Article

Identification of Novel Biomarker Candidates for the Immunohistochemical Diagnosis of Cholangiocellular Carcinoma

Authors:
  • Protagen Protein Services, Heilbronn, Germany
  • Selma-Lagerlöf Sekundarschule
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Abstract

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer which arises from the epithelial cells of bile ducts and is characterised by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analysed by two-dimensional differential in-gel electrophoresis (2D-DIGE) and by mass spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins which were found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p-value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell type-specific expression profile by immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma (SFN) and stress-induced phosphoprotein 1 (STIP1) in tumorous cholangiocytes in comparison to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Since STIP1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases as well as metastatic malignancies in the liver.

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... The set for the proteome analysis comprised 11 CCC and 9 PDAC samples (cohort 1). Experience from similar studies conducted previously has proven sample sizes in this range sufficient to detect reproducible proteome patterns and therefore reveal potential biomarkers which were successfully verified in subsequent experiments (10)(11)(12). From each sample, three technical replicates were analysed by LC-MS/MS to improve overall protein identification and quantification accuracy. ...
... The immunohistochemical staining of tissue sections was evaluated using an immuno-reactive score (IRS) as described previously (11). Briefly, the proportion of positively stained cells was graded as 0 (0%), 1 (1-5%), 2 (6-10%), 3 (11-50%) or 4 (51-100%) and the staining intensity was expressed as 0 (none), 1 (weak), 2 (intermediate) or 3 (strong). ...
... We therefore chose a global unbiased approach in form of LC-MS-based shotgun proteomics for the discovery phase. This has previously proven to be a suitable method for biomarker discovery studies (10,11) and incidentally holds the advantage of enabling subsequent re-or meta-analysis of the data for, e.g., functional studies. ...
Article
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Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types which arise from epithelial cells of the pancreatobiliary system. Owing to their histological and morphological similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. The detection of biomarkers with high specificity and sensitivity for the differentiation of these tumour types would therefore be a valuable tool. Here, we address this problem by comparing microdissected CCC and PDAC tumour cells from nine and eleven cancer patients, respectively, in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of 73 CCC, 78 primary and 18 metastatic PDAC tissue sections. In the proteome analysis, we found 180 proteins with a significantly differential expression between CCC and PDAC cells (p-value < 0.05, absolute fold change > 2). Nine candidate proteins were chosen for an immunohistochemical verification out of which three showed very promising results. These were the annexins ANXA1, ANXA10 and ANXA13. For the correct classification of PDAC, ANXA1 showed a sensitivity of 84% and a specificity of 85% and ANXA10 a sensitivity of 90% at a specificity of 66%. ANXA13 was higher abundant in CCC. It presented a sensitivity of 84% at a specificity of 55%. In metastatic PDAC tissue ANXA1 and ANXA10 showed similar staining behaviour as in the primary PDAC tumours (13/18 and 17/18 positive, respectively). ANXA13, however, presented positive staining in eight out of eighteen secondary PDAC tumours and was therefore not suitable for the differentiation of these from CCC. We conclude that ANXA1 and ANXA10 are promising biomarker candidates with high diagnostic values for the differential diagnosis of intrahepatic CCC and metastatic liver tumours deriving from PDAC.
... STIP1 is located at 11q13, and copy number gain of this region has been found in cancers and linked to poor prognosis [17][18][19][20]. STIP1 has been reported to be up-regulated in various types of cancer, including hepatocellular carcinoma [21], pancreatic cancer [22], ovarian cancer [23,24], colon cancer [25], and cholangiocellular carcimoma [26]. Whether STIP1 involved in the regulation of GC metastasis remains unknown. ...
... Matrix meralloproteinases was reported to play an important role in cell migration and invasion since cell invasion involves degradation of basement membrane extracellular matrix proteins [28]. Previous studies have showed that STIP1 was associated with disease progression and poor prognosis in various types of cancers, particularly for those with advanced lymph node metastasis [21][22][23][24][25][26]. In this study, we found that STIP1 increased invasiveness and metastatic potential by in vitro and in vivo assays. ...
Article
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Background Stress-Inducible Protein-1 (STIP1) is a co-chaperone that associates directly with heat shock proteins, and regulates motility of various types of cancer. In the present study, we investigated the role of STIP1 on metastasis of gastric cancer (GC). Methods In vivo metastatic experimental model was employed to investigate the effect of STIP1 on metastasis of GC cells. Loss-of-function and gain-of-function experiments were performed to examine the role of STIP1 on metastasis of GC cells. Western blot, immunofluorescence staining, migration and invasion assays, microarray and KEGG pathway analysis were applied to explore the underlying mechanism. ResultsIn current study, we demonstrated that STIP1 promoted lung metastasis of GC cells in vivo. Furthermore, STIP1 significantly enhanced migration and invasion abilities of GC cells. In contrast, knock-down of STIP1 yielded the opposite effects on these phenotypes in vitro. STIP1 promoted tumor metastasis through inducing epithelial-to-mesenchymal transition in GC cells. Mechanistically, STIP1 promoted GC metastasis via up-regulation of targeted genes in Wnt/β-catenin signaling pathway, including c-Myc and Cyclin D1, and accompanied with nuclear translocation of β-catenin. Conclusions Our findings indicate that elevated expression of STIP1 exhibited a metastasis-promoting effect in GC cells through activation of Wnt/β-catenin signaling pathway. STIP1 may be served as a potential therapeutic target for preventing GC metastasis.
... Knockout mice lacking STIP1 are embryonic lethal, suggesting a key developmental role for this molecule 6 . Growing evidence also indicates that STIP1 is markedly overexpressed in various human solid malignancies [7][8][9][10][11][12][13] . Conversely, its repression blocks both tumor cell proliferation 11 and migration 14 . ...
... Upregulation of STIP1 in various cancers [7][8][9]11,12,14,[36][37][38] is recently found to be regulated by both RAS activation and p53 inhibition 39 . Nuclear HSP90 chaperone complexes are involved in transcriptional regulation in nucleus 40 . ...
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Stress-induced phosphoprotein 1 (STIP1)-a co-chaperone of heat shock proteins-promotes cell proliferation and may act as an oncogenic factor. Similarly, glycogen synthase kinase-3 beta (GSK3β)-mediated phosphorylation of lysine-specific demethylase 1 (LSD1)-an epigenetic regulator-can contribute to the development of an aggressive cell phenotype. Owing to their ability to tether different molecules into functional complexes, scaffold proteins have a key role in the regulation of different signaling pathways in tumorigenesis. Here, we show that STIP1 acts as a scaffold promoting the interaction between LSD1 and GSK3β. Specifically, the TPR1 and TPR2B domains of STIP1 are capable of binding with the AOL domain of LSD1, whereas the TPR2A and TPR2B domains of STIP1 interact with the kinase domain of GSK3β. We also demonstrate that STIP1 is required for GSK3β-mediated LSD1 phosphorylation, which promoted LSD1 stability and enhanced cell proliferation. After transfection of cancer cells with double-mutant (S707A/S711A) LSD1, subcellular localization analysis revealed that LSD1 was translocated from the nucleus to the cytoplasm. In vitro experiments also showed that the LSD1 inhibitor SP2509 and the GSK3β inhibitor LY2090314 acted synergistically to induce cancer cell death. Finally, the immunohistochemical expression of STIP1 and LSD1 showed a positively correlation in human cancer specimens. In summary, our data provide mechanistic insights into the role of STIP1 in human tumorigenesis by showing that it serves as a scaffold for GSK3β-mediated LSD1 phosphorylation. The combination of LSD1 and GSK3β inhibitors may exert synergistic antitumor effects and deserves further scrutiny in preclinical studies.
... In a previous study, we identified potential biomarkers for ICC in a label-free and gel-based proteomic approach. 12 In the present study, these were evaluated in malignant, benign and reactive bile duct lesions in an effort to determine which markers are suitable in regard to the differential diagnosis of malignant and non-malignant bile duct lesions. Subsequently, the results were compared with the above-mentioned, previously proposed biomarkers. ...
... In a previous study, tumorous tissue and adjacent non-tumorous liver tissue from eight patients with ICC were analysed by twodimensional differential in-gel electrophoresis and by mass spectrometry-based label-free proteomics. 12 In this study, we further investigated the most promising candidate proteins (stress-induced phosphoprotein 1 (STIP1), SerpinH1, 14-3-3Sigma) regarding their usefulness in distinguishing cholangiocarcinoma from benign bile duct lesions. Subsequently, the results were compared with markers already published for the discrimination of malignant and benign bile duct lesions (CD56, HSP27, HSP70, BCL2, p53, ki67). ...
Article
Aims: The distinction between intrahepatic cholangiocarcinoma (ICC) and benign bile duct lesions can be challenging. Using our previously identified potential biomarkers for ICC, we examined whether these are useful for the differential diagnosis of ICC, bile duct adenoma and reactive bile duct proliferations in an immunohistochemical approach and identified a diagnostic marker panel including known biomarkers. Methods: Subjects included samples from 77 patients with ICC, 33 patients with bile duct adenoma and 47 patients with ductular reactions in liver cirrhosis. Our previously identified biomarkers (stress-induced phosphoprotein 1 (STIP1), SerpinH1, 14-3-3Sigma) were tested immunohistochemically following comparison with candidates from the literature (cluster of differentiation 56, heat shock protein (HSP)27, HSP70, B-cell-lymphoma2, p53, ki67). Results: The expression of SerpinH1 and 14-3-3Sigma was significantly higher in ICC than in bile duct adenomas and ductular reactions (p<0.05), whereas STIP1 expression was significantly higher (p<0.05) in ICC than in ductular reactions, but the difference to the bile duct adenoma group was not significant. A panel of the biomarker SerpinH1, 14-3-3Sigma and ki67 (≥2 marker positive) showed a high diagnostic accuracy (sensitivity 87.8%, specificity 95.9%, accuracy 91.8%) in the differential diagnosis of ICC versus non-malignant bile duct lesions. Conclusions: This suggests that 14-3-3Sigma and SerpinH1 may be useful in the differential diagnosis of malignant, benign and reactive bile duct lesions in addition to ki67 where a cut-off of >5% might be used for the distinction of malignant and non-malignant lesions.
