells acquire distinctive metabolic and bioenergetic features that enable them to survive in resource-limited conditions, mainly by harnessing alternative nutrients. Several recent studies have explored the metabolic plasticity of cancer cells with the aim of identifying new druggable targets, while therapeutic strategies to limit the access to nutrients have been successfully applied to the treatment of some tumours. Cholangiocarcinoma (CCA), a highly heterogeneous tumour, is the second most common form of primary liver cancer. It is characterised by resistance to chemotherapy and poor prognosis, with 5-year survival rates of below 20%. Deregulation of metabolic pathways have been described during the onset and progression of CCA. Increased aerobic glycolysis and glutamine anaplerosis provide CCA cells with the ability to generate biosynthetic intermediates. Other metabolic alterations involving carbohydrates, amino acids and lipids have been shown to sustain cancer cell growth and dissemination. In this review, we discuss the complex metabolic rewiring that occurs during CCA development and leads to unique nutrient addiction. The possible role of therapeutic interventions based on metabolic changes is also thoroughly discussed.

Paclitaxel Restores Sensitivity to Chemotherapy in Preclinical Models of Multidrug-Resistant Intrahepatic Cholangiocarcinoma.

The treatment of unresectable cholangiocarcinoma (CCA) is limited by the development of resistance to conventional first-line chemotherapy based on gemcitabine (GEM). In addition, a prior treatment with GEM frequently induces cross-resistance to other drugs employed in the second-line. Paclitaxel (PTX) is now emerging as an alternative option for the management of advanced/metastatic CCA. In the present work, we evaluate the antitumor activity of PTX in preclinical models of multidrug-resistant intrahepatic cholangiocarcinoma (iCCA). In vitro, PTX decreases tumor cell viability by affecting the cell cycle and inducing apoptosis and impairs the stem cell compartment. In vivo, a therapeutic regimen containing albumin-bound nanoparticle (Nab)-PTX overcomes drug resistance resulting in delayed tumor growth, impaired organization of the tumor vasculature, and reduced glucose uptake. Together, our results provide a rationale to consider PTX-based regimens in patients with iCCA who became refractory to conventional therapies.

Macrophage MerTK promotes profibrogenic cross-talk with hepatic stellate cells via soluble mediators.

BACKGROUND & AIMS: Activation of Kupffer cells and recruitment of monocytes are key events in fibrogenesis. These cells release soluble mediators which induce the activation of hepatic stellate cells (HSCs), the main fibrogenic cell type within the liver. Mer tyrosine kinase (MerTK) signaling regulates multiple processes in macrophages and has been implicated in the pathogenesis of non-alcoholic steatohepatitis-related fibrosis. In this study, we explored if MerTK activation in macrophages influences the profibrogenic phenotype of HSCs. METHODS: Macrophages were derived from THP-1 cells or differentiated from peripheral blood monocytes towards MerTK+/CD206+/CD163+/CD209- macrophages. The role of MerTK was assessed by pharmacologic and genetic inhibition. HSC migration was determined in Boyden chambers, viability was measured by the MTT assay, and proliferation was evaluated by the BrdU incorporation assay. RESULTS: Gas-6 induced MerTK phosphorylation and Akt activation in macrophages, and these effects were inhibited by UNC569. During polarization, MerTK+/CD206+/CD163+/CD209- macrophages exhibited activation of STAT3, ERK1/2, p38 and increased expression of VEGF-A. Activation of MerTK in THP-1 macrophages induced a secretome which promoted a significant increase in migration, proliferation, viability and expression of profibrogenic factors in HSCs. Similarly, conditioned medium from MerTK+ macrophages induced a significant increase in cell migration, proliferation, STAT3 and p38 phosphorylation and upregulation of IL-8 expression in HSCs. Moreover, conditioned medium from Gas-6-stimulated Kupffer cells induced a significant increase in HSC proliferation. These effects were specifically related to MerTK expression and activity in macrophages, as indicated by pharmacologic inhibition and knockdown experiments. CONCLUSIONS: MerTK activation in macrophages modifies the secretome to promote profibrogenic features in HSCs, implicating this receptor in the pathogenesis of hepatic fibrosis. LAY SUMMARY: Fibrosis represents the process of scarring occurring in patients with chronic liver diseases. This process depends on production of scar tissue components by a specific cell type, named hepatic stellate cells, and is regulated by interaction with other cells. Herein, we show that activation of MerTK, a receptor present in a population of macrophages, causes the production of factors that act on hepatic stellate cells, increasing their ability to produce scar tissue.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. METHODS: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+ ) cells in immune-deficient NOD-SCID/IL2Rgnull  (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. RESULTS: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (ß-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene ß-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. CONCLUSION: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

