Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer

Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum samples from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum samples were processed either by linear (pH 4-7) or narrow range (pH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear pH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum samples collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.     

卵巢高/低级别浆液性癌的定量蛋白质组学比较研究

目的:探讨卵巢高级别浆液性癌和低级别浆液性癌的差异表达蛋白,为阐明卵巢癌发生机制及寻找诊断和预后标志物的提供线索。方法:收集卵巢癌新鲜组织标本冻存于液氮中,经病理学确诊为高级别浆液性癌和低级别浆液性癌,两种类型各收集15例。应用i TRAQ定量蛋白质组学技术筛选及鉴定高/低级别浆液性癌的差异表达蛋白,并进行生物信息学分析。结果:卵巢高级别和低级别浆液性癌组织的定量蛋白质组学比较研究鉴定出差异表达蛋白314个,其中与低级别浆液性癌组比较,高级别浆液性癌组上调蛋白有97种,下调蛋白有217种。GO分析显示这些差异蛋白在分子功能、生物学功能、细胞成分方面均具有一定分布特点。KEGG分析显示这些差异蛋白涉及复杂的信号通路。结论:高/低级别浆液性癌之间存在差异表达蛋白,这些蛋白涉及复杂的功能和信号通路可能在两型卵巢癌发生机制及肿瘤生物学行为差异中具有重要意义。     

Identification of differentially expressed proteins in spontaneous thymic lymphomas from knockout mice with deletion of p53

Background

Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two-dimensional gel electrophoresis (2D-PAGE).

Results

Protein spots excised from 2D-PAGE gels, were subjected to in-gel tryptic digestion and identified by liquid chromatography – tandem mass spectrometry. A total of 47 protein spots were identified. Immunological verification was performed for several of the differentially regulated proteins where suitable antibodies could be obtained. Functional annotation clustering revealed similarities as well as differences between the tumours. Twelve proteins that changed similarly in both tumours included up-regulation of rho GDP-dissociation inhibitor 2, proteasome subunit α type 3, transforming acidic coiled-coil containing protein 3, mitochondrial ornithine aminotransferase and epidermal fatty acid binding protein and down-regulation of adenylosuccinate synthetase, tubulin β-3 chain, a 25 kDa actin fragment, proteasome subunit β type 9, cofilin-1 and glia maturation factor γ.

Conclusion

Some of the commonly differentially expressed proteins are also differentially expressed in other tumours and may be putative diagnostic and/or prognostic markers for lymphomas.     

Evaluation of changes in serum protein profiles during neoadjuvant chemotherapy in HER2‐positive breast cancer using an LC‐MALDI‐TOF/MS procedure

Comparison of protein profiles of sera acquired before and after preoperative chemotherapy for breast cancer may reveal tumor markers that could be used to monitor tumor response. In this study, we analyzed pre‐ and post‐chemotherapy protein profiles of sera from 39 HER2‐postive breast cancer patients (n=78 samples) who received 6 months of preoperative chemotherapy using LC‐MALDI‐TOF/MS technology. We detected qualitative and quantitative differences in pair‐wise comparison of pre‐ and post chemotherapy samples that were different in patients who achieved pathological complete response (pCR, n=21) compared with those with residual disease (n=18). We identified 2329 and 3152 peaks as differentially expressed in the pre‐chemotherapy samples of the responders and non‐responders. Comparison of matching pre‐ and post‐chemotherapy samples identified 34 (32 decreased, two increased) and 304 peaks (157 decreased, 147 increased) that significantly changed (p<0.01, false discovery rate ≤20%) after treatment in responders and non‐responders, respectively. The top 11 most significantly altered peptide peaks with the greatest change in intensity were positively identified. These corresponded to eight proteins including α‐2‐macroglobulin, complement 3, hemopexin, and serum amyloid P in the responder group and chains C and A of apolipoprotein A‐I, hemopexin precursor, complement C, and amyloid P component in the non‐responding groups. All proteins decreased after therapy, except chain C apolipoprotein A and hemopexin precursor that increased. These results suggest that changes in serum protein levels occur in response to chemotherapy and these changes partly appear different in patients who are highly sensitive to chemotherapy compared with those with lesser response.     