... For quantitative proteome analysis 350 ng tryptically digested peptides of each sample were analyzed on an Orbitrap Elite or QExactive instrument online coupled to an Ultimate 3000 RSLCnano system (both from Thermo Scientific). Details regarding the chromatographic and mass-spectrometric settings have been published earlier 45,46 . For protein and peptide identification, Proteome Discoverer Software (ver. ...
... 4.0, Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK). A detailed description of each step of quantification has been published earlier 45,46 . Briefly, the previously identified peptides were matched to the quantified LC-MS features. ...
Article
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In terms of infected human individuals, herpesviruses range among the most successful virus families. Subclinical herpesviral infections in healthy individuals contrast with life-threatening syndromes under immunocompromising and immunoimmature conditions. Based on our finding that cytomegaloviruses interact with Cullin Roc ubiquitin ligases (CRLs) in the context of interferon antagonism, we systematically assessed viral dependency on CRLs by utilizing the drug MLN4924. CRL activity is regulated through the conjugation of Cullins with the ubiquitin-like molecule Nedd8. By inhibiting the Nedd8-activating Enzyme (NAE), MLN4924 interferes with Nedd8 conjugation and CRL activity. MLN4924 exhibited pronounced antiviral activity against mouse and human cytomegalovirus, herpes simplex virus (HSV)- 1 (including multi-drug resistant clinical isolates), HSV-2, adeno and influenza viruses. Human cytomegalovirus genome amplification was blocked at nanomolar MLN4924 concentrations. Global proteome analyses revealed that MLN4924 blocks cytomegaloviral replication despite increased IE1 amounts. Expression of dominant negative Cullins assigned this IE regulation to defined Cullin molecules and phenocopied the antiviral effect of MLN4924.
... There is a strong interest in the identification of cell biomarkers of proliferative liver lesions in humans. The cell-cycle regulator protein 14-3-3s has become a very promising human liver tumour biomarker but, in animal species, has only been investigated in normal canine liver where it is not constitutively expressed (Su arez-Bonnet et al., 2010;Padden et al., 2014). E-cadherin, the main cell-adhesion protein, regulates cell differentiation, maintains cell structure and its loss is associated with tumour invasiveness, metastasis and a poor prognosis (Berretta et al., 2017). ...
... There is neoexpression of 14-3-3s in human CCA and HCC, which is in agreement with our findings of absence of 14-3-3s in normal hepatocytes and bile ducts, but strong and homogeneous neoexpression in CCA. Several groups recommend the use of 14-3-3s as a novel and reliable biomarker for liver neoplasia (Wu et al., 2012;Padden et al., 2014;Reis et al., 2015) but the utility of this marker in NHPs has not previously been explored. Although the underlying mechanism of action is not well understood, neoplastic cell migration, invasion and anoikis resistance were reduced in 14-3-3s knock-out CCA cell lines, suggesting that this protein could be a promising therapeutic target (Khongmanee et al., 2013;Yang et al., 2017). ...
Article
We present a unique case of metastatic cholangiocarcinoma with concurrent abdominal cestodiasis in an African green monkey (Chlorocebus pygerythrus) that presented with respiratory insufficiency and abdominal discomfort. There were multiple white–grey masses in the liver and colonic serosa alongside intra-abdominal parasitic cysts. Histopathologically, the liver masses were composed of poorly-differentiated epithelial cells that formed densely cellular solid areas and trabeculae. The neoplastic cells were strongly immunopositive for CK7 but negative for Hep-Par1 antigen, which confirmed a diagnosis of cholangiocarcinoma. Interestingly, there was strong and diffuse neoexpression in the tumour of the cell cycle regulator 14-3-3σ, which is not constitutively expressed in normal liver. There was aberrantly strong expression of E-cadherin, a key cell–cell adhesion protein, in neoplastic cells with evidence of cytoplasmic internalization. This is the first immunohistochemical analysis of 14-3-3σ and E-cadherin in a liver neoplasm in an animal species and the use of these markers requires further investigation in animal liver neoplasms.
... The challenging diagnosis and limited treatment options have revealed the need of new biomarkers to improve diagnosis. Padden and colleagues identified STI1 as a novel diagnostic biomarker for CCC by analyzing the increase in protein expression in a cohort of 60 patients using immunohistochemistry studies complemented with 2D-Dige and massspectrometry-based label-free proteomics [88]. ...
... In another study, using an HCC mouse model, deletion of STI1 in HCC cells reduced intrahepatic and lung metastasis. Remarkably, in human metastatic tissue and serum samples, STI1 had higher expression than in non-metastatic controls [88]. Besides, STI1 activates Wnt/β-catenin pathway, promoting growth and migration of HCC cells [8]. ...
Article
Stress inducible protein 1 (STI1) is a co-chaperone acting with Hsp70 and Hsp90 for the correct client proteins’ folding and therefore for the maintenance of cellular homeostasis. Besides being expressed in the cytosol, STI1 can also be found both in the cell membrane and the extracellular medium playing several relevant roles in the central nervous system (CNS) and tumor microenvironment. During CNS development, in association with cellular prion protein (PrPc), STI1 regulates crucial events such as neuroprotection, neuritogenesis, astrocyte differentiation and survival. In cancer, STI1 is involved with tumor growth and invasion, is undoubtedly a pro-tumor factor, being considered as a biomarker and possibly therapeutic target for several malignancies. In this review, we discuss current knowledge and new findings on STI1 function as well as its role in tissue homeostasis, CNS and tumor progression.
... Several malignancies including hepatocellular carcinoma [11], pancreatic cancer [12], ovarian cancer [13,14], colon cancer [15], and cholangiocellular carcinoma [16] are characterized by STIP1 overexpression. In cancer cells, knockdown of STIP1 expression has been shown to reduce tumor invasiveness through the downregulation of matrix metalloproteinase-2 [17] and RhoC GTPase and related inhibition of pseudopodia formation [18]. ...
... Interestingly, clinical studies demonstrated that an increased STIP1 protein expression portends adverse outcomes in ovarian cancer [13]. STIP1 may also serve as a potential biomarker for cholangiocellular carcinoma [16] and hepatocellular carcinoma [11]. ...
Article
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Overexpression of stress-induced phosphoprotein 1 (STIP1) - a co-chaperone of heat shock protein (HSP) 70/HSP90 - and activation of the JAK2-STAT3 pathway occur in several tumors. Combined treatment with a HSP90 inhibitor and a JAK2 inhibitor exert synergistic anti-cancer effects. Here, we show that STIP1 stabilizes JAK2 protein in ovarian and endometrial cancer cells. Knock-down of endogenous STIP1 decreased JAK2 and phospho-STAT3 protein levels. The N-terminal fragment of STIP1 interacts with the N-terminus of JAK2, whereas the C-terminal DP2 domain of STIP1 mediates the interaction with HSP90 and STAT3. A peptide fragment in the DP2 domain of STIP1 (peptide 520) disrupted the interaction between STIP1 and HSP90 and induced cell death through JAK2 suppression. In an animal model, treatment with peptide 520 inhibited tumor growth. In summary, STIP1 modulates the function of the HSP90-JAK2-STAT3 complex. Peptide 520 may have therapeutic potential in the treatment of JAK2-overexpressing tumors.
... In China, 90% of cases are ESCC, compared to only 26% in the United States [3]. Despite many advances in the treatments of patients with EC, the 5-year survival rate remains poor (e.g., 17.4% in the United States) [4]. The survival rate of EC could reach up to 85% when diagnosed at an early stage but is no more than 10% if diagnosed at an advanced stage [5]. ...
... STIP1 is one of the cochaperones that are most extensively studied and contains three tetratricopeptide repeat (TPR) domains, which can simultaneously bind Hsp70 and Hsp90 [13,14]. STIP1 was identified to be overexpressed in several kinds of cancers, such as colorectal carcinoma (CRC) [15], pancreatic cancer [16], cholangiocellular carcinoma (CCC) [17], ovarian cancer [18], and so on. Moreover, increased expression of STIP1 may indicate poor survival outcome in cancer patients [18,19]. ...
Article
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Esophageal squamous cell carcinoma (ESCC) remains one of the leading causes of cancer-related mortality around the world. The identification of novel serum biomarkers is required for early detection of ESCC. This study was designed to elucidate whether autoantibodies against STIP1 could be a diagnostic biomarker in ESCC. An enzyme-linked immunosorbent assay was performed to detect serum levels of STIP1 autoantibodies in a training cohort (148 ESCC patients and 111 controls) and a validation cohort (60 ESCC patients and 40 controls). Mann–Whitney’s U test showed that ESCC patients in two cohorts have higher levels of autoantibodies against STIP1 when compared to controls ( P<0.001 ). According to receiver operating characteristic analysis, the sensitivity, specificity, and area under the curve (AUC) of autoantibodies against STIP1 in ESCC were 41.9%, 90.1%, and 0.682 in the training cohort and 40.0%, 92.5%, and 0.710 in the validation cohort, respectively. Moreover, detection of autoantibodies against STIP1 could discriminate early-stage ESCC patients from controls, with sensitivity, specificity, and AUC of 35.7%, 90.1%, and 0.684 in the training cohort and 38.5%, 92.5%, and 0.756 in the validation cohort, respectively. Our findings indicated that autoantibodies against STIP1 might be a useful biomarker for early-stage ESCC detection.
... Recent studies have suggested an important relationship between STIP1 and cancer. For example, STIP1 is overexpressed in a number of tumors, such as papillary thyroid carcinoma (PTC), pancreatic cancer, ovarian cancer and cholangiocellular carcinoma (11)(12)(13)(14). Recent studies show that high STIP1 expression correlates with the prognosis of metastatic ovarian cancer and with a poor prognosis in PTC, though the function of STIP1 is little known (11,15). ...
... In our study, high expression of STIP1 was positively associated with tumor size and stage, which was similar to what was reported by Tsai et al. Similarly, one independent group used two-dimensional differential ingel electrophoresis and by mass spectrometry-based labelfree proteomics and reported that the expression of STIP1 was higher in cholangiocellular carcinoma than in normal hepatocytes and non-tumorous cholangiocytes (14). In our study, the expression of STIP1 was higher in breast cancer tissues than in adjacent normal tissues, which was in agreement with the results of Padden et al. ...
