Autophagy initiation triggers p150Glued-AP-2ß interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

A Multi-Faceted Analysis Showing CRNDE Transcripts and a Recently Confirmed Micropeptide as Important Players in Ovarian Carcinogenesis.

CRNDE is considered an oncogene expressed as long non-coding RNA. Our previous paper is the only one reporting CRNDE as a micropeptide-coding gene. The amino acid sequence of this micropeptide (CRNDEP) has recently been confirmed by other researchers. This study aimed at providing a mass spectrometry (MS)-based validation of the CRNDEP sequence and an investigation of how the differential expression of CRNDE(P) influences the metabolism and chemoresistance of ovarian cancer (OvCa) cells. We also assessed cellular localization changes of CRNDEP, looked for its protein partners, and bioinformatically evaluated its RNA-binding capacities. Herein, we detected most of the CRNDEP sequence by MS. Moreover, our results corroborated the oncogenic role of CRNDE, portraying it as the gene impacting carcinogenesis at the stages of DNA transcription and replication, affecting the RNA metabolism, and stimulating the cell cycle progression and proliferation, with CRNDEP being detected in the centrosomes of dividing cells. We also showed that CRNDEP is located in nucleoli and revealed interactions of this micropeptide with p54, an RNA helicase. Additionally, we proved that high CRNDE(P) expression increases the resistance of OvCa cells to treatment with microtubule-targeted cytostatics. Furthermore, altered CRNDE(P) expression affected the activity of the microtubular cytoskeleton and the formation of focal adhesion plaques. Finally, according to our in silico analyses, CRNDEP is likely capable of RNA binding. All these results contribute to a better understanding of the CRNDE(P) role in OvCa biology, which may potentially improve the screening, diagnosis, and treatment of this disease.

ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling.

Sortilin is a neuronal receptor for apolipoprotein E (apoE). Sortilin-dependent uptake of lipidated apoE promotes conversion of polyunsaturated fatty acids (PUFA) into neuromodulators that induce anti-inflammatory gene expression in the brain. This neuroprotective pathway works with the apoE3 variant but is lost with the apoE4 variant, the main risk factor for Alzheimer's disease (AD). Here, we elucidated steps in cellular handling of lipids through sortilin, and why they are disrupted by apoE4. Combining unbiased proteome screens with analyses in mouse models, we uncover interaction of sortilin with fatty acid-binding protein 7 (FABP7), the intracellular carrier for PUFA in the brain. In the presence of apoE3, sortilin promotes functional expression of FABP7 and its ability to elicit lipid-dependent gene transcription. By contrast, apoE4 binding blocks sortilin-mediated sorting, causing catabolism of FABP7 and impairing lipid signaling. Reduced FABP7 levels in the brain of AD patients expressing apoE4 substantiate the relevance of these interactions for neuronal lipid homeostasis. Taken together, we document interaction of sortilin with mediators of extracellular and intracellular lipid transport that provides a mechanistic explanation for loss of a neuroprotective lipid metabolism in AD.

The interactome of multifunctional HAX1 protein suggests its role in the regulation of energy metabolism, de-aggregation, cytoskeleton organization and RNA-processing.

HCLS1-associated protein X-1 (HAX1) is a multifunctional protein involved in many cellular processes, including apoptosis, cell migration and calcium homeostasis, but its mode of action still remains obscure. Multiple HAX1 protein partners have been identified, but they are involved in many distinct pathways, form different complexes and do not constitute a coherent group. By characterizing HAX1 protein interactome using targeted approach, we attempt to explain HAX1 multiple functions and its role in the cell. Presented analyses indicate that HAX1 interacts weakly with a wide spectrum of proteins and its interactome tends to be cell-specific, which conforms to a profile of intrinsically disordered protein (IDP). Moreover, we have identified a mitochondrial subset of HAX1 protein partners and preliminarily characterized its involvement in the cellular response to oxidative stress and aggregation.

HAX1 impact on collective cell migration, cell adhesion, and cell shape is linked to the regulation of actomyosin contractility.

HAX1 protein is involved in the regulation of apoptosis, cell motility and calcium homeostasis. Its overexpression was reported in several tumors, including breast cancer. This study demonstrates that HAX1 has an impact on collective, but not single-cell migration, thus indicating the importance of cell-cell contacts for the HAX1-mediated effect. Accordingly, it was shown that HAX1 knockdown affects cell-cell junctions, substrate adhesion, and epithelial cell layer integrity. As demonstrated here, these effects can be attributed to the modulation of actomyosin contractility through changes in RhoA and septin signaling. Additionally, it was shown that HAX1 does not influence invasive potential in the breast cancer cell line, suggesting that its role in breast cancer progression may be linked instead to collective invasion of the epithelial cells but not single-cell dissemination.

