CCA Metastasis Research Articles

Increased EphB2 expression predicts cholangiocarcinoma metastasis.

The activation of Ephrin (Eph) receptors, the largest tyrosine kinase families of cell surface receptor, has recently been addressed in human cholangiocarcinoma (CCA). Therefore, the present study aimed to investigate the role of Eph receptors and its ligands in CCA. Of all 50 cases of human CCA tested, immunohistochemical staining demonstrated that EphB2, EphB4, ephrinB1, and ephrinB2 were 100 % positive in CCA tissues with overexpressions of the above proteins as 56, 56, 70, and 48 % of cases, respectively. High expression of EphB2 was significantly correlated with the metastatic status of patients (P = 0.027). We also found that the high co-expression level of EphB2/ephrinB1 or EphB2/ephrinB2 were significantly correlated with the metastatic status of the patients (P = 0.034 and P = 0.024). Furthermore, we showed that the high co-expression level of EphB4/MVD and ephrinB1/MVD were significantly correlated with the metastasis status of CCA patients (P = 0.012 and P = 0.029). We further demonstrated that the EphB2 suppression using siRNA significantly reduced CCA cell migration by decreasing the phosphorylation of focal adhesion kinase (FAK) and paxillin. In conclusion, the upregulation of EphB2 receptors and its specific ligands (ephrinB1 and ephrinB2) leads to CCA metastasis. Suppression of EphB2 expression as well as inhibition of its downstream signaling proteins might serve as possible therapeutic strategies in human CCA.

Loss of E-cadherin promotes migration and invasion of cholangiocarcinoma cells and serves as a potential marker of metastasis.

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. E-cadherin, a cell adhesion molecule, is frequently downregulated and has been proposed as an important mediator in epithelial-mesenchymal transition (EMT) in tumors. In this study, we investigated the expression of E-cadherin and its association with cancer invasion and prognosis in cholangiocarcinoma (CCA). Immunohistochemistry results demonstrated a statistically significant association between the positive metastasis status with low E-cadherin protein expression in human CCA tissues (P = 0.04). Statistical trends were identified for low E-cadherin level and shorter survival time (P = 0.08). Targeting the E-cadherin expression in CCA cells with siRNA caused upregulation of vimentin, a mesenchymal marker, and disappearance of the E-cadherin/β-catenin adhesion complex from cell membranes. Moreover, migration and invasion abilities of the cells were increased under this condition. These findings suggest that reduction of E-cadherin contributes to CCA progression by attenuating the strength of cellular adhesion, which affects motility as well as regulating the expression of EMT-related genes during CCA invasion and metastasis. Thus, E-cadherin can act as a central modulator of tumor cell phenotype and is a potential metastasis marker in CCA.

Tumor necrosis factor-α modulates epithelial mesenchymal transition mediators ZEB2 and S100A4 to promote cholangiocarcinoma progression.

The epithelial-mesenchymal transition (EMT) process strongly contributes to cancer metastasis. This study was to investigate the alteration of EMT-related proteins (ZEB1, ZEB2 and S100A4) in cholangiocarcinoma (CCA) tissues. The effect of tumor necrosis factor-α (TNF-α) on the expression of those molecules in CCA cells was investigated. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used to quantify ZEB1, ZEB2 and S100A4 mRNA levels in 50 CCA tissues and related its expression to clinicopathological data. ZEB2 protein immunostaining was investigated in 165 CCA tissues. The effect of TNF-α on EMT-related CCA cell migration was evaluated using qRT-PCR, immunofluorescence and transwell migration assays. ZEB2 and S100A4 mRNA levels were found to be higher in CCA tissues. High levels of S100A4 mRNA and ZEB2 protein were significantly associated with CCA metastasis (P = 0.04 and P = 0.03). Moreover, a trend toward statistical association was found with high levels of both ZEB2 mRNA and protein with shorter survival time (P = 0.10 and P = 0.19). In addition, TNF-α induced CCA cell migration by the induction of transforming growth factor-β (TGF-β) resulting in ZEB2 and S100A4 mRNA and protein activation. These studies demonstrate that TNF-α plays crucial role in the progression of CCA by activating TGF-β signaling and the induction of ZEB2 and S100A4, EMT-related proteins expression.

