Extrahepatic Cholangiocarcinoma Cells Research Articles

The AKR1C1-CYP1B1-cAMP signaling axis controls tumorigenicity and ferroptosis susceptibility of extrahepatic cholangiocarcinoma.

Extrahepatic cholangiocarcinoma (ECC), a highly malignant type of cancer with increasing incidence, has a poor prognosis due to limited treatment options. Based on genomic analysis of ECC patient samples, here we report that aldo-keto reductase family 1 member C1 (AKR1C1) is highly expressed in human ECC tissues and closely associated with ECC progression and poor prognosis. Intriguingly, we show that inducible AKR1C1 knockdown triggers ECC cells to undergo ferroptosis. Mechanistically, AKR1C1 degrades the protein stability of the cytochrome P450 family member CYP1B1, a newly discovered mediator of ferroptosis, via ubiquitin-proteasomal degradation. Additionally, AKR1C1 decreases CYP1B1 mRNA level through the transcriptional factor aryl-hydrocarbon receptor (AHR). Furthermore, the AKR1C1-CYP1B1 axis modulates ferroptosis in ECC cells via the cAMP-PKA signaling pathway. Finally, in a xenograft mouse model of ECC, AKR1C1 depletion sensitizes cancer cells to ferroptosis and synergizes with ferroptosis inducers to suppress tumor growth. Therefore, the AKR1C1-CYP1B1-cAMP signaling axis is a promising therapeutic target for ECC treatment, especially in combination with ferroptosis inducers.

HOXA5 inhibits the proliferation of extrahepatic cholangiocarcinoma cells by enhancing MXD1 expression and activating the p53 pathway

Homeobox A5 (HOXA5) is a transcription factor in mammalian and can regulate cell differentiation, proliferation, and apoptosis as well as tumorigenesis. However, little is known on whether and how HOXA5 can regulate the malignant behaviors of cholangiocarcinoma. The methylation levels of HOXA5 were evaluated by methylation microarray and bisulfite sequencing PCR. HOXA5 expression in tissue samples was examined by immunohistochemistry and Western blot. The proliferation of tumor cells was assessed by CCK-8, EdU, and nude mouse tumorigenicity assays. The invasion, apoptosis and cell cycling of tumor cells were evaluated by Wound healing assay and flow cytometry. The interaction between HOXA5 and the MXD1 promoter was examined by CUT & Tag assay, luciferase reporter assay and chromatin immunoprecipitation. Hypermethylation in the HOXA5 promoter down-regulated HOXA5 expression in extrahepatic cholangiocarcinoma (ECCA) tissues, which was correlated with worse overall survival. HOXA5 overexpression significantly inhibited the proliferation and tumor growth. HOXA5 overexpression enhanced MXD1 expression by directly binding to the MXD1 promoter in ECCA cells. MXD1 overexpression inhibited the proliferation and tumor growth while MXD1 silencing abrogated the HOXA5-mediated proliferation inhibition. HOXA5 overexpression increased p53 protein expression in an MXD1-dependent manner. HOXA5 and MXD1 acted as tumor suppressors to inhibit the mitosis of ECCA cells by enhancing the p53 signaling. Our findings may uncover molecular mechanisms by which the HOXA5/MXD1 axis regulates the progression of ECCA, suggesting that the HOXA5/MXD1 may be therapeutic targets for ECCA.

Correlation of apparent diffusion coefficient and intravoxel incoherent motion imaging parameters with Ki-67 expression in extrahepatic cholangiocarcinoma