Mass Spectrometry-Based Comprehensive Analysis of Pancreatic Cyst Fluids.

Pancreatic cyst fluids (PCFs) enriched in tumour-derived proteins are considered a potential source of new biomarkers. This study aimed to determine compositional and quantitative differences between the degradome and proteome of PCFs aspirated from different types of pancreatic cyst lesions (PCLs). 91 patients who underwent endoscopic ultrasound-fine needle aspiration under routine clinical diagnosis of PCLs were enrolled. Four cysts were malignant (CAs), and 87 were nonmalignant and consisted of 18 intraductal papillary mucinous neoplasms (IPMNs), 14 mucinous cystic neoplasms (MCNs), nine serous cystic neoplasms (SCNs), 29 pseudocysts (PCs), and 17 unclassified. Profiles of the <5 kDa fraction, the degradome, and the trypsin-digested proteome were analysed using an LTQ-Orbitrap Elite mass spectrometer coupled with a nanoACQUITY LC system. Qualitative analyses identified 796 and 366 proteins in degradome and proteome, respectively, and 689 (77%) and 285 (78%) of them were present in the Plasma Proteome Database. Gene Ontology analysis showed a significant overrepresentation of peptidases and peptidases inhibitors in both datasets. In the degradome fraction, quantitative values were obtained for 6996 peptides originating from 657 proteins. Of these, 2287 peptides were unique to a single type, and 515 peptides, derived from 126 proteins, were shared across cyst types. 32 peptides originating from 12 proteins had differential (adjusted p-value ≤0.05, FC ≥1.5) abundance in at least one of the five cysts types. In proteome, relative expression was measured for 330 proteins. Of them, 33 proteins had significantly (adjusted p-value ≤0.05, FC ≥1.5) altered abundance in at least one of the studied groups and 19 proteins appeared to be unique to a given cyst type. PCFs are dominated by blood proteins and proteolytic enzymes. Although differences in PCF peptide composition and abundance could aid classification of PCLs, the unpredictable inherent PCF proteolytic activity may limit the practical applications of PCF protein profiling.

Limited prolonged effects of rifaximin treatment on irritable bowel syndrome-related differences in the fecal microbiome and metabolome.

Irritable bowel syndrome (IBS) is a chronic functional disorder and its development may be linked, directly and indirectly, to intestinal dysbiosis. Here we investigated the interactions between IBS symptoms and the gut microbiome, including the relation to rifaximin (1200 mg daily; 11.2 g per a treatment). We recruited 72 patients, including 31 with IBS-D (diarrhea), 11 with IBS-C (constipation), and 30 with IBS-M (mixed constipation and diarrhea) and 30 healthy controls (HCs). Of them, 68%, 64%, and 53% patients with IBS-D, IBS-C, and IBS-M, respectively, achieved 10-12 week-term improvement after the rifaximin treatment. Stool samples were collected before and after the treatment, and fecal microbiotic profiles were analyzed by deep sequencing of 16S rRNA, while stool metabolic profiles were studied by hydrogen 1-nuclear magnetic resonance ((1)H-NMR) and gas chromatography-mass spectrometry (GC-MS). Of 26 identified phyla, only Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria were consistently found in all samples. Bacteroidetes was predominant in fecal samples from HCs and IBS-D and IBS-M subjects, whereas Firmicutes was predominant in samples from IBS-C subjects. Species richness, but not community diversity, differentiated all IBS patients from HCs. Metabolic fingerprinting, using NMR spectra, distinguished HCs from all IBS patients. Thirteen metabolites identified by GC-MS differed HCs and IBS patients. However, neither metagenomics nor metabolomics analyses identified significant differences between patients with and without improvement after treatment.

Histone H1 Variants in Arabidopsis Are Subject to Numerous Post-Translational Modifications, Both Conserved and Previously Unknown in Histones, Suggesting Complex Functions of H1 in Plants.