Gankyrin promotes tumor growth and metastasis through activation of IL-6/STAT3 signaling in human cholangiocarcinoma

Although gankyrin is involved in the tumorigenicity and metastasis of some malignancies, the role of gankyrin in cholangiocarcinoma (CCA) is unclear. In this study we investigated the expression of gankyrin in human CCA tissues and cell lines. The effects of gankyrin on CCA tumor growth and metastasis were determined both in vivo and in vitro. The results showed that gankyrin was overexpressed in CCA tissues and cell lines. Gankyrin expression was associated with CCA histological differentiation, TNM stage, and metastasis. The multivariate Cox analysis revealed that gankyrin was an independent prognostic indicator for overall survival. Gankyrin overexpression promoted CCA cell proliferation, migration, and invasion, while gankyrin knockdown inhibited CCA tumor growth, metastasis, and induced Rb-dependent senescence and G1 phase cell cycle arrest. Gankyrin increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and promoted the nuclear translocation of p-STAT3. Suppression of STAT3 signaling by small interfering RNA (siRNA) or STAT3 inhibitor interfered with gankyrin-mediated carcinogenesis and metastasis, while interleukin (IL)-6, a known upstream activator of STAT3, could restore the proliferation and migration of gankyrin-silenced CCA cells. The IL-6 level was decreased by gankyrin knockdown, while increased by gankyrin overexpression. Gankyrin regulated IL-6 expression by way of facilitating the phosphorylation of Rb; meanwhile, rIL-6 treatment increased the expression of gankyrin, suggesting that IL-6 was regulated by a positive feedback loop involving gankyrin in CCA. In the xenograft experiments, gankyrin overexpression accelerated tumor formation and increased tumor weight, whereas gankyrin knockdown showed the opposite effects. The in vivo spontaneous metastasis assay revealed that gankyrin promoted CCA metastasis through IL-6/STAT3 signaling pathway. Gankyrin is crucial for CCA carcinogenesis and metastasis by activating IL-6/STAT3 signaling pathway through down-regulating Rb protein.

Milk fat globule epidermal growth factor 8 serves a novel biomarker of opisthorchiasis-associated cholangiocarcinoma

Milk fat globule epidermal growth factor 8 (MFG-E8) is a pleiotropic secreted glycoprotein to play roles in mediating immune tolerance and homeostasis maintenance and enhancing angiogenesis. To evaluate its value as a biomarker in opisthorchiasis-associated cholangiocarcinoma (CCA), the present study investigated MFG-E8 expression kinetics during the tumorigenesis in Opisthorchis viverrini infection-induced CCA, and demonstrated its expression in the tumor tissues of CCA patients and its serum level among them. During the tumorigenesis of CCA, MFG-E8 expression was increased in a time-dependent manner with the pathological processes. Absolutely higher expression of MFG-E8 messenger RNA was detected in the tumor tissues from CCA patients, compared with those in adjacent tissues. Immunobiochemical analysis showed that more than 90% CCA cases were positive and the positive reaction located in the membrane and cytoplasm of the tumor cells. Moreover, the average serum level in the CCA patients was significantly higher than that in healthy individuals and those with O. viverrini infection or other parasitosis. Correlation analysis of MFG-E8 expression with CCA clinicopathology revealed that a high expression of MFG-E8 protein was significantly bound with a poor differentiation, pathological advanced stage, and metastasis of CCA. The multivariation analysis indicated that MFG-E8 was an independent prognostic factor. In addition, short hairpin RNA-mediated MFG-E8 knockdown in CCA cell line obviously suppressed the cell proliferation. Our results strongly suggested that MFG-E8 is a promising biomarker for the diagnosis, prognosis, and therapy target of opisthorchiasis-associated CCA.

Suppression of thymosin β10 increases cell migration and metastasis of cholangiocarcinoma