Article
Background: Recent studies suggested an important relationship between tumor stress-induced phosphoprotein 1 (STIP1) and cancer. However, the expression of STIP1 in breast cancer tissues and its relationship with clinical characteristics and survival have not been investigated in humans. The aim of our work was to evaluate the association of STIP1 and the prognosis of breast cancer patients. Methods: The included patients were followed-up by telephone and through a review of their outpatient records. The expression of STIP1 was assessed by immunohistochemistry (IHC). The 5-year recurrence-free survival (RFS) rate and the 5-year overall survival (OS) rate were the prognostic indicators evaluated by the Kaplan-Meier method. Univariate and multivariate analyses employing a Cox regression model were used to calculate hazard ratios (HRs). Results: The rate of high expression of STIP1 was 55.3% (126/228) in breast cancer tissues and 14.9% (34/228) in adjacent normal tissues (χ2=81.495, P<0.001). High expression of STIP1 was associated with tumor size, stage and human epidermal growth factor receptor 2 (HER-2) status. The 5-year RFS rate was 75.4% in the STIP1 high expression group and 87.3% in the STIP1 low expression group (χ2=5.721, P=0.017). The 5-year OS rate was 84.1% in the STIP1 high expression group and 94.1% in the STIP1 low expression group (χ2=5.814, P=0.016). STIP1 was found to be an independent relapse predictor for the adjusted HR is 1.983 (95% CI, 1.031-3.815). Conclusions: High expression of STIP1 is associated with the poor prognosis of breast cancer patients and HER-2 positive expression. STIP1 may therefore serve as a prognostic biomarker for breast cancer patients.
... Nonlinear Dynamics, Newcastle upon Tyne, U.K.). A detailed description of the single steps of quantification has been recently published (28,29). ...
Article
Arabinogalactan (AG) isolated from dust of a traditional farm prevents disease in murine models of allergy. However, it is unclear whether this polysaccharide has immune regulatory properties in humans. The aim of this study was to test the influence of AG on the immune-stimulating properties of human dendritic cells (DCs). Moreover, we sought to identify the receptor to which AG binds. AG was produced from plant callus tissue under sterile conditions to avoid the influence of pathogen-associated molecular patterns in subsequent experiments. The influence of AG on the human immune system was investigated by analyzing its impact on monocyte-derived DCs. To analyze whether the T cell stimulatory capacity of AG-stimulated DCs is altered, an MLR with naive Th cells was performed. We revealed that AG reduced T cell proliferation in a human MLR. In the search for a molecular mechanism, we found that AG binds to the immune modulatory receptors DC-specific ICAM-3 -: grabbing non integrin (DC-SIGN) and macrophage mannose receptor 1 (MMR-1). Stimulation of these receptors with AG simultaneously with TLR4 stimulation with LPS increased the expression of the E3 ubiquitin-protein ligase tripartite motif -: containing protein 21 and decreased the phosphorylation of NF-κB p65 in DCs. This led to a reduced activation profile with reduced costimulatory molecules and proinflammatory cytokine production. Blocking of MMR-1 or DC-SIGN with neutralizing Abs partially inhibits this effect. We conclude that AG dampens the activation of human DCs by LPS via binding to DC-SIGN and MMR-1, leading to attenuated TLR signaling. This results in a reduced T cell activation capacity of DCs.
... Several other immunohistochemical markers have been studied in cholangiocarcinoma, but these are primarily aimed at differentiating between various intrahepatic malignant processes (ie, differentiation of cholangiocarcinoma from hepatocellular carcinoma or metastatic adenocarcinoma) [18,19]. A recent study identified "significant increased expression" of 14-3-3Sigma and SerpinH1 proteins in intrahepatic cholangiocarcinoma, and the authors advocate use of immunohistochemical stains for these proteins, together with the Ki-67 proliferative index (with a 5% proliferative index cutoff), as part of a 3-stain panel for distinction between benign and malignant biliary lesions [20]. ...
Article
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Differentiation between benign and malignant lesions of the hepatic biliary tree may pose a diagnostic problem, as well-differentiated intrahepatic cholangiocarcinoma may mimic biliary hamartoma, bile duct adenoma, or parenchymal extinction. We evaluated Ki-67 proliferative index and p53 status by immunohistochemical staining to aid in exclusion of cholangiocarcinoma. 14 biliary hamartomas, 21 bile duct adenomas, and 11 livers with parenchymal extinction were compared to 26 intrahepatic cholangiocarcinomas (16 well-differentiated and 10 moderate- or poorly- differentiated tumors). We found an increased proliferative index in intrahepatic cholangiocarcinomas compared to benign biliary proliferations (average 23.0% in cholangiocarcinoma vs. 1.4% in all benign biliary lesions, n 26 vs. 46, P<.001). No difference in average proliferative index was observed between well-differentiated and moderately/poorly differentiated cholangiocarcinomas (average 22.7% vs. 23.3%, n 16 vs. 10, P=.92). Average proliferation indices of benign biliary lesions were uniformly low (biliary hamartoma 1.2%; bile duct adenoma 2%; parenchymal extinction 0.4%). The majority of cholangiocarcinomas (23/26, 88.5%), but none of the benign lesions (0/46, 0%) had proliferative indices greater than 10%. Strong nuclear p53 immunohistochemical staining was only seen in cholangiocarcinomas (9/26, 34.6%) and not in benign biliary lesions (0/46, 0%), though many of the benign lesions showed weak to moderate staining Immunohistochemical staining for Ki67 facilitates distinction between benign and malignant lesions of the intrahepatic biliary tree, whereas p53 immunohistochemical staining, is less helpful.
... Our analysis revealed that some genes in the NOS target module were potent prognostic elements including SERPINH1, CYP1B1, DPYSL3, KCNMB4, MCTS1, and SEMA3C. SERPINH1 was considered as a potential diagnostic biomarker for cholangiocellular carcinoma (CCC) with a specificity of 98% and a sensitivity of 64% (Padden et al. 2014). Similarly, this gene has been identified to be involved in the progression of head and neck carcinomas and gastric cancer (Ollins et al. 2002;Zhang et al. 2010). ...
Article
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Osteosarcoma (OSA) is one of the most common malignancies in humans (especially children) and dogs. Canine OSA is considered an ideal model of human OSA because OSA in both species shares comparable oncogenic properties—including histopathological features, tumor biology, therapeutic approaches, and molecular mechanisms. In this study, we sought to compare the expression profiles of embryonic stem cell (ES) gene signatures in canine and human OSA tissues and cell lines. The expression patterns of three major ES gene signatures (modules or gene sets)—namely Myc, Core, and PRC modules—were primarily analyzed through the gene set enrichment analysis method in three gene expression datasets. For verification of the primary results, an additional 13 ES gene sets which were categorized into four groups—namely NOS targets, ES expressed, Myc targets, and Polycomb targets—were evaluated in expression datasets. Additionally, the prognostic efficacy of the gene sets with a similar enrichment pattern in humans and dogs was evaluated using the Cox proportional hazard model. Our results revealed that there were similar ES module expression patterns in the human OSA tissue and cell line, where the MYC modules, Myc targets, ES exp1, and NOS target modules were upregulated and the Polycomb target modules were downregulated in both entities. The canine OSA cell line showed ES enrichment patterns more similar to the patterns in its human counterpart, where we were able to detect the upregulation of the MYC modules, Myc targets, ES exp1, and NOS target modules and the downregulation of the Polycomb targets, including H3K27 bound and PRC2 targets. However, the canine OSA tissues presented a similar enrichment pattern at a lower degree than the canine OSA cell line. Furthermore, survival analysis identified the NOS targets as a more robust prognostic gene signature that efficiently predicted survival time in the human and canine OSA samples.
... Serpin H1 (also known as heat shock protein 47, Hsp 47) is a 47 kDa endoplasmic reticulum (ER) protein which can specifically bind to collagen and works as a chaperone in collagen synthesis (Ito and Nagata 2017). Hsp 47 (encoded by SERPINH1), a member from serpin family, participated in progression of cancer such as hepatocellular carcinoma (HCC) (Naboulsi et al. 2016) and cholangiocellular carcinoma (Padden et al. 2014). Overexpression of Hsp 47 was also reported in micro-vessels from invasive ductal carcinoma (IDC) of breast cancer (vs. ...
Article
Currently, drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis. Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improvement of prognosis. Here we present our N-glycoproteomics study of putative N-glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7 (MCF-7/ADR) relative to parental michigan cancer foundation-7 (MCF-7) cells. Intact N-glycopeptides (IDs) from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), labeled with stable isotopic diethylation (SIDE), and analyzed with C18-RPLC-MS/MS (HCD with stepped normalized collision energies); these IDs were identified with database search engine GPSeeker, and the differentially expressed intact N-glycopeptides (DEGPs) were quantified with GPSeekerQuan. With target-decoy searches and control of spectrum-level FDR ≤ 1%, 322 intact N-glycopeptides were identified; these intact N-glycopeptides come from the combination of 249 unique peptide backbones (corresponding to 234 intact N-glycoproteins) and 90 monosaccharide compositions (corresponding to 248 putative N-glycosites). The sequence structures of 165 IDs were confirmed with structure-diagnostic fragment ions. With the criteria of observation at least twice among the three technical replicates, ≥ 1.5-fold change and p value < 0.05, 20 DEGPs were quantified, where five of them were up-regulated and 15 of them were down-regulated; the corresponding intact N-glycoproteins as putative markers of drug resistance were discussed.
... Stress-induced phosphoprotein 1 (STIP1, Gene ID:10963)-an adaptor protein of the HSP70/HSP90 chaperone heterocomplex [1,2]-contains a nuclear localization signal regulated by cell cycle kinase phosphorylation [3] and plays a critical role in cell proliferation [4]. Significant increases in the expression of STIP1 have been reported in several solid tumors, including hepatocellular carcinoma [5], pancreatic cancer [6], ovarian cancer [7,8], colon cancer [9], breast cancer [10], and cholangiocellular carcinoma [11]. Conversely, targeted downregulation of endogenous STIP1 expression resulted in a decreased proliferation of pancreatic cancer, ovarian cancer, and osteosarcoma cells [12][13][14][15]. ...
