Increased Secretory Leukocyte Protease Inhibitor (SLPI) Production by Highly Metastatic Mouse Breast Cancer Cells

The precise molecular mechanisms enabling cancer cells to metastasize from the primary tumor to different tissue locations are still largely unknown. Secretion of some proteins by metastatic cells could facilitate metastasis formation. The comparison of secreted proteins from cancer cells with different metastatic capabilities in vivo might provide insight into proteins involved in the metastatic process. Comparison of the secreted proteins from the mouse breast cancer cell line 4T1 and its highly metastatic 4T1.2 clone revealed a prominent differentially secreted protein which was identified as SLPI (secretory leukocyte protease inhibitor). Western blotting indicated higher levels of the protein in both conditioned media and whole cell lysates of 4T1.2 cells. Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining. A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level. Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.     

A Dry Powder Formulation of Liposome-Encapsulated Recombinant Secretory Leukocyte Protease Inhibitor (rSLPI) for Inhalation: Preparation and Characterisation

Inhaled recombinant secretory leukocyte protease inhibitor (rSLPI) has shown potential for the treatment of inflammatory lung conditions. Rapid inactivation of rSLPI by cathepsin L (Cat L) and rapid clearance from the lungs has limited clinical efficacy to date. Previous studies by us have shown that encapsulation of rSLPI within1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]/cholesterol (DOPS/Chol) liposomes protects rSLPI against Cat L inactivation in vitro. Liquid DOPS–rSLPI preparations were found to be unstable upon long-term storage and nebulisation. The aim of this study was therefore to develop a method of manufacture for preparing DOPS–rSLPI liposomes as a dry powder for inhalation. DOPS–rSLPI dry powders were lyophilised and subsequently micronised with a novel micronisation aid. The effects of formulation and processing on rSLPI stability, activity, and uniformity of content within the powders were characterised. Using D-mannitol as the micronisation aid, dry powder particles in the inhalable size range (<5 μm) were prepared. By optimising process parameters, up to 54% of rSLPI was recovered after micronisation, of which there was no significant loss in anti-neutrophil elastase activity and no detectable evidence of protein degradation. Aerosolisation was achieved using a dry powder inhaler, and mass median aerodynamic diameter (MMAD) was evaluated after collection in a cascade impactor. Aerosolisation of the DOPS–rSLPI dry powder yielded 38% emitted dose, with 2.44 μm MMAD. When challenged with Cat L post-aerosolisation, DOPS–rSLPI dry powder was significantly better at retaining a protective function against Cat L-induced rSLPI inactivation compared to the aqueous DOPS–rSLPI liposome dispersion and was also more stable under storage.     

Network Analysis Identifies SOD2 mRNA as a Potential Biomarker for Parkinson's Disease

Increasing evidence indicates that Parkinson''s disease (PD) and type 2 diabetes (T2DM) share dysregulated molecular networks. We identified 84 genes shared between PD and T2DM from curated disease-gene databases. Nitric oxide biosynthesis, lipid and carbohydrate metabolism, insulin secretion and inflammation were identified as common dysregulated pathways. A network prioritization approach was implemented to rank genes according to their distance to seed genes and their involvement in common biological pathways. Quantitative polymerase chain reaction assays revealed that a highly ranked gene, superoxide dismutase 2 (SOD2), is upregulated in PD patients compared to healthy controls in 192 whole blood samples from two independent clinical trials, the Harvard Biomarker Study (HBS) and the Diagnostic and Prognostic Biomarkers in Parkinson''s disease (PROBE). The results from this study reinforce the idea that shared molecular networks between PD and T2DM provides an additional source of biologically meaningful biomarkers. Evaluation of this biomarker in de novo PD patients and in a larger prospective longitudinal study is warranted.     