BackgroundThymosin β10 (Tβ10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of Tβ10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of Tβ10 in CCA tumor tissues and cell lines as well as molecular mechanisms of Tβ10 in tumor metastasis of CCA cell lines.MethodsTβ10 expression was determined by real time RT-PCR or immunocytochemistry. Tβ10 silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell migration was assessed using modified Boyden chamber and wound healing assay. The effect of silencing Tβ10 on CCA tumor metastasis was determined in nude mice. Phosphorylation of ERK1/2 and the expression of EGR1, Snail and matrix metalloproteinases (MMPs) were studied.ResultsTen pairs of CCA tissues (primary and metastatic tumors) and 5 CCA cell lines were studied. With real time RT-PCR and immunostaining analysis, Tβ10 was highly expressed in primary tumors of CCA; while it was relatively low in the metastatic tumors. Five CCA cell lines showed differential expression levels of Tβ10. Silence of Tβ10 significantly increased cell migration, invasion and wound healing of CCA cells in vitro; reversely, overexpression of Tβ10 reduced cell migration compared with control cells (P<0.05). In addition, silence of Tβ10 in CCA cells increased liver metastasis in a nude mouse model of CCA implantation into the spleen. Furthermore, silence of Tβ10 activated ERK1/2 and increased the expression of Snail and MMPs in CCA cell lines. Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked Tβ10 silence-associated ERK1/2 activation, Snail expression and cell migration.ConclusionsLow expression of Tβ10 is associated with metastatic phenotype of CCA in vitro and in vivo, which may be mediated by the activation of Ras, ERK1/2 and upregulation of Snail and MMPs. This study suggests a new molecular pathway of CCA pathogenesis and a novel strategy to treat or prevent CCA metastasis.

Oncogenic Activity of Retinoic Acid Receptor γ Is Exhibited through Activation of the Akt/NF-κB and Wnt/β-Catenin Pathways in Cholangiocarcinoma

Aberrant expression and function of retinoic acid receptor γ (RARγ) are often involved in the progression of several cancers. However, the role of RARγ in cholangiocarcinoma (CCA), chemoresistant bile duct carcinoma with a poor prognosis, remains unclear. In the present study, we found that RARγ was frequently overexpressed in human CCA specimens. Its overexpression was associated with poor differentiation, lymph node metastasis, high serum carbohydrate antigen 19-9 level, and poor prognosis of CCA. Downregulation of RARγ reduced CCA cell proliferation, migration, invasion, and colony formation ability in vitro and tumorigenic potential in nude mice. RARγ knockdown resulted in upregulation of cell cycle inhibitor P21, as well as downregulation of cyclin D1, proliferating cell nuclear antigen, and matrix metallopeptidase 9, in parallel with suppression of the Akt/NF-κB pathway. Furthermore, overexpression of RARγ contributed to the multidrug chemoresistance of CCA cells, at least in part due to upregulation of P glycoprotein via activation of the Wnt/β-catenin pathway. Molecular mechanism studies revealed that RARγ interacted with β-catenin and led to β-catenin nuclear translocation. Taken together, our results suggested that RARγ plays an important role in the proliferation, metastasis, and chemoresistance of CCA through simultaneous activation of the Akt/NF-κB and Wnt/β-catenin pathways, serving as a potential molecular target for CCA treatment.

Diethyldithiocarbamate Suppresses an NF-κB Dependent Metastatic Pathway in Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) is a tumor of biliary ducts, which has a high mortality rate and dismal prognosis. Constitutively activation of the transcription factor nuclear factor kappa-B (NF-κB) has been previously demonstrated in CCA. It is therefore a potential target for CCA treatment. Effects of diethyldithiocarbamate (DDTC) on NF-κB-dependent apoptosis induction in cancer have been reported; however, anti-metastasis has never been addressed. Therefore, here the focus was on DDTC effects on CCA migration and adhesion. Anti-proliferation, anti-migration and anti-adhesion activities were determined in CCA cell lines, along with p65 protein levels and function. NF-κB target gene expression was determined by quantitative RT-PCR. DDTC inhibited CCA cell proliferation. Suppression of migration and adhesion were observed prior to anti-CCA proliferation. These effects were related to decreased p65, reduction in NF-κB DNA binding, and impaired activity. Moreover, suppression of ICAM-1 expression supported NF-kB-dependent anti-metastatic effects of DDTC. Taken together, DDTC suppression of CCA migration and adhesion through inhibition of NF-κB signaling pathway is suggested from the current study. This might be a promising treatment choice against CCA metastasis.

