Prediction of CIAPIN1 (Cytokine-Induced Apoptosis Inhibitor 1) Signaling Pathway and Its Role in Cholangiocarcinoma Metastasis.

Cholangiocarcinoma (CCA), a malignancy of the biliary epithelium, can arise at any point in the biliary system. We previously reported that CIAPIN1 is detectable in the sera and that its overexpression was associated with poor prognosis and metastasis of CCA patients. In this study, we investigated further its expression in CCA tissues, biological functions, and related signaling pathways in CCA cells. First, we examined CIAPIN1 expression in CCA tissues of 39 CCA patients using immunohistochemistry (IHC). Then, CIAPIN1-related proteins expressed in CCA cells were identified using RNA interference (siRNA) and liquid chromatography-mass spectrometry (LC-MS/MS). To predict the functions and signaling pathways of CIAPIN1 in CCA cells, the identified proteins were analyzed using bioinformatics tools. Then, to validate the biological functions of CIAPIN1 in the CCA cell line, transwell migration/invasion assays were used. CIAPIN1 was overexpressed in CCA tissues compared with adjacent noncancerous tissues. Its overexpression was correlated with lymph node metastasis. Bioinformatic analyses predicted that CIAPIN1 is connected to the TGF-ß/SMADs signaling pathway via nitric oxide synthase 1 (NOS1) and is involved in the metastasis of CCA cells. In fact, cell migration and invasion activities of the KKU-100 CCA cell line were significantly suppressed by CIAPIN1 gene silencing. Our results unravel its novel function and potential signaling pathway in metastasis of CCA cells. CIAPIN1 can be a poor prognostic factor and can be a promising target molecule for CCA chemotherapy.

Bioinformatic Prediction of Novel Signaling Pathways of Apoptosis-inducing Factor, Mitochondrion-associated 3 (AIFM3) and Their Roles in Metastasis of Cholangiocarcinoma Cells.

BACKGROUND/AIM: We previously demonstrated that a mitochondrial protein, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) is over-expressed in cholangiocarcinoma (CCA) and its serum levels can be a prognostic biomarker for CCA. To elucidate the functional roles of AIFM3 in CCA progression, we aimed to determine the signaling pathways of AIFM3 in CCA. MATERIALS AND METHODS: AIFM3 gene in CCA cells was silenced and AIFM3-related proteins were identified using mass spectrometry and bioinformatics tools. The relationships between AIFM3 and 441 related proteins were explored. To validate the functions of AIFM3, transwell migration/invasion assays were used. RESULTS: Bioinformatic analyses predicted that AIFM3 interacts with formin-like protein 3 (FMNL3) and is involved in tumor cell motilities. Online database analysis revealed higher AIFM3 mRNA expression levels in CCA, particularly with lymph node metastasis. After AIFM3 gene silencing, CCA cell migration/invasion was significantly decreased (p<0.001). Furthermore, AIFM3 expression levels were significantly associated with lymph node metastasis (p=0.0009) and shorter survival time (p=0.020). CONCLUSION: The AIFM3 signaling pathway is mediated via FMNL3 and involved in metastasis, suggesting that AIFM3 might be a molecular target to prevent CCA metastasis.

Serum Levels of Cytokine-Induced Apoptosis Inhibitor 1 (CIAPIN1) as a Potential Prognostic Biomarker of Cholangiocarcinoma.

The mortality rate of cholangiocarcinoma (CCA) is high since there is a lack of a non-invasive technique to accurately detect tumors at the early stage. CCA biomarkers are consistently needed for various purposes including screening, early diagnosis, prognosis and follow-up. Herein, using bioinformatic analysis of our mitochondrial proteome database of CCA tissues, we identified cytokine-induced apoptosis inhibitor 1 (CIAPIN1) as a potential prognostic biomarker for CCA. CIAPIN1 levels in the sera of 159 CCA patients and 93 healthy controls (HC) were measured using a dot blot assay. The median level ± quartile deviation of CIAPIN1 level in the sera of CCA patient group was 0.5144 ± 0.34 µg/µL, which was significantly higher than 0.2427 ± 0.09 µg/µL of the HC group (p < 0.0001). In CCA patients, higher serum CIAPIN1 level was significantly associated with lymph node metastasis (p = 0.024) and shorter overall survival time (p = 0.001, Kaplan-Meier test). Cox regression analysis showed that the serum CIAPIN1 level can be an independent prognostic indicator for the survival of CCA patients. Moreover, for the prediction of CCA prognosis, CIAPIN1 is superior to CEA, CA19-9 and ALP. In conclusion, CIAPIN1 can be a serum biomarker candidate for the poor prognosis of CCA.