Laminin,gamma 2 (LAMC2): A Promising New Putative Pancreatic Cancer Biomarker Identified by Proteomic Analysis of Pancreatic Adenocarcinoma Tissues

In pancreatic cancer, the incidence and mortality curves coincide. One major reason for this high mortality rate in pancreatic ductal adenocarcinoma (PDAC) patients is the dearth of effective diagnostic, prognostic, and disease-monitoring biomarkers. Unfortunately, existing tumor markers, as well as current imaging modalities, are not sufficiently sensitive and/or specific for early-stage diagnosis. There is, therefore, an urgent need for improved serum markers of the disease. Herein, we performed Orbitrap® mass spectrometry proteomic analysis of four PDAC tissues and their adjacent benign tissues and identified a total of 2190 nonredundant proteins. Sixteen promising candidates were selected for further scrutiny using a systematic scoring algorithm. Our preliminary serum verification of the top four candidates (DSP, LAMC2, GP73, and DSG2) in 20 patients diagnosed with pancreatic cancer and 20 with benign pancreatic cysts, showed a significant (p < 0.05) elevation of LAMC2 in pancreatic cancer serum. Extensive validation of LAMC2 in healthy, benign, and PDAC sera from geographically diverse cohorts (n = 425) (Japan, Europe, and USA) demonstrated a significant increase in levels in early-stage PDAC compared with benign diseases. The sensitivity of LAMC2 was comparable to CA19.9 in all data sets, with an AUC value greater than 0.85 in discriminating healthy patients from early-stage PDAC patients. LAMC2 exhibited diagnostic complementarity with CA19.9 by showing significant (p < 0.001 in two out of three cohorts) elevation in PDAC patients with clinically low CA19.9 levels.Pancreatic ductal adenocarcinoma (PDAC)¹ is one of the most devastating cancers and the fourth leading cause of cancer-related deaths in North America (1). Ninety-five percent of patients will not survive beyond five years; this high mortality rate is primarily attributed to the lack of effective diagnostic techniques and treatment regimens. The hallmark features of pancreatic cancer (PC) are late presentation and aggressive metastatic progression (2, 3). The National Cancer Institute statistics estimate that approximately $1.9 billion is being spent in the United States alone each year on PC diagnosis and treatment. PDAC is classified into resectable (∼10–20%), locally advanced unresectable (∼30–40%), and metastatic (∼50%) (3). PDAC diagnosed at resectable stage can possibly be cured with complete surgical removal. This could improve the survival rates and considerably lower treatment costs. It is projected that 20–40% of patients with resectable PDAC survive more than five years after complete surgical removal, highlighting the importance of early-stage diagnosis. Unfortunately, carbohydrate antigen 19–9 (CA19.9), the current standard serum tumor marker for PDAC, has certain limitations as an early detection biomarker (its sensitivity for small tumors {<3 cm} is ∼50% and it is significantly elevated in many benign conditions (e.g. biliary obstruction, hepatic cirrhosis, chronic pancreatitis)) (4, 5). In light of the scarcity of other, more reliable markers, CA19.9 is currently used in the clinic as a prognostic and surveillance marker. Undoubtedly, the need for a more reliable consistent biomarker (or biomarker panel) for early PDAC diagnosis remains unmet. In pursuit of novel PDAC biomarker candidates, we have previously delineated the proteomes of malignant pancreatic ascitic fluids, pools of pancreatic juice, and pancreatic cancer cell lines (BxPC3, CAPAN, CFPAC1, MIA-Paca2, PANC1, and SU.86.86). We identified a panel of five potential candidate biomarkers, which, in combination, slightly outperformed CA19.9 in a pilot verification study (40 individuals; 20 healthy, and 20 PDAC) (6).From a different perspective, in the current study, we deployed a comparative quantitative tissue proteomic methodology to compare the proteome of malignant pancreatic tissues with that of their adjacent normal counterparts. A total of 2190 nonredundant proteins were identified, which were further scrutinized using a systematic scoring algorithm based on their quantified cancer-versus-normal ratios, on their identification in malignant pancreatic ascites fluid, on their cancer-specific nature, and on their tissue-expression profiles. Our analysis resulted in sixteen promising candidate biomarkers, which fulfilled our criteria and selected for further validation studies. In a multistep validation approach, the selected candidates were first verified in serum samples obtained from 20 patients with benign pancreatic diseases and 20 patients with pancreatic cancer, using commercially available ELISA kits. The best candidate (LAMC2) was further tested in three geographically diverse cohorts from Germany, Japan, and the US composed of 435 serum samples from healthy, benign, and early and late stage cancer patients. Our approach brought to light a previously unknown, promising PDAC candidate biomarker, LAMC2.     