AOSP21 GENETIC POLYMORPHISMS OF DRUG-METABOLISING ENZYMES AND CHEMOTHERAPY RESISTANCE IN PATIENTS WITH LYMPHOPROLIFERATIVE DISEASES

s, 5th AOS and 9th Annual Conference of OOTR, 2013 / European Journal of Cancer 49S1 (2013) S1−S22 S13 AOSP19 ANTIMETASTATIC EFFECTS OF DIETHYLDITHIOCARBAMATE ON CHOLANGIOCARCINOMA CELLS P. Srikoona, R. Kariyaa, K. Vaeteewoottacharna,b, E. Kudoa, H. Gotoa, M. Tauraa, S. Wongkhamb, S. Okadaa *. aDivision of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto, Japan, bDepartment of Biochemistry, Faculty of Medicine and Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand Introduction. Cholangiocarcinoma (CCA) is a malignant tumour of biliary ductswith poor prognosis andhighmortality rate. Highly constitutive activated transcription factor nuclear factor uB (NF-uB) is found generally in CCA cells. NF-uB is well known to play roles in different tumour genesis processes. Diethyldithiocarbamate (DDTC) has been reported to inhibit the NF-uB pathway, inducing cancer cell apoptosis. However, the effects of DDTC on cancer cell metastasis have not reported yet. Methods. Human CCA cell lines (KKU-M213, KKU-M214, and KKU-OCA17) were used to screen the inhibition of cell viability by DDTC treatment. Inhibitions of cell metastasis were performed using the KKU-M213 cell line. Cells treated with DDTC were compared with untreated (control) samples. Antimigration activities were investigated by wound healing assay and Transwell assay. Cell adhesion was observed by adhesion assay. NF-uB transcriptional activity was investigated by luciferase assay. Protein-DNA binding affinity was determined by electrophoresis mobility shift assay (EMSA). Western blot analysis was analysed to determine NF-uB protein levels. The levels of mRNA expression of NF-uB target genes related to cancer cellmetastasiswere determined by quantitative real-time PCR. Results. DDTC inhibited cell migration and adhesion in KKU-M213 cells in dose-dependent and time-dependent manners. Luciferase assay indicated that DDTC inhibit NF-uB transcriptional activity. The protein-DNA binding affinity was clearly suppressed, while protein levels showed the attenuated active p65 with DDTC treatment. The strong suppressionof intercellular adhesionmolecule-1 (ICAM1) gene expression suggested that CCA cell adhesion was inhibited by DDTC via the inhibition of NF-uB pathway. Discussion. These findings suggest that DDTC has an antimetastatic effect on CCA cells through the suppression of the NF-uB signalling pathway, which suggests therapeutic potential against human CCA metastasis. Funding. Grants-in-Aid for Science Research (No. 23107725) from the Ministry of Education, Science, Sports, and Culture of Japan. The authors declared no conflicts of interest. AOSP20 Abstract withdrawn AOSP21 GENETIC POLYMORPHISMS OF DRUG-METABOLISING ENZYMES AND CHEMOTHERAPY RESISTANCE IN PATIENTS WITH LYMPHOPROLIFERATIVE DISEASES E. Gorbachenkoa *, O. Gorevaa, A. Grishanovaa, E. Vigovskayab. aInstitute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk, Russian Federation, bMunicipal Clinical Hospital No. 2, Hematology Center, Novosibirsk, Russian Federation Introduction. One of the molecular mechanisms of multidrug resistance may be xenobiotic metabolism, including anticancer drugs. Cytochromes P450 (CYPs) and glutathioneS-transferases (GSTs) are important enzymes of phase I and phase II xenobiotic metabolism, respectively. CYPs and GSTP1 genes are polymorphic and can underlie the individual reactions of patients to chemotherapy. Our study analysed the association of functionally significant gene polymorphisms with chemotherapy resistance in patients with lymphoproliferative diseases. Methods. We determined CYP2C8*2, CYP2C8*3, CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP3A5*3, GSTP1 313A→G and GSTP1 341C→T genetic polymorphisms in blood samples of 79 patients (mean age 57 years, SD 13) with lymphoproliferative diseases (non-Hodgkin lymphoma, chronic lymphocytic leukaemia) by PCR-RFLP assays. All patients received chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisolone). The odds ratio (OR) was used to estimate association of CYP genotypes with chemotherapy resistance risk and was calculated for the combined group (mutant homozygous and heterozygous) in comparison with the wild genotype group. Results.Wehave found the followingmutant allele frequencies in patients with chronic lymphoproliferative diseases: CYP2C8*2, 0.00; CYP2C8*3, 0.14; CYP2C9*2, 0.11; CYP2C9*3, 0.05; CYP2C19*2, 0.13; CYP2C19*3, 0.00; CYP3A5*3, 0.93; GSTP1 313A→G, 0.21; and GSTP1 341C→T, 0.12. Patients were divided in two groups according to the response to chemotherapy: effect was estimated as positive (n=57) in cases of complete or partial remission or stabilisation and as negative (n=22) in cases of progression of disease. The risk of resistance to chemotherapy for patients had one or two CYP2C8*3 or CYP2C9*2 allele was 7.05-fold significantly (p=0.002) and 4.1fold significantly (p=0.02) higher than for wild genotype carriers, respectively. There were no significant associations of CYP2C9*3 (OR 0.85, p=0.99), CYP2C19*2 (OR 0.88, p=0.99), CYP3A5*3 (OR 1.58, p=0.72), GSTP1 313A→G (OR 1.38, p=0.74), and GSTP1 341C→T (OR 1.21, p=0.97) genetic polymorphisms with chemotherapy response. Discussion. Our results suggest that CYP2C8*3 and CYP2C9*2 polymorphisms are associated with chemotherapy resistance in patients with lymphoproliferative diseases. Funding. None declared. The authors declared no conflicts of interest.

