Human Biliary Tract Cancer Cell Research Articles

Exosomal miR-141-3p Induces Gemcitabine Resistance in Biliary Tract Cancer Cells.

Gemcitabine (GEM)-based chemotherapy has been established as the core multimodal therapy for biliary tract cancer (BTC). However, the prognosis of BTC is unfavorable because of its resistance to GEM. Exosomes play important roles in the regulation of tumor progression and metastasis, immune dysregulation, and chemoresistance. This study investigated the effects of exosomes on GEM resistance in BTC. The human intrahepatic cholangiocarcinoma cell line CC-LP-1, its GEM-resistant (GR) derivative cell line CC-LP-1-GR, and the human intrahepatic cholangiocarcinoma cell lines HuCCA-1 and HuCCT1, were used. GEM resistance was examined by measuring cell viability in the presence of GEM using an MTS assay. Exosomes were isolated using ultracentrifugation and quantified using ELISA. Comprehensive expression analysis was performed using RNA sequencing. The effects of microRNAs were examined by miRNA mimic transfection. The conditioned medium and exosomes derived from CC-LP-1-GR cells enhanced the GEM resistance of parental CC-LP-1 cells. In the presence of GEM, the p53 pathway was negatively enriched in CC-LP-1-GR and CC-LP-1 cells treated with exosomes from CC-LP-1-GR (rExo) compared to CC-LP-1 cells. The expression of miR-141-3p was higher in rExos than in CC-LP-1 cells. CC-LP-1 cells transfected with miR-141-3p mimic showed significantly (p<0.05) increased viability in the presence of GEM. A GEM-resistant human BTC cell line, CC-LP-1-GR, may acquire resistance to GEM by exosomes containing miR-141-3p.

Abstract 1097: Surufatinib improved the radiation sensitivity of biliary cancer growth by reprograming of TAM and anti-angiogenesis

Abstract Background: Patients with Biliary tract cancer (BTC) have poor clinical outcomes. Radiation therapy is an adjunct strategy throughout biliary tract cancer treatment. The aim of the study was to explore the efficacy and mechanism of the radiosensitization of surufatinib(SF), an oral tyrosine kinase inhibitor with anti-angiogenic and immunomodulatory activities. Methods: The NOZ and TFK1 human BTC cell lines were treated with SF and/or radiation. Effects on cell viability and cell cycle progression were measured by CCK8, a clonogenic survival assay, and flow cytometry. Finally, the in vivo effect on the growth of NOZ xenografts was assessed in nude mice. F4/80, CD206, Ki67 and CD31 expression were employed in tumor samples. Results: SF inhibited the proliferation and enhanced the radiosensitivity in NOZ and TFK1 cells lines. SF combined with radiation increased cell cycle arrest in the G2/M phase compared to the other treatments in the NOZ cell line (P&amp;lt;0.05). Similarly, cell cycle arrest occurred in the TFK1 cell line, (P&amp;lt;0.05). In vivo, SF plus radiation significantly inhibited the tumor growth compared to either agent independently. Conclusions: SF significantly increased the radiosensitivity of NOZ and TFK1 cells in vitro and in vivo. Radiation combined with SF inhibited tumor proliferation, increased apoptosis, prolonged G2/M arrest. SF decreased the number of blood vessels and reprogrammed the macrophages from M2 to M1. These data support the clinical trials of biologically targeted and conventional therapies in the treatment of cancer. Citation Format: Hong Ma, Ai Huang, Yong Xiao, Jing Yao, Ningyu Wang, Xiangping Mei, Shaoshi Wen, Xing Lv. Surufatinib improved the radiation sensitivity of biliary cancer growth by reprograming of TAM and anti-angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1097.

ACAT-1-Regulated Cholesteryl Ester Accumulation Modulates Gemcitabine Resistance in Biliary Tract Cancer.