Linker histones (H1s) are conserved and ubiquitous structural components of eukaryotic chromatin. Multiple non-allelic variants of H1, which differ in their DNA/nucleosome binding properties, co-exist in animal and plant cells and have been implicated in the control of genetic programs during development and differentiation. Studies in mammals and Drosophila have revealed diverse post-translational modifications of H1s, most of which are of unknown function. So far, it is not known how this pattern compares with that of H1s from other major lineages of multicellular Eukaryotes. Here, we show that the two main H1variants of a model flowering plant Arabidopsis thaliana are subject to a rich and diverse array of post-translational modifications. The distribution of these modifications in the H1 molecule, especially in its globular domain (GH1), resembles that occurring in mammalian H1s, suggesting that their functional significance is likely to be conserved. While the majority of modifications detected in Arabidopsis H1s, including phosphorylation, acetylation, mono- and dimethylation, formylation, crotonylation and propionylation, have also been reported in H1s of other species, some others have not been previously identified in histones.

The putative oncogene, CRNDE, is a negative prognostic factor in ovarian cancer patients.

The CRNDE gene seems to play an oncogenic role in cancers, though its exact function remains unknown. Here, we tried to assess its usefulness as a molecular prognostic marker in ovarian cancer. Based on results of our microarray studies, CRNDE transcripts were further analyzed by Real-Time qPCR-based profiling of their expression. The qPCR study was conducted with the use of personally designed TaqMan assays on 135 frozen tissue sections of ovarian carcinomas from patients treated with platinum compounds and either cyclophosphamide (PC, N = 32) or taxanes (TP, N = 103). Elevated levels of two different CRNDE transcripts were a negative prognostic factor; they increased the risk of death and recurrence in the group of patients treated with TP, but not PC (DNA-damaging agents only). Higher associations were found for overexpression of the short CRNDE splice variant (FJ466686): HR 6.072, 95% CI 1.814-20.32, p = 0.003 (the risk of death); HR 15.53, 95% CI 3.812-63.28, p < 0.001 (the risk of recurrence). Additionally, accumulation of the TP53 protein correlated with decreased expression of both CRNDE transcripts in tumor cells. Our results depict CRNDE as a potential marker of poor prognosis in women with ovarian carcinomas, and suggest that its significance depends on the therapeutic regimen used.

Beads-free protein immunoprecipitation for a mass spectrometry-based interactome and posttranslational modifications analysis.

BACKGROUND: Protein immunoprecipitation (IP) coupled with MS provides means to interrogate protein complexes and their posttranslational modifications (PTMs). In a typical protein IP assay antibodies are conjugated to protein A/G beads requiring large amounts of antibodies, tube transfers and centrifugations. RESULTS: As an alternative, we present Matrix-IP, beads-free microplate-based platform with surface-immobilized antibodies. Assay utilizes standard 96-well polypropylene PCR plates that are laboratory-fabricated with UV-C light and then protein A/G coated prior to IP reaction. We demonstrate application of Matrix-IP platform in MS analysis of heterogeneous nuclear ribonucleoprotein K (hnRNP K) interactome and PTMs. CONCLUSION: Matrix-IP is time-saving, easy to use high throughput method adaptable for low sample amounts and automation.

The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues.

CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene.

jChIP: a graphical environment for exploratory ChIP-Seq data analysis.

BACKGROUND: Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) provides a powerful tool for discovering protein-DNA interactions. Still, the computational analysis of the great amount of ChIP-Seq data generated, involving mapping of raw data to reference genome, has been a bottle neck for most of researchers in the transcriptional and epigenetic fields. Thus, user-friendly ChIP-Seq processing method sare much needed to enable greater community of computational and bench biologists to exploit the power of ChIP-Seq technology . FINDINGS: jChIP is a graphical tool that was developed to analyze and display ChIP-Seq data. It matches reads to the corresponding loci downloaded from Ensembl Genes or Ensembl Regulation databases. jChIP provides a friendly interface for exploratory analysis of mapped reads as well as peak calling data. The built-in functions for graphical display of reads distribution allows to evaluate the quality and meaning of ChIP-Seq data. CONCLUSION: jChIP is a user-friendly GUI-based software for the analysis of ChIP-Seq data within context of known genomic features. Further, jChIP provides tools for discovering new and refining known genome-wide protein binding patterns.

Histone H3 lysine 27 acetylation is altered in colon cancer.