AOSP19 ANTIMETASTATIC EFFECTS OF DIETHYLDITHIOCARBAMATE ON CHOLANGIOCARCINOMA CELLS

s, 5th AOS and 9th Annual Conference of OOTR, 2013 / European Journal of Cancer 49S1 (2013) S1−S22 S13 AOSP19 ANTIMETASTATIC EFFECTS OF DIETHYLDITHIOCARBAMATE ON CHOLANGIOCARCINOMA CELLS P. Srikoona, R. Kariyaa, K. Vaeteewoottacharna,b, E. Kudoa, H. Gotoa, M. Tauraa, S. Wongkhamb, S. Okadaa *. aDivision of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto, Japan, bDepartment of Biochemistry, Faculty of Medicine and Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand Introduction. Cholangiocarcinoma (CCA) is a malignant tumour of biliary ductswith poor prognosis andhighmortality rate. Highly constitutive activated transcription factor nuclear factor uB (NF-uB) is found generally in CCA cells. NF-uB is well known to play roles in different tumour genesis processes. Diethyldithiocarbamate (DDTC) has been reported to inhibit the NF-uB pathway, inducing cancer cell apoptosis. However, the effects of DDTC on cancer cell metastasis have not reported yet. Methods. Human CCA cell lines (KKU-M213, KKU-M214, and KKU-OCA17) were used to screen the inhibition of cell viability by DDTC treatment. Inhibitions of cell metastasis were performed using the KKU-M213 cell line. Cells treated with DDTC were compared with untreated (control) samples. Antimigration activities were investigated by wound healing assay and Transwell assay. Cell adhesion was observed by adhesion assay. NF-uB transcriptional activity was investigated by luciferase assay. Protein-DNA binding affinity was determined by electrophoresis mobility shift assay (EMSA). Western blot analysis was analysed to determine NF-uB protein levels. The levels of mRNA expression of NF-uB target genes related to cancer cellmetastasiswere determined by quantitative real-time PCR. Results. DDTC inhibited cell migration and adhesion in KKU-M213 cells in dose-dependent and time-dependent manners. Luciferase assay indicated that DDTC inhibit NF-uB transcriptional activity. The protein-DNA binding affinity was clearly suppressed, while protein levels showed the attenuated active p65 with DDTC treatment. The strong suppressionof intercellular adhesionmolecule-1 (ICAM1) gene expression suggested that CCA cell adhesion was inhibited by DDTC via the inhibition of NF-uB pathway. Discussion. These findings suggest that DDTC has an antimetastatic effect on CCA cells through the suppression of the NF-uB signalling pathway, which suggests therapeutic potential against human CCA metastasis. Funding. Grants-in-Aid for Science Research (No. 23107725) from the Ministry of Education, Science, Sports, and Culture of Japan. The authors declared no conflicts of interest. AOSP20 Abstract withdrawn AOSP21 GENETIC POLYMORPHISMS OF DRUG-METABOLISING ENZYMES AND CHEMOTHERAPY RESISTANCE IN PATIENTS WITH LYMPHOPROLIFERATIVE DISEASES E. Gorbachenkoa *, O. Gorevaa, A. Grishanovaa, E. Vigovskayab. aInstitute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk, Russian Federation, bMunicipal Clinical Hospital No. 2, Hematology Center, Novosibirsk, Russian Federation Introduction. One of the molecular mechanisms of multidrug resistance may be xenobiotic metabolism, including anticancer drugs. Cytochromes P450 (CYPs) and glutathioneS-transferases (GSTs) are important enzymes of phase I and phase II xenobiotic metabolism, respectively. CYPs and GSTP1 genes are polymorphic and can underlie the individual reactions of patients to chemotherapy. Our study analysed the association of functionally significant gene polymorphisms with chemotherapy resistance in patients with lymphoproliferative diseases. Methods. We determined CYP2C8*2, CYP2C8*3, CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP3A5*3, GSTP1 313A→G and GSTP1 341C→T genetic polymorphisms in blood samples of 79 patients (mean age 57 years, SD 13) with lymphoproliferative diseases (non-Hodgkin lymphoma, chronic lymphocytic leukaemia) by PCR-RFLP assays. All patients received chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisolone). The odds ratio (OR) was used to estimate association of CYP genotypes with chemotherapy resistance risk and was calculated for the combined group (mutant homozygous and heterozygous) in comparison with the wild genotype group. Results.Wehave found the followingmutant allele frequencies in patients with chronic lymphoproliferative diseases: CYP2C8*2, 0.00; CYP2C8*3, 0.14; CYP2C9*2, 0.11; CYP2C9*3, 0.05; CYP2C19*2, 0.13; CYP2C19*3, 0.00; CYP3A5*3, 0.93; GSTP1 313A→G, 0.21; and GSTP1 341C→T, 0.12. Patients were divided in two groups according to the response to chemotherapy: effect was estimated as positive (n=57) in cases of complete or partial remission or stabilisation and as negative (n=22) in cases of progression of disease. The risk of resistance to chemotherapy for patients had one or two CYP2C8*3 or CYP2C9*2 allele was 7.05-fold significantly (p=0.002) and 4.1fold significantly (p=0.02) higher than for wild genotype carriers, respectively. There were no significant associations of CYP2C9*3 (OR 0.85, p=0.99), CYP2C19*2 (OR 0.88, p=0.99), CYP3A5*3 (OR 1.58, p=0.72), GSTP1 313A→G (OR 1.38, p=0.74), and GSTP1 341C→T (OR 1.21, p=0.97) genetic polymorphisms with chemotherapy response. Discussion. Our results suggest that CYP2C8*3 and CYP2C9*2 polymorphisms are associated with chemotherapy resistance in patients with lymphoproliferative diseases. Funding. None declared. The authors declared no conflicts of interest.

Aberrant expression of GATA binding protein 6 correlates with poor prognosis and promotes metastasis in cholangiocarcinoma

AimGATA6, a zinc-finger transcription factor, functions as a tumour promoter or suppresser according to different tumour origins. We investigated the clinical significance of GATA6 and its role in invasion and metastasis in cholangiocarcinoma (CCA). MethodsExpression of GATA6 in 87 cancerous, 24 paracancerous, 32 lymph-node metastatic and 8 liver metastatic samples from 87 CCA patients undergoing surgical resection was detected by immunohistochemistry. Impact of GATA6 on invasion, metastasis and 67kDa laminin receptor expression (67LR) was evaluated in CCA cells by shRNA lentivirus or expressed-plasmid transfection. ResultsAberrant expression of GATA6 in CCAs was significantly associated with lymph-node metastasis. GATA6 expression was higher in lymph-node and liver metastatic tissues compared with primary cancerous tissues. Kaplan–Meier analysis showed GATA6 expression correlated with poor overall survival and early recurrence in CCAs. Cox analysis suggested GATA6 was an independent prognostic marker for overall survival and recurrence-free survival. CCA cell invasion and migration were decreased by GATA6 knockdown and enhanced by GATA6 overexpression in vitro. Knockdown of GATA6 reduced CCA cell metastasis by xenotransplantation into nude mice. 67LR, which is overexpressed in CCAs and promotes invasion and metastasis through several pathways, positively correlated with GATA6 expression in 87 CCAs. Both mRNA and protein levels of 67LR were regulated by GATA6 in CCA cells. Moreover, ChIP analysis showed GATA6 bound to 67LR gene promoter in CCA cells. ConclusionAberrant expression of GATA6 correlates with poor prognosis and promotes invasion and metastasis in CCA.