Biliary tract cancer (BTC) has few choices of chemotherapy, including gemcitabine, therefore exploring the mechanisms of gemcitabine resistance is important. We focused on lipid metabolism because biliary tract epithelial cells are essential in cholesterol and bile acid metabolism and the messenger RNA (mRNA) microarray analysis showed high acyl coenzyme A: cholesterol acyltransferase 1 (ACAT-1) expression in BTC gemcitabine-resistant (GR) cell lines. We hypothesized that aberrant accumulation of cholesteryl ester (CE) regulated by ACAT-1 could modulate GR in BTC. CE accumulations were measured in human BTC cell lines, and the relationships between CE levels, ACAT-1 expressions, and gemcitabine sensitivity were analyzed. We performed a small-interfering RNA (siRNA)-mediated knockdown and biochemical inhibition of ACAT-1 in BTC cell lines and alterations of gemcitabine sensitivity were evaluated. To evaluate the clinical significance of ACAT-1 in regard to GR, immunohistochemistry was performed and ACAT-1 expressions were analyzed in resected BTC specimens. CE levels were correlated with ACAT-1 expressions and GR in four human BTC cell lines. siRNA-mediated knockdown of ACAT-1 in two independent GR cell clones as well as ACAT-1 inhibitor treatment significantly increased gemcitabine sensitivity; knockdown of ACAT-1: 5.63- and 8.02-fold; ACAT-1 inhibitor: 8.75- and 9.13-fold, respectively. ACAT-1 expression in resected BTC specimens revealed that the disease-free survival of the ACAT-1 low-intensity group (median 2.3 years) had a significantly better outcome than that of the ACAT-1 high-intensity group (median 1.1years) under gemcitabine treatment after surgery (*p<0.05). Our findings suggest that CE and ACAT-1 might be a novel therapeutic target for GR in BTC.

Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease

BackgroundBTC is an aggressive disease exacerbated by inflammation and immune suppression. Expansion of immunosuppressive cells occurs in biliary tract cancer (BTC), yet the role of BTC-derived cytokines in this process is unclear.MethodsActivated signalling pathways and cytokine production were evaluated in a panel of human BTC cell lines. Human peripheral blood mononuclear cells (PBMCs) were cultured with BTC supernatants, with and without cytokine neutralising antibodies, and analysed by flow cytometry or immunoblot. A human BTC tissue microarray (TMA, n = 69) was stained for IL-6, GM-CSF, and CD33+S100a9+ cells and correlated with clinical outcomes.ResultsImmunomodulatory factors (IL-6, GM-CSF, MCP-1) were present in BTC supernatants. BTC supernatants expanded CD33dimCD11b+HLA-DRlow/− myeloid-derived suppressor cells (MDSCs) from human PBMCs. Neutralisation of IL-6 and GM-CSF in BTC supernatants inhibited activation of STAT3/5, respectively, in PBMCs, with heterogeneous effects on MDSC expansion in vitro. Staining of a BTC TMA revealed a positive correlation between IL-6 and GM-CSF, with each cytokine and more CD33+S100a9+ cells. Increased CD33+S100a9+ staining positively correlated with higher tumour grade, differentiation and the presence of satellite lesions.ConclusionBTC-derived factors promote suppressive myeloid cell expansion, and higher numbers of CD33+S100a9+ cells in resectable BTC tumours correlates with more aggressive disease.

Therapeutic Targeting of the DNA Damage Response Using an ATR Inhibitor in Biliary Tract Cancer.

PurposeThe DNA damage response (DDR) is a multi-complex network of signaling pathways involved in DNA damage repair, cell cycle checkpoints, and apoptosis. In the case of biliary tract cancer (BTC), the strategy of DDR targeting has not been evaluated, even though many patients have DNA repair pathway alterations. The purpose of this study was to test the DDR-targeting strategy in BTC using an ataxia-telangiectasia and Rad3-related (ATR) inhibitor.Materials and MethodsA total of nine human BTC cell lines were used for evaluating anti-tumor effect of AZD6738 (ATR inhibitor) alone or combination with cytotoxic chemotherapeutic agents through MTT assay, colony-forming assays, cell cycle analyses, and comet assays. We established SNU478-mouse model for in vivo experiments to confirm our findings.ResultsAmong nine human BTC cell lines, SNU478 and SNU869 were the most sensitive to AZD6738, and showed low expression of both ataxia-telangiectasia mutated (ATM) and p53. AZD6738 blocked p-Chk1 and p-glycoprotein and increased γH2AX, a marker of DNA damage, in sensitive cells. AZD6738 significantly increased apoptosis, G2/M arrest and p21, and decreased CDC2. Combinations of AZD6738 and cytotoxic chemotherapeutic agents exerted synergistic effects in colony-forming assays, cell cycle analyses, and comet assays. In our mouse models, AZD6738 monotherapy decreased tumor growth and the combination with cisplatin showed more potent effects on growth inhibition, decreased Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling than monotherapy with each drug.ConclusionIn BTC, DDR targeting strategy using ATR inhibitor demonstrated promising antitumor activity alone or in combination with cytotoxic chemotherapeutic agents. This supports further clinical development of DDR targeting strategy in BTC.