PurposeTo investigate the correlation of magnetic resonance imaging (MRI), apparent diffusion coefficient (ADC) and intravoxel incoherent motion imaging parameters with Ki-67 expression in cholangiocarcinoma. MethodsA total of 42 extrahepatic cholangiocarcinoma (EHCC) cases confirmed by surgical pathology were analyzed retrospectively. Subjects underwent MRI at 3.0 T and intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) sequential scanning prior to surgery, and postoperative Ki-67 expression was recorded by immunohistochemistry (IHC). The patients were divided into 4 groups (I–IV) based on increasing Ki-67 expression from − to +++. ADC values and IVIM-DWI parameters were calculated, including true diffusion coefficient (D), perfusion fraction (f), and pseudo-diffusion coefficient (D*). The comparison among groups was analyzed by univariate ANOVA (normal distribution) or Kruskal-Wallis H (non-normal distribution). Spearman correlation analysis was used to analyze the correlation of each parameter with Ki-67 expression. The diagnostic efficiency of each parameter was compared using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve. ResultsExcept for D*, other values had statistically significant differences between groups (P < 0.05). ADC, D and f values had negative correlations with Ki-67 expression (r values were −0.607, −0.795, −0.531, respectively, P < 0.05). The AUCs were 0.701, 0.880, 0.623, respectively (P < 0.0001). ConclusionIVIM-DWI technology can reflect the proliferative activity of EHCC cells to a certain extent, and has clinical value for predicting the degree of malignancy of a tumor.

Silencing of Kangai 1 C-terminal interacting tetraspanin suppresses progression of cholangiocarcinoma

Cholangiocarcinoma (CC) is the second most common primary hepatic malignancy. CC treatment options are very limited especially for patients with distant metastasis. Kangai 1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in numerous cancers, but the role of KITENIN in CC remains unknown. Here, we have investigated for the first time the function of KITENIN in human CC cell lines (TFK-1, SZ-1), tissues and a CC mouse model (Alb-Cre/LSL-KRASG12D/p53L/L). KITENIN was expressed in 92.2% of human CC tissues, in murine CC samples and also in human CC cell lines. Knockdown of KITENIN by small interfering RNA (siRNA) effectively reduced proliferation, migration, invasion and colony formation in both intra- and extra-hepatic CC cells. The expression of epithelial-mesenchymal transition (EMT) markers like N-cadherin, Vimentin, Snail and Slug were suppressed in KITENIN knockdown CC cells. Our results indicate that KITENIN is crucial for cholangiocarcinogenesis and it might become a potential therapeutic target for human CC treatment.

L1 Cell Adhesion Molecule Promotes Migration and Invasion via JNK Activation in Extrahepatic Cholangiocarcinoma Cells with Activating KRAS Mutation.

Extrahepatic cholangiocarcinoma (ECC), a malignant tumor of biliary origin, has a poor prognosis with limited treatment options. The KRAS oncogene is the most commonly mutated gene in ECC and one of the factors that predicts a poor prognosis and low survival rate. L1 cell adhesion molecule (L1CAM) is expressed in ECC cells and acts as an independent poor prognostic factor in predicting patient survival. In this study we investigate the functional significance of L1CAM in ECC cells with activating KRAS mutation. We selected an ECC cell line, EGI-1, with activating KRAS mutation, and then confirmed its expression of L1CAM by RT-PCR, western blot analysis, and flow cytometry. The suppression of L1CAM expression (using a specific lentivirus-delivered shRNA) significantly decreased the migratory and invasive properties of EGI-1 cells, without altering their proliferation or survival. Analyses of signaling effectors in L1CAM-depleted and control EGI-1 cells indicated that L1CAM suppression decreased the levels of both phosphorylated MKK4 and total MKK4, together with c-Jun N-terminal kinase (JNK) phosphorylation. Further, exposure to a JNK inhibitor (SP600125) decreased migration and invasion of EGI-1 cells. These results suggest that L1CAM promotes cellular migration and invasion via the induction of MKK4 expression, leading to JNK activation. Our study is the first to demonstrate a functional role for L1CAM in ECC carrying the activating KRAS mutation. Given that KRAS is the most commonly mutated oncogene in ECC, L1CAM may serve as an attractive therapeutic target for ECC cells with activating KRAS mutation.

Stathmin1 regulates p27 expression, proliferation and drug resistance, resulting in poor clinical prognosis in cholangiocarcinoma

Patients with extrahepatic cholangiocarcinoma (EHCC) have a poor prognosis; postoperative survival depends on cancer progression and therapeutic resistance. The mechanism of EHCC progression needs to be clarified to identify ways to improve disease prognosis. Stathmin1 (STMN1) is a major cytosolic phosphoprotein that regulates microtubule dynamics and is associated with malignant phenotypes and chemoresistance in various cancers. Recently, STMN1 was reported to interact with p27, an inhibitor of cyclin-dependent kinase complexes. Eighty EHCC cases were studied using immunohistochemistry and clinical pathology to determine the correlation between STMN1 and p27 expression; RNA interference to analyze the function of STMN1 in an EHCC cell line was also used. Cytoplasmic STMN1 expression correlated with venous invasion (P = 0.0021) and nuclear p27 underexpression (P = 0.0011). Patients in the high-STMN1-expression group were associated with shorter recurrence-free survival and overall survival than those in the low-expression group. An in vitro protein-binding assay revealed that cytoplasmic STMN1 bound to p27 in the cytoplasm, but not in the nucleus of EHCC cells. Moreover, p27 accumulated in EHCC cells after STMN1 suppression. STMN1 knockdown inhibited proliferation and increased the sensitivity of EHCC cells to paclitaxel. STMN1 contributes to a poor prognosis and cancer progression in EHCC patients. Understanding the regulation of p27 by STMN1 could provide new insights for overcoming therapeutic resistance in EHCC.

Périartérite noueuse

Cholangiocarcinoma (CC) is the second most common primary hepatic malignancy. CC treatment options are very limited especially for patients with distant metastasis. Kangai 1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in numerous cancers, but the role of KITENIN in CC remains unknown. Here, we have investigated for the first time the function of KITENIN in human CC cell lines (TFK-1, SZ-1), tissues and a CC mouse model (Alb-Cre/LSL-KRASG12D/p53L/L). KITENIN was expressed in 92.2% of human CC tissues, in murine CC samples and also in human CC cell lines. Knockdown of KITENIN by small interfering RNA (siRNA) effectively reduced proliferation, migration, invasion and colony formation in both intra- and extra-hepatic CC cells. The expression of epithelial-mesenchymal transition (EMT) markers like N-cadherin, Vimentin, Snail and Slug were suppressed in KITENIN knockdown CC cells. Our results indicate that KITENIN is crucial for cholangiocarcinogenesis and it might become a potential therapeutic target for human CC treatment.

WWOX Suppresses Cell Growth and Induces Cell Apoptosis via Inhibition of P38 Nuclear Translocation in Cholangiocarcinoma

Background: Extrahepatic cholangiocarcinoma (EHCC) is the second most common primary hepatic malignancy, and is associated with high morbidity and mortality. We previously reported the decreased expression of WWOX in EHCC samples, but the underlying mechanism remained unclear. Methods: Immunoprecipitation and immunofluorescence were performed to examine the interaction of WWOX and P38 MAPK. Western blot was carried out to detect the expression of ATF2 and eIF-4E. MTT, colony formation, and Annexin V-FITC assays were performed to detect the cell proliferation and apoptosis. IHC was performed to detect the protein expression in clinical samples. Results: WWOX interacted with P38 and modulated its sub-cellular localization, leading to the cytoplasmic retention of P38. WWOX over-expression inhibited the phosphorylation of ATF2 and eIF-4E, while exogenous P38 reversed this reduction in phosphorylation. Ectopic expression of WWOX in EHCC cells led to inhibited proliferation and stimulated apoptosis in a P38 MAPK-dependent manner. In addition, we found a negative association of WWOX with nuclear localization of P38 and expression of phosphorylated ATF2 in EHCC samples. Conclusion: Our data demonstrated the role of WWOX in EHCC progression, revealing the potential of WWOX/p-ATF2 as a novel diagnostic and prognostic marker, and therapeutic target for EHCC.

