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.     

Anthrax edema toxin has cAMP-mediated stimulatory effects and high-dose lethal toxin has depressant effects in an isolated perfused rat heart model

While anthrax edema toxin produces pronounced tachycardia and lethal toxin depresses left ventricular (LV) ejection fraction in in vivo models, whether these changes reflect direct cardiac effects as opposed to indirect ones related to preload or afterload alterations is unclear. In the present study, the effects of edema toxin and lethal toxin were investigated in a constant pressure isolated perfused rat heart model. Compared with control hearts, edema toxin at doses comparable to or less than a dose that produced an 80% lethality rate (LD(80)) in vivo in rats (200, 100, and 50 ng/ml) produced rapid increases in heart rate (HR), coronary flow (CF), LV developed pressure (LVDP), dP/dt(max), and rate-pressure product (RPP) that were most pronounced and persisted with the lowest dose (P ≤ 0.003). Edema toxin (50 ng/ml) increased effluent and myocardial cAMP levels (P ≤ 0.002). Compared with dobutamine, edema toxin produced similar myocardial changes, but these occurred more slowly and persisted longer. Increases in HR, CF, and cAMP with edema toxin were inhibited by a monoclonal antibody blocking toxin uptake and by adefovir, which inhibits the toxin's intracellular adenyl cyclase activity (P ≤ 0.05). Lethal toxin at an LD(80) dose (50 ng/ml) had no significant effect on heart function but a much higher dose (500 ng/ml) reduced all parameters (P ≤ 0.05). In conclusion, edema toxin produced cAMP-mediated myocardial chronotropic, inotropic, and vasodilatory effects. Vasodilation systemically with edema toxin could contribute to shock during anthrax while masking potential inotropic effects. Although lethal toxin produced myocardial depression, this only occurred at high doses, and its relevance to in vivo findings is unclear.     

Non-enzymatic depolymerization of cotton cellulose by fungal mimicking metabolites

Small, low molecular weight, non-enzymatic compounds have been linked to the early stages of brown rot decay as the enzymes involved with holocellulose degradation are too large to penetrate the S3 layer of intact wood cells. We investigated the most notable of these compounds, i.e. hydrogen peroxide, iron, and oxalic acid. The former two are involved in the Fenton reaction in which they react to form hydroxyl radicals, which cause an accelerated depolymerization in cotton cellulose. We found the same reaction to be caused by both iron Fe³⁺ and Fe²⁺. A 10 mM oxalic acid solution showed significant depolymerization effect on cotton cellulose. An oxalic acid/sodium oxalate buffered pH gradient had an inhibitory effect on the reduction of cellulose polymers at increased pH values. The organic iron chelator, EDTA, was found to promote depolymerization of cellulose in combination with Fenton’s reagents, but inhibited the effect of oxalic acid in the absence of iron and hydrogen peroxide. Manganese was tested to see if metals other than iron could generate a significant impact on the degree of polymerization (DP) in cotton cellulose. Depolymerizing properties comparable to iron were seen. The results confirm that low molecular weight metabolites are capable of depolymerizing cellulose and suggest an importance of these mechanisms during incipient decay by brown rot fungi.     

Evaluating Methods for Transplanting Endangered Elkhorn Corals in the Virgin Islands

Restoration of rare corals is desirable and restoration projects are fairly common, but scientific evaluation of this approach is limited. We tested several techniques for transplant and restabilization of Acropora palmata (the elkhorn coral), an ecologically important Caribbean coral whose populations have suffered severe declines. In rough weather, fragments break‐off colonies of branching corals like A. palmata as a normal form of asexual reproduction. We transplanted naturally produced coral fragments from remnant populations to nearby restoration sites. Untouched control fragments at the donor site died faster and grew slower than fragments attached to the reef, so attaching fragments to the reef is beneficial. Transplanted fragments grew and died at a rate similar to fragments left at the donor site (both groups were attached to the reef), so there were no effects of moving fragments or differences in habitat quality between donor and restoration sites. Growth and survival were similar using four methods of attaching fragments to the reef: cable ties, two types of epoxy resin, and hydrostatic cement. Corals sometimes compete with the macroalgae that dominate degraded reefs, and clearing surrounding algae improved the growth of fragments. After 4 years, transplanted fragments grew to 1,450 cm² in area and so were potentially sexually active. Because the methods tested are simple and cheap, they could be used by volunteer recreational divers to restore locally extirpated A. palmata populations or to enhance reefs where natural recovery is slow.     

Injectable polyHIPEs as high-porosity bone grafts

Polymerization of high internal phase emulsions (polyHIPEs) is a relatively new method for the production of high-porosity scaffolds. The tunable architecture of these polyHIPE foams makes them attractive candidates for tissue engineered bone grafts. Previously studied polyHIPE systems require either toxic diluents or high cure temperatures which prohibit their use as an injectable bone graft. In contrast, we have developed an injectable polyHIPE that cures at physiological temperatures to a rigid, high-porosity foam. First, a biodegradable macromer, propylene fumarate dimethacrylate (PFDMA), was synthesized that has appropriate viscosity and hydrophobicity for emulsification. The process of surfactant selection is detailed with particular focus on the key structural features of both polymer (logP values, hydrogen bond acceptor sites) and surfactant (HLB values, hydrogen bond donor sites) that enable stable HIPE formation. Incubation of HIPEs at 37 °C was used to initiate radical cross-linking of the unsaturated double bond of the methacrylate groups to polymerize the continuous phase and lock in the emulsion geometry. The resulting polyHIPEs exhibited ~75% porosity, pore sizes ranging from 4 to 29 μm, and an average compressive modulus and strength of 33 and 5 MPa, respectively. These findings highlight the great potential of these scaffolds as injectable, tissue engineered bone grafts.     

Water integration in the clonal emergent hydrophyte, Justicia americana: benefits of acropetal water transfer from mother to daughter ramets

Clonal plant growth is common in aquatic freshwater plants and their success is largely attributed to the sharing of resources (e.g., photosynthates, minerals, and water) within the greater genet. The flow of materials within the clone is largely driven by source-sink dynamics, and in established genets may involve acropetal and/or basipetal flow. During the production of new ramets, however, the mother often provides needed resources to the daughter through acropetal flow. In this study, we consider the role of water sharing in a clonal hydrophyte, Justicia americana, when soil moisture levels around the daughter plant declined. The results of this study indicate that acropetal water sharing is an important function between mother and daughter ramets in this species, as indicated by sustained growth and higher survival in connected daughters residing in water-deprived soils. Interestingly, mother plants, when connected to a drought daughter, began to develop similar xeromorphic features (e.g., greater leaf succulence) even though the parent remained in flooded conditions. We suspect that some physical or chemical signal was conveyed from the daughter to the mother that ??forecast?? water scarcities, which could prepare the entire genet for potential drought conditions.     

Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination.