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.

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 coiled-coil domain containing 25 protein as a potential screening/diagnostic biomarker for cholangiocarcinoma.

Coiled-coil domain containing 25 (CCDC25) was previously reported to be upregulated in cholangiocarcinoma (CCA) tissues compared with adjacent normal tissues. The present study investigated whether serum CCDC25 level may be used as a potential marker for the diagnosis of CCA. Bioinformatics tools were used to reveal that CCDC25 is secreted into plasma/serum via a non-conventional pathway, which secretes proteins independently from the endoplasmic reticulum/golgi complex, but is yet to be fully elucidated. Subsequently, the CCDC25 levels in the sera of patients with CCA (n=141), patients with benign biliary disease (BBD; n=53) and healthy controls (HC; n=72) were measured using a quantitative dot blot assay based on the standard curve created using recombinant CCDC25 protein. The results demonstrated that the serum CCDC25 level in the CCA group (0.28±0.06 ng/µl) was significantly higher compared with that in the BBD (0.15±0.03 ng/µl) or HC (0.0017±0.0008 ng/µl) groups. Serum CCDC25 level provided an improved resolution (P=0.0001) compared with carcinoembryonic antigen (P=0.098) or carbohydrate antigen 19-9 (P=0.271) for the differential diagnosis between BBD and CCA. Receiver operating characteristic curve analysis revealed high sensitivity and specificity of serum CCDC25 level to differentiate between patients with CCA and HC (93.0 and 100%, respectively), and also to differentiate between patients with CCA and patients with BBD (75.0 and 84.0%, respectively). CCDC25 expression was further investigated in 23 CCA tissues, and CCDC25 expression in cancer tissues was moderately correlated with the serum CCDC25 level (r2=0.52, P=0.01). Among patients with CCA, serum CCDC25 level was significantly higher in patients with non-metastatic CCA compared with patients with metastatic CCA. Correspondingly, a higher serum CCDC25 level was associated with a longer overall survival time in patients with CCA. In conclusion, serum CCDC25 level may be a promising screening and diagnostic marker for the differential diagnosis of CCA.

Serum pyruvate dehydrogenase kinase as a prognostic marker for cholangiocarcinoma.

Pyruvate dehydrogenase kinase (PDK) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase and serves a key role in aerobic glycolysis, which is a hallmark of cancer cells. The present study determined the PDK expression in cholangiocarcinoma (CCA) tissues and sera to evaluate their applicability as a biomarker for CCA. Using proteomic analysis, PDK was revealed to be the most overexpressed mitochondrial protein in CCA tissues. Then, the expression of PDK isoforms in CCA tissues was examined in 15 CCA cases by immunohistochemistry. The PDK3 isoform levels in the sera were measured using a dot blot assay for 39 patients with CCA, 20 patients with benign biliary disease and 19 healthy volunteers. The results revealed a 27-fold overexpression of PDK3 in cancerous tissues when compared with adjacent non-cancerous tissues. The immunohistochemical results demonstrated that the PDK1, 2 and 3, but not the PDK4, isoforms were overexpressed in cancerous tissues. When the PDK3 levels in the sera were examined, they were significantly higher in CCA when compared with the BBD and healthy groups. The specificity and sensitivity of PDK3 as a marker for CCA were 97.5 and 33.0%, respectively, and high PDK3 levels in the sera were correlated with a short survival time for CCA. In conclusion, PDK3 can be used as a diagnostic/prognostic marker for CCA.

High expression of apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) in human cholangiocarcinoma.

Prognosis of cholangiocarcinoma (CCA) patients is absolutely poor in spite of extensive efforts for the development of chemotherapy. Mitochondrial proteins play key roles in carcinogenesis of various cancers. Therefore, mitochondria are considered as the target organelles for chemotherapy of several cancers including CCA. The purpose of this study is to identify potential candidate proteins for chemotherapy using mitochondrial proteome analysis for CCA tissues. A shotgun proteomic approach using SDS-PAGE coupled with LC-MS/MS was applied to compare the expression of mitochondrial proteins in CCA and the adjacent non-cancerous tissues. Using the proteomic analysis for the pooled mitochondrial proteins purified from three each of papillary and non-papillary types of CCA and their adjacent tissues, 281 proteins were identified as mitochondrial proteins, and 105 of them have significantly different expression levels compared with the corresponding counterparts. Among the 105 proteins, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) was a unique protein commonly over-expressed in both papillary and non-papillary types of CCA tissues but not in the adjacent non-cancerous tissues. In conclusion, AIFM3 was aberrantly expressed only in the mitochondria of CCA tissues. This finding suggests that AIFM3 could be a potential target molecule for CCA chemotherapy.