Development and In Vitro Proof-of-Concept of Interstitially Targeted Zinc- Phthalocyanine Liposomes for Photodynamic Therapy

Photodynamic therapy (PDT) has been successfully used to treat various solid tumors. However, some cancer types respond poorly to PDT, including urothelial carcinomas, nasopharyngeal carcinomas, and extrahepatic cholangiocarcinomas. The therapeutic recalcitrance is in part due to the use of photosensitizers with suboptimal optical/ photochemical properties and unfavorable pharmacokinetics. To circumvent these drawbacks, a second-generation photosensitizer with improved optical/photochemical properties, zinc phthalocyanine (ZnPC), was encapsulated in interstitially targeted, polyethylene glycol-coated liposomes (ITLs) intended for systemic administration. The ZnPC-ITLs were examined for reactive oxygen species (ROS) generation and oxidation capacity and validated for tumoricidal efficacy in human extrahepatic cholangiocarcinoma (Sk-Cha1) cells. ZnPC-ITL uptake and the mechanism and mode of PDT-induced cell death were studied. The ITL formulation was optimized on the basis of fluorescence spectroscopy and photon correlation spectroscopy. The extent of ROS generation, protein oxidation, and membrane oxidation were determined by the 2',7'-dichlorodihydrofluorescein, tryptophan oxidation, and calcein leakage assays, respectively. PDT efficacy was evaluated by measuring mitochondrial activity and apoptosis-/necrosis-specific staining in combination with flow cytometry. The uptake of fluorescently labeled ITLs was assayed by confocal microscopy, flow cytometry, and fluorescence spectroscopy. ZnPC-ITLs exhibited maximum ROS-generating and oxidation potential at a ZnPC:lipid molar ratio of 0.003. PDT of Sk-Cha1 cells incubated with ZnPC-ITLs induced cell death in a lipid concentration- dependent manner. The mode of PDT-induced cell death comprised both apoptosis and necrosis, with necrotic cell death predominating. Post-PDT cell death was attributable to pre-PDT ZnPC-ITL uptake by cancer cells, which was more efficient at smaller ITL diameters and a more positive surface charge. ZnPC-ITLs constitute a nanoparticulate photosensitizer delivery system capable of inducing apoptosis and necrosis in cultured extrahepatic cholangiocarcinoma cells by PDT-mediated oxidative processes. PDT-induced cell death is dependent on the extent of ITL uptake, which in turn relies on ITL size and zeta potential.

Quantitative analysis of VEGF-C mRNA of extrahepatic cholangiocarcinoma with real-time PCR using samples obtained during endoscopic retrograde cholangiopancreatography

Objective. Vascular endothelial growth factor (VEGF)-C overexpression in extrahepatic cholangiocarcinoma (ECC) has been shown to be correlated with lymph node metastasis. The intensity of immunohistochemical staining of VEGF-C protein in surgical samples has been used as index of VEGF-C overexpression in previous studies. The aim of the study was to examine if VEGF-C overexpression in ECC could be preoperatively detected by using samples obtained during ERCP. Methods. Consecutive patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) for biliary stricture during the study period were prospectively analyzed. VEGF-C mRNA was quantified by real-time PCR methods using endoscopic samples obtained during ERCP. The high intensity of immunohistochemical staining of VEGF-C protein in surgical samples was used for the reference standard of VEGF-C overexpression. The level of S100P mRNA which was a novel diagnostic marker of ECC was also quantified to evaluate whether the endoscopic samples contained ECC cells. Results. Twenty-five patients were enrolled in this study. Eighteen patients were diagnosed as ECC and seven patients were diagnosed as benign biliary structure. Nine of eighteen patients with ECC, who showed positive S100P mRNA in endoscopic samples and received surgical resection, were finally analyzed. Receiver operating characteristics analysis yielded VEGF-C mRNA cut-off value of 3.85 for detection of VEGF-C overexpression, and the diagnostic performance of VEGF-C mRNA measurement in the endoscopic sample for VEGF-C overexpression reached sensitivity of 75.0%, specificity of 100%, and accuracy of 88.9%. Conclusion. The quantification of VEGF-C mRNA of ECC with real-time PCR using endoscopic samples was useful for preoperative detection of VEGF-C overexpression.