Article
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Stress-induced phosphoprotein-1 (STIP1)—a heat shock protein (HSP)70/HSP90 adaptor protein—is commonly overexpressed in malignant cells, where it controls proliferation via multiple signaling pathways, including JAK2/STAT3. We have previously shown that STIP1 stabilizes the protein tyrosine kinase JAK2 in cancer cells via HSP90 binding. In this study, we demonstrate that STIP1 may act as a substrate for JAK2 and that phosphorylation of tyrosine residues 134 and 152 promoted STIP1 protein stability, induced its nuclear-cytoplasmic shuttling, and promoted its secretion into the extracellular space. We also found that JAK2-mediated STIP1 phosphorylation enhanced cell viability and increased resistance to cisplatin-induced cell death. Conversely, interference STIP1 with JAK2 interaction—attained either through site-directed mutagenesis or the use of cell-penetrating peptides—decreased JAK2 protein levels, ultimately leading to cell death. On analyzing human ovarian cancer specimens, JAK2 and STIP1 expression levels were found to be positively correlated with each other. Collectively, these results indicate that JAK2-mediated phosphorylation of STIP-1 is critical for sustaining the JAK2/STAT3 signaling pathway in cancer cells.
... 4 Thus, the search continues for a useful circulating biomarker for CCA. Several approaches are available, 5 among which one is based on proteomics of samples such as plasma, 6 serum, 7 extracellular vesicles, 8 bile, 9 membrane protein, 10 secreted protein from tumor cell lines, 11 tumor tissue, 12,13 and microdissected cells from tumor tissue. 14 Recently, tumor interstitial fluid (TIF) has been proposed as a possible source of biomarkers when investigated using proteomic-based approaches. ...
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Tumor interstitial fluid contains tumor-specific proteins that may be useful biomarkers for cancers. In this study, we identified proteins present in cholangiocarcinoma interstitial fluid. Proteins derived from three samples of tumor interstitial fluid and paired samples of adjacent normal interstitial fluid from cholangiocarcinoma patients were subjected to two-dimensional liquid chromatography with tandem mass spectrometry. Candidate proteins were selected based on a greater than twofold change in expression levels between tumor interstitial fluid and normal interstitial fluid. Upregulation of six proteins in tumor interstitial fluid, including S100 calcium binding protein A6 (S100A6), S100 calcium binding protein A9, aldo-keto reductase family 1 member C4, neuropilin-1, 14-3-3 zeta/delta, and triosephosphate isomerase was assessed by western blot and immunohistochemistry. Their potential as markers was evaluated in human cholangiocarcinoma tissue arrays, and in serum using enzyme-linked immunosorbent assay. Expression of S100A6 was higher in tumor interstitial fluid than in normal interstitial fluid and showed the highest positive rate (98.96%) in cholangiocarcinoma tissues. Serum levels of S100A6 did not differ between cholangitis and cholangiocarcinoma patients, but were significantly higher than in healthy individuals (p < 0.0001). In cholangiocarcinoma cases, S100A6 level was associated with vascular invasion (p = 0.007) and could distinguish cholangiocarcinoma patients from healthy individuals as effectively as the carbohydrate antigen 19-9. In addition, potential for drug treatment targeting S100A6 and other candidate proteins was also demonstrated using STITCH analysis. In conclusion, proteomics analysis of tumor interstitial fluid could be a new approach for biomarker discovery, and S100A6 is a potential risk marker for screening of cholangiocarcinoma.
... Stress-induced phosphoprotein 1 (STIP1), also known as HOP/P60/STI1, is a chaperone protein that comprises three tetratricopeptide repeat domains, which can simultaneously bind HSP70s and HSP90s. STIP1 is a tumour-associated antigen (TAA) 33 ; its overexpression has been identified in numerous cancers, including colorectal carcinoma 34 , pancreatic cancer 35 , cholangiocellular carcinoma 36 and ovarian cancer 37 , and it is possibly associated with poor survival outcomes in patients with cancer 38 . ST13 encodes ST13 HSP70s-interacting protein (HIP)/putative tumour suppressor ST13/ suppression of tumorigenicity 13 protein, which is also involved in the regulation of heat shock factor 1 (HSF1)mediated heat shock response and which mediates the association of HSP70s and HSP90s. ...
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The tumourigenesis of early lung adenocarcinomas, including adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and lepidic predominant invasive adenocarcinoma (LPA), remains unclear. This study aimed to capture disease-related molecular networks characterising each subtype and tumorigenesis by assessing 14 lung adenocarcinomas (AIS, five; MIA, five; LPA, four). Protein–protein interaction networks significant to the three subtypes were elucidated by weighted gene co-expression network analysis and pairwise G-statistics based analysis. Pathway enrichment analysis for AIS involved extracellular matrix proteoglycans and neutrophil degranulation pathway relating to tumour growth and angiogenesis. Whereas no direct networks were found for MIA, proteins significant to MIA were involved in oncogenic transformation, epithelial-mesenchymal transition, and detoxification in the lung. LPA was associated with pathways of HSF1-mediated heat shock response regulation, DNA damage repair, cell cycle regulation, and mitosis. Genomic alteration analysis suggested that LPA had both somatic mutations with loss of function and copy number gains more frequent than MIA. Oncogenic drivers were detected in both MIA and LPA, and also LPA had a higher degree of copy number loss than MIA. Our findings may help identifying potential therapeutic targets and developing therapeutic strategies to improve patient outcomes.
... Several previous studies have employed various MSbased proteomics approaches to identify biomarkers of cholangiocarcinoma through analysis of resected human tissues [72,[77][78][79][80][81][82][83][84][85][86][87][88]. The majority of these previous studies have investigated iCCA or a mixture of different CCA subtypes. ...
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... Clinical studies have shown that increased STIP1 protein expression may be a sign of ovarian cancer (Chao et al., 2013). STIP1 can also be used as a potential biomarker of bile duct cell carcinoma and hepatocellular carcinoma (Padden et al., 2014;Sun et al., 2007). ...
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... Fisher suggested 5% (α=0.05) level could be used for concluding that fairly strong evidence exists against H0. Based on the definition of statistically significant differences and the method of referring to a large number of relevant literatures [48][49][50][51][52][53], we also defined the differential genes as | Fold Change | > 1.5, P-value < 0.05. ...
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... Sample preparation for protein identification by mass spectrometry was carried out as described in Schrötter et al. (44). LC-MS/MS Analysis and data processing were performed as described by Padden et al (45). ...
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Proteins designated for peroxisomal proteins import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, while Pex18p is especially important for oleate-induced import of PTS2-proteins. The glycerol-producing PTS2 protein glycerol-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol and in the nucleus under osmotic stress conditions. We show (i) that Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD salvage pathway, Pnc1p, and (ii) that Pnc1p, a nicotinamidase without functional PTS2 is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
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Chemotherapeutic treatment regimens often take advantage of synergistic effects of drug combinations. Anticipating that synergistic effects on cell biological level likely manifest on proteome level, the analysis of proteome modulations represents an appropriate strategy to study drug combinations on molecular level. More specifically, the detection of single proteins exhibiting synergistic abundance changes could be helpful to shed light on key molecules, which contribute in mechanisms facilitating the synergistic interaction and therefore represent potential targets for specific therapeutic approaches. In the reported study, we investigated the drug combination of cisplatin and the neddylation inhibitor MLN4924 in HCT-116 cells via cell biological analyses and mass spectrometry-based quantitative proteomics. From 1,789 proteins quantified with two unique peptides, activated RNA polymerase II transcriptional coactivator p15 (SUB1) was highlighted as most synergistically regulated protein using a synergistic scoring approach. Western blotting and analyses of cellular processes associated with this protein (DNA damage, oxidative stress and apoptosis) revealed supporting evidence for the synergistic regulation. Whereas the distinct role of SUB1 in the investigated drug combination needs to be elucidated in future studies, the presented results demonstrated the benefit and feasibility of synergistic scoring of proteome alterations to highlight proteins that likely contribute to the underlying molecular mechanisms of synergistic effects. Data are available via ProteomeXchange with identifier PXD009185.
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Objective Alterations in sphingolipid and ceramide metabolism have been associated with various diseases, including nonalcoholic fatty liver disease (NAFLD). Acid sphingomyelinase (ASM) converts the membrane lipid sphingomyelin to ceramide, thereby affecting membrane composition and domain formation. We investigated the ways in which the Asm knockout (Smpd1−/−) genotype affects diet-induced NAFLD. Methods Smpd1−/− mice and wild type controls were fed either a standard or Western diet (WD) for 6 weeks. Liver and adipose tissue morphology and mRNA expression were assessed. Quantitative proteome analysis of liver tissue was performed. Expression of selected genes was quantified in adipose and liver tissue of obese NAFLD patients. Results Although Smpd1−/− mice exhibited basal steatosis with normal chow, no aggravation of NAFLD-type injury was observed with a Western diet. This protective effect was associated with the absence of adipocyte hypertrophy and the increased expression of genes associated with brown adipocyte differentiation. In white adipose tissue from obese patients with NAFLD, no expression of these genes was detectable. To further elucidate which pathways in liver tissue may be affected by Smpd1−/−, we performed an unbiased proteome analysis. Protein expression in WD-fed Smpd1−/− mice indicated a reduction in Rictor (mTORC2) activity; this reduction was confirmed by diminished Akt phosphorylation and altered mRNA expression of Rictor target genes. Conclusion These findings indicate that the protective effect of Asm deficiency on diet-induced steatosis is conferred by alterations in adipocyte morphology and lipid metabolism and by reductions in Rictor activation.
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Context and objective: Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of novel marker candidates by quantitative proteomics. Methods and results: Proteomic differences between HCC and nontumorous liver tissue were studied by mass spectrometry. Among several significantly upregulated proteins, translocator protein 18 (TSPO) and Ras-related protein Rab-1A (RAB1A) were selected for verification by immunohistochemistry in an independent cohort. For RAB1A, a high accuracy for the discrimination of HCC and nontumorous liver tissue was observed. Conclusion: RAB1A was verified to be a potent biomarker candidate for HCC.