The Role of the Hedgehog Pathway in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a poorly treatable type of cancer and, along with hepatocellular carcinoma (HCC), is the predominant type of primitive liver cancer in adults. The lack of understanding of CCA biology has slowed down the identification of novel targets and the development of effective treatments. While tumors share some general characteristics, detailed knowledge of specific features is essential for the development of effectively tailored therapeutic approaches. The Hedgehog (HH) signaling cascade regulates stemness biology, embryonal development, tissue homeostasis, and cell proliferation and differentiation. Its aberrant activation has been associated with a variety of solid and hematological human malignancies. Several HH-inhibiting compounds have been indeed developed as potential anticancer agents in different types of tumors, with Smoothened and GLI inhibitors showing the most promising results. Beside its well-established function in other tumors, findings regarding the HH signaling in CCA are still controversial. Here we will give an overview of the most important clinical and molecular features of cholangiocarcinoma, and we will discuss the available evidence of the crosstalk between the HH signaling pathway and the cholangiocarcinoma cell biology.

Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA. APPROACH AND RESULTS: ERK5 expression was detected in two established (HuCCT-1 and CCLP-1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT-1 and CCLP-1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5-silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP-1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA. CONCLUSIONS: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment.

Establishment and characterization of a new spontaneously immortalized ER-/PR-/HER2+ human breast cancer cell line, DHSF-BR16.

Invasive ductal carcinoma (IDC) constitutes the most frequent malignant cancer endangering women's health. In this study, a new spontaneously immortalized breast cancer cell line, DHSF-BR16 cells, was isolated from the primary IDC of a 74-years old female patient, treated with neoadjuvant chemotherapy and disease-free 5-years after adjuvant chemotherapy. Primary breast cancer tissue surgically removed was classified as ER-/PR-/HER2+, and the same phenotype was maintained by DHSF-BR16 cells. We examined DHSF-BR16 cell morphology and relevant biological and molecular markers, as well as their response to anticancer drugs commonly used for breast cancer treatment. MCF-7 cells were used for comparison purposes. The DHSF-BR16 cells showed the ability to form spheroids and migrate. Furthermore, DHSF-BR16 cells showed a mixed stemness phenotype (i.e. CD44+/CD24-/low), high levels of cytokeratin 7, moderate levels of cytokeratin 8 and 18, EpCAM and E-Cadh. Transcriptome analysis showed 2071 differentially expressed genes between DHSF-BR16 and MCF-7 cells (logFC > 2, p-adj < 0.01). Several genes were highly upregulated or downregulated in the new cell line (log2 scale fold change magnitude within - 9.6 to + 12.13). A spontaneous immortalization signature, mainly represented by extracellular exosomes-, plasma membrane- and endoplasmic reticulum membrane pathways (GO database) as well as by metabolic pathways (KEGG database) was observed in DHSF-BR16 cells. Also, these cells were more resistant to anthracyclines compared with MCF-7 cells. Overall, DHSF-BR16 cell line represents a relevant model useful to investigate cancer biology, to identify both novel prognostic and drug response predictive biomarkers as well as to assess new therapeutic strategies.

A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient.

Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α-fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.

Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma.

BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.

Role of Chemokines in the Biology of Cholangiocarcinoma.

Cholangiocarcinoma (CCA), a heterogeneous tumor with poor prognosis, can arise at any level in the biliary tree. It may derive from epithelial cells in the biliary tracts and peribiliary glands and possibly from progenitor cells or even hepatocytes. Several risk factors are responsible for CCA onset, however an inflammatory milieu nearby the biliary tree represents the most common condition favoring CCA development. Chemokines play a key role in driving the immunological response upon liver injury and may sustain tumor initiation and development. Chemokine receptor-dependent pathways influence the interplay among various cellular components, resulting in remodeling of the hepatic microenvironment towards a pro-inflammatory, pro-fibrogenic, pro-angiogenic and pre-neoplastic setting. Moreover, once tumor develops, chemokine signaling may influence its progression. Here we review the role of chemokines in the regulation of CCA development and progression, and the modulation of angiogenesis, metastasis and immune control. The potential role of chemokines and their receptors as possible biomarkers and/or therapeutic targets for hepatobiliary cancer is also discussed.

Cholangiocarcinoma 2020: the next horizon in mechanisms and management.

Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted.

Multifaceted Aspects of Metabolic Plasticity in Human Cholangiocarcinoma: An Overview of Current Perspectives.

Cholangiocarcinoma (CCA) is a deadly tumor without an effective therapy. Unique metabolic and bioenergetics features are important hallmarks of tumor cells. Metabolic plasticity allows cancer cells to survive in poor nutrient environments and maximize cell growth by sustaining survival, proliferation, and metastasis. In recent years, an increasing number of studies have shown that specific signaling networks contribute to malignant tumor onset by reprogramming metabolic traits. Several evidences demonstrate that numerous metabolic mediators represent key-players of CCA progression by regulating many signaling pathways. Besides the well-known Warburg effect, several other different pathways involving carbohydrates, proteins, lipids, and nucleic acids metabolism are altered in CCA. The goal of this review is to highlight the main metabolic processes involved in the cholangio-carcinogeneis that might be considered as potential novel druggable candidates for this disease.

The protein kinase CK2 contributes to the malignant phenotype of cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a particularly aggressive hepatobiliary malignancy, for which the molecular mechanisms underlying the malignant phenotype are still poorly understood, and novel and effective therapeutic strategies are limited. The pro-survival protein kinase CK2 is frequently overexpressed in cancer and is receiving increasing interest as an anti-tumor drug target. Its precise role in CCA biology is still largely unknown. Here we show that expression of the CK2α and α' catalytic subunits and of the ß regulatory subunit is increased in human CCA samples. Increased expression of CK2 subunits was shown in CCA cell lines compared to non-transformed cholangiocytes. We used chemical inhibition of CK2 and genetic modification by CRISPR/Cas9 to explore the contribution of CK2 to the malignant phenotype of CCA cells. Disruption of CK2 activity results in cell death through apoptosis, reduced invasion and migration potential, and G0/G1 cell cycle arrest. Importantly, CCA cells with a reduced CK2 activity are more sensitive to chemotherapy. Altogether, our results demonstrate that CK2 significantly contributes to increased proliferative potential and augmented growth of CCA cells and indicate the rationale for its targeting as a promising pharmacologic strategy for cholangiocarcinoma.

Antitumor Activity of a Novel Fibroblast Growth Factor Receptor Inhibitor for Intrahepatic Cholangiocarcinoma.

Fibroblast growth factor receptor 2 (FGFR2) might have an important role in the pathogenesis and biology of cholangiocarcinoma (CCA). We examined FGFR expression in CCA tumor specimens obtained from patients and CCA cell lines, and then determined the effects of the novel FGFR inhibitor, derazantinib (DZB; formally, ARQ 087), which is currently in clinical phase 2 trials for intrahepatic CCA. DZB inhibited the growth of CCA cell lines in a dose-dependent manner, and extracellular signal-regulated kinase 1/2 and AKT. It also activated apoptotic and cell growth arrest signaling. DZB reduced the in vitro invasiveness and the expression of key epithelial-mesenchymal transition genes. The in vitro data correlated with the expression of FGFRs in human CCA specimens by immunohistochemistry (FGFR1, 30% positive; and FGFR2, 65% positive) and the CCA cell lines assayed by Western blot analysis. These correlated in vitro studies suggest that FGFR may play an important role in the pathogenesis and biology of CCA. Our findings support the notion that FGFR inhibitors, like DZB, should be further evaluated at the clinical stage as targeted therapy for CCA treatment.

