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    Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia. Mizuguchi Yoshiaki,Specht Susan,Isse Kumiko,Sasatomi Eizaburo,Lunz John G,Takizawa Toshihiro,Demetris Anthony J Journal of hepatology BACKGROUND & AIMS:Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers. METHODS:Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a. RESULTS:BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers. CONCLUSION:BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment. 10.1016/j.jhep.2015.02.047
    The new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant ROS1-G2032R mutation in preclinical models. Katayama Ryohei,Gong Bo,Togashi Noriko,Miyamoto Masaya,Kiga Masaki,Iwasaki Shiho,Kamai Yasuki,Tominaga Yuichi,Takeda Yasuyuki,Kagoshima Yoshiko,Shimizu Yuki,Seto Yosuke,Oh-Hara Tomoko,Koike Sumie,Nakao Naoki,Hanzawa Hiroyuki,Watanabe Kengo,Yoda Satoshi,Yanagitani Noriko,Hata Aaron N,Shaw Alice T,Nishio Makoto,Fujita Naoya,Isoyama Takeshi Nature communications ROS1 gene rearrangement was observed in around 1-2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1-rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1-rearranged cancers or NTRK-rearranged cancers in vitro and in vivo. Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors. 10.1038/s41467-019-11496-z
    Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study. Marabelle Aurelien,Le Dung T,Ascierto Paolo A,Di Giacomo Anna Maria,De Jesus-Acosta Ana,Delord Jean-Pierre,Geva Ravit,Gottfried Maya,Penel Nicolas,Hansen Aaron R,Piha-Paul Sarina A,Doi Toshihiko,Gao Bo,Chung Hyun Cheol,Lopez-Martin Jose,Bang Yung-Jue,Frommer Ronnie Shapira,Shah Manisha,Ghori Razi,Joe Andrew K,Pruitt Scott K,Diaz Luis A Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Genomes of tumors that are deficient in DNA mismatch repair (dMMR) have high microsatellite instability (MSI-H) and harbor hundreds to thousands of somatic mutations that encode potential neoantigens. Such tumors are therefore likely to be immunogenic, triggering upregulation of immune checkpoint proteins. Pembrolizumab, an anti‒programmed death-1 monoclonal antibody, has antitumor activity against MSI-H/dMMR cancer. We report data from the phase II KEYNOTE-158 study of pembrolizumab in patients with previously treated, advanced noncolorectal MSI-H/dMMR cancer. PATIENTS AND METHODS:Eligible patients with histologically/cytologically confirmed MSI-H/dMMR advanced noncolorectal cancer who experienced failure with prior therapy received pembrolizumab 200 mg once every 3 weeks for 2 years or until disease progression, unacceptable toxicity, or patient withdrawal. Radiologic imaging was performed every 9 weeks for the first year of therapy and every 12 weeks thereafter. The primary end point was objective response rate per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, as assessed by independent central radiologic review. RESULTS:Among 233 enrolled patients, 27 tumor types were represented, with endometrial, gastric, cholangiocarcinoma, and pancreatic cancers being the most common. Median follow up was 13.4 months. Objective response rate was 34.3% (95% CI, 28.3% to 40.8%). Median progression-free survival was 4.1 months (95% CI, 2.4 to 4.9 months) and median overall survival was 23.5 months (95% CI, 13.5 months to not reached). Treatment-related adverse events occurred in 151 patients (64.8%). Thirty-four patients (14.6%) had grade 3 to 5 treatment-related adverse events. Grade 5 pneumonia occurred in one patient; there were no other treatment-related fatal adverse events. CONCLUSION:Our study demonstrates the clinical benefit of anti-programmed death-1 therapy with pembrolizumab among patients with previously treated unresectable or metastatic MSI-H/dMMR noncolorectal cancer. Toxicity was consistent with previous experience of pembrolizumab monotherapy. 10.1200/JCO.19.02105
    YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors. Marti Patricia,Stein Claudia,Blumer Tanja,Abraham Yann,Dill Michael T,Pikiolek Monika,Orsini Vanessa,Jurisic Giorgia,Megel Philippe,Makowska Zuzanna,Agarinis Claudia,Tornillo Luigi,Bouwmeester Tewis,Ruffner Heinz,Bauer Andreas,Parker Christian N,Schmelzle Tobias,Terracciano Luigi M,Heim Markus H,Tchorz Jan S Hepatology (Baltimore, Md.) UNLABELLED:The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature. CONCLUSIONS:These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins. 10.1002/hep.27992
    Prohibitin 1 suppresses liver cancer tumorigenesis in mice and human hepatocellular and cholangiocarcinoma cells. Fan Wei,Yang Heping,Liu Ting,Wang Jiaohong,Li Tony W H,Mavila Nirmala,Tang Yuanyuan,Yang JinWon,Peng Hui,Tu Jian,Annamalai Alagappan,Noureddin Mazen,Krishnan Anuradha,Gores Gregory J,Martínez-Chantar Maria L,Mato José M,Lu Shelly C Hepatology (Baltimore, Md.) Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c-MYC interacts directly with both proteins. Furthermore, c-MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c-MYC and PHB1's role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down-regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each other's expression, whereas PHB1 negatively regulates the expression of c-MYC, MAFG, and c-MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E-box element, and repress E-box promoter activity. PHB1 promoter contains a repressive E-box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c-MYC, c-MAF, and MAFG in cancer cells and human HCC/CCA. All 8-month-old liver-specific Phb1 knockout mice developed HCC, and one developed CCA. Five-month-old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. CONCLUSION:We have identified that PHB1, down-regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E-box and positively regulates MAT1A while suppressing c-MYC, MAFG, and c-MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis-induced CCA. (Hepatology 2017;65:1249-1266). 10.1002/hep.28964
    Derazantinib for intrahepatic cholangiocarcinoma. Gourd Elizabeth The Lancet. Oncology 10.1016/S1470-2045(18)30891-X
    Mitogen-activated protein kinase kinase kinase 4 deficiency in intrahepatic cholangiocarcinoma leads to invasive growth and epithelial-mesenchymal transition. Yang Liu-Xiao,Gao Qiang,Shi Jie-Yi,Wang Zhi-Chao,Zhang Yong,Gao Ping-Ting,Wang Xiao-Ying,Shi Ying-Hong,Ke Ai-Wu,Shi Guo-Ming,Cai Jia-Bin,Liu Wei-Ren,Duan Meng,Zhao Ying-Jun,Ji Yuan,Gao Dong-Mei,Zhu Kai,Zhou Jian,Qiu Shuang-Jian,Cao Ya,Tang Qi-Qun,Fan Jia Hepatology (Baltimore, Md.) UNLABELLED:The molecular pathogenesis of intrahepatic cholangiocarcinoma (iCCA) is poorly understood, and its incidence continues to increase worldwide. Deficiency of mitogen-activated protein kinase kinase kinase 4 (MAP3K4) has been reported to induce the epithelial-mesenchymal transition (EMT) process of placental and embryonic development, yet its role in human cancer remains unknown. MAP3K4 has somatic mutation in iCCA so we sequenced all exons of MAP3K4 in 124 iCCA patients. We identified nine somatic mutations in 10 (8.06%) patients, especially in those with lymph node metastasis and intrahepatic metastasis. We also showed that messenger RNA and protein levels of MAP3K4 were significantly reduced in iCCA versus paired nontumor tissues. Furthermore, knockdown of MAP3K4 in cholangiocarcinoma cells markedly enhanced cell proliferation and invasiveness in vitro and tumor progression in vivo, accompanied by a typical EMT process. In contrast, overexpression of MAP3K4 in cholangiocarcinoma cells obviously reversed EMT and inhibited cell invasion. Mechanistically, MAP3K4 functioned as a negative regulator of EMT in iCCA by antagonizing the activity of the p38/nuclear factor κB/snail pathway. We found that the tumor-inhibitory effect of MAP3K4 was abolished by inactivating mutations. Clinically, a tissue microarray study containing 322 iCCA samples from patients revealed that low MAP3K4 expression in iCCA positively correlated with aggressive tumor characteristics, such as vascular invasion and intrahepatic or lymph node metastases, and was independently associated with poor survival and increased recurrence after curative surgery. CONCLUSIONS:MAP3K4, significantly down-regulated, frequently mutated, and potently regulating the EMT process in iCCA, was a putative tumor suppressor of iCCA. 10.1002/hep.28149