Thesis
Kumulative Habilitationsschrift zur Erlangung der Venia legendi für das Fach "Immunologie"
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Background: Dependent on the extent of adenosine triphosphate (ATP) hydrolysis and/or ATP/ADP exchange, the stress-induced phosphoprotein 1 (STIP1) mediates molecular interaction and complex formation between the molecular chaperones heat shock protein (Hsp)70 and Hsp90. The overexpression of STIP1 is increasingly being documented in various human malignancies, including ovarian, cholangiocellular, renal and gastric cancers. However, the role of STIP1 in pancreatic cancer (PANC) and probable molecular mechanism remains largely unexplored. Methods & results: In the present study, using clinical samples (n = 88) and human PANC cell lines PANC-1, Capan-2, SW1990, and BxPC-3, we demonstrated that STIP1 is aberrantly expressed in human PANC tissues or cell lines compared to adjacent non-tumor pancreas samples or human pancreatic duct epithelial cells (HPDEC), respectively. Clinicopathological correlation studies revealed significant positive correlation between high STIP1 expression and lymph node involvement (p = 0.001), cancer metastasis (p = 0.002), microvascular invasion (p = 0.002), advance TNM stage (p = 0.024), perineural invasion (PNI; p = 0.013), and cancer-related death (p = 0.002) among patients with PANC. Univariate and multivariate analyses indicate that STIP1overexpression is an independent prognostic factor of PANC. Furthermore, STIP1 knockdown significantly inhibit the migration and invasive ability of PANC-1 and SW1990 cells, while downregulating N-cadherin and Vimentin, but upregulating E-cadherin mRNA expression levels, concurrently. We also demonstrated that STIP1 knockdown suppressed p-FAK, p-AKT, MMP2, MMP9, and Slug protein and mRNA expression levels, thus, indicating, at least in part, a role for STIP1 in the activation of FAK/AKT/MMP signaling. Conclusion: Taken together, our results demonstrate a critical role for STIP1 in cancer metastasis, disease progression and poor prognosis, as well as, provide evidence suggestive of the therapeutic efficacy of STIP1-mediated targeting of the FAK/AKT/MMP signaling axis in patients with PANC.
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Objective: Cerebral vasospasm (CV) and associated secondary brain injury are major contributors to death and disability after aneurysmal subarachnoid hemorrhage (aSAH). Microparticles (MP) are small vesicular micro-molecules released by red and white blood cells, platelets and endothelial cells that can change rapidly and specifically depending on the type of cellular insult. They may serve as useful tools to target a specific pool of proteins associated with the development of CV post aSAH. In these studies, our goal was to use targeted MP-derived protein isolation to find reliable biomarkers indicating increased risk for the development of CV. We hypothesize that there are specific early changes in MP-derived protein expression in CV patients. These proteins may be useful as biomarkers for CV and may help us to further understand the mechanism for the development of CV. Patients Adult male and female patients with angiographically confirmed aSAH and an external ventricular drain (EVD) placed for medical or surgical needs were included in this study. Patients were closely monitored for CV development. Cerebrospinal fluid (CSF) was collected daily until EVD was removed. Methods: Microparticles were isolated using serial ultra centrifugation. Differential protein expression in CSF microparticles was analyzed by a mass spectroscopy based system using isotopically-tagged peptides to profile proteins and determine their relative concentrations in individual patient samples. These proteins were correlated with the patient's clinical data and used to identify candidates for biomarkers predictive of CV. Results: Over 140 proteins were isolated from CSF microparticles. Proteomic and molecular pathways analysis revealed marked differential expression of proteins in patients with CV. We identified specific candidate proteins that could potentially serve as early biomarkers for CV. ApoE, ApoD, synaptic nuclear envelope protein 1, clusterin, α-1-acid glycoprotein, plasma protease C1 inhibitor, and prostaglandin H2 D isomerase were downregulated in patients who developed CV post aSAH. Haptoglobin, fibrinogen α and γ chain, synaptic nuclear envelope protein 2, and hemoglobin subunits α and β were upregulated. Some of these proteins are associated with immune and metabolic processes and some have been specifically associated with cerebrovascular disease states. Conclusions: This is the first preliminary demonstration that there is differential protein expression in CSF microparticles from CV patients. Alone or in combination, these and other proteins may be useful as reliable biomarkers to guide in stratifying patients into categories of risk to develop CV post aSAH. These results will deepen our understanding of the mechanisms of cerebral vasospasm and potentially facilitate the development of safer and more effective therapies therapies for cerebral vasospasm.
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Stress-induced phosphoprotein 1 (STIP1) has been recently identified as a released biomarker in human ovarian cancer. In addition, STIP1 secreted by human ovarian cancer cells has been shown to promote tumor cell proliferation by binding to ALK2 (activin A receptor, type II-like kinase 2) and activating the SMAD-ID3 signaling pathways. In this study, a total of 330 ovarian cancer tumor samples were evaluated for STIP1 expression by immunohistochemistry and analyzed for a possible correlation with patient characteristics and survival. The quantification of immunoreactivity was accomplished by applying an immunohistochemical scoring system (histoscore). Patients with high-level STIP1 expression (histoscore ≥169) had a significantly worse survival (high STIP1, mean survival time = 76 months; low STIP1, mean survival time = 112 months; <0.0001). Moreover, STIP1 histoscores were significantly higher in high-grade tumors (grade 3) than in low-grade (grade 1-2) malignancies (<0.0001), suggesting that STIP1 may be a proxy for tumor aggressiveness. The results of multivariable analysis revealed that high STIP1 histoscores, advanced stages, histologic types, and the presence of residual disease (≥2 cm) were independent predictors of poor prognosis. The addition of STIP1 histoscores improved the prediction of overall and progression-free survival rates in the multivariable Cox proportional hazard model. The treatment of ovarian cancer cells with recombinant STIP1 stimulated cell proliferation and migration, but co-treatment with anti-STIP1 antibodies abrogated this effect. Our findings suggest that STIP1 expression may be related to prognosis and that the STIP1 pathway may represent a novel therapeutic target for human ovarian cancer.
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Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary gel-based and LC-MS-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 x HCC vs. 7 x non-tumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of MVP and BHMT were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but non-significant trends were detectable in this independent cohort. Based on these results we assume that MVP and BHMT have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.
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The PRoteomics IDEntifications (PRIDE, http://www.ebi.ac.uk/pride) database at the European Bioinformatics Institute is one of the most prominent data repositories of mass spectrometry (MS)-based proteomics data. Here, we summarize recent developments in the PRIDE database and related tools. First, we provide up-to-date statistics in data content, splitting the figures by groups of organisms and species, including peptide and protein identifications, and post-translational modifications. We then describe the tools that are part of the PRIDE submission pipeline, especially the recently developed PRIDE Converter 2 (new submission tool) and PRIDE Inspector (visualization and analysis tool). We also give an update about the integration of PRIDE with other MS proteomics resources in the context of the ProteomeXchange consortium. Finally, we briefly review the quality control efforts that are ongoing at present and outline our future plans.
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Stress-induced phosphoprotein 1 (STIP1), a cochaperone that organizes other chaperones, heat shock proteins (HSPs), was recently shown to be secreted by human ovarian cancer cells. In neuronal tissues, binding to prion protein was required for STIP1 to activate the ERK (extracellular-regulated MAP kinase) signaling pathways. However, we report that STIP1 binding to a bone morphogenetic protein (BMP) receptor, ALK2 (activin A receptor, type II-like kinase 2), was necessary and sufficient to stimulate proliferation of ovarian cancer cells. The binding of STIP1 to ALK2 activated the SMAD signaling pathway, leading to transcriptional activation of ID3 (inhibitor of DNA binding 3), promoting cell proliferation. In conclusion, ovarian-cancer-tissue-secreted STIP1 stimulates cancer cell proliferation by binding to ALK2 and activating the SMAD-ID3 signaling pathways. Although animal studies are needed to confirm these mechanisms in vivo, our results may pave the way for developing novel therapeutic strategies for ovarian cancer.
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The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses – the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferroni-type procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.
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Cholangiocarcinoma is the most frequent biliary malignancy. It is difficult to diagnose owing to its anatomic location, growth patterns and lack of definite diagnostic criteria. Currently, cholangiocarcinoma is classified into the following types according to its anatomic location along the biliary tree: intrahepatic, perihilar or distal extrahepatic cholangiocarcinoma. These cholangiocarcinoma types differ in their biological behavior and management. The appropriate stratification of patients with regard to the anatomic location and stage of cholangiocarcinoma is a key determinate in their management. Staging systems can guide this stratification and provide prognostic information. In addition, staging systems are essential in order to compare and contrast the outcomes of different therapeutic approaches. A number of staging systems exist for cholangiocarcinoma-several early ones have been updated, and new ones are being developed. We discuss the emerging diagnostic criteria as well as the different staging systems for cholangiocarcinoma, and provide a critical appraisal regarding these advances in biliary tract malignancies.
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Receiver operating characteristic (ROC) curves are useful tools to evaluate classifiers in biomedical and bioinformatics applications. However, conclusions are often reached through inconsistent use or insufficient statistical analysis. To support researchers in their ROC curves analysis we developed pROC, a package for R and S+ that contains a set of tools displaying, analyzing, smoothing and comparing ROC curves in a user-friendly, object-oriented and flexible interface. With data previously imported into the R or S+ environment, the pROC package builds ROC curves and includes functions for computing confidence intervals, statistical tests for comparing total or partial area under the curve or the operating points of different classifiers, and methods for smoothing ROC curves. Intermediary and final results are visualised in user-friendly interfaces. A case study based on published clinical and biomarker data shows how to perform a typical ROC analysis with pROC. pROC is a package for R and S+ specifically dedicated to ROC analysis. It proposes multiple statistical tests to compare ROC curves, and in particular partial areas under the curve, allowing proper ROC interpretation. pROC is available in two versions: in the R programming language or with a graphical user interface in the S+ statistical software. It is accessible at http://expasy.org/tools/pROC/ under the GNU General Public License. It is also distributed through the CRAN and CSAN public repositories, facilitating its installation.
