Discovery of Colorectal Cancer Biomarker Candidates by Membrane Proteomic Analysis and Subsequent Verification using Selected Reaction Monitoring (SRM) and Tissue Microarray (TMA) Analysis

Recent advances in quantitative proteomic technology have enabled the large-scale validation of biomarkers. We here performed a quantitative proteomic analysis of membrane fractions from colorectal cancer tissue to discover biomarker candidates, and then extensively validated the candidate proteins identified. A total of 5566 proteins were identified in six tissue samples, each of which was obtained from polyps and cancer with and without metastasis. GO cellular component analysis predicted that 3087 of these proteins were membrane proteins, whereas TMHMM algorithm predicted that 1567 proteins had a transmembrane domain. Differences were observed in the expression of 159 membrane proteins and 55 extracellular proteins between polyps and cancer without metastasis, while the expression of 32 membrane proteins and 17 extracellular proteins differed between cancer with and without metastasis. A total of 105 of these biomarker candidates were quantitated using selected (or multiple) reaction monitoring (SRM/MRM) with stable synthetic isotope-labeled peptides as an internal control. The results obtained revealed differences in the expression of 69 of these proteins, and this was subsequently verified in an independent set of patient samples (polyps (n = 10), cancer without metastasis (n = 10), cancer with metastasis (n = 10)). Significant differences were observed in the expression of 44 of these proteins, including ITGA5, GPRC5A, PDGFRB, and TFRC, which have already been shown to be overexpressed in colorectal cancer, as well as proteins with unknown function, such as C8orf55. The expression of C8orf55 was also shown to be high not only in colorectal cancer, but also in several cancer tissues using a multicancer tissue microarray, which included 1150 cores from 14 cancer tissues. This is the largest verification study of biomarker candidate membrane proteins to date; our methods for biomarker discovery and subsequent validation using SRM/MRM will contribute to the identification of useful biomarker candidates for various cancers. Data are available via ProteomeXchange with identifier PXD000851.Recent advances in proteomic technology have contributed to the identification of biomarkers for various diseases. Improvements in LC-MS technology have led to an increase in the number of proteins that have been identified. In addition, a stable isotopic labeling method using isobaric tag for relative and absolute quantitation (iTRAQ)¹ and stable isotope labeling by amino acids in cell culture has enabled the quantitative analysis of multiple samples (1, 2). Therefore, a large number of proteins have already been identified as biomarker candidates; however, only a few of these have been used in practical applications because most have not yet progressed to the validation stage, in which potential biomarker candidates are quantified on a large scale. The validation of biomarker candidates is generally accomplished using Western blotting and enzyme-linked immunosorbent assays (ELISA) if specific and well-characterized antibodies for these candidates are available. However, highly specific antibodies are not currently available for most novel biomarker candidate proteins, and it takes a significant amount of time and money to obtain these antibodies and optimize ELISA assay systems for many candidates; therefore, another validation assay system needs to be developed. Selected (or multiple) reaction monitoring (SRM or MRM) was previously shown to be a potentially effective method for the validation of biomarker candidates (3–5). The SRM/MRM assay can measure multiple targets at high sensitivity and throughput without antibodies; hence, it is useful for initial quantitative evaluations and the large-scale validation of biomarker candidates, which defines validation of hundreds of biomarker candidate proteins simultaneously.In addition to these technical improvements, the fractionation process also plays an important role in proteome analysis for biomarker discovery. This procedure very effectively analyzes the proteomes of specific cellular compartments or organelles in detail, which reduces sample complexity. The preparation of a membrane fraction was previously shown to be useful for identifying membrane proteins that are generally expressed at relatively low levels. Membrane proteins play critical roles in many biological functions, such as signal transduction, cell-cell interactions, and ion transport, account for ∼38% of all proteins encoded by the mammalian genome and more than one-third of biomarker candidates, and are also potential targets for drug therapy (6, 7). Therefore, membrane proteome analysis is important for biomarker discovery. However, difficulties have been associated with extracting and solubilizing membrane proteins and subsequent protease digestion. Many procedures have consequently been developed to improve the solubilization and digestion of membrane proteins (8–11), and a protocol using phase transfer surfactant (PTS) was shown to be suitable for membrane proteomics using LC-MS/MS (12, 13).The selection of a control group for comparisons is also important for identifying potential biomarkers. Tissue samples from cancer patients have been used in many studies to discover biomarker candidates by proteomic analysis. Previous studies, including our own, attempted to compare cancer tissues with matched normal tissue (14–17). However, marked differences have been reported in the histology, genetics, and proteomics of normal and cancer tissues, and many biomarker candidates have been identified, by making it difficult to narrow down more reliable candidates for further validation. Lazebnik recently emphasized that the features of malignant, but not benign tumors could be used as a hallmark of cancer (18), and also that premalignant lesions were more appropriate controls for cancer tissue than normal tissue for the identification of biomarker candidates involved in cancer progression. Moreover, comparisons of cancer with and without metastasis may also assist in the discovery of biomarker candidates involved in cancer metastasis. Therefore, the identification of biomarker candidates that can be used to diagnose and determine the prognosis of cancer should become more effective by comparing cancer tissues at different stages, including benign tumors.We performed a shotgun proteomic analysis of membrane fractions prepared from colorectal cancer tissue and benign polyps in the present study to identify biomarker candidates for the diagnosis and treatment of cancer. We identified a large number of biomarker candidate proteins associated with the progression of colon cancer by using membrane protein extraction with PTS followed by iTRAQ labeling. SRM/MRM confirmed the altered expression of these biomarker candidates, and these results were further verified using an independent set of tissue samples. A protein with uncharacterized function, C8orf55, was also validated with a tissue microarray that included various types of cancers.     