Anti-tumor effects of NVP-BKM120 alone or in combination with MEK162 in biliary tract cancer

There are currently no clinically validated therapeutic targets for biliary tract cancer (BTC). Despite promising results in other cancers, compounds targeting the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, alone or in combination with Ras/Raf/MEK pathway inhibitors, have not been evaluated in BTC. Here, we examined the effects of a pan-PI3K inhibitor (BKM120) with or without a MEK inhibitor (MEK162), on eight human BTC cell lines carrying mutations in K-Ras and/or the PI3K catalytic subunit, PI3KCA. BKM120 inhibited the colony-forming ability and migration of BTC cells carrying wild-type (WT) PI3KCA and either mutant (MT) or WT K-Ras, but not of cells carrying mutations in both genes. In K-Ras-WT cells, BKM120 decreased the phosphorylation of Akt, its downstream effector kinase p70S6K, and the translational repressor 4E-BP1. Interestingly, BKM120 did not induce cell cycle arrest or suppress PI3K signaling via restoration of p-4E-BP1 in cells with PIK3CA and K-Ras double mutations. Notably, the resistance of dual K-Ras/PI3KCA-mutant cells to BKM120 was overcome by treatment with a combination of BKM120 and MEK162. Our findings thus support the clinical development of BKM120 monotherapy or BKM120/MEK162 combination therapy for the treatment of BTC.

IMP2/IGF2BP2 expression, but not IMP1 and IMP3, predicts poor outcome in patients and high tumor growth rate in xenograft models of gallbladder cancer.

Overexpression of the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2/IGF2BP2) has been described in different cancer types. Gallbladder carcinoma (GBC) is a rare but highly aggressive cancer entity with late clinical detection and poor prognosis.The aim of this study was to investigate the role of IMP2 in human GBC.Tissue microarrays (TMAs) of an international multi-center GBC sample collection from n = 483 patients were analyzed by immunohistochemistry. IMP2 immunoreactivity was found in 74.3% of the tumor samples on TMA, of which 14.0% showed strong and 86.0% low staining intensity. 72.4% of the tumor samples were IMP1 positive, but IMP1 showed lower expression in tumor tissue compared to control tissues. IMP3 immunoreactivity was observed in 92.7% of all tumors, of which 53.6% revealed strong IMP3 expression. Kaplan-Meier analysis linked high IMP2 expression to shorter survival time (p = 0.033), whereas neither IMP1 nor IMP3 expression was linked to a decreased survival time. Eight different human biliary tract cancer (BTC) cell lines were evaluated for tumor growth kinetics in mouse xenografts. Cell lines with high IMP2 expression levels showed the fastest increase in tumor volumes in murine xenografts. Furthermore, IMP2 expression in these cells correlated with the generation of reactive oxygen species (ROS) and RAC1 expression in BTC cells, suggesting RAC1-induced ROS generation as a potential mechanism of IMP2-promoted progression of GBC.In conclusion, IMP2 is frequently overexpressed in GBC and significantly associated with poor prognosis and growth rates in vivo. IMP2 might therefore represent a new target for the treatment of advanced GBC.