Expression of Smad7 in Cholangiocarcinoma: Prognostic Significance and Implications for Tumor Metastasis

There are few molecular markers known to predict cholangiocarcinoma (CCA) prognosis. Smad7 has a certain relationship with epithelial-mesenchymal transition (EMT), but its relevance to CCA in unclear. Therefore expression and clinical significance of Smad7 in CCA was the focus of this study. Expression of Smad7, E-cadherin and vimentin was assessed in 41 patients with CCA by immunohistochemistry and analyzed for associations with clinical parameters. Smad7 and vimentin expression in the CCA tissue was dramatically higher than that in adjacent tissues. In addition, Smad7, vimentin and E-cadherin expression was significantly associated with CCA lymph node metastasis and perineural invasion(P ≤ 0.05), but not other factors, such as gender, age, tumor location, tumor type and tumor differentiation degree (P>0.05). The overall survival and relapse-free survival rate was significantly higher in patients with negative Smad7 expression than those with positive Smad7 expression. EMT phenomena may occur in the process of CCA invasion and metastasis. Smad7, which was highly expressed in CCA, may be considered to be one feedback regulator in late stages and could have potential as a prognostic indicator for clinical assessment.

Expression of oxysterol binding protein isoforms in opisthorchiasis-associated cholangiocarcinoma: A potential molecular marker for tumor metastasis

Oxysterols are oxygenated derivatives of cholesterol generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by inflammation-associated non-enzymatic reactions. Oxysterol binding proteins (OSBPs) are cytosolic high affinity receptors for oxysterols. We previously found that OSBPL-8 is upregulated in liver fluke ( Opisthorchis viverrini)-induced hamster cholangiocarcinoma (CCA). Our aims were to determine the expression patterns of OSBP isoforms in human CCA tissues and to evaluate whether OSBPs could be used as molecular markers for the identification of blood-borne CCA metastasis. Expression levels of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 in CCA tissues were detected using qRT-PCR and immunohistochemistry. Expression of OSBPs at mRNA level in the blood of CCA patients was also investigated. We confirmed increased expression of OSBPL-8 in O. viverrini -induced hamster CCA tissues. Moreover, increased expression of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 was seen in human CCA tissues. Notably, a significant increased level of OSBPL-7 mRNA was observed in tumor compared to non-tumor liver tissue. Immunohistochemistry supported the mRNA results, in that OSBPL-7 protein was over-expressed in cancer cells and hepatocytes but not in normal biliary cells and surrounding inflammatory cells. Interestingly, in our preliminary results, significantly higher levels of OSBP2 and OSBPL-7 mRNA were seen in blood samples from CCA patients than in healthy controls. These results suggest that OSBP2 and OSBPL-7 might serve as molecular markers for the identification of CCA metastasis in the bloodstream.

Myristoylated alanine‐rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C‐dependent pathway

Myristoylated alanine-rich C kinase substrate (MARCKS), a substrate of protein kinase C (PKC) has been suggested to be implicated in cell adhesion, secretion, and motility through the regulation of the actin cytoskeletal structure. The quantitative real-time-polymerase chain reaction analysis revealed that MARCKS is significantly overexpressed in Opisthorchis viverrini-associated cholangiocarcinoma (CCA) (P = 0.001) in a hamster model, which correlated with the results of mRNA in situ hybridization. An immunohistochemical analysis of 60 CCA patients revealed a significant increase of MARCKS expression. Moreover, the log-rank analysis indicated that CCA patients with a high MARCKS expression have significantly shorter survival times than those with a low MARCKS expression (P = 0.02). This study investigated whether MARCKS overexpression is associated with CCA metastasis. Using a confocal microscopic analysis of CCA cell lines that had been stimulated with the PKC activator, 12-0-tetradecanoyl phorbol-13-acetate (TPA), MARCKS was found to be translocated from the plasma membrane to the perinuclear area. In addition, phosphorylated MARCKS (pMARCKS) became highly concentrated in the perinuclear area. Moreover, an adhesion assay demonstrated that the exogenous overexpression of MARCKS remarkably promoted cell attachment. Interestingly, after TPA stimulation, the CCA cell line-depleted MARCKS showed a decrease in migration and invasion activity. It can be concluded that in non-stimulation, MARCKS promotes cell attachment to the extracellular matrix. After TPA stimulation, PKC phosphorylates MARCKS leading to cell migration or invasion. Taken together, the results of this study reveal a prominent role for MARCKS as one of the key players in the migration of CCA cells and suggest that cycling between MARCKS and pMARCKS can regulate the metastasis of biliary cancer cells.