Quantitative proteome analysis of ovarian cancer tissues using a iTRAQ approach

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Here, we comparatively analyzed the proteome profiles of ovarian cancer tissues and normal ovarian epithelial tissues. Using the high‐throughput proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ)‐coupled with two‐dimensional‐liquid chromatography‐tandem mass spectrometry, 1,259 unique proteins were identified. Of those, 205 were potentially differentially expressed between ovarian cancer and normal ovarian tissues. Several of the potentially differentially expressed proteins were validated by Western blotting and real‐time quantitative RT‐PCR analyses. Furthermore, up‐regulation of KRT8, PPA1, IDH2, and S100A11 were validated in ovarian tissue microarrays by immunohistochemistry. Silencing of S100A11 expression suppressed the migration and invasion properties of ovarian cancer cells in vitro. Our study represents the successful application of iTRAQ technology to an investigation of ovarian cancer. Many of the potentially differentially expressed proteins identified had not been linked to ovarian cancer before, and provide valuable novel insights into the underlying mechanisms of carcinogenesis in human ovarian cancer. J. Cell. Biochem. 113: 3762–3772, 2012. © 2012 Wiley Periodicals, Inc.     

Identification of Mu-Class Glutathione Transferases M2-2 and M3-3 as Cytosolic Prostaglandin E Synthases in the Human Brain

Cytosolic prostaglandin (PG) E synthase was purified from human brain cortex. The N-terminal amino acid sequence, PMTLGYXNIRGL, was identical to that of the human mu-class glutathione transferase (GST) M2 subunit. Complementary DNAs for human GSTM2, GSTM3, and GSTM4 subunits were cloned, and recombinant proteins were expressed as homodimers in Escherichia coli. The recombinant GSTM2-2 and 3-3 catalyzed the conversion of PGH₂ to PGE₂ at the rates of 282 and 923 nmol/min/mg of protein, respectively, at the optimal pH of 8, whereas GSTM4-4 was inactive; although all three enzymes showed GST activity. The PGE synthase activity depended on thiols, such as glutathione, dithiothreitol, 2-mercaptoethanol, or L-cysteine. Michaelis-Menten constants and turnover numbers for PGH₂ were 141 M and 10.8 min^–1 for GSTM2-2 and 1.5 mM and 130 min^–1 for GSTM3-3, respectively. GSTM2-2 and 3-3 may play crucial roles in temperature regulation, nociception, and sleep-wake regulation by producing PGE₂ in the brain.     

Proteomic analysis of low‐ to high‐grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non‐neoplastic brain tissue as control using 2‐DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up‐regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down‐regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real‐time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.     

Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines

Chemoresistance is a major therapeutic obstacle in cancer patients, and the mechanisms of drug resistance are not fully understood. In the present study, we established platinum-resistant human ovarian cancer cell lines and identified differentially expressed proteins related to platinum resistance. The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP) human ovarian cancer cell lines were isolated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In total, 57 differential protein spots were identified, and five proteins, including annexin A3, destrin, cofilin 1, Glutathione-S-transferase omega 1 (GSTO1-1), and cytosolic NADP+-dependent isocitrate dehydrogenase (IDHc), were found to be co-instantaneous significance compared with their parental cells. The expression of the five proteins was validated by quantitative PCR and western blot, and the western blot results showed complete consistency with proteomic techniques. The five proteins are hopeful to become candidates for platinum resistance. These may be useful for further study of resistance mechanisms and screening of resistant biomarkers.     

Proteomic analysis of the soluble and the lysosomal + mitochondrial fractions from rat pancreas: Implications for cerulein-induced acute pancreatitis

Alterations in protein expression within the initiation phase of acute pancreatitis (AP) might play an important role in the development of this disease, lysosomes being involved in its pathophysiology. The use of pancreatic subcellular fractions in proteomic analysis, simplifies protein maps and helps in the identification of new protein changes and biomarkers characterizing tissue damage. The present study aims to determine the differentially expressed acidic proteins in the pancreatic soluble and lysosomal + mitochondrial (L + M) fractions from rats during the early phase of the experimental model of cerulein (Cer)-induced AP. Subcellular pancreatic extracts from diseased and control rats were analyzed by 2-DE (3–5.6 pH range) and MALDI-TOF/TOF MS. Comparative analysis afforded the conclusive identification of 13 (soluble fraction) and 7 (L + M fraction) proteins or protein fragments ocurring in different amounts between diseased and control pancreas, some of them being newly described in AP. In the soluble fraction, we detected changes related to inflammation and apoptosis (α1-inhibitor-3, α-1 antitrypsin, α-1 macroglobulin, haptoglobin, STRAP), oxidative stress and stress response (peroxiredoxin-2, thioredoxin-like 1, GRP94/TRA1, heat shock cognate 71 kDa protein), digestive proteases (elastase 3B), serine protease inhibition (serpins B6 and A3L) and translation processes (EF 1-δ). In the L + M fraction, we detected changes mainly related to energy generation or cellular metabolism (ATP synthase β subunit, chymotrypsinogen B, triacylglycerol lipase), cell redox homeostasis (iodothyronine 5´monodeiodinase) and digestive proteases (carboxypeptidase B1). The data should provide valuable information for unraveling the early pathophysiologic mechanisms of Cer-induced AP.     