Clinical implications of APEX1 and Jagged1 as chemoresistance factors in biliary tract cancer

PurposeBiliary cancer is a highly malignant neoplasm with poor prognosis and most patients need to undergo palliative chemotherapy, however major clinical problem associated with the use of chemotherapy is chemoresistance. So far, we aimed at investigating clinical implications of apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and Jagged1 as chemoresistance factors in biliary tract cancer.MethodsWe used 5 human biliary tract cancer cell lines (SNU-245, SNU-308, SNU-478, SNU-1079, and SNU-1196), and investigated the chemosensitivity of APEX1 and Jagged1 through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and Western blot. Alternately, the 10 patients of advanced biliary cancer consist of 2 group according to the chemotherapy response examined by immunohistochemistry using APEX1 and Jagged1 antibody, and protein expression level was scored for staining intensity and percent positive cell.ResultsThe result of MTT assay after APEX1 knockdown showed that strong coexpression of APEX1 and Jagged1 cell line (SNU-245, SNU-1079, and SNU-1196) showed a greater decrease in IC50 of chemotherapeutic agent (5-fluorouracil, gemcitabine and cisplatin). The Western blot analysis of APEX1 and Jagged1 expression in biliary cancer cell lines after APEX1 knockdown definitively demonstrated decreased Jagged1 expression. The APEX1 and Jagged1expression level of immunohistochemistry represented that chemorefractory patients had higher than chemoresponsive patients.ConclusionThese results demonstrate that simultaneous high expression of APEX1 and Jagged1 is associated with chemoresistance in biliary cancer and suggest that is a potential therapeutic target for chemoresistance in advanced biliary cancer.

Src as a Therapeutic Target in Biliary Tract Cancer.

Src, a nonreceptor tyrosine kinase, is involved in a number of cancer-related signaling pathways and aberrantly activated in biliary tract cancer (BTC). This study aimed to elucidate the potential role of Src as a therapeutic target in BTC. We tested bosutinib, an orally active c-Src/Abl kinase inhibitor, alone or in combination with cytotoxic agents using 9 human BTC cell lines: SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079, SNU-1196, HuCCT1, TFK-1, and EGI-1. Of these, SNU-308 and SNU-478 were relatively sensitive to bosutinib. Bosutinib abrogated phosphorylation of Src and its downstream molecules, and significantly increased G1 cell-cycle arrest and apoptosis. Bosutinib significantly inhibited cell migration and invasion and decreased epithelial-mesenchymal transition markers. Bosutinib combined with gemcitabine or cisplatin showed synergistic antiproliferative and antimigratory effects. In addition, this combination further inhibited phosphorylation of Src and its downstream molecules and decreased epithelial-mesenchymal transition marker expression compared with bosutinib alone. We established a SNU-478 xenograft model for in vivo experiments, because SNU-478 was more tumorigenic than SNU-308. Bosutinib combined with gemcitabine or cisplatin showed significantly more potent antitumor effects than bosutinib alone. Bosutinib combined with gemcitabine further decreased Ki-67 expression and Src phosphorylation, and further increased TUNEL expression. Our data suggest that Src might be a potential therapeutic target in BTC. Bosutinib demonstrated promising antitumor activity alone or in combination with gemcitabine or cisplatin in BTC cells, which supports further clinical development in patients with advanced BTC. Mol Cancer Ther; 15(7); 1515-24. ©2016 AACR.

Bile acid accelerates erbB2-induced pro-tumorigenic activities in biliary tract cancer.

Although very few studies have addressed the molecular and cellular mechanisms underlying the development of biliary tract cancer (BTC), several lines of evidence suggest a role for the erbB receptor family. Overexpression and activation of erbB2 has been reported in a significant percentage of human BTC. Further, we previously reported that overexpression of erbB2 basal epithelial cells of the biliary tract (BK5.erbB2 mouse) led to the development of BTC. However, the mechanisms by which erbB2 overexpression led to the spontaneous development of tumors specifically in the biliary tract are not completely understood. The goals of the current study were to (1) determine whether a cooperative relationship between bile acid exposure and erbB2 activation exists during biliary tract carcinogenesis and (2) to characterize the mechanism(s) underlying bile acid-mediated biliary tract carcinogenesis in cells with activated erbB2. In this study, we demonstrated that the secondary conjugated bile acid, taurochenodeoxycholic acid (TCDC), increased proliferation of primary cultured gallbladder epithelial cells from BK5.erbB2 mice and human BTC cells. TCDC treatment activated EGFR/erbB2 and downstream signaling molecules in both primary cultured cells and human BTC cells. TCDC also increased the expression of epidermal growth factor receptor (EGFR) ligands and TACE activity in human BTC cells. Inhibition of src activation led to attenuation of bile-induced upregulation of TACE activity as well as signaling through the EGFR/erbB2, suggesting that during the development of BTC erbB2 overexpression/activation accelerates the bile acid-induced signaling cascade: bile acid → src → TACE → EGFR/erbB2 → downstream signaling. We also provide direct evidence that bile acids possess tumor promoting capacity in epithelial cells overexpressing erbB2 using the two-stage skin carcinogenesis model. Collectively these findings suggest cooperative roles for bile acid and erbB2 activation in epithelial cell proliferation; bile acid appears to accelerate erbB2-induced pro-tumorigenic activities in the biliary tract and skin.