Importance of Both Fatness and Aerobic Fitness on Metabolic Syndrome Risk in Japanese Children

Associations between body mass index (BMI), peak oxygen consumption (VO_2peak), and metabolic syndrome (MetS) risk factors have not been adequately studied in Japanese children. Here the relationships between these parameters and the threshold aerobic fitness level necessary for low MetS risk were determined. The participants (299 children; 140 boys and 159 girls, aged 9.1 ± 0.3 years) were divided into four groups using the medians of predicted VO_2peak (_pVO_2peak) and BMI. MetS risk scores were calculated using z-scores. Receiver Operating Characteristic analysis was used to determine the threshold aerobic fitness level necessary for low MetS risk. The MetS risk score of the High BMI group was significantly higher than that of the Low BMI group for both sexes (p < 0.0001). However, the High BMI/High Fitness group had a significantly lower MetS risk score than the High BMI/Low Fitness group for both sexes. The _pVO_2peak cut-off values for low MetS risk were 47.9 and 44.9 ml/kg/min for boys and girls, respectively. Our results suggest that improvements in both fatness and aerobic fitness are important for decreasing MetS risk. We also confirmed the _pVO_2peak of cut-off values necessary for low MetS risk in Japanese children.     

Demonstration of different metal ion-induced calcineurin conformations using a monoclonal antibody

It has been suggested that calcineurin, a calmodulin-stimulated phosphatase, may exist in different metal ion-dependent conformational states (Pallen, C.J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141). Evidence in favor of this hypothesis comes from studies involving a monoclonal antibody, VA1, which is specific for the small (beta) subunit of calcineurin. This antibody inhibits Ni2+-stimulated but not Mn2+-stimulated phosphatase activity against p-nitrophenyl phosphate and phosphorylase kinase. Inhibition is not due to competition of the antibody with substrate or to interference with metal ion binding to the enzyme. Complex formation between the antibody and calcineurin can be demonstrated either in the presence of Mn2+ or Ni2+ or in the absence of metal ion activators. These results indicate that the active conformational states of calcineurin are metal ion dependent, that the monoclonal antibody VA1 affects the Ni2+-induced conformational change of the enzyme, and that the beta subunit of calcineurin plays a critical role in the expression of Ni2+-stimulated phosphatase activity.     

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1 ⁺ gene was present in the introduced leu1::ura4 ⁺ DNA fragment; but a decrease in length of homology leads to an increase in the ratio of nonhomologous to homologous recombination events. The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination. The results suggested that there are multiple modes of integration; most events simply involve both ends of the fragments, while in other cases, fragments were integrated in a more complicated manner, probably via circularization or multimerization. To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reaction (IPCR) and their nucleotide sequences were determined. There was no obvious homology between introduced DNA and chromosomal DNA at these recombination sites. Furthermore it was found that each terminal region of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA. Two models are proposed to explain the mechanism of nonhomologous integration.     