BACKGROUND: Histone post-translational modifications (PTMs) play an important role in the regulation of the expression of genes, including those involved in cancer development and progression. However, our knowledge of PTM patterns in human tumours is limited. METHODS: MS-based analyses were used to quantify global alterations of histone PTMs in colorectal cancer (CRC) samples. Histones isolated from 12 CRCs and their corresponding normal mucosa by acidic extraction were separated by SDS-PAGE and analysed by liquid chromatography-mass spectrometry. RESULTS: Among 96 modified peptides, 41 distinct PTM sites were identified, of which 7, 13, 11, and 10 were located within the H2A, H2B, H3, and H4 sequences, respectively, and distributed among the amino-terminal tails and the globular domain of the four histones. Modification intensities were quantified for 33 sites, of which 4 showed significant (p-value ≤ 0.05) differences between CRC tissues and healthy mucosa samples. We identified histone H3 lysine 27 acetylation (H3K27Ac) as a modification upregulated in CRC, which had not been shown previously. CONCLUSIONS: The present results indicate the usefulness of a bottom-up proteomic approach for the detection of histone modifications at a global scale. The differential abundance of H3K27Ac mark in CRC, a PTM associated with active enhancers, suggests its role in regulating genes whose expression changes in CRC.

Mitochondrial-related proteomic changes during obesity and fasting in mice are greater in the liver than skeletal muscles.

Although mitochondrial dysfunction is implicated in the pathogenesis of obesity, the molecular mechanisms underlying obesity-related metabolic abnormalities are not well established. We performed mitochondrial quantitative proteomic and whole transcriptome analysis followed by functional annotations within liver and skeletal muscles, using fasted and non-fasted 16- and 48-week-old high-fat diet (HFD)-fed and normal diet-fed (control group) wild-type C56BL/6J mice, and hyperphagic ob/ob and db/db obese mice. Our study identified 1,675 and 704 mitochondria-associated proteins with at least two peptides in liver and muscle, respectively. Of these, 221 liver and 44 muscle proteins were differentially expressed (adjusted p values ≤ 0.05) between control and all obese mice, while overnight fasting altered expression of 107 liver and 35 muscle proteins. In the liver, we distinguished a network of 27 proteins exhibiting opposite direction of expression changes in HFD-fed and hyperphagic mice when compared to control. The network centered on cytochromes P450 3a11 (Cyp3a11) and 4a14 (Cyp4a14), and fructose-bisphosphate aldolase B (Aldob) proteins which bridged proteins cluster involved in Metabolism of xenobiotics with proteins engaged in Fatty acid metabolism and PPAR signaling pathways. Functional annotations revealed that most of the hepatic molecular alterations, which characterized both obesity and fasting, related to different aspects of energy metabolism (such as Fatty acid metabolism, Peroxisome, and PPAR signaling); however, only a limited number of functional annotations could be selected from skeletal muscle data sets. Thus, our comprehensive molecular overview revealed that both obesity and fasting states induce more pronounced mitochondrial proteome changes in the liver than in the muscles.

Pre-analytical-related variability influencing serum peptide profiles demonstrated in a mass spectrometry-based search for colorectal and prostate cancer biomarkers.

Although the degradome, which comprises proteolytic fragments of blood proteins, presents a potential source of diagnostic biomarkers, studies on cancer peptide biomarkers have provided inconsistent conclusions. In the present study, we reevaluated the usefulness of serum degradome analyses for searching peptide cancer biomarker candidates. Particular attention was paid to pre-analytical factors influencing the variability of determined peptide levels, including clotting time and control group selection. Studies were conducted on 44 and 86 serum samples collected from cancer patients and healthy individuals, respectively, using liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS)-based analyses. We identified 1373 unique peptides, nearly 40% of which originated from five blood proteins: fibrinogen alpha chain, apolipoprotein A-IV (APOA4), complement C3, apolipoprotein A-I, and alpha-1-antitrypsin. A set of 118 and 88 peptides exhibited highly significant differences (adjusted p-value ≤ 0.01 and fold change ≥ 2) in pair-wise comparisons of control vs. prostate cancer and control vs. colorectal cancer, respectively, with 37 peptides displaying a consistent direction of change for these pair-wise comparisons. The levels of 67 peptides differed significantly in serum samples collected from healthy individuals immediately prior to colonoscopy and those who underwent colonoscopic examination at least four weeks earlier. Of them, 49 peptides originated from APOA4. Whereas earlier studies, including ours, have utilized fragments of fibrinopeptide A (FPA) to distinguish cancer from healthy cases, here we show that their absolute abundance is a sensitive indicator of clotting time. These observations may have implications for future serum peptidome studies since these issues have not previously been recognized.

Diffprot - software for non-parametric statistical analysis of differential proteomics data.