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Proteins that exhibit different expression levels in normal and malignant lung cells are good candidate biomarkers to improve early diagnosis and intervention. We used a quantitative approach and compared the proteome of microdissected cells from normal human bronchial epithelium and squamous cell carcinoma tumors of histopathological grades G2 and G3. DIGE analysis and subsequent MS-based protein identification revealed that 32 non-redundant proteins were differentially regulated between the respective tissue types. These proteins are mainly involved in energy pathways, cell growth or maintenance mechanisms, protein metabolism, and the regulation of DNA and RNA metabolism. The expression of some of these proteins was analyzed by immunohistochemistry using tissue microarrays containing tissue specimen of 55 patients, including normal bronchial epithelium, squamous cell carcinomas, adenocarcinomas, and large cell carcinomas. The results of the immunohistochemical studies correlated with the proteome study data and revealed that particularly HSP47 and a group of cytokeratins (i.e. cytokeratins 6a, 16, and 17) are significantly co-regulated in squamous cell carcinoma. Furthermore cytokeratin 17 showed significantly higher abundance in G2 grade compared with G3 grade squamous cell carcinomas in both the gel-based and the immunohistochemical analysis. Therefore this protein might be used as a marker for stratification between different tumor grades.
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The 14-3-3 sigma gene has been implicated in G2/M cell cycle arrest by p53. Frequent inactivation of the 14-3-3 sigma gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of this study was to examine the methylation status of CpG islands of the 14-3-3 sigma gene in hepatocellular carcinoma (HCC). The methylation status of the 14-3-3 sigma gene was evaluated in four normal liver tissues and 19 paired specimens of carcinoma and adjacent non-tumorous liver tissues using bisulfite-single strand conformation polymorphism (bisulfite-SSCP), a combination of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. The 14-3-3 sigma protein expression was examined by immunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 sigma gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samples analysed by immunohistochemistry showed loss of 14-3-3 sigma expression, while both of the methylation-negative HCC samples retained the expression, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumorous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis tissues), but the 14-3-3 sigma expression was retained in all of these tissues. In a methylation-positive HCC cell line, HLE, 5-aza-2'-deoxycytidine (5-aza-dC)-induced demethylation of CpG islands led to reactivation of gene expression, indicating that hypermethylation plays a causal role in inactivation of the 14-3-3 sigma gene in HCC. Hypermethylation and the resulting loss of expression of the 14-3-3 sigma gene corresponds to one of the most common abnormalities reported to date in HCC, suggesting their crucial role in the development and/or progression of HCC.
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### 1.1 Development of guidelines There is currently no clear national consensus for the optimal diagnosis and treatment of cholangiocarcinoma. The need for these guidelines was highlighted following the annual meeting of the British Association for the Study of the Liver (BASL) in September 2000. During their development these guidelines were presented at a BASL Liver Cancer Workshop in January 2001. They were also circulated to BASL members and the Liver Section of the British Society of Gastroenterology (BSG) Committee members, including gastroenterologists, hepatologists, gastroenterological surgeons, pathologists, radiologists, and epidemiologists for comments before the final consensus document was drawn up. ### 1.2 Strategy The guidelines are based on comprehensive literature surveys including results from randomised controlled trials, systematic reviews and meta-analyses, and cohort, prospective, and retrospective studies. On issues where no significant study data were available, evidence was obtained from expert committee reports or opinions. Where possible, specific recommendations have been graded, based on the quality of evidence available (section 2.4). ### 1.3 Context and intent These guidelines are intended to bring consistency and improvement in the patient’s management from first suspicion of cholangiocarcinoma through to confirmation of the diagnosis and subsequent management. As stated in previous BSG guidelines, patient preferences must be sought and decisions made jointly by the patient and health carer, based on the risks and benefits of any intervention. Furthermore, the guidelines should not necessarily be regarded as the standard of care for all patients. Individual cases must be managed on the basis of all clinical data available for that case. The guidelines are subject to change in light of future advances in scientific knowledge. Mortality rates from intrahepatic cholangiocarcinoma have risen steeply and steadily over the past 30 years and since the mid 1990s more deaths have been coded annually in England and Wales as being due to this tumour than to hepatocellular carcinoma.1 In 1997 and …
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Hepatozelluläre Karzinome (HCC) und Gallengangskarzinome (CCC) haben eine schlechte Prognose, da sie häufig erst in fortgeschrittenen Stadien entdeckt werden und es bislang keine wirksame adjuvante Therapie gibt. Zur frühzeitigen Erkennung dieser Tumoren können Serummarker (z. B AFP, CA19-9) eingesetzt werden, die jedoch nur eine mäßige Sensitivität oder Spezifität aufweisen. Das Golgi-Apparat-assoziierte Protein GOLPH2 wurde im Gewebe und Serum von HCC- und CCC-Patienten nachgewiesen und kann für die Früherkennung dieser Tumoren eingesetzt werden. Der Goldstandard der Diagnose von HCC und CCC ist immer noch die Biopsie. Eine sichere Diagnosestellung ist jedoch bei zirrhotisch oder entzündlich verändertem Gewebe nicht immer rein morphologisch möglich. Neue immunhistochemische Marker können bei dieser wichtigen Differenzialdiagnose sehr hilfreich sein. Studien zeigten, dass das onkofetale IGF-II-mRNA-bindende Protein 3 (IMP3), die Zelladhäsionsmoleküle P-Cadherin und CD24, das Cancer-Testis-Antigen MAGE-C2/CT-10 sowie das Protein Periostin als Gewebsmarker in der Diagnostik für die Früherkennung von HCC und CCC sehr gut einsetzbar sind.
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A new approach to problems of multiple significance testing was presented in Benjamini and Hochberg (1995), which calls for controlling the expected ratio of the number of erroneous rejections to the number of rejections–the False Discovery Rate (FDR). The procedure given there was shown to control the FDR for independent test statistics. When some of the hypotheses are in fact false, that procedure is too conservative. We present here an adaptive procedure, where the number of true null hypotheses is estimated first as in Hochberg and Benjamini (1990), and this estimate is used in the procedure of Benjamini and Hochberg (1995). The result is still a simple stepwise procedure, to which we also give a graphical companion. The new procedure is used in several examples drawn from educational and behavioral studies, addressing problems in multi-center studies, subset analysis and meta-analysis. The examples vary in the number of hypotheses tested, and the implication of the new procedure on the conclusions. In a large simulation study of independent test statistics the adaptive procedure is shown to control the FDR and have substantially better power than the previously suggested FDR controlling method, which by itself is more powerful than the traditional family wise error-rate controlling methods. In cases where most of the tested hypotheses are far from being true there is hardly any penalty due to the simultaneous testing of many hypotheses.
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To investigate the association of expression status of α-enolase (ENO1) and clinicopathological outcomes of CCA patients. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to compare differential expressed protein profiles of four human CCA cell lines and H69, a non-malignant biliary cell line, as a control. Immunohistochemical analysis was carried out in tissue-microarray of human CCA tissues (n=301). We identified ENO1 in all CCA cell lines but not H69 by proteomics based. About 75% of patients with CCA showed over-expression of ENO1 in hyperplastic bile duct and the tumors compared with that in tumor-adjacent normal tissue counterparts. Moreover, over-expression of ENO1 is significantly associated with poor prognosis and tumor invasion of CCA patients. ENO1 may serve as a prognostic marker to monitor the disease progression of these patients.
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We performed a comparative proteomic analysis of protein expression profiles in 4 cholangiocarcinoma cell lines: K100, M156, M213, and M139. The H69 biliary cell line was used as a control. Peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 were selected for further validation by immunohistochemistry using a cholangiocarcinoma tissue microarray (n = 301) to assess their prognostic value in this cancer. Both peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 were overexpressed in cholangiocarcinoma tissues compared with normal liver tissues. Of the 301 cholangiocarcinoma cases, overexpression of peroxiredoxin 1 in 103 (34.3%) was associated with an age-related effect in young patients (P = .011) and the absence of cholangiocarcinoma in lymphatic vessels and perineural tissues (P = .004 and P = .037, respectively). Expression of radixin-moesin-binding phosphoprotein 50 correlated with histopathologic type, with 180 (59.8%) of moderately or poorly differentiated tumors (P = .039) being higher, and was associated with the presence of cholangiocarcinoma in lymphatic and vascular vessels (P < .001 and P < .001, respectively). The high expression of radixin-moesin-binding phosphoprotein 50 and the low expression of peroxiredoxin 1 correlated with reduced survival by univariate analysis (P = .017 and P = .048, respectively). Moreover, the impact of peroxiredoxin 1 and radixin-moesin-binding phosphoprotein 50 expression on patient survival was an independent predictor in multivariate analyses (P = .004 and P = .025, respectively). Therefore, altered expression of peroxiredoxin 1 and radixin-moesin-binding phosphoprotein 50 may be used as prognostic markers in cholangiocarcinoma.
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Cholangiocarcinoma is one of the deadliest malignancies worldwide. Recent studies reported that treatment with gemcitabine was effective in prolonging survival. However, as the treatment only benefited a limited subset of patients, selection of patients before treatment is required. To discover biomarkers predictive of the response to gemcitabine treatment in cholangiocarcinoma, we examined the proteome of three types of material resource; ten cell lines, nine xenografts and nine surgically resected primary tumors from patients who exhibited different response to gemcitabine treatment. Two-dimensional difference gel electrophoresis generated quantitative protein expression profiles including 3571 protein spots. We detected 172 protein spots with significant correlation with response to gemcitabine treatment. All proteins corresponding to these 172 protein spots were identified by mass spectrometry. We found that the macrophage-capping protein (CapG) was associated with response to gemcitabin treatment in all three types of material source. Immunohistochemical validation in an additional set of 196 cholangiocarcinoma cases revealed that CapG expression was associated with lymphatic invasion status and overall survival. Multivariate analysis showed that CapG protein expression was an independent prognostic factor for overall survival. In conclusion, CapG was identified as a novel candidate biomarker to predict response to gemcitabine treatment and survival in cholangiocarcinoma.