Aminolevulinic acid derivatives-based photodynamic therapy in human intra- and extrahepatic cholangiocarcinoma cells

Hexyl-aminolevulinic acid (HALA) was compared with aminolevulinic acid (ALA) in terms of improving ALA-based photodynamic therapy (PDT) for human intra- and extrahepatic cholangiocarcinoma (CCA) HuCC-T1 and SNU1196 cells. Because of the different uptake mechanisms of HALA, a relatively higher amount of protoporphyrin IX (PpIX) was induced in the both CCA cell types at low concentrations of HALA. Furthermore, higher expression of porphobilinogen deaminase, coproporphyrinogen III oxidase, and protoporphyrinogen oxidase, the key enzymes for synthesizing PpIX in the heme biosynthetic pathway, facilitated the exuberant generation of PpIX in HuCC-T1 cells. PpIX accumulation with ALA was markedly different between the two CCA cell types. Even at lower concentrations of ALA, SNU1196 cell successfully synthesized PpIX, due to the higher expression of the ALA transporter, mammalian H +/peptide co-transporter PEPT1. Considering the difference of PEPT1 or key enzyme expression, HALA could be a very effective substitute for ALA in doing PDT for cure of CCA.

Genetic and epigenetic alterations are involved in the regulation of TPM1 in cholangiocarcinoma

Cholangiocarcinoma is a malignant tumor originating from biliary epithelial cells. The tumor suppressor gene tropomyosin 1 (TPM1) is downregulated in several human cancer types; however, its expression status in cholangiocarcinoma is still unknown. We elucidated TPM1 expression and its regulation mechanism in cholangiocarcinoma. Real-time (RT)-PCR, western blot analysis and immunohistochemistry were performed to examine TPM1 expression levels in cholangiocarcinoma cell lines and tumor tissues. Cell lines were treated with lentiviral vector containing the miR-21 knockdown and inhibitors of genetic and epigenetic mechanisms (manumycin A, LY294002, U0126, DAC and TSA), and the TPM1 expression change was observed by RT-PCR and western blot analyses. Cell proliferation, apoptosis and migration were evaluated by water-soluble tetrazolium salt (WST-1) assay, flow cytometry and wound healing experiments, respectively. TPM1 was downregulated in the intrahepatic cholangiocarcinoma cells (HuCCT1) and upregulated in the extrahepatic cholangiocarcinoma cells (QBC939) compared with normal intrahepatic biliary epithelial cells (HIBEC). TPM1 stained negative in the intrahepatic cholangiocarcinoma tissues, as revealed by immunohistochemistry, although there was no significant difference in staining of the intrahepatic cholangiocarcinoma tissues and adjacent non-cancer tissues. RAS and two important downstream signaling pathways (RAS/PI3K/AKT and RAS/MEK/ERK) were involved in TPM1 regulation and inhibition of the epigenetic mechanisms such as DNA methylation, histone deacetylation and miR-21 upregulation upregulated TPM1 expression. Inhibitors of genetic and epigenetic mechanisms (manumycin A, LY294002, U0126, DAC and TSA) inhibited cell proliferation and migration and induced apoptosis. These data indicated that TPM1 is downregulated in HuCCT1 cells and that the Ras signaling pathway as well as DNA methylation, histone deacetylation and miR-21 upregulation play important roles in the suppression of TPM1 expression in HuCCT1 cells. Thus, compounds that inhibit genetic and epigenetic mechanisms may be promising agents in treating cholangiocarcinoma.