Abstract C266: Evaluation of Src as a therapeutic target in biliary tract cancer.

Abstract Background: In biliary tract cancer (BTC), no molecular therapeutic target has been validated so far. Therefore, cytotoxic chemotherapy is the mainstay of treatment of advanced BTC and the prognosis is poor and there is a huge unmet medical need. Src is a nonreceptor tyrosine kinase involved in the crosstalk and mediation of many signaling pathways that promote cell proliferation, invasion and angiogenesis. Elevation of Src activity has been reported in many types of cancers including BTC. The purpose of this study is to evaluate of Src as a therapeutic target in BTC and to elaborate the therapeutic potential of bosutinib, an orally active small molecule c-Src/abl kinase inhibitor. Methods: Eight human biliary tract cancer cell lines (SNU-245, 308, 478, 869, 1079, 1196, HuCCT1, TFK1) were used. MTT assay, colony formation assay and 3D culture were done for determining growth inhibitory effect of bosutinib, alone or in combination with chemotherapeutic agents (gemcitabine, cisplatin). Cell cycle analysis was done by FACS Calibur flow cytometer. Matrigel invasion assay and wound healing assay were done. The methods described by Chou and Talalay were used to determine whether a synergistic effect existed between drug combination. Tumor xenograft model was made and used for in vivo test of bosutinib/gemcitabine. Results: Bosutinib inhibited the growth of BTC cells, especially SNU308, SNU478 and HuCCT1 (IC50 0.6-0.8 µmol/L). Bosutinib induced G1 arrest and decreased pSrc, pFAK, pAKT and pERK in these cells. Inhibition of pSrc was accompanied with increase of pSTAT3 in SNU308. Bosutinib inhibited the cell migration and invasion. Interestingly, bosutinib showed synergistic antiproliferative effects with gemcitabine. In the xenograft model of SNU478, bosutinib decreased tumor growth compared to control and bosutinib in combination of gemcitabine showed the synergistic antitumor effect. Conclusion: These preclinical evaluations support that Src could be a potential therapeutic target in biliary tract cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C266. Citation Format: Do-Youn Oh, Ah-Rong Nam, Tae Yong Kim, Sae-Won Han, Sang-Hyun Song, Seock-Ah Im, Tae-You Kim, Yung-Jue Bang. Evaluation of Src as a therapeutic target in biliary tract cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C266.

Abstract C171: Novel therapeutic strategies targeting the hedgehog signaling pathway and mTOR pathway in biliary tract cancer (BTC).

Abstract Background: BTC represents the second most common primary cancer of the hepatobiliary system and is associated with a poor prognosis. Activation of PI3K/mTOR and hedgehog pathways has been demonstrated in biliary cancer. Activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenesis through S6K1-mediated Gli phosphorylation. Recently, synergistic antitumor efficacy was demonstrated with the combination of hedgehog and mTOR inhibitors, particularly in cases with mTOR/S6K pathway in esophageal cancer (Cancer Cell 2012; 21(3):374-87). Methods: Gene expression profiling was performed on 10 human biliary tract cancer cell lines (MMNK-1, Mz-ChA-1, Sk-ChA-1, KKU-M213, KKU-M214, KKU-M055, KKU-M139, M213L5H, and M213LOH) by real-time qRT-PCR. CellTiter-Glo® luminescent cell viability assay, colony formation, and stem cells morphology spheres were conducted to examine the effects of the combination of Rapamycin and Vismodegib on cell proliferation. Western-blot assay was used to examine the related protein expression. Fluorescence-activated cell sorting (FACS) was used to examine the effects of the combination on BTC stem cells. Results: Based on Gli1 and S6K gene expression, the Mz-ChA-1 and Sk-ChA-1 cell lines were chosen to investigate the effects of the combination of Rapamycin and Vismodegib on BTC (Table 1). Combination of Rapamycin and Vismodegib exhibited the synergistic antiproliferative effect on Mz-ChA-1 cells. The combination of Rapamycin and Vismodegib inhibited the Mz-ChA-1 cell proliferation, colony formation and stem cell sphere formation. This combination therapy decreases the NANOG, OTC3/4, and E-Cadherin gene expression in Mz-ChA-1 stem cells. Furthermore, the drug combination can decrease the phospho-p70S6K and Gli1 protein and increase cleaved caspase-3 expression in Mz-ChA-1 cells. No such effect was observed in Sk-ChA-1 cells.Conclusions: Crosstalk between the mTOR pathway and HH pathway provides a new avenue for combination therapy in BTC with hedgehog and mTOR inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C171. Citation Format: Mingxin Zuo, Asif Rashid, Chaitanya Churi, Mien-Chie Hung, Milind Javle. Novel therapeutic strategies targeting the hedgehog signaling pathway and mTOR pathway in biliary tract cancer (BTC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C171.