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Hepatocellular carcinomas (HCC) and bile duct carcinomas (BDC) have a poor prognosis since they are often detected at advanced stages and respond poorly to adjuvant therapy. Serum markers (e.g. AFP, CA19-9, etc.) can be used for early detection of these tumours but have only moderate sensitivity and specificity. The Golgi-associated protein GOLPH2 was found in the tissue and serum of patients with HCC and CCC and might be used to detect these tumours in time. The biopsy still remains the gold standard in the diagnosis of HCC and CCC. When biopsies are taken from these tumours they are often fragmented and contain reactive changes. Therefore immunohistochemical markers can aid in excluding or ascertaining malignancy. Studies have shown that the oncofetal protein "IGF-II mRNA-binding protein 3" (IMP3), the cell adhesion molecules P-cadherin and CD24, the cancer testis antigen MAGE-C2/CT-10 as well as the protein periostin can be used as tissue markers in the diagnosis of HCC and CCC.
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To date, protein profiles for hepatocellular carcinomas and cholangiocarcinomas have not been systematically evaluated and compared with each other in an unbiased way. Thirty-six hepatocellular carcinomas and adjacent normal tissue samples were analyzed using histology-directed, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Four cholangiocarcinomas and adjacent normal tissue samples were also evaluated. Tissue samples were sectioned at 10 µm, with 1-3 sections thaw-mounted on a conductive indium tin oxide-coated glass slide. Sinapinic acid was manually deposited on areas of each tissue section enriched by epithelial cells, either tumor or normal, and mass spectra were acquired using a MALDI-time of flight instrument. According to class prediction analysis, average prediction accuracy in test sets (composed of 18 hepatocellular carcinoma-normal pairs) ranged from 93.0 to 95.8%. Cholangiocarcinomas and hepatocellular carcinomas had different protein profiles, as evidenced by average prediction accuracy of >95% in the test set for all classifiers. Permutation P-values for 0.632 + bootstrap cross validated misclassification rates (at feature selection P < 0.001) were less than 0.05 for predicting p53 immunostaining status. We conclude that MALDI MS profiles may be useful in assisting with the diagnosis and the differential diagnosis of primary liver cancers.
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The 14-3-3sigma gene has been implicated in G2/M cell cycle arrest by p53, and the loss of 14-3-3sigma protein expression has been reported in diverse human cancers. However, the role of 14-3-3sigma in the signaling pathway of the cell cycle in the progression of intrahepatic cholangiocarcinoma has not been well understood. To clarify the role of 14-3-3sigma, we examined the protein expressions of 14-3-3sigma, cyclin B1, and p53 in 93 cases of intrahepatic cholangiocarcinoma by immunohistochemical staining. We also examined the correlation between these expressions and survival rate and clinicopathologic factors such as sex, age, tumor grade (ie, pathologic differentiation, tumor size, lymphatic permeation, vascular invasion, perineural invasion, lymph node metastasis), and tumor stage. Positive 14-3-3sigma protein expression (>30% of tumor cells) was observed in 67.7% (63/93) of cases of intrahepatic cholangiocarcinoma and was inversely correlated with cyclin B1 expression. No correlation was found between 14-3-3sigma expression and p53 expression or clinicopathologic factors; however, decreased 14-3-3sigma expression was an independent prognostic factor by multivariate survival analysis (P = .0282). Extensive methylation of 14-3-3sigma was found by methylation-specific polymerase chain reaction and sequence; however, no significant correlation was detected between methylation states and protein expression. These results indicate that depressed 14-3-3sigma protein is involved in the uncontrolled cell cycle in intrahepatic cholangiocarcinoma and that the decreased expression of 14-3-3sigma protein is a significant indicator of poor prognosis for patients with intrahepatic cholangiocarcinoma.
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Cholangiocarcinoma is an intractable cancer for which there is no effective therapy other than surgical resection, and many patients are not candidates for this treatment. Even for patients who undergo surgical resection, the 5-year survival rate is low. One reason for this is that the disease is often detected in late stages. Thus, there is a clear need for better biomarkers to facilitate early diagnosis and prognostication. During the biomarker discovery phase of our study, we used LC-MS-based proteomics with spectral counting, a semiquantitative approach to differential expression profiling, in paired cancerous and normal bile duct tissue samples from two cases. In total, 38 proteins up-regulated in the cancer samples were identified. These were verified using a SILAC method for MS-based validation. The results led to the identification of well-characterized proteins and proteins of unknown function that are up-regulated in cholangiocarcinoma. We used immunoblot analysis to validate four candidate biomarkers, actinin-1, actinin-4, protein DJ-1 and cathepsin B, with the test case samples and four additional cholangiocarcinoma case samples. Each of the four candidate proteins was overexpressed in a subset of five of the six cases tested. By immunohistochemistry, we further confirmed that expression of these proteins was elevated in cancer cells as compared with normal bile duct cells. Thus, we successfully identified several proteins up-regulated in cholangiocarcinoma. These proteins are candidate biomarkers and may also help to provide new insights into our understanding of the disease.
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Two types of fatty acid-binding protein (FABP) were isolated from human kidney by gel filtration and ion-exchange chromatography. Northern-blot analysis showed the presence of two FABP transcripts in total kidney RNA, hybridizing with cDNA of human liver and muscle FABP respectively. Characterisation based on molecular mass, isoelectric point, fluorescence with dansylaminoundecanoic acid and immunological cross-reactivity showed that one, type B, was fairly similar to human heart FABP. The other, type A, showed, like human liver FABP, a high fluorescence enhancement and a wavelength shift with dansylaminoundecanoic acid as well as the binding of a variety of ligands. Antibodies raised against FABP type A and against liver FABP markedly cross-reacted in e.l.i.s.a., in Western blotting and in indirect immunoperoxidase staining on kidney and liver sections. Differences in amino acid composition and isoelectric points, however, indicate that type A is a new kidney-specific FABP type. The FABP type A is more abundant in kidney than the B type and is predominantly localized in the cortex, especially in the cells of the proximal tubules. The FABP type B is mainly present in the cells of the distal tubules. In conclusion, this study shows the presence of two types of FABP in the kidney. One type seems to be related to heart FABP, while the other type resembles, but is not identical with, liver FABP. Both types have a characteristic cellular distribution along the nephron.
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An antiserum to carcinoembryonic antigen (CEA) and a monoclonal antibody to cytokeratin 19 (CK 19) were studied for their suitability as diagnostic reagents for the differential diagnosis of primary and secondary malignant epithelial tumours of the liver, on paraffin sections. With the antiserum to CEA, positive bile canalicular structures were found in 60 per cent of the hepatocellular carcinomas. All the cholangiocarcinomas and 66.6 per cent of the metastatic carcinomas were positive for CEA, without displaying a canalicular staining pattern. All the hepatocellular carcinomas were negative for CK 19. All the cholangiocellular carcinomas and the metastatic carcinomas were positive for CK 19. This staining profile may prove helpful in difficult diagnostic cases.
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Methods of evaluating and comparing the performance of diagnostic tests are of increasing importance as new tests are developed and marketed. When a test is based on an observed variable that lies on a continuous or graded scale, an assessment of the overall value of the test can be made through the use of a receiver operating characteristic (ROC) curve. The curve is constructed by varying the cutpoint used to determine which values of the observed variable will be considered abnormal and then plotting the resulting sensitivities against the corresponding false positive rates. When two or more empirical curves are constructed based on tests performed on the same individuals, statistical analysis on differences between curves must take into account the correlated nature of the data. This paper presents a nonparametric approach to the analysis of areas under correlated ROC curves, by using the theory on generalized U-statistics to generate an estimated covariance matrix.
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The major components of Reichert's membrane (laminin, type IV procollagen, entactin and heparan sulphate proteoglycan) are all synthesized by the parietal endoderm cells of the mouse embryo. Fibronectin is found mainly on the trophoblast side of Reichert's membrane and does not appear to be a major structural component. Parietal endoderm cells are thought to differentiate and migrate as individual cells from the margins of the epithelial visceral endoderm layer. They may interact with the type IV collagen in Reichert's membrane via a surface-associated protein of Mr = 47000 known as 'colligin'. Parietal endoderm cells are a rich source of mRNAs for basement membrane components and have been used to prepare a cDNA library in the expression vectors pUC8 and pUC9 from which cDNAs for type IV collagen and laminin B chains have been isolated.
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Gelatin coupled to Sepharose has been used to isolate [35S]methionine-labeled polypeptides of Mr = 47,000, 56,000, 62,000, and 65,000 from the 12,000 X g supernatant of detergent extracts of mouse embryo parietal endoderm cells. The polypeptides can also be recovered from various established cell lines which synthesize type IV procollagen, and in these cells the Mr = 47,000 polypeptide is the major gelatin-binding component. Several lines of evidence, including the results of continuous labeling and pulse-chase experiments, show that the polypeptides are not derived by proteolytic cleavage of larger precursors and are distinct from fragments of fibronectin. The Mr = 47,000, 62,000, and 65,000 polypeptides all contain N-linked oligosaccharide side chains, as judged by their labeling with [3H]mannose and their sensitivity to tunicamycin. The Mr = 62,000 and 65,000 polypeptides can be resolved by two-dimensional gel electrophoresis into species with different isoelectric points, and the Mr = 47,000 has a pI of 7.5-8.0. None of the gelatin-binding polypeptides appear to accumulate in the culture medium, and the Mr = 47,000, 62,000, and 65,000 species are labeled in a lactoperoxidase-catalyzed iodination of intact cells, suggesting that they are associated with the cell surface. Only the Mr = 47,000 glycoprotein binds to native type IV collagen. Possible functions for these surface components in vivo are discussed.
Article
cDNA encoding human 4-aminobutyrate aminotransferase (aminobutyrate:2-oxoglutarate aminotransferase) was prepared by polymerase chain reaction using mRNA from human neuroblastoma cells as the template and oligonucleotides synthesized on the basis of the information obtained from direct protein sequencing. The cDNA-deduced sequence enabled peptides, sequenced by automated Edman degradation, to be aligned for confirmation of the complete primary structure. The results are compared with the recently published sequence of the rat enzyme deduced entirely from DNA sequencing [Medina-Kauwe, L. K., Tillakaratne, N. J. K., Wu, J.-Y. & Tobin, A. J. (1994) J. Neurochem. 62, 1267-1275]. Although the sequences are almost identical for most of their length, they differ in a segment of 36 residues. Almost complete identity of the two sequences is established if it is assumed that a frame-shift error was introduced into the reported rat cDNA sequence. The human cDNA was used to probe for the presence of 4-aminobutyrate aminotransferase mRNA in human tissues and a significant transcript was found in heart, placenta and in tissues usually associated with the expression of this enzyme.