基于电化学发光检测技术的乳腺癌生物标记物EZH2表达定量分析

Zeste同源染色体2增强子基因(EZH2)在人类乳腺癌中过度表达,它可以被视为一个检测肿瘤的发展和转移的生物标记物。传统技术检测或定量特异性基因表达存在一些缺点,因此,本研究拟开发电致化学发光(ECL)技术来检测和量化EZH2 mRNA的表达量。在本研究中,用生物素和三(2,2-联吡啶)钌(II)(TBR)分别标记在PCR引物的5’末端上,用作扩增靶基因,扩增产物用ECL系统进行检测。我们用癌细胞作为模型分析了该方法的有效性和灵敏度,并且将其应用于25例乳腺癌的临床样本中EZH2基因表达量的检测。检测结果表明,EZH2基因在肿瘤细胞系中过量表达,而在正常血细胞中则低表达。最重要的是,在25例临床乳腺癌样品中发现10例样品(40%)的EZH2 mRNA过度表达。此方法提供了一种新的工具来评估EZH2基因在乳腺癌中的表达水平,且有可能成为一种快速、简便和灵敏的乳腺癌检测和诊断方法。     

Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer

Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current–based MS1 workflow to quantify ～6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and ‘drug response’ mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.     

Spatiotemporal Proteomic Analyses during Pancreas Cancer Progression Identifies Serine/Threonine Stress Kinase 4 (STK4) as a Novel Candidate Biomarker for Early Stage Disease