Kallikrein-11, in Association with Coiled-Coil Domain Containing 25, as a Potential Prognostic Marker for Cholangiocarcinoma with Lymph Node Metastasis.

Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes. Currently, the treatment and prognosis for CCA are mostly poor. Recently, we have reported that coiled-coil domain containing 25 (CCDC25) protein level in the sera may be a diagnostic marker for CCA. Subsequently, we identified three binding proteins of CCDC25 and found that kallikrein-11 (KLK11) expression was highest among those binding proteins. In this study, we investigated CCDC25 and KLK11 expression in CCA and adjacent normal tissues (n = 18) using immunohistochemistry. The results demonstrated that the expressions of CCDC25 and KLK11 in CCA tissues were both significantly higher than the adjacent tissues (p < 0.001 and p = 0.001, respectively). Then, using GEPIA bioinformatics analysis, KLK11 mRNA was significantly overexpressed in CCA tumor tissues compared with normal tissues (p < 0.05). Moreover, CCDC25 expression was positively correlated with KLK11 expression in CCA with lymph node metastasis (p = 0.028, r = 0.593). An analysis for the interaction of KLK11 with CCDC25 and other proteins, using STRING version 11.0, revealed that CCDC25 and KLK11 correlated with metastasis-related proteins. In addition, Kaplan-Meier survival curve analysis revealed that a high expression of KLK11 was associated with the poor prognosis of CCA. In conclusion, KLK11 is, as a binding protein for CCDC25, possibly involved in the metastatic process of CCA. KLK11 may be used as a prognostic marker for CCA.

Bioinformatic Prediction of Signaling Pathways for Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and Its Role in Cholangiocarcinoma Cells.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) is involved in the DNA damage repair pathways and associates with the metastasis of several human cancers. However, the signaling pathway of APEX1 in cholangiocarcinoma (CCA) has never been reported. In this study, to predict the signaling pathways of APEX1 and related proteins and their functions, the effects of APEX1 gene silencing on APEX1 and related protein expression in CCA cell lines were investigated using mass spectrometry and bioinformatics tools. Bioinformatic analyses predicted that APEX1 might interact with cell division cycle 42 (CDC42) and son of sevenless homolog 1 (SOS1), which are involved in tumor metastasis. RNA and protein expression levels of APEX1 and its related proteins, retrieved from the Gene Expression Profiling Interactive Analysis (GEPIA) and the Human Protein Atlas databases, revealed that their expressions were higher in CCA than in the normal group. Moreover, higher levels of APEX1 expression and its related proteins were correlated with shorter survival time. In conclusion, the signaling pathway of APEX1 in metastasis might be mediated via CDC42 and SOS1. Furthermore, expression of APEX1 and related proteins is able to predict poor survival of CCA patients.

Interleukin 25 (IL-25) expression in cholangiocarcinoma.

Various cytokines are involved in carcinogenesis and tumor progression. Some tumor cells produce cytokines by themselves. Using secretome analysis, a high expression of APEX-1 was found in cholangiocarcinoma (CCA) cell lines. During this secretome analysis, it was found that CCA cell lines overexpressed some cytokines and related molecules, including interleukin 25 (IL-25). In the present study, we first performed precise secretome analysis on cytokines and related molecules in CCA cell lines and identified that IL-25 was overexpressed in CCA cell lines. Then, using immunohistochemical methods, we investigated the expression of IL-25 in the cancer tissues from 20 CCA patients in Northeast Thailand. Correlation between IL-25 expression levels and patients' clinical parameters were analyzed. The results showed that IL-25 expression was significantly (P<0.0001) higher in cancerous tissues than in the normal bile ducts and in the adjacent tissues. Overexpression of IL-25 protein in CCA tissue was confirmed using western blot analysis. Moreover, IL-25 expression in cancerous tissues was significantly (P<0.0015) higher in CCA patients with metastasis than in CCA patients without metastasis. Survival analysis revealed that a high expression of IL-25 was correlated with shorter survival time of CCA patients (P=0.0260). Aberrant expression of IL-25 in CCA tissue was associated with tumor metastasis and poor prognosis, suggesting that IL-25 is a potential prognostic biomarker. Biological roles of IL-25 in CCA genesis and progression should be explored in future.

Serum Cystatin C as a Potential Marker for Glomerular Filtration Rate in Patients with Cholangiocarcinoma.

Background: Cholangiocarcinoma (CCA) is the second most common primary hepatobiliary cancer. These patients have meager prognosis and short-term survival. Precise assessment of glomerular filtration rate is a fundamental aspect of clinical care in cancer patients. Cystatin C has been proposed to be superior to creatinine, a well-known marker of renal function. This study aimed to evaluate cystatin C as a marker of GFR calculation in CCA patients. Materials and Methods: One hundred thirty serum samples from CCA patients and 32 from controls were included in this study. Serum cystatin C was measured using immunoturbidity assay. Estimated glomerular filtration rate was calculated by three equations established by chronic kidney disease epidemiology collaboration (based on creatinine and/or cystatin C). Results: Serum cystatin C in CCA patients was higher than that of controls (p=0.0002). Cystatin C was positively correlated with BUN in CCA group (p=0.019). eGFR based on cystatin C and based on both cystatin C and creatinine in CCA was low with significantly different from those of control (p<0.001). Although there was no difference in eGFR using three equations in control, creatinine based eGFR was high with significantly different from eGFR based on cystatin C and on both creatinine and cystatin C in CCA (P=0.000). Proportion in each eGFR stage by three equations showed a high sensitivity with significantly different in CCA (p<0.05). Conclusion: There was a high sensitivity of cys C with significant difference between creatinine and/or cystatin C based eGFR in CCA patients. It should be taken into consideration of mild changes in eGFR by cystatin C which is important in managing drug dosage for CCA patients.

Apoptosis-Inducing Factor, Mitochondrion-Associated 3 (AIFM3) Protein Level in the Sera as a Prognostic Marker of Cholangiocarcinoma Patients.

Prognosis of cholangiocarcinoma (CCA) patients is absolutely poor. Since improvement of prognosis and/or response to treatment by personalized and precision treatments requires earlier and precise diagnostic markers, discovery of prognostic markers attracts more attention. Apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) is highly expressed in several cancers including CCA. The present study investigated whether the serum AIFM3 level can be used as a potential marker for CCA prognosis. For this purpose, we first determined secretory protein nature of AIFM3 using bioinformatic tools. The results show that although AIFM3 lacks signal peptide, it can be secreted into plasma/serum via an unconventional pathway. Then, the AIFM3 levels in the sera of 141 CCA patients and 70 healthy controls (HC) were measured using a semi-quantitative dot blot assay. The results show that the AIFM3 level in the sera of CCA group was significantly higher than that of HC. When correlation between serum AIFM3 levels and the clinicopathological parameters of CCA patients were examined, serum AIFM3 levels correlated significantly with lymph node metastasis, age, and the patients' overall survival (OS). Higher AIFM3 levels were significantly associated with shorter OS, and only AIFM3 was an independent prognostic marker for CCA. In conclusion, AIFM3 can be used as a prognostic marker for CCA.

Serum Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) Level as a Potential Biomarker of Cholangiocarcinoma.

Diagnostic and/or prognostic biomarkers for cholangiocarcinoma (CCA) are still insufficient with poor prognosis of patients. To discover a new CCA biomarker, we constructed our secretome database of three CCA cell lines and one control cholangiocyte cell line using GeLC-MS/MS. We selected candidate proteins by five bioinformatics tools for secretome analysis. The inclusion criteria were as follows: having predicted signal peptide or being predicted as non-classically secreted protein; together with having no transmembrane helix and being previously detected in plasma and having the highest number of signal peptide cleavage sites. Eventually, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) was selected for further analysis. To validate APEX1 as a bio-marker for CCA, serum APEX1 levels of 80, 39, and 40 samples collected from CCA, benign biliary diseases (BBD), and healthy control groups, respectively, were measured using dot blot analysis. The results showed that serum APEX1 level in CCA group was significantly higher than that in BBD or healthy control group. Among CCA patients, serum APEX1 level was significantly higher in patients having metastasis than in those without metastasis. The higher level of serum APEX1 was correlated with the shorter survival time of the patients. Serum APEX1 level might be a diagnostic and prognostic biomarker for CCA.