Article
Expression of heat-shock protein 47 in intact and fibrotic liver and in hepatic constituent cells was investigated in mice. Immunohistochemical study of intact liver and Western blot analysis of the protein from isolated liver cells revealed that stellate cells and smooth muscle cells of interlobular vessels, but not hepatocytes, Kupffer cells, or endothelial cells, expressed heat-shock protein 47. The protein was found in both vitamin-A-storing stellate cells and myofibroblast-like cells. The amount of the protein in cultured stellate cells was reduced by dexamethasone but was not regulated by quercetin, transforming growth factor beta, interferon gamma, or retinoic acid. In CCl4-treated or bile-duct-ligated mouse liver, the number of cells positive for heat-shock protein 47 markedly increased in the centrilobular area or around the periportal area, respectively, and the level of heat-shock protein 47 also increased.
Article
The cell cycle checkpoint plays an important role in maintaining the integrity of cells. Recently, one of the 14-3-3 protein family members, 14-3-3sigma, was shown to be regulated by p53 and to play a role in the G2-M-phase checkpoint. To determine whether 14-3-3sigma is inactivated in human cancers, the methylation status of the 5' region of 14-3-3sigma was investigated in a series of gastric, colorectal, and hepatocellular cancer cell lines. Of 22 cell lines examined, 6 showed aberrant methylation. The methylation status of 14-3-3sigma was found to be correlated with loss of expression, which was restored by 5-aza-2'-deoxycytidine treatment. Furthermore, normal G2 arrest after DNA damage was not demonstrated in the cell lines with methylation. In primary gastric cancers, 14-3-3sigma hypermethylation was observed frequently in 26 of 60 (43%) cases and observed more frequently in poorly differentiated adenocarcinomas (P = 0.0017). Our findings suggest that 14-3-3sigma is inactivated by aberrant methylation of the 5' region in various human cancers and that it might play an important role in the development of undifferentiated gastric cancers.
Article
It has been known for at least 50 years that alterations in methionine metabolism occur in human liver cirrhosis. However, the molecular basis of this alteration is not completely understood. In order to gain more insight into the mechanisms behind this condition, mRNA levels of methionine adenosyltransferase (MAT1A), glycine methyltransferase (GNMT), methionine synthase (MS), betaine homocysteine methyltransferase (BHMT) and cystathionine beta-synthase (CBS) were examined in 26 cirrhotic livers, five hepatocellular carcinoma (HCC) tissues and ten control livers. The expression of the above-mentioned genes was determined by quantitative RT-PCR analysis. Methylation of MAT1A promoter was assessed by methylation-sensitive restriction enzyme digestion of genomic DNA. When compared to normal livers MAT1A, GNMT, BHMT, CBS and MS mRNA contents were significantly reduced in liver cirrhosis. Interestingly, MAT1A promoter was hypermethylated in the cirrhotic liver. HCC tissues also showed decreased mRNA levels of these enzymes. These findings establish that the abundance of the mRNA of the main genes involved in methionine metabolism is markedly reduced in human cirrhosis and HCC. Hypermethylation of MAT1A promoter could participate in its reduced expression in cirrhosis. These observations help to explain the hypermethioninemia, hyperhomocysteinemia and reduced hepatic glutathione content observed in cirrhosis.
Article
Betaine-homocysteine S-methyltransferase (BHMT) has been shown to be expressed at high levels in the livers of all vertebrate species tested. It has also been shown to be abundant in primate and pig kidney but notably very low in rat kidney and essentially absent from the other major organs of monogastric animals. We recently showed by enzyme activity and Western analysis that pig kidney BHMT was only expressed in the cortex and was absent from the medulla. Using immunohistochemical detection, we report here that in human, pig, and rat kidney, BHMT is expressed in the proximal tubules of the cortex. Immunohistochemical staining for BHMT in human, pig, and rat liver indicate high expression in hepatocytes. The staining patterns are consistent with cytosolic expression in both organs.
Article
One isoform of the 14-3-3 family, 14-3-3sigma, plays a crucial role in the G2 checkpoint by sequestering Cdc2-cyclinB1 in the cytoplasm, and the expression of 14-3-3sigma is frequently lost in breast cancers. This loss of expression is thought to cause a G2 checkpoint defect, resulting in chromosomal aberrations. Since lung cancers frequently carry numerous chromosomal aberrations, we examined the DNA methylation status and expression level of the 14-3-3sigma gene in 37 lung cancer cell lines and 30 primary lung tumor specimens. We found that small cell lung cancer (SCLC) cell lines frequently showed DNA hypermethylation (9 of 13 lines, 69%), and subsequent silencing of the 14-3-3sigma gene. Among non-small cell lung cancers (NSCLC), large cell lung cancer cell lines showed frequent hypermethylation and silencing of 14-3-3sigma (4 or 7 lines, 57%). In contrast, in other NSCLC cell lines, hypermethylation occurred very rarely (1 of 17 lines, 6%). All eight primary SCLC specimens examined also showed a loss or significant reduction in 14-3-3sigma expression in vivo, while a loss or reduction of 14-3-3sigma expression was very rare in primary NSCLC specimens (1 of 22 tissues, 5%). This is the first description that indicates lung cancers frequently show significant inactivation of the 14-3-3sigma gene mainly due to DNA hypermethylation in SCLC, but rarely in NSCLC, suggesting involvement of the 14-3-3sigma gene in lung tumorigenesis in a histological type-specific manner.
Article
The age-standardized mortality rate for hepatocellular carcinoma is increasing in several countries. However, in England and Wales we previously reported an increase in mortality rates from intrahepatic cholangiocarcinoma. Trends in cholangiocarcinoma in most other industrialized countries are unknown. To further study trends in hepatobiliary and pancreatic tumours, we analysed mortality data from the United States, Japan, Australia and Europe. Age-standardized mortality rates for men and women for subcategories of liver tumours, tumours of the gall bladder and extrahepatic biliary tree and pancreas from 1979 to 1998 were obtained from the World Health Organization mortality database. We confirmed previously reported increases in hepatocellular carcinoma, but also found increases in other countries, particularly Australia (3-year average rise from 1.20 to 2.27, men). Mortality for intrahepatic cholangiocarcinoma increased in men in all countries studied, with the largest increases in Australia (from 0.10 to 0.70) and England and Wales (from 0.20 to 0.83). We present a hitherto unreported rise in age-standardized mortality rates from intrahepatic cholangiocarcinoma across four continents. The cause remains uncertain. An impact on the observed trends of improved diagnostic techniques and death certificate misclassification cannot be completely ruled out. Future research should include epidemiological studies to examine possible case-clustering and investigation of potential aetiological and host factors.
Article
Intestinal-type fatty acid-binding protein (I-FABP) has been proposed as plasma marker for the detection of acute intestinal injury. However, intestinal mucosa also expresses liver-type FABP (L-FABP). We have investigated the tissue distribution of I-FABP and L-FABP in segments of the human intestine along the duodenal to colonal axis and the potential of both proteins to serve as plasma marker for the diagnosis of intestinal injury. I-FABP and L-FABP were measured with specific immunoassays in autopsy samples of the intestine (duodenum, jejunum, ileum and colon) of 23 subjects and in plasma samples from patients (n = 51) with intestinal and/or hepatic disease. Plasma reference values were established in normal healthy individuals (n = 92). The I-FABP tissue contents in duodenum, jejunum, ileum, proximal colon and distal colon amounted to 2.22, 4.79, 1.04, 0.27 and 0.25 mug/g ww, respectively. L-FABP tissue contents were markedly higher, amounting to 124 and 198 mug/g ww in duodenum and jejunum, and to 58, 26 and 44 mug/g ww in ileum, proximal colon and distal colon, respectively. Elevated plasma levels of both I-FABP and L-FABP were found in patients suffering from intestinal diseases, while only L-FABP was increased in cases of purely hepatocellular injury. I-FABP and L-FABP show a similar pattern of tissue distribution along the duodenal to colonal axis with highest tissue contents found in the jejunum but in each intestinal segment a >40-fold higher content of L-FABP than of I-FABP. Accordingly, besides I-FABP, also L-FABP is a useful plasma marker for the detection of intestinal injury, especially in patients undergoing intestinal surgery.
Article
Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countries, such as Thailand. Distinguishing CCA from hepatocellular carcinoma (HCC) of the liver often requires the use of histochemistry, so molecular markers for diagnosis and prognosis are still required. In this study, the two-dimensional (2-D) protein map of a Thai human bile duct epithelial carcinoma cell line (HuCCA-1) has been compared to human hepatocellular carcinoma cell lines (HepG2 and HCC-S102) and a human breast epithelial cancer cell line (MCF-7). Our results show that HuCCA-1 expressed a unique pattern of proteins. Forty-three major proteins were identified by matching to the map of MCF-7, and by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). Cytokeratins CK8 and CK18 were overexpressed in both HuCCA-1 and HCC, while CK7 and CK19 were only expressed in HuCCA-1. Four specific proteins with MW/pI 57.2/5.21 (U1, vimentin), 42.2/6.20 (U2), 43.2/6.20 (U3, EF-TU), and 42.2/6.40 (U4, unidentified) were absent from HepG2. U2 showed high expression in HuCCA-1, while U1 and U4 showed high expression in HCC-S102. U2 could be separated in 2 proteins, U2/1 (alpha-enolase) and U2/2 (not identified) by using IPG pH 4-7. Galectin-3 showed high expression level in HuCCA-1 by 1-DE immunodetection, and gave only one spot with MW 32.9 kDa and pI 8.29 on 2-DE immunoblotting, Thus, certain proteins, namely CK7, CK19, U2/2 and galectin-3, may be good markers useful for differential diagnosis of cholangiocarcinoma compared to hepatocellular carcinoma.