Two coupled oscillators as a model for the coordinated finger tapping by both hands

Recently, it was found that rhythmic movements (e.g. locomotion, swimmeret beating) are controlled by mutually coupled endogeneous neural oscillators (Kennedy and Davis, 1977; Pearson and Iles, 1973; Stein, 1974; Shik and Orlovsky, 1976; Grillner and Zangger, 1979). Meanwhile, it has been found out that the phase resetting experiment is useful to investigate the interaction of neural oscillators (Perkel et al., 1963; Stein, 1974). In the preceding paper (Yamanishi et al., 1979), we studied the functional interaction between the neural oscillator which is assumed to control finger tapping and the neural networks which control some tasks. The tasks were imposed on the subject as the perturbation of the phase resetting experiment. In this paper, we investigate the control mechanism of the coordinated finger tapping by both hands. First, the subjects were instructed to coordinate the finger tapping by both hands so as to keep the phase difference between two hands constant. The performance was evaluated by a systematic error and a standard deviation of phase differences. Second, we propose two coupled neural oscillators as a model for the coordinated finger tapping. Dynamical behavior of the model system is analyzed by using phase transition curves which were measured on one hand finger tapping in the previous experiment (Yamanishi et al., 1979). Prediction by the model is in good agreement with the results of the experiments. Therefore, it is suggested that the neural mechanism which controls the coordinated finger tapping may be composed of a coupled system of two neural oscillators each of which controls the right and the left finger tapping respectively.     

Effect of chlordimeform on nerve-muscle system of the larva of the waxmoth, Galleria mellonella

The action of chlordimeform on the nerve-muscle preparation of the larvae of the waxmoth Galleria mellonella has been studied by means of microelectrodes. Exitatory junction potential evoked by nerve stimulation is reversibly suppressed by 2 × 10^?3 M chlordimeform, and spike-like component is abolished. The resting membrane potential of the muscle fibre and the action potential from the nerve terminal are not affected at 5 × 10^?3 M chlordimeform. The depolarizing membrane response caused by outward current and the effective membrane resistance are not appreciably affected. It appears that chlordimeform exerts its blocking action on the neuromuscular junction rather than the conductance mechanism of muscle fibre membrane.     

Ontogenesis of hypothalamic immunoreactive ACTH cells in vivo and in vitro: role of Rathke's pouch

The ontogenesis of immunoreactive (ir) ACTH cells and ir alpha-MSH cells in rat hypothalamus was studied in vivo and in vitro. Ir ACTH cells first appeared in the neuroepithelial cell layer lining the floor of the third ventricle on Day 13.5 of gestation, whereas ir alpha-MSH first appeared in the cytoplasm of several ir ACTH cells in the basal part of the arcuate nucleus of the hypothalamus on Day 19.5. When the medial-basal hypothalamus of 12.5-day embryos was cultured alone, a few ir ACTH cells were found after culture for 10 days, but not 3 days, and no ir alpha-MSH cells were observed in the cultures. When the hypothalamus was cultured with Rathke's pouch (intact or without the intermediate lobe anlage), ir ACTH cells appeared within 3 days. In these cultures on Days 6 and 10, long beaded fibers were seen projecting from cells in the neuronal tissue, and some cells showed immunolabeling for alpha-MSH. When the hypothalamus was cocultured with oral epithelium instead of Rathke's pouch, the appearance of neuronal ir ACTH cells was like that in cultures of hypothalamus alone. These in vitro findings suggest that stimulus from the anterior lobe anlage of the pituitary is necessary for normal development of ir ACTH/alpha-MSH cells in the hypothalamus.     