    SOX17 regulates cholangiocyte differentiation and acts as a tumor suppressor in cholangiocarcinoma. Merino-Azpitarte Maite,Lozano Elisa,Perugorria María J,Esparza-Baquer Aitor,Erice Oihane,Santos-Laso Álvaro,O'Rourke Colm J,Andersen Jesper B,Jiménez-Agüero Raúl,Lacasta Adelaida,D'Amato Mauro,Briz Óscar,Jalan-Sakrikar Nidhi,Huebert Robert C,Thelen Kristen M,Gradilone Sergio A,Aransay Ana M,Lavín José L,Fernández-Barrena Maite G,Matheu Ander,Marzioni Marco,Gores Gregory J,Bujanda Luis,Marin José J G,Banales Jesús M Journal of hepatology BACKGROUND & AIMS:Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS:SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. RESULTS:SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/β-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. CONCLUSIONS:SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. LAY SUMMARY:Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker. 10.1016/j.jhep.2017.02.017
    MicroRNA (miR)-433 and miR-22 dysregulations induce histone-deacetylase-6 overexpression and ciliary loss in cholangiocarcinoma. Mansini Adrian P,Lorenzo Pisarello Maria J,Thelen Kristen M,Cruz-Reyes Maetzin,Peixoto Estanislao,Jin Sujeong,Howard Brynn N,Trussoni Christy E,Gajdos Gabriella B,LaRusso Nicholas F,Perugorria Maria J,Banales Jesus M,Gradilone Sergio A Hepatology (Baltimore, Md.) Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6-targeting miRNAs, miR-433 and miR-22, are down-regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin-5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin-5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin-5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. CONCLUSION:These data suggest that down-regulated Exportin-5 impairs the nuclear export of miR-433 and miR-22 precursor forms, causing a decrease in levels of mature miR-433 and miR-22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018). 10.1002/hep.29832
    FoxO3 inactivation promotes human cholangiocarcinoma tumorigenesis and chemoresistance through Keap1-Nrf2 signaling. Guan Li,Zhang Lei,Gong Zhicheng,Hou Xiaonan,Xu Yuxiu,Feng Xinhua,Wang Hongyang,You Han Hepatology (Baltimore, Md.) UNLABELLED:FoxO transcription factors have been reported to play pivotal roles in tumorigenesis and drug resistance. The mechanisms underlying the tumor suppression function of FoxOs in human cancers remain largely unknown. Aberrant expression and activation of Nrf2 often correlate with chemoresistance and poor prognosis. Here, we report that FoxO3 directs the basal transcription of Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein that bridges Nrf2 to Cul3 for degradation. FoxO3 depletion resulted in Keap1 down-regulation, thereby activating Nrf2 signaling. We further demonstrated that inhibition of the FoxO3-Keap1 axis accounts for Nrf2 induction and activation induced by constitutively active AKT signaling or tumor necrosis factor α treatment. Unlike previous findings, FoxO3 silencing led to decreased reactive oxygen species production, therefore protecting cells from oxidative stress-induced killing in an Nrf2-dependent manner. Importantly, FoxO3 deficiency strongly potentiated tumor formation in nude mice and rendered cholangiocarcinoma xenografts resistant to cisplatin-induced cell death by activating Nrf2. Additionally, we found that clinical cholangiocarcinoma samples displayed FoxO3-Keap1 down-regulation and Nrf2 hyperactivation, underscoring the essential roles of these proteins in cholangiocarcinoma development. CONCLUSION:Our results unravel a unique mechanism underlying the tumor suppressor function of FoxO3 through constraining Nrf2 signaling. (Hepatology 2016;63:1914-1927). 10.1002/hep.28496
    Global trends in mortality from intrahepatic and extrahepatic cholangiocarcinoma. Khan Shahid A,Genus Tracey,Morement Helen,Murphy Andrew,Rous Brian,Tataru Daniela Journal of hepatology 10.1016/j.jhep.2019.07.024
    MicroRNA-191 acts as a tumor promoter by modulating the TET1-p53 pathway in intrahepatic cholangiocarcinoma. Li Hao,Zhou Zun-Qiang,Yang Zhang-Ru,Tong Da-Nian,Guan Jiao,Shi Bao-Jie,Nie Jia,Ding Xian-Ting,Li Bin,Zhou Guang-Wen,Zhang Zheng-Yun Hepatology (Baltimore, Md.) Current treatment of intrahepatic cholangiocarcinoma (ICC) remains ineffective because knowledge of ICC carcinogenesis is unclear. Increasing evidence suggests that microRNAs (miRNAs), including miR-191, play an important role in tumorigenesis; but expression and biological functions of miR-191 in ICC remain to be established. This study investigated the functions and underlying mechanisms of miR-191 in ICC. ICC miRNA profiles were generated in five pairs of ICC and matched to normal bile duct tissues by next-generation sequencing technology; ICC miRNA profiles were verified in 18 pairs of ICC tissues and normal bile duct tissues by quantitative RT-PCR. The miR-191-associated mechanisms in ICC were investigated in vitro and in vivo, and clinical outcomes associated with miR-191 were correlated in 84 patients. Our results showed that miR-191 expression was significantly increased in ICC compared with the adjacent normal bile duct tissues (P < 0.001). Overexpression of miR-191 promoted proliferation, invasion, and migration of cholangiocarcinoma cells in vitro and in vivo. The elevated miR-191 expression reduced the expression level of ten-eleven translocation 1 (TET1)-a direct target gene of miR-191 in ICC, which catalyzes demethylation. The reduced TET1 expression level allowed the methylated CpG-rich regions at the p53 gene transcription start site stay methylated, leading to reduced p53 expression level, which compromises p53's anticancer vigor. Finally, miR-191 was found to be an independent risk factor for poor prognosis in patients with ICC (overall survival, hazard ratio = 3.742, 95% confidence interval 2.080-6.733, P < 0.001; disease-free survival, hazard ratio = 2.331, 95% confidence interval 1.346-4.037, P = 0.003). CONCLUSION:Our results suggest that overexpressed miR-191 is associated with ICC progression through the miR-191/TET1/p53 pathway. (Hepatology 2017;66:136-151). 10.1002/hep.29116
    Towards greater clarity in the treatment of cholangiocarcinoma. de la Fouchardiere Christelle The Lancet. Oncology 10.1016/S1470-2045(20)30214-X
    Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma. Razumilava Nataliya,Gradilone Sergio A,Smoot Rory L,Mertens Joachim C,Bronk Steven F,Sirica Alphonse E,Gores Gregory J Journal of hepatology 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. 10.1016/j.jhep.2013.11.005
    Dependency of Cholangiocarcinoma on Cyclin D-Dependent Kinase Activity. Sittithumcharee Gunya,Suppramote Orawan,Vaeteewoottacharn Kulthida,Sirisuksakun Chumphon,Jamnongsong Supawan,Laphanuwat Phatthamon,Suntiparpluacha Monthira,Matha Arriya,Chusorn Porncheera,Buraphat Pongsakorn,Kakanaporn Chumpot,Charngkaew Komgrid,Silsirivanit Atit,Korphaisarn Krittiya,Limsrichamrern Somchai,Tripatara Pinpat,Pairojkul Chawalit,Wongkham Sopit,Sampattavanich Somponnat,Okada Seiji,Jirawatnotai Siwanon Hepatology (Baltimore, Md.) Cholangiocarcinoma (CCA) is a bile duct cancer with a very poor prognosis. Currently, there is no effective pharmacological