Pancreas cancer, or pancreatic ductal adenocarcinoma, is the deadliest of solid tumors, with a five-year survival rate of <5%. Detection of resectable disease improves survival rates, but access to tissue and other biospecimens that could be used to develop early detection markers is confounded by the insidious nature of pancreas cancer. Mouse models that accurately recapitulate the human condition allow disease tracking from inception to invasion and can therefore be useful for studying early disease stages in which surgical resection is possible. Using a highly faithful mouse model of pancreas cancer in conjunction with a high-density antibody microarray containing ∼2500 antibodies, we interrogated the pancreatic tissue proteome at preinvasive and invasive stages of disease. The goal was to discover early stage tissue markers of pancreas cancer and follow them through histologically defined stages of disease using cohorts of mice lacking overt clinical signs and symptoms and those with end-stage metastatic disease, respectively. A panel of seven up-regulated proteins distinguishing pancreas cancer from normal pancreas was validated, and their levels were assessed in tissues collected at preinvasive, early invasive, and moribund stages of disease. Six of the seven markers also differentiated pancreas cancer from an experimental model of chronic pancreatitis. The levels of serine/threonine stress kinase 4 (STK4) increased between preinvasive and invasive stages, suggesting its potential as a tissue biomarker, and perhaps its involvement in progression from precursor pancreatic intraepithelial neoplasia to pancreatic ductal adenocarcinoma. Immunohistochemistry of STK4 at different stages of disease revealed a dynamic expression pattern further implicating it in early tumorigenic events. Immunohistochemistry of a panel of human pancreas cancers confirmed that STK4 levels were increased in tumor epithelia relative to normal tissue. Overall, this integrated approach yielded several tissue markers that could serve as signatures of disease stage, including early (resectable), and therefore clinically meaningful, stages.In 2013 the American Cancer Society estimated there were 45,220 new cases of cancer of the pancreas, the most common form of which is pancreatic ductal adenocarcinoma (PDA),¹ with an estimated 38,430 deaths (1). The incidence of PDA increased on average by 1.5% per year from 2004 to 2008, a trend that is expected to continue. Because of the frequently asymptomatic character of PDA at early stages, diagnosis generally occurs only after locally advanced or metastatic spread, and the majority of patients therefore present with unresectable disease. This fact, combined with the notable resistance of PDA to chemotherapy, usually yields a very poor prognosis, with less than 20% survival past 12 months after diagnosis (2).Surgical resection with curative intent improves five-year survival rates from less than 5% to ∼20% (3), yet resection is possible in only 10% to 15% of patients because of the low probability of identifying resectable disease in asymptomatic patients (4). Accurate diagnosis of PDA can be further confounded by difficulties in distinguishing it from chronic pancreatitis (CP) and the associated fibrosis; conversely, pancreatitis can also be initiated by a tumor causing ductal obstruction (5). A non-invasive blood-based test would be ideal for early detection and diagnosis, but no validated markers currently exist with sufficient sensitivity and specificity to improve clinical decision making. Early stage tissue markers could complement current imaging (MRI, computed tomography, endoscopic ultrasound) and CA19.9 plasma screening modalities (6) and improve the identification of resectable disease, as well as provide further insight into pancreas cancer etiology.Mouse models that clinically, histologically, and molecularly recapitulate human PDA have been instrumental in elucidating the molecular mechanisms underlying PDA etiology and progression (7–11). Studies in these systems have shown that pancreas-specific expression of mutant Kras can initiate precursor pancreatic intraepithelial neoplasms (PanINs) that progress to invasive adenocarcinoma (7) and that the course, rate of progression, and metastatic capability of PDA are affected by the additional loss and/or mutation of key tumor suppressor genes (e.g. Dpc4/Smad4, Cdnk2a/ink4a, and Trp53), as well as the relative timing of these events (8, 10, 11). Accurate mouse models can also complement static genomic and proteomic studies of human samples because they enable spatiotemporal monitoring of markers from disease initiation to metastasis. Although many static analyses of putative biomarkers have been performed on resected tissues (12, 13), fewer studies have taken advantage of genetically engineered mouse models of PDA that progress from well-characterized precursor lesions to invasive and metastatic disease to discover protein biomarkers and chronicle their distribution and expression during disease progression (14, 15).Proteomic technologies enable the molecular characterization of biological samples at the structural and functional level (16), and multiplex antibody microarray platforms allow the characterization of multiple analytes simultaneously. Antibody microarrays have the added benefits of requiring minimal sample volumes and the ability to identify their cognate analytes with pico- to femtomolar sensitivities (17, 18). Given their low cost, immunoassay-based diagnostics are thought to be the most applicable proteomic technology in a clinical setting (19, 20).The KPC mouse model of PDA employs conditional pancreas-specific, endogenous expression of Kras^G12D and Trp53^R172H and spontaneously develops precursor PanINs with 100% penetrance that progress to invasive and metastatic PDA (8). We thus found this an ideal model to use in conjunction with a novel, high-dimensional, pancreas-cancer-tailored antibody microarray to assay for proteomic changes during disease progression (18, 21, 22). The microarray consisted of ∼2500 antibodies and represents, to our knowledge, the largest antibody array platform produced. Our goal was to follow the evolving primary tumor proteome from PanIN to invasive PDA. Using this approach, we identified and validated a panel of tissue markers differentiating early stages of tumorigenesis from both the normal organ and chronic pancreatitis. We identified the serine/threonine kinase STK4 as a novel early marker of PDA progression and confirmed its expression in human PDA.     