G?ttingen miniature swine as a model for diet-induced atherosclerosis

Twenty-four G?ttingen Miniature Swine/csk, in order to evaluate their potential usefulness as a model for experimental atherosclerosis studies, were fed diets of three types, a high-fat plus high cholesterol diet, a high-fat diet, and a commercial diet. Each group consisted of 4 males and 4 females. Swine fed the experimental diet were investigated by gross, microscopic and serum biochemical examination on the 1st, 3rd, 6th and 9th month after start of experimentation. Lesions of atherosclerosis were observed in the high-fat plus high-cholesterol diet group. After a month on the experimental diet, intimal thickening was detected in the abdominal aorta just above the origin of internal iliac artery, left coronary artery and ascending aorta by microscopic examination. Thereafter, on the 9th month after the start, there was more extensive and severe atherosclerosis. These lesions were classified into two types by the difference in the histologic architecture of arterial wall. One was fatty streaks that were in thoracic aorta belonging to the elastic type and the other was fibrous plaques that were in abdominal aorta and iliac artery and so on, belong to the transitional or muscular type. High-fat plus high-cholesterol diet feeding led to elevated serum cholesterol and beta-lipoprotein levels, and had an effect on several kinds of metabolism. All of the swine fed high-fat or commercial diet had little gross, microscopic lesions and had no change in serum cholesterol and beta-lipoprotein levels. Hypercholesterolemia and hyper-beta-lipoproteinemia had a close relation to the development and acceleration of atherosclerosis. It was possible to show that the diet induced atherosclerosis was similar in quality to that observed in humans, and that the G?ttingen miniature swine was a suitable animal for the study of atherosclerosis.     

Respiratory chain deficiency slows down cell-cycle progression via reduced ROS generation and is associated with a reduction of p21CIP1/WAF1

We have used HeLa cells without mitochondrial DNA (rho0-cells) and transient rho0-phenocopies, obtained from wild-type cells by short-term treatment with ethidium bromide, to analyze how the absence of a functional mitochondrial respiratory chain slows down proliferation. We ruled out an energetic problem (ATP/ADP content) as well as defective synthesis of pyrimidine, iron-sulfur clusters or heme as important causes for the proliferative defect. Flow cytometric analysis revealed that reactive oxygen species were reduced in rho0-cells and in rho0-phenocopies, and that, quite unusually, all stages of the cell cycle were slowed down. Specific quenching of mitochondrial ROS with the ubiquinone analog MitoQ also resulted in slower growth. Some important cell-cycle regulators were reduced in rho0-cells: cyclin D3, cdk6, p18INK4C, p27KIP1, and p21CIP1/WAF1. In the rho0-phenocopies, the expression pattern did not fully duplicate the complex response observed in rho0-cells, and mainly p21CIP1/WAF1 was downregulated. Activities of the growth regulatory PKB/Akt and MAPK/ERK-signaling pathways did not correlate with proliferation rates of rho0-cells and rho0-phenocopies. Our study demonstrates that loss of a functional mitochondrial electron transport chain inhibits cell-cycle progression, and we postulate that this occurs through the decreased concentration of reactive oxygen species, leading to downregulation of p21CIP1/WAF1.     

Cytochemical and ultrastructural examination of apoptotic odontoclasts induced by bisphosphonate administration

Since odontoclasts share similar characteristics with osteoclasts, this study has examined whether odontoclasts exhibit cytological alteration after treatment with bisphosphonate, which induces apoptosis of osteoclasts. After the administration of bisphosphonate to 6-day-old rabbits, many odontoclasts detached from the dentine surface of the deciduous teeth, resulting in the reduction of tartrate-resistant acid phosphatase (TRAP-ase) and immunoreactivity for cathepsin K. Transmission electron microscopy revealed a number of odontoclasts showing poorly developed or a lack of ruffled borders, a Golgi apparatus markedly reduced in size, and numerous cytoplasmic vesicles. The bisphosphonate-treated odontoclasts displayed fragmented DNA in the pyknotic nuclei evidenced by terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick-end labeling, indicating that bisphosphonate can induce apoptosis of the odontoclasts. Ultrastructural observations of the apoptotic odontoclasts revealed condensed heterochromatin at the margin of the nuclear envelope, assembled arrays of rough endoplasmic reticulum, and many vacuoles and vesicles. Some apoptotic odontoclasts showed ladder-like structures between the adjacent nuclear envelopes, enlargement of the nuclear envelopes, and the formation of a ribosome-like granular structure in the nuclei. Thus, odontoclasts are able to undergo apoptosis after bisphosphonate treatment; this results in cytological alterations, including reduced resorption activity and the inhibition of protein synthesis/transport as indicated by the diminished TRAPase and cathepsin K and the poorly developed Golgi apparatus, respectively. Nuclear alteration as evidenced by the appearance of ladder-like and ribosome-like structures was characteristic of apoptotic odontoclasts.