Abstract 2081: Evaluation of Src as a therapeutic target in biliary tract cancer using bosutinib, a c-Src/Abl kinase inhibitor.

Abstract Background: Src is a nonreceptor tyrosine kinase involved in the crosstalk and mediation of many signaling pathways that promote cell proliferation, invasion and angiogenesis. Elevation of Src activity has been reported in many types of cancers including biliary tract cancer (BTC). In BTC, no molecular therapeutic target has been validated so far. Therefore, the prognosis of advanced BTC is limited and there is a huge unmet medical need in this area. The purpose of this study is to evaluate of Src as a therapeutic target in BTC and to elucidate the therapeutic potential and action mechanism of bosutinib, an orally active small molecule c-Src/Abl kinase inhibitor, alone or in combination with chemotherapy for the treatment of BTC. Methods: Experiments were conducted using 8 human biliary tract cancer cell lines (SNU-245, 308, 478, 869, 1079, 1196, HuCCT1, TFK1). MTT assay, colony formation assay and 3D culture were done for determining growth inhibitory effect of bosutinib, alone or in combination with chemotherapeutic agents (gemcitabine, cisplatin). Cell cycle distribution was analyzed by FACS Calibur flow cytometer. Matrigel invasion assay and wound healing assay were done. The methods described by Chou and Talalay were used to determine whether a synergistic effect existed between drug combination. Results: pSrc was highly expressed in most BTC cells. Bosutinib inhibited the growth of BTC cells, especially SNU308, SNU478 and HuCCT1 (IC50, 0.6-0.8 μmol/L). Bosutinib induced G1 arrest (decrease of cyclin D, cyclin E, cyclin A, cyclin B and increase of p27) in SNU308 and SNU478. It decreased pSrc, pFAK, pAKT and pERK in these cells. Bosutinib also inhibited the cell migration and invasion in parallel with decrease of snail, not only in SNU308, SNU478 cells but also in several other cells. Interestingly, bosutinib showed synergistic antiproliferative effects (Combination Index value &amp;lt;1) with gemcitabine in 4 out of 8 BTC cells. Bosutinib was downregulated TS (thymidine synthase) expression, one of resistance mechanisms to gemcitabine. Bosutinib also showed synergistic anti-invasion activity in combination of gemcitabine or cisplatin. Conclusion: Taken together, these preclinical evaluations reveal that Src could be a potential therapeutic target in biliary tract cancer and support clinical development of bosutinib alone or in combination with chemotherapy in biliary tract cancer. Citation Format: Ah-Rong Nam, Ahrum Min, Sae-Won Han, Sang-hyun Song, Do-Youn Oh, Seock-Ah Im, Tae-You Kim, Youg-Jue Bang. Evaluation of Src as a therapeutic target in biliary tract cancer using bosutinib, a c-Src/Abl kinase inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2081. doi:10.1158/1538-7445.AM2013-2081

Aberrant expression of the PHF14 gene in biliary tract cancer cells

DNA copy number aberrations in human biliary tract cancer (BTC) cell lines were investigated using a high-density oligonucleotide microarray. A novel homozygous deletion was detected at chromosomal region 7p21.3 in the OZ cell line. Further validation experiments using genomic PCR revealed a homozygous deletion of a single gene, plant homeodomain (PHD) finger protein 14 (PHF14). No PHF14 mRNA or protein expression was detected, thus demonstrating the absence of PHF14 expression in the OZ cell line. Although the PHD finger protein is considered to be involved in chromatin-mediated transcriptional regulation, little is known about the function of PHF14 in cancer. The present study observed that the knock down of PHF14 using small interfering RNA (siRNA) enhanced the growth of the BTC cells. These observations suggest that aberrant PHF14 expression may have a role in the tumorigenesis of BTC.

Tumor growth effects of rapamycin on human biliary tract cancer cells

BackgroundLiver transplantation is an important treatment option for patients with liver-originated tumors including biliary tract carcinomas (BTCs). Post-transplant tumor recurrence remains a limiting factor for long-term survival. The mammalian target of rapamycin-targeting immunosuppressive drug rapamycin could be helpful in lowering BTC recurrence rates. Therein, we investigated the antiproliferative effect of rapamycin on BTC cells and compared it with standard immunosuppressants.MethodsWe investigated two human BTC cell lines. We performed cell cycle and proliferation analyses after treatment with different doses of rapamycin and the standard immunosuppressants, cyclosporine A and tacrolimus.ResultsRapamycin inhibited the growth of two BTC cell lines in vitro. By contrast, an increase in cell growth was observed among the cells treated with the standard immunosuppressants.ConclusionsThese results support the hypothesis that rapamycin inhibits BTC cell proliferation and thus might be the preferred immunosuppressant for patients after a liver transplantation because of BTC.

Association of stem cell marker expression pattern and survival in human biliary tract cancer

The aim of this study was to investigate the molecular and protein expression pattern of markers of stemness phenotype and its clinicopathological significance in human biliary tract cancer (BTC). Human BTC cell lines (CCLP-1, Egi-1, MzChA-1, MzChA-2, SkChA-1, TFK-1 and GBC) were analyzed in vitro and in xenotransplanted animals for expression of markers of stemness and compared to tissue microarrays (TMA) of 34 cases of human BTC with complete pathomorphological and clinical data (survival). Molecular analyses on the mRNA and protein level included makers of stemness and progenitor (Bmi-1, Sox-2, Nestin, CD133, CD44 and Nanog), proliferation and differentiation (cell cycle proteins, intermediate filaments). The investigated BTC samples showed a low to moderate and partially significantly different expression pattern of the stem cell markers in vitro, in vivo and in TMA. Hierarchical cluster analysis identified subgroups with homogenous expression of stem cell markers significantly differing with respect to cytokeratin expression in xenografts and Ki67 proliferation marker in human TMA, respectively - thus indicating possible heterogeneous carcinogenesis pathways in BTC. Additionally, these stem cell markers could be linked to morphology and molecular markers of proliferation and differentiation on the mRNA and protein level. Finally, survival analysis identified the combination of CD133 and CD44 as an independent prognostic factor yet their value as prognostic factors need testing in prospective study design.

The therapeutic effect of histone deacetylase inhibitor PCI-24781 on gallbladder carcinoma in BK5.erbB2 mice

Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat, demonstrating the need for more effective therapeutic strategies. In our current study, we examined the therapeutic effects of the histone deacetylase (HDAC) inhibitor PCI-24781 against GBCa that developed in BK5.erbB2 mice. PCI-24781 [50 mg/kg/day] and control solutions were administered to BK5.erbB2 mice for 4 weeks. The therapeutic effect of PCI-24781 was evaluated by ultrasound biomicroscopy (USBM) throughout the experiment and histological analyses at the end of the experiment. To investigate potential mechanisms underlining the therapeutic effects of PCI-24781 on GBCa in BK5.erbB2 mice, PCI-24781-treated gallbladders were subjected to Western blot and RT-PCR analysis. The inhibitory effect of PCI-24781 on the growth of BTC cells was compared to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and gemcitabine. To study the role of miRNAs in GBCa tumorigenesis, the expression profile of 368 miRNAs in GBCas from BK5.erbB2 (both treated and untreated) and wild type mice was analyzed. Treatment of BK5.erbB2 mice with PCI-24781 for 1 month prevented 79% of GBCa cases from progression and showed a clinical effect in 47% of cases. We also confirmed a potent inhibitory effect on tumor cell growth in human BTC cell lines treated with PCI-24781. This effect was associated with downregulation of ErbB2 mRNA and ErbB2 protein/activity and upregulation of acetylated histone and acetylated tubulin. Treatment with PCI-24781 resulted in decreased expression of Muc4, an intramembrane ligand for ErbB2, in BTC cells. PCI-24781 had more effects on growth inhibition of BTC cells than SAHA. In addition, PCI-24781 effectively inhibited the growth of gemcitabine-resistant cells. miRNA profiling revealed that the expression of several miRNAs was significantly altered in GBCa in the BK5.erbB2 mouse compared to normal gallbladder, including upregulated miR21, which was downregulated by PCI-24781. These results indicate that PCI-24781 potently inhibits the growth of BTC cells by decreasing ErbB2 expression and activity as well as regulating altered miRNA expression. PCI-24781 may have a potential value as a novel chemotherapeutic agent against human BTC in which ErbB2 is overexpressed.

Influence of Five Potential Anticancer Drugs on Wnt Pathway and Cell Survival in Human Biliary Tract Cancer Cells

The role of Wnt signalling in carcinogenesis suggests compounds targeting this pathway as potential anti-cancer drugs. Several studies report activation of Wnt signalling in biliary tract cancer (BTC) thus rendering Wnt inhibitory drugs as potential candidates for targeted therapy of this highly chemoresistant disease. In this study we analysed five compounds with suggested inhibitory effects on Wnt signalling (DMAT, FH535, myricetin, quercetin, and TBB) for their cytotoxic efficiency, mode of cell death, time- and cell line-dependent characteristics as well as their effects on Wnt pathway activity in nine different BTC cell lines. Exposure of cancer cells to different concentrations of the compounds results in a clear dose-dependent reduction of viability for all drugs in the order FH535 > DMAT > TBB > myricetin > quercetin. The first three substances show high cytotoxicity in all tested cell lines, cause a direct cytotoxic effect by induction of apoptosis and inhibit pathway-specific signal transduction in a Wnt transcription factor reporter activity assay. Selected target genes such as growth-promoting cyclin D1 and the cell cycle progression inhibitor p27 are down- and up-regulated after treatment, respectively. Taken together, these data demonstrate that the small molecular weight inhibitors DMAT, F535 and TBB have a considerable cytotoxic and possibly Wnt-specific effect on BTC cell lines in vitro. Further in vivo investigation of these drugs as well as of new Wnt inhibitors may provide a promising approach for targeted therapy of this difficult-to-treat tumour.

Different expression pattern of stem cell markers in xenografts of human biliary tract cancer cell lines

Different expression pattern of stem cell markers in xenografts of human biliary tract cancer cell lines

Uptake and phototoxicity of meso-tetrahydroxyphenyl chlorine are highly variable in human biliary tract cancer cell lines and correlate with markers of differentiation and proliferation

Photodynamic therapy (PDT) using Photofrin and, recently, Foscan has gained broad acceptance for palliative treatment of non-resectable cholangiocarcinoma (CC). No information, however, is available whether the phenotype of CC tumour cells has an effect on the efficiency of the treatment. Using a well-characterised set of n = 9 biliary tract cancer cell lines this study investigated the uptake, phototoxicity, and release of meso-tetrahydroxyphenyl chlorine (mTHPC, Foscan) after incubation with 200 or 400 ng ml(-1) mTHPC. For uptake of mTHPC we found great variations between the individual cell lines (up to a factor 2), resulting in even more pronounced differences in phototoxicity. Based on statistical classification by hierarchical cluster analysis, two groups of cell lines can be distinguished which are characterised by either high or low susceptibility towards mTHPC-based photodynamic treatment. Correlation analysis with previously established immunochemical parameters showed that cells with a low cytokeratin-19 (ductal differentiation), high vimentin (mesenchymal marker), and high proliferative phenotype preferentially show higher uptake of mTHPC and subsequent phototoxicity. These results demonstrate high variability of biliary tract cancer cells when subjected to mTHPC-based photodynamic treatment and identify possible markers that could be used in the clinical setting in order to predict the efficiency of PDT and adjust the dose for complete tumour elimination.