Proteomic Profiling of Ovarian Cancer Plasma using Immunoaffinity Depleted Plasma and Two-Dimensional PAGE

Objective

The aim of this study was to evaluate a multiple immunoaffinity protein depletion (multiple affinity removal system, MARS) pre-treatment strategy with subsequent two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and peptide mass finger printing analysis for the detection of ovarian cancer-associated plasma proteins.

Materials and Methods

Following immunoaffinity depletion, total plasma protein content was reduced by 84.2?±?1.8% (mean?±?SE, n?=?32). The number of proteins detected in the control and ovarian cancer groups was 349 and 357, respectively. This represented an increase in spot detection of almost twofold when compared to 2D PAGE displays of untreated plasma (174 spots). Of the proteins displayed, post-depletion, 300 (control) and 302 (ovarian cancer, OC) were common within each group. PDQuest analysis indicated that 109 protein spots were statistically different between the two groups and, of these, 59 exhibited greater than or equal to twofold difference in spot density (Student’s t test, p?=?0.01). Thirty-nine of these proteins were successfully identified with reliable confidence.

Results and Discussion

The data obtained in this study demonstrates that immunodepletion of plasma before 2D PAGE profiling have generated identifiable plasma proteins that are differentially expressed in the high-grade ovarian cancer sample set compared to controls. This approach, therefore, may be useful in identifying candidate biomarkers for inclusion in multi-marker tests for ovarian cancer that may exhibit greater sensitivity and specificity than those currently available. It was evident, however, from the predominant identification of host response proteins that immunodepletion did not generate sufficient levels of enrichment of lower abundance tumor-specific proteins to facilitate detection.     

Proteomic analysis of cisplatin resistance in human ovarian cancer using 2-DE method

Platinum-based chemotherapy, such as cisplatin, is the primary treatment for human ovarian cancer. However, overcoming drug resistance has become an important issue in cancer chemotherapy. In this study, we performed 2-DE and ESI-Q-TOF MS/MS analysis to identify differential proteins expression between cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780-CP) ovarian cancer cell lines. Of the 14 spots identified as differentially expressed (±over twofold, P < 0.05) between the two cell lines, ten spots (corresponding to ten unique proteins) were positively identified by ESI-Q-TOF MS/MS analysis. These proteins include capsid glycoprotein, fructose-bisphosphate aldolase C, heterogeneous nuclear ribonucleoproteins A2/B1, putative RNA-binding protein 3, Ran-specific GTPase-activating protein, ubiquitin carboxyl-terminal hydrolase isozyme L1, stathmin, ATPSH protein, chromobox protein homolog3 and phosphoglycerate kinase 1. The proteins identified in this study would be useful in revealing the mechanisms underlying cisplatin resistance and also provide some clues for further research.     

Three-dimensional in vitro cell biology models of ovarian and endometrial cancer

Objectives:  This study aims to establish three-dimensional (3D) cell culture models of human ovarian and endometrial cancers and to compare biological and morphological characteristics of these models with those of two-dimensional (2D) models of the same cell lines and the primary tumours.
Methods:  3D models of ovarian and endometrial cancer cell cultures were established using a Rotary Cell Culture System. Immunohistochemical profiling and differential proteomics were used to characterize biological characteristics of multicellular spheroids (MCS) formed from these cultures. These were compared to characteristics of the same cells established in 2D and of the primary tumours from which the cell lines were derived.
Results:  MCSs from 3D cell cultures appeared histologically similar to the primary tumours. Immunohistochemical profiling of multiple markers, including CA125, BCL2 and p53, showed that patterns of protein expression in MCSs resemble those of the primary tumours. Proteomic profiling identified several differentially expressed protein markers between 2D and 3D cultures. These included prohibitin, which was down-regulated in 3D cultures suggesting cells proliferate less compared to 2D cultures; and VDAC1 and annexin 4, which were up-regulated in 3D cultures suggesting greater levels of apoptosis in 3D compared to 2D models.
Conclusion:  Establishing 3D models of cancer cell lines is likely to be of value for studying the molecular and biological mechanisms of ovarian/endometrial tumour progression and for testing novel molecular targets for cancer therapy.     

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.     