treatment available for it. We showed that CCA ubiquitously relies on cyclin-dependent kinases 4 and 6 (CDK4/6) activity to proliferate. Primary CCA tissues express high levels of cyclin D1 and the specific marker of CDK4/6 activity, phospho-RB Ser780. Treatment of a 15-CCA cell line collection by pharmacological CDK4/6 inhibitors leads to reduced numbers of cells in the S-phase and senescence in most of the CCA cell lines. We found that expression of retinoblastoma protein (pRB) is required for activity of the CDK4/6 inhibitor, and that loss of pRB conferred CDK4/6 inhibitor-drug resistance. We also identified that sensitivity of CCA to CDK4/6 inhibition is associated with the activated KRAS signature. Effectiveness of CDK4/6 inhibition for CCA was confirmed in the three-dimensional spheroid-, xenograft-, and patient-derived xenograft models. Last, we identified a list of genes whose expressions can be used to predict response to the CDK4/6 inhibitor. Conclusion: We investigated a ubiquitous dependency of CCA on CDK4/6 activity and the universal response to CDK4/6 inhibition. We propose that the CDK4/6-pRB pathway is a suitable therapeutic target for CCA treatment. 10.1002/hep.30704
    Identification of a Pan-Gamma-Secretase Inhibitor Response Signature for Notch-Driven Cholangiocarcinoma. O'Rourke Colm J,Matter Matthias S,Nepal Chirag,Caetano-Oliveira Rui,Ton Phuongnga T,Factor Valentina M,Andersen Jesper B Hepatology (Baltimore, Md.) Cholangiocarcinoma (CCA) mortality rates are increasing as a result of rising incidence and limited curative treatment(s) for patients with advanced disease. NOTCH pathway reactivation has been reported in biliary malignancies to conflicting degrees, hindering prioritization of key therapeutic targets within the network and identification of candidate responder patients for NOTCH-directed therapies. We analyzed genomic data from 341 patients with CCA and identified NOTCH1 significantly increased in a subgroup characterized by distinct stromal infiltration. Network-wide imbalance of the NOTCH pathway was seen in CCA, including correlation of NOTCH1 with NOTCH3 and NOTCH ligands. Given the diversity of observed NOTCH receptor engagement, γ-secretase modulation was rationalized as a therapeutic option. Indeed, subcutaneous transplantation of sensitive and resistant CCA cell lines pretreated with a γ-secretase inhibitor (GSi) cocktail demonstrated the antineoplastic effects of GSi in a subset of CCA and led to the development of a 225-gene responder signature. This signature was validated in an independent cohort of 119 patients. Further, this signature was enriched in liver tumors initiated by hydrodynamic injections of activated-NOTCH as compared with the AKT-RAS-driven tumors. Candidate GSi-responder patients were characterized by distinct transcriptomes overlapping with previous hepatobiliary metastasis and stemness, unique stromal properties, and dysfunctional intratumoral immune infiltration. Pan-cancer analysis identified 41.9% of cancer types to harbor prospective GSi-responder patients, which was adapted into a 20-gene GSi-sensitivity score metric capable of discriminating nanomolar versus micromolar sensitivity to a cell-permeable GSi (Z-LLNle-CHO) across 60 diverse tumor lines (area under the curve = 1). Conclusion: We have established a GSi-responder signature with evidence across several patient cohorts, as well as in vitro and in vivo models, to enable precision medicine application of NOTCH-directed therapy in CCA as well as prospectively across diverse malignancies. 10.1002/hep.30816
    Epithelial Transforming Growth Factor-β Signaling Does Not Contribute to Liver Fibrosis but Protects Mice From Cholangiocarcinoma. Mu Xueru,Pradere Jean-Philippe,Affò Silvia,Dapito Dianne H,Friedman Richard,Lefkovitch Jay H,Schwabe Robert F Gastroenterology BACKGROUND & AIMS:Transforming growth factor-β (TGFβ) exerts key functions in fibrogenic cells, promoting fibrosis development in the liver and other organs. In contrast, the functions of TGFβ in liver epithelial cells are not well understood, despite their high level of responsiveness to TGFβ. We sought to determine the contribution of epithelial TGFβ signaling to hepatic fibrogenesis and carcinogenesis. METHODS:TGFβ signaling in liver epithelial cells was inhibited by albumin-Cre-, K19-CreERT-, Prom1-CreERT2-, or AAV8-TBG-Cre-mediated deletion of the floxed TGFβ receptor II gene (Tgfbr2). Liver fibrosis was induced by carbon tetrachloride, bile duct ligation, or disruption of the multidrug-resistance transporter 2 gene (Mdr2). Hepatocarcinogenesis was induced by diethylnitrosamine or hepatic deletion of PTEN. RESULTS:Deletion of Tgfbr2 from liver epithelial cells did not alter liver injury, toxin-induced or biliary fibrosis, or diethylnitrosamine-induced hepatocarcinogenesis. In contrast, epithelial deletion of Tgfbr2 promoted tumorigenesis and reduced survival of mice with concomitant hepatic deletion of Pten, accompanied by an increase in tumor number and a shift from hepatocellular carcinoma to cholangiocarcinoma. Surprisingly, both hepatocyte- and cholangiocyte-specific deletion of Pten and Tgfbr2 promoted the development of cholangiocarcinoma, but with different latencies. The prolonged latency and the presence of hepatocyte-derived cholangiocytes after AAV8-TBG-Cre-mediated deletion of Tgfbr2 and Pten indicated that cholangiocarcinoma might arise from hepatocyte-derived cholangiocytes in this model. Pten deletion resulted in up-regulation of Tgfbr2, and deletion of Tgfbr2 increased cholangiocyte but not hepatocyte proliferation, indicating that the main function of epithelial TGFBR2 is to restrict cholangiocyte proliferation. CONCLUSIONS:Epithelial TGFβ signaling does not contribute to the development of liver fibrosis or formation of hepatocellular carcinomas in mice, but restricts cholangiocyte proliferation to prevent cholangiocarcinoma development, regardless of its cellular origin. 10.1053/j.gastro.2015.11.039
    Targeting tumor-associated macrophages and granulocytic myeloid-derived suppressor cells augments PD-1 blockade in cholangiocarcinoma. Loeuillard Emilien,Yang Jingchun,Buckarma EeeLN,Wang Juan,Liu Yuanhang,Conboy Caitlin,Pavelko Kevin D,Li Ying,O'Brien Daniel,Wang Chen,Graham Rondell P,Smoot Rory L,Dong Haidong,Ilyas Sumera The Journal of clinical investigation Immune checkpoint blockade (ICB) has revolutionized cancer therapeutics. Desmoplastic malignancies, such as cholangiocarcinoma (CCA), have an abundant tumor immune microenvironment (TIME). However, to date, ICB monotherapy in such malignancies has been ineffective. Herein, we identify tumor-associated macrophages (TAMs) as the primary source of programmed death-ligand 1 (PD-L1) in human and murine CCA. In a murine model of CCA, recruited PD-L1+ TAMs facilitated CCA progression. However, TAM blockade failed to decrease tumor progression due to a compensatory emergence of granulocytic myeloid-derived suppressor cells (G-MDSCs) that mediated immune escape by impairing T cell response. Single-cell RNA sequencing (scRNA-Seq) of murine tumor G-MDSCs highlighted a unique ApoE G-MDSC subset enriched with TAM blockade; further analysis of a human scRNA-Seq data set demonstrated the presence of a similar G-MDSC subset in human CCA. Finally, dual inhibition of TAMs and G-MDSCs potentiated ICB. In summary, our findings highlight the therapeutic potential of coupling ICB with immunotherapies targeting immunosuppressive myeloid cells in CCA. 10.1172/JCI137110