Free episomal and integrated HBV DNA in HBsAg-negative patients with intrahepatic cholangiocarcinoma.

There is evidence that chronic hepatitis B virus (HBV) infection is associated with an increased risk of intrahepatic cholangiocarcinoma (ICC) development, and it has been hypothesized an etiological role of HBV in the development of this tumor. Very little is known about occult HBV infection (OBI) in ICC. Aims of the study were to investigate the OBI prevalence and to characterize the HBV molecular status at intrahepatic level in OBI-positive cases with ICC. Frozen liver tumor specimens from 47 HBV surface-antigen-negative patients with ICC and 41 paired non-tumor liver tissues were tested for OBI by 4 different HBV-specific nested PCR. Covalently closed circular HBV DNA (HBV cccDNA) and viral integrations were investigated in OBI-positive cases. HBV DNA was detected in tumor and/or non-tumor specimens from 29/47 (61.7%) ICC patients. HBV cccDNA was found in tissues from 5/17 (34.5%) cases examined. HBV integration was detected in 4/10 (40%) tumor tissues tested and involved HBx and HBV-core gene sequences in 3 and 1 cases, respectively. Viral integration occurred: (a) 9,367 nucleotides upstream of the cat-eye-syndrome critical region protein-5-isoform coding sequence; (b) within the cystinosin isoform-1-precursor gene; (c) within the thromboxane-A-synthase-1 gene; (d) within the ATPase phospholipid transporting 9B gene. Occult HBV infection is highly prevalent in patients with ICC. Both free viral genomes and integrated HBV DNA can be present in these cases. These results suggest an involvement of HBV in the carcinogenic process leading to ICC development even in cases with occult infection.

Role of Myeloid-Epithelial-Reproductive Tyrosine Kinase and Macrophage Polarization in the Progression of Atherosclerotic Lesions Associated With Nonalcoholic Fatty Liver Disease.

Recent lines of evidence highlight the involvement of myeloid-epithelial-reproductive tyrosine kinase (MerTK) in metabolic disease associated with liver damage. MerTK is mainly expressed in anti-inflammatory M2 macrophages where it mediates transcriptional changes including suppression of proinflammatory cytokines and enhancement of inflammatory repressors. MerTK is regulated by metabolic pathways through nuclear sensors including LXRs, PPARs, and RXRs, in response to apoptotic bodies or to other sources of cholesterol. Nonalcoholic fatty liver disease (NAFLD) is one of the most serious public health problems worldwide. It is a clinicopathological syndrome closely related to obesity, insulin resistance, and oxidative stress. It includes a spectrum of conditions ranging from simple steatosis, characterized by hepatic fat accumulation with or without inflammation, to nonalcoholic steatohepatitis (NASH), defined by hepatic fat deposition with hepatocellular damage, inflammation, and accumulating fibrosis. Several studies support an association between NAFLD and the incidence of cardiovascular diseases including atherosclerosis, a major cause of death worldwide. This pathological condition consists in a chronic and progressive inflammatory process in the intimal layer of large- and medium-sized arteries. The complications of advanced atherosclerosis include chronic or acute ischemic damage in the tissue perfused by the affected artery, leading to cellular death. By identifying specific targets influencing lipid metabolism and cardiovascular-related diseases, the present review highlights the role of MerTK in NAFLD-associated atherosclerotic lesions as a potential innovative therapeutic target. Therapeutic advantages might derive from the use of compounds selective for nuclear receptors targeting PPARs rather than LXRs regulating macrophage lipid metabolism and macrophage mediated inflammation, by favoring the expression of MerTK, which mediates an immunoregulatory action with a reduction in inflammation and in atherosclerosis.