Proteomic analysis of proteins regulated by TRPS1 transcription factor in DU145 prostate cancer cells

The aim of the present study was to identify proteins differentially regulated by TRPS1 in human prostate cancer cells in order to better understand the role of TRPS1 in prostate cancer development. The proteomes of androgen-independent DU145 prostate cancer cells, that do not express TRPS1 and of genetically engineered DU145 cells that stable and inducible express recombinant TRPS1 protein, were compared. Using two-dimensional electrophoresis followed by mass spectrometric analysis, 13 proteins that were differentially expressed between these two cell lines were identified. These proteins represent a dominant reduction of expression of antioxidant proteins, including superoxide dismutase, protein disulfide isomerase A3 precursor, endoplasmin precursor and annexin A2. Furthermore, regulation was observed for mitochondrion-associated proteins, glycolytic enzymes, a cytoskeleton-associated protein, a nuclear protein and proteins involved in apoptosis. Our data indicate that overexpression of TRPS1 protein is correlated with reduced protein expression of certain antioxidants. This suggests a possible involvement of TRPS1 in oxidative stress, and possibly in apoptosis in androgen-independent DU145 prostate cancer cells.     

Investigation of serum proteome alterations in human glioblastoma multiforme

Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and lethal adult malignant brain tumor. The present study was conducted to investigate the alterations in the serum proteome in GBM patients compared to healthy controls. Comparative proteomic analysis was performed employing classical 2DE and 2D‐DIGE combined with MALDI TOF/TOF MS and results were further validated through Western blotting and immunoturbidimetric assay. Comparison of the serum proteome of GBM and healthy subjects revealed 55 differentially expressed and statistically significant (p <0.05) protein spots. Among the identified proteins, haptoglobin, plasminogen precursor, apolipoprotein A‐1 and M, and transthyretin are very significant due to their functional consequences in glioma tumor growth and migration, and could further be studied as glioma biomarkers and grade‐specific protein signatures. Analysis of the lipoprotein pattern indicated elevated serum levels of cholesterol, triacylglycerol, and low‐density lipoproteins in GBM patients. Functional pathway analysis was performed using multiple software including ingenuity pathway analysis (IPA), protein analysis through evolutionary relationships (PANTHER), database for annotation, visualization and integrated discovery (DAVID), and GeneSpring to investigate the biological context of the identified proteins, which revealed the association of candidate proteins in a few essential physiological pathways such as intrinsic prothrombin activation pathway, plasminogen activating cascade, coagulation system, glioma invasiveness signaling, and PI3K signaling in B lymphocytes. A subset of the differentially expressed proteins was applied to build statistical sample class prediction models for discrimination of GBM patients and healthy controls employing partial least squares discriminant analysis (PLS‐DA) and other machine learning methods such as support vector machine (SVM), Decision Tree and Naïve Bayes, and excellent discrimination between GBM and control groups was accomplished.     

Identification of hemagglutinin structural domain and polymorphisms which may modulate swine H1N1 interactions with human receptor

Background

Mesothelin is a 40 kDa protein present on the surface of normal mesothelial cells and overexpressed in many human tumours, including mesothelioma and ovarian and pancreatic adenocarcinoma. It forms a strong and specific complex with MUC16, which is also highly expressed on the surface of mesothelioma and ovarian cancer cells. This binding has been suggested to be the basis of ovarian cancer metastasis. Knowledge of the structure of this protein will be useful, for example, in building a structural model of the MUC16-mesothelin complex. Mesothelin is produced as a precursor, which is cleaved by furin to produce the N-terminal half, which is called the megakaryocyte potentiating factor (MPF), and the C-terminal half, which is mesothelin. Little is known about the function of mesothelin and there is no information on its possible three-dimensional structure. Mesothelin has been reported to be homologous to the deafness-related inner ear proteins otoancorin and stereocilin, for neither of which the three-dimensional structure is known.

Results

The BLAST and PSI-BLAST searches confirmed that mesothelin and mesothelin precursor proteins are remotely homologous to stereocilin and otoancorin and more closely homologous to the hypothetical protein MPFL (MPF-like). Secondary structure prediction servers predicted a predominantly helical structure for both mesothelin and mesothelin precursor proteins and also for stereocilin and otoancorin. Three-dimensional structure prediction servers INHUB and I-TASSER produced structural models for mesothelin, which consisted of superhelical structures with ARM-type repeats in conformity with the secondary structure predictions. Similar ARM-type superhelical repeat structures were predicted by 3D-PSSM server for mesothelin precursor and for stereocilin and otoancorin proteins.

Conclusion

The mesothelin superfamily of proteins, which includes mesothelin, mesothelin precursor, megakaryocyte potentiating factor, MPFL, stereocilin and otoancorin, are predicted to have superhelical structures with ARM-type repeats. We suggest that all of these function as superhelical lectins to bind the carbohydrate moieties of extracellular glycoproteins.