    Expert consensus document: Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA). Banales Jesus M,Cardinale Vincenzo,Carpino Guido,Marzioni Marco,Andersen Jesper B,Invernizzi Pietro,Lind Guro E,Folseraas Trine,Forbes Stuart J,Fouassier Laura,Geier Andreas,Calvisi Diego F,Mertens Joachim C,Trauner Michael,Benedetti Antonio,Maroni Luca,Vaquero Javier,Macias Rocio I R,Raggi Chiara,Perugorria Maria J,Gaudio Eugenio,Boberg Kirsten M,Marin Jose J G,Alvaro Domenico Nature reviews. Gastroenterology & hepatology Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the "European Network for the Study of Cholangiocarcinoma" (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted. 10.1038/nrgastro.2016.51
    ID3 Promotes Stem Cell Features and Predicts Chemotherapeutic Response of Intrahepatic Cholangiocarcinoma. Huang Lifeng,Cai Jie,Guo Han,Gu Jinyang,Tong Ying,Qiu Bijun,Wang Chenchen,Li Meng,Xia Lei,Zhang Jianjun,Wu Hailong,Kong Xiaoni,Xia Qiang Hepatology (Baltimore, Md.) Cancer stem cells contribute to a high rate of recurrence and chemotherapeutic resistance in many types of cancer, including intrahepatic cholangiocarcinoma (ICC). Inhibitor of differentiation 3 (ID3) has been reported to promote cancer stem cells, but its role in ICC is obscure. In this study, we identified that ID3 is highly expressed in human ICC tissues compared with matched normal tissues and correlates with poor prognosis. Functional studies demonstrate that ID3 is required for stemness maintenance in cholangiocarcinoma both in vitro and in vivo. Consistent with the regulation of cancer stem cell features by ID3, transgenic expression of ID3 enhances chemoresistance of cholangiocarcinoma cells. Moreover, we found that ICC patients with low ID3 levels benefited from postoperative transarterial chemoembolization, whereas patients with high ID3 levels did not, indicating the significance of ID3 in individualized ICC therapy. Mechanistically, ID3 could interact with E47 and block E47 recruitment to the promoter of β-catenin, which leads to activation of Wnt/β-catenin signaling. Conclusion: Our results show that ID3 could promote the stemness of ICC by increasing the transcriptional activity of β-catenin and could serve as a biomarker in predicting ICC patients' response to adjuvant chemotherapeutics. 10.1002/hep.30404
    Nab-Paclitaxel and Gemcitabine as First-line Treatment of Advanced or Metastatic Cholangiocarcinoma: A Phase 2 Clinical Trial. Sahai Vaibhav,Catalano Paul J,Zalupski Mark M,Lubner Sam Joseph,Menge Mark R,Nimeiri Halla Sayed,Munshi Hidayatullah G,Benson Al Bowen,O'Dwyer Peter J JAMA oncology Importance:Gemcitabine with platinum has limited efficacy for treatment of advanced cholangiocarcinoma, necessitating an evaluation of alternative drug combinations. Recent evidence suggests that paclitaxel may potentiate gemcitabine activity. Objective:To evaluate whether gemcitabine plus nanoparticle albumin-bound (nab)-paclitaxel is safe and effective for treatment of advanced cholangiocarcinoma. Design, Setting, and Participants:This single-arm, 2-stage, phase 2 clinical trial was conducted at 23 community and academic centers across the United States and Europe. Patients aged 18 years or older enrolled between September 2014 and March 2016 had confirmed advanced or metastatic cholangiocarcinoma without prior systemic therapy, and had an Eastern Cooperative Oncology Group Performance Status score of 0 to 1 and a Child-Pugh score less than 8. Previous surgery, radiation, or liver-directed therapies were permitted. Interventions:Patients received intravenous nab-paclitaxel, 125 mg/m2, followed by gemcitabine, 1000 mg/m2, on days 1, 8, and 15 of each 28-day treatment cycle until disease progression or unacceptable toxic effects. Main Outcomes and Measures:The primary outcome was improvement in 6-month progression-free survival (PFS) rate (null and alternative hypotheses of 55% and 70%, respectively) in the evaluable population. Secondary outcomes included median overall survival (OS), PFS, time to progression, best overall response rate, disease control rate, safety and toxicity, and association of change in carbohydrate antigen 19-9 with survival. Results:Seventy-four patients with a median age of 62 (range, 36-87) years, including 44 women (60%), were enrolled. Patients received a median of 6 (range, 1-18) treatment cycles, and the median follow-up was 10.2 (range, 0.6-27.3) months. The observed 6-month PFS rate of 61% (95% CI, 48%-73%) did not favor the alternative hypothesis. Median PFS was 7.7 (95% CI, 5.4-13.1) months, median OS was 12.4 (95% CI, 9.2-15.9) months, and median time to progression was 7.7 (95% CI, 6.1-13.1) months. The confirmed best overall response rate and disease control rate were 30% and 66%, respectively. Hazard ratios for an association between a change in serum carbohydrate antigen 19-9 and median PFS as well as median OS were 2.02 (95% CI, 0.86-4.75) (P = .10) and 1.54 (95% CI, 0.64-3.71) (P = .34), respectively. The most common treatment-related hematologic and nonhematologic adverse events at grade 3 or higher were neutropenia (43%) and fatigue (14%), respectively. Conclusions and Relevance:Although the trial did not meet its primary efficacy end point, the results indicate that a nab-paclitaxel plus gemcitabine regimen was well tolerated and may be an alternative option to current therapeutic approaches for advanced cholangiocarcinoma. Trial Registration:ClinicalTrials.gov identifier: NCT02181634. 10.1001/jamaoncol.2018.3277
    Global trends in mortality from intrahepatic and extrahepatic cholangiocarcinoma. Bertuccio Paola,Malvezzi Matteo,Carioli Greta,Hashim Dana,Boffetta Paolo,El-Serag Hashem B,La Vecchia Carlo,Negri Eva Journal of hepatology BACKGROUND & AIMS:Intrahepatic (ICC) and extrahepatic cholangiocarcinoma (ECC) have rarely been studied individually, probably due to difficulties in their diagnosis and certification. Mortality trends from these 2 neoplasms have been inconsistent over the last decades. The aim of this study was to analyze worldwide trends in mortality from ICC and ECC in selected countries. METHODS:We extracted death certification data for ICC and ECC, and population estimates from the World Health Organization and Pan American Health Organization databases for 32 selected countries from Europe, the Americas, and Australasia from 1995 to 2016. We computed age-standardized (world population) mortality rates from ICC and ECC, and performed joinpoint regression analysis. RESULTS:Mortality rates from ICC increased in all countries considered, with a levelling off over recent years in Germany (women), Italy (men), Argentina (men), the USA (men), Hong Kong (men), and Japan (both sexes). The highest rates in 2010-2014 (1.5-2.5/100,000 in men and 1.2-1.7/100,000 in women) were registered in Hong Kong, France, Austria, Spain, the UK, and Australia. The lowest rates (0.2-0.6/100,000 in both sexes) were registered in Latin American and eastern European countries. Mortality from ECC decreased in most of the countries considered, with rates below 1/100,000 in both sexes between 2010 and 2014, with the only exception being Japan (2.8/100,000 in men and 1.4/100,000 in women). CONCLUSIONS:Increasing mortality from ICC was observed globally, due to trends in risk factors and possibly, in part, due to better disease classification. Mortality from ECC levelled off or decreased, most likely following the increased use of laparoscopic cholecystectomy. LAY SUMMARY:Biliary tract cancers include intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC), however there are some differences in their risk factors. Consequently, the distinction between ICC and ECC is important. Over the last few decades, mortality from ICC has tended to rise in several areas of the world, following the increased prevalence of its major risk factors. In contrast, mortality from ECC tended to decrease in most countries, following the increased use of laparoscopic cholecystectomy. 10.1016/j.jhep.2019.03.013