Proteomic Analysis of Temporally Stimulated Ovarian Cancer Cells for Biomarker Discovery

While ovarian cancer remains the most lethal gynecological malignancy in the United States, there are no biomarkers available that are able to predict therapeutic responses to ovarian malignancies. One major hurdle in the identification of useful biomarkers has been the ability to obtain enough ovarian cancer cells from primary tissues diagnosed in the early stages of serous carcinomas, the most deadly subtype of ovarian tumor. In order to detect ovarian cancer in a state of hyperproliferation, we analyzed the implications of molecular signaling cascades in the ovarian cancer cell line OVCAR3 in a temporal manner, using a mass-spectrometry-based proteomics approach. OVCAR3 cells were treated with EGF¹, and the time course of cell progression was monitored based on Akt phosphorylation and growth dynamics. EGF-stimulated Akt phosphorylation was detected at 12 h post-treatment, but an effect on proliferation was not observed until 48 h post-exposure. Growth-stimulated cellular lysates were analyzed for protein profiles between treatment groups and across time points using iTRAQ labeling and mass spectrometry. The protein response to EGF treatment was identified via iTRAQ analysis in EGF-stimulated lysates relative to vehicle-treated specimens across the treatment time course. Validation studies were performed on one of the differentially regulated proteins, lysosomal-associated membrane protein 1 (LAMP-1), in human tissue lysates and ovarian tumor tissue sections. Further, tissue microarray analysis was performed to demarcate LAMP-1 expression across different stages of epithelial ovarian cancers. These data support the use of this approach for the efficient identification of tissue-based markers in tumor development related to specific signaling pathways. LAMP-1 is a promising biomarker for studies of the progression of EGF-stimulated ovarian cancers and might be useful in predicting treatment responses involving tyrosine kinase inhibitors or EGF receptor monoclonal antibodies.Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, and the fifth leading cause of cancer-related deaths in women (1). Epithelial ovarian cancers are extensively heterogeneous; histological sub-classification by cell type includes serous, endometrioid, clear-cell, mucinous, transitional, squamous, and undifferentiated (2). Serous epithelial cancers are the most commonly diagnosed epithelial ovarian cancer subtype and are associated with the majority of ovarian-cancer-related deaths (1).From a molecular perspective, the basic characteristic of any cancerous cell is its ability to grow uncontrollably. As a cell proliferates, a cascade of molecular and morphological changes occurs, including the activation of signaling cascades that modulate cytoskeletal dynamics, cell cycle progression, and angiogenesis (3–5). In addition to the unrestrained aberrant proliferation of cancer cells, other processes are required for disease progression, including changes in cellular adhesion to endothelial cells and in the extracellular microenvironment (6). It is important to note, however, that cancer cell progression is not an instantaneous event, and the demarcation between non-cancer and cancer is not static. It is postulated that epithelial cancer cells transition to a highly motile and invasive mesenchymal cell type, and this epithelial-to-mesenchymal transition is a critical molecular mechanism in tumor progression and metastasis (6). Several important signaling cascades have been implicated in this transition, including those mediated by EGF, PDGF, and TGFβ and those involving PI3K/Akt activation (7, 8). Thus, biomarkers of cancer progression can serve as indicators of disease etiology and potential staging, as well as predictive markers of therapeutic regimen responses. The identification of differentially expressed proteins during cancer metastasis has the potential to be utilized both prognostically with regard to metastatic development and predictively, through the implementation of pathway-specific therapies.Molecular analyses indicate the oncogenic role of the epidermal growth factor receptor (EGFR) in several human cancers, including lung cancers and Her2-amplified breast cancers (9). However, less is known regarding the implications of aberrant EGFR expression in ovarian cancer progression, particularly in terms of increased activation of downstream signaling cascades and efficacious therapeutic regimens. Studies illustrate overamplification of the EGFR gene in between 4% and 22% of ovarian cancers, with aberrant protein expression in up to 60% of ovarian malignancies (10–12). Aberrant EGFR expression has been associated with high tumor grade, increased cancerous cell proliferation, and poorer patient outcomes (12–15). Gene amplification and the overexpression of other EGFR family members such as Her2 and ErbB3 have also been reported in epithelial ovarian cancers (15). Further, studies performed in vitro illustrate the ability of EGF to induce DNA synthesis and stimulate cell growth in OVCAR3 cells (16).Although EGFR and downstream EGF-regulated signaling cascades have been implicated in ovarian malignancies, the treatment of ovarian tumors with anti-EGFR agents has induced minimal response. Targeted EGFR therapies fall into two categories: monoclonal antibodies that target the receptor extracellular domain to prevent ligand binding, and tyrosine kinase inhibitors (TKIs), which aim to prevent the activation of downstream signaling cascades. Although EGFR inhibitors exhibit modest success in vitro, no agents have been approved by the U.S. Food and Drug Administration for the treatment of malignant ovarian tumors (17). Among other therapeutic approaches, studies have looked at the potential efficacy of the TKIs erlotinib and gefitinib in the treatment of ovarian cancers; unfortunately, neither drug was effective in eliciting a significant response in ovarian tumor treatment (12, 15, 18, 19). However, the identification of markers of pathway-stimulated processes might help to stratify disease and select patients with EGF signaling activation. The identified markers might facilitate the prediction of treatment responses.MS-based proteomic studies have been heavily implemented in the identification of candidate biomarkers in a variety of specimen sources ranging from epithelial ovarian cancer tissue to immortalized cell lines and cultured media (20–22). The human adenocarcinoma OVCAR3 cell line is derived from an epithelial ovarian cancer with a high grade serous cell type and exhibits many of the molecular and morphological aspects of serous epithelial cancers (23, 24). This cell line can be stimulated to promote or inhibit cellular proliferation using various molecular agonists and antagonists (23–25). Because of the molecular and morphological similarities between the OVCAR3 cell line and ovarian adenocarcinoma cells, it serves as an appropriate high-throughput surrogate for candidate biomarker identification. Further, the analysis of a single cell line allows for the identification of temporal protein regulation within a single homogeneous cell population using an orthogonal approach.In the present study, the OVCAR3 cell line was treated with the hyperproliferative molecule EGF or the PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 over a 48-h time course. Three time points were analyzed for biochemical and molecular changes, including Akt phosphorylation status and increased proliferation. Additionally, growth-stimulated and growth-inhibited cellular lysates were analyzed using quantitative proteomics with iTRAQ and MS/MS, and these analyses illustrated comparable global protein profiles between treatment groups and across time points. Differentially expressed proteins were identified in growth-stimulated cells as opposed to control (vehicle-treated) cells. One of the differentially regulated proteins, lysosomal-associated membrane protein 1 (LAMP-1, also known as CD107a), was further verified via immunoblotting and immunohistochemical analyses in normal and ovarian cancer tissues, in addition to tissue microarray analysis. This study demonstrates that through the use of a growth-stimulated cell culture model using EGF, the rapid identification of differentially regulated proteins as proliferation progresses may be achieved via large-scale proteomic analyses. The identification of regulated proteins along the pathway of increased cellular growth and proliferation might serve a predictive role in treatment outcomes.     

Heritability of a Quantitative and Qualitative Protease Inhibitor Polymorphism in Nicotiana attenuata

Abstract: Many plant species contain chemical defenses that protect them against herbivores. Despite the benefit of these chemical defenses, not all individuals contain high levels of these compounds. In the native tobacco Nicotiana attenuata we found that plants from three natural populations differed considerably in their ability to produce trypsin protease inhibitors (PIs), which are defensive proteins that reduce herbivore damage to plants. Plants from a Utah (U) population produced high levels, whereas plants from Arizona (A) contained no detectable PI levels. Californian (C) plants had intermediate levels. The PI-producing U and C plants thus differ quantitatively from each other, whereas they both differ qualitatively from PI-deficient A plants. Here we analyze how PI production is inherited in N. attenuata with the ultimate goal of better understanding how the quantitative and qualitative differences between the three populations have evolved. Using a series of classical crossing designs, we determined that the ability to produce PIs is inherited as a dominant Mendelian trait. PI-deficient plants contain two non-functional recessive alleles, whereas heterozygous plants or homozygous dominant plants both are able to produce PIs. Similarly, the level of constitutive PIs may be determined by its genotype, either by an interaction between a functional and a non-functional allele in heterozygotes, or by a factor on the PI allele itself in homozygous C plants. Based on these data and on previous studies with A and U plants we postulate that the PI-deficient A plants may have originated from a mutant that lost its ability to produce PIs. The fitness loss due to reduced herbivore resistance may be offset by the fitness gain associated with increased competitive ability, a trade-off which may maintain this mutation in the Arizona population.     