Su1205 Epidermal Growth Factor Upregulates the mRNA and Protein Levels of Skp2/Cks1 and P27kip1 via the PI3K/AKT Pathway in Human Extrahepatic Cholangiocarcinoma Cells

Su1205 Epidermal Growth Factor Upregulates the mRNA and Protein Levels of Skp2/Cks1 and P27kip1 via the PI3K/AKT Pathway in Human Extrahepatic Cholangiocarcinoma Cells

Isolation and characterization of tumorigenic extrahepatic cholangiocarcinoma cells with stem cell‐like properties

Recent studies suggest that the ability to form and grow tumors specifically resides in a small cell population called cancer stem cells (CSCs). These studies were conducted mainly on various human cancers; however, isolation and characterization of stem cells from cholangiocarcinoma have not been attempted. The molecular markers CD24, CD44, CD34, and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of CSCs. In this study, we used these markers to identify a subpopulation of cells in extrahepatic cholangiocarcinoma (ECC) with cancer stem/progenitor cell-like properties. We found that CD24(+) CD44(+) EpCAM(high) cells (0.39-2.27%) were present in human ECC tissues. The expression of a CD24(+) CD44(+) EpCAM(high) subpopulation was consistent with primary cancers and could be duplicated during serial in vivo passaging in NOD/SCID mice. CD24(+) CD44(+) EpCAM(high) cells isolated from 3 cholangiocarcinoma xenografts showed high tumorigenic potential compared with CD24(-) CD44(-) EpCAM(low/-) cells. These tumorigenic ECC cells exhibited the stem cell properties of self-renewal and ability to produce heterogeneous progeny. We report the identification of a CSC population in ECC characterized by CD24, CD44 and EpCAM phenotypes. Our findings could provide new insight into the tumorigenesis of cholangiocarcinoma and offer a potential target for anti-cancer therapy.

Comment on: Isolation and characterization of tumorigenic extrahepatic cholangiocarcinoma cells with stem cell-like properties

Comment on: Isolation and characterization of tumorigenic extrahepatic cholangiocarcinoma cells with stem cell-like properties

Difference in Biological Characteristics and Sensitivity to Chemotherapy and Radiotherapy Between Intrahepatic and Extrahepatic Cholangiocarcinoma Cells in Vitro

To investigate and compare the biological characteristics and sensitivity to chemotherapy and radiotherapy of intrahepatic and extrahepatic cholangiocarcinoma cells in vitro. The intrahepatic and extrahepatic cholangiocarcinoma cell lines were established, and cells with steady passage were chosen to study the biological characteristics including morphology, growth dynamics, chromosome, and levels of cancer antigen (CA) 125, CA19-9, alpha-fetoprotein (AFP), and carcino-embryonic antigen (CEA). Meanwhile, MTT assay was used to determine the sensitivity of both kinds of cells to 6 chemotherapeutic drugs, including cisplatin, paclitaxel, harringtonine, 5-fluorouracil, vincristine, and aclacimomycin, and the inhibitory rate of cells under the irradiation of 10 Gy ray was also measured. The intrahepatic cholangiocarcinoma cells were mostly fusiform in shape, and extrahepatic cholangiocarcinoma cells were mostly round or polygon in shape. Their doubling time was 26. 3 hours and 23. 1 hours, respectively. Their average number of chromosomes was 59 (range, 38-84) and 67 (range, 49-103), respectively. The chromosome karyotypes of most intrahepatic cholangiocarcinoma cells were hyperdiploid and hypotriploid, while hypertriploid was predominant in extrahepatic cholangiocarcinoma cells. The level of CA 125 in supernatant of extrahepatic cholangiocarcinoma cells increased obviously, while levels of other determined tumor markers in both kinds of cells were all within normal range. The intrahepatic cholangiocarcinoma cells were low sensitive to cisplatin and paclitaxel, but not sensitive to the other 4 chemotherapeutic drugs. The extrahepatic cholangiocarcinoma cells were high sensitive to cisplatin, but not sensitive to the other 5 drugs. Both kinds of cells had poor sensitivity to radiotherapy. Intrahepatic and extrahepatic cholangiocarcinoma cells show differences in shape, doubling time, chromosome karyotype, tumor marker level, and chemosensitivity, whereas they both have poor radiosensitivity. Though they are similar in histopathology, they have different growth characteristics and have discrepancy in treatment and prognosis.