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.     

Quantitative Proteomic Analysis of Niemann-Pick Disease,Type C1 Cerebellum Identifies Protein Biomarkers and Provides Pathological Insight

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.     

Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

Introduction

Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method

We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results

Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions

Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.     

Proteomic Profiling of Paired Interstitial Fluids Reveals Dysregulated Pathways and Salivary NID1 as a Biomarker of Oral Cavity Squamous Cell Carcinoma,

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Highlights

• •Proteome profiling of tissue interstitial fluids (TIFs) of oral cancer.
• •Pathway of aminoacyl tRNA biosynthesis enriched in proteome of TIFs.
• •Verification of nidogen-1 as a salivary biomarker of oral cancer.
• •High correlation between elevated tissue level of nidogen-1 and poor survival.

     

Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery

Background

The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.

Methodology/Principal Findings

We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.

Conclusions/Significance

Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.     

Cross-Sectional Analysis of Obesity and Serum Analytes in Males Identifies sRAGE as a Novel Biomarker Inversely Associated with Diverticulosis

Diverticulosis can lead to diverticulitis, a colon condition involving inflammation and other complications. Diverticulosis can result from biological, behavioral, or genetic causes. However, the etiology of diverticulosis is unknown. Although diet is associated with diverticulosis, recent studies suggest other factors influence risk. We sought to identify anthropometric or serum markers that were associated with the presence of diverticulosis. To determine these associations, 126 asymptomatic men (48–65 yr) were recruited at the time of preventative screening colonoscopy. Anthropometric measures were taken, and blood was collected for serum protein analysis. Data were analyzed by logistic regression and factor analysis. Obese individuals (BMI >30) were 7.8 (CI: 2.3–26.3) times more likely than normal weight (BMI <25) individuals to have diverticulosis. The relationship was similar for waist circumference. Individuals with a waist circumference >45 inches were 8.1 (CI: 2.8–23.8) times more likely to have diverticulosis than those with a waist circumference <38 inches. Leptin was also positively associated with diverticulosis (OR = 5.5, CI: 2.0–14.7). Both low molecular weight adiponectin (LMW, OR = 0.50; CI: 0.3–0.8) and the soluble receptor for advanced glycation end products (sRAGE, OR = 0.4, CI: 0.3–0.7) were inversely related to the presence of diverticulosis. sRAGE levels were not correlated with leptin or C-peptide concentrations. The pattern of high BMI, waist circumference, leptin and C-peptide increased the odds of diverticulosis while the pattern of high levels of sRAGE and LMW adiponectin decreased the odds of diverticulosis. Associations between diverticulosis and anthropometric or serum markers may elucidate the origins of diverticulosis and may enable physicians to identify individuals at risk for diverticulitis.     

Retrospective Proteomic Analysis of a Novel,Cancer Metastasis-Promoting RGD-Containing Peptide

Patients who undergo surgical extirpation of a primary liver carcinoma followed by radiotherapy and chemotherapy leading to complete remission are nevertheless known to develop cancerous metastases 3–10 years later. We retrospectively examined the blood sera collected over 8 years from 30 patients who developed bone metastases after the complete remission of liver cancer to identify serum proteins showing differential expression compared to patients without remission. We detected a novel RGD (Arg-Gly-Asp)-containing peptide derived from the C-terminal portion of fibrinogen in the sera of metastatic patients that appeared to control the EMT (epithelial-mesenchymal transition) of cancer cells, in a process associated with miR-199a-3p. The RGD peptide enhanced new blood vessel growth and increased vascular endothelial growth factor levels when introduced into fertilized chicken eggs. The purpose of this study was to enable early detection of metastatic cancer cells using the novel RGD peptide as a biomarker, and thereby develop new drugs for the treatment of metastatic cancer.