Mass spectrometry-based global proteomics experiments generate large sets of data that can be converted into useful information only with an appropriate statistical approach. We present Diffprot - a software tool for statistical analysis of MS-derived quantitative data. With implemented resampling-based statistical test and local variance estimate, Diffprot allows to draw significant results from small scale experiments and effectively eliminates false positive results. To demonstrate the advantages of this software, we performed two spike-in tests with complex biological matrices, one label-free and one based on iTRAQ quantification; in addition, we performed an iTRAQ experiment on bacterial samples. In the spike-in tests, protein ratios were estimated and were in good agreement with theoretical values; statistical significance was assigned to spiked proteins and single or no false positive results were obtained with Diffprot. We compared the performance of Diffprot with other statistical tests - widely used t-test and non-parametric Wilcoxon test. In contrast to Diffprot, both generated many false positive hits in the spike-in experiment. This proved the superiority of the resampling-based method in terms of specificity, making Diffprot a rational choice for small scale high-throughput experiments, when the need to control the false positive rate is particularly pressing.

Comparative kinome analysis to identify putative colon tumor biomarkers.

Kinase domains are the type of protein domain most commonly found in genes associated with tumorigenesis. Because of this, the human kinome (the protein kinase component of the genome) represents a promising source of cancer biomarkers and potential targets for novel anti-cancer therapies. Alterations in the human colon kinome during the progression from normal colon (NC) through adenoma (AD) to adenocarcinoma (AC) were investigated using integrated transcriptomic and proteomic datasets. Two hundred thirty kinase genes and 42 kinase proteins showed differential expression patterns (fold change ≥ 1.5) in at least one tissue pair-wise comparison (AD vs. NC, AC vs. NC, and/or AC vs. AD). Kinases that exhibited similar trends in expression at both the mRNA and protein levels were further analyzed in individual samples of NC (n = 20), AD (n = 39), and AC (n = 24) by quantitative reverse transcriptase PCR. Individual samples of NC and tumor tissue were distinguishable based on the mRNA levels of a set of 20 kinases. Altered expression of several of these kinases, including chaperone activity of bc1 complex-like (CABC1) kinase, bromodomain adjacent to zinc finger domain protein 1B (BAZ1B) kinase, calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D), serine/threonine-protein kinase 24 (STK24), vaccinia-related kinase 3 (VRK3), and TAO kinase 3 (TAOK3), has not been previously reported in tumor tissue. These findings may have diagnostic potential and may lead to the development of novel targeted therapeutic interventions for colorectal cancer.

Gene expression alterations induced by low molecular weight heparin during bowel anastomosis healing in rats.

Colon anastomosis is therapeutically challenging because multiple, usually undetectable factors influence a spectrum of repair mechanisms. We hypothesized that low molecular weight heparins, routinely administered perioperatively, may differentially affect gene expression related to colon healing. Twenty pairs of untreated and enoxaparin-treated rats underwent left-side hemicolectomy with a primary end-to-end anastomosis. Normal colon and anastomotic bowel segments were resected on day 0 and on days 1, 3, 5, and 7 after surgery, respectively. Serial anastomosis transverse cross-sections were evaluated microscopically and by microarray (Rat Genome 230 2.0, Affymetrix). Differentially expressed probe sets were annotated with Gene Ontology. We also examined the influence of enoxaparin on fibroblast proliferation and viability in vitro. Among the 5476 probe sets, we identified differential expression at each healing time point, yielding 79 subcategories. Most indicated genes were involved in wound healing, including multicellular organismal development, locomotory behavior, immune response, cell adhesion, inflammatory response, cell-cell signaling, blood vessel development, and tissue remodeling. Although we found no intensity differences in histological features of healing between enoxaparin-treated and control rats, treatment did induce significant expression changes during early healing. Of these changes, 83 probe sets exhibited at least twofold changes and represented different functional annotations, including inflammatory response, regulation of transcription, regulation of apoptosis, and angiogenesis. Fibroblast culture confirmed an anti-viability effect of enoxaparin. Enoxaparin affects colon wound-related gene expression profiles, but further studies will resolve whether heparin treatment is a risk factor after intestinal surgery, at least in some patients.

Integrating proteomic and transcriptomic high-throughput surveys for search of new biomarkers of colon tumors.

To the search of new colon tumor biomarkers in the transition from normal colon (NC) mucosa to adenoma (AD) and adenocarcinoma (AC), we integrated microarray data with the results of a high-throughput proteomic workflow. In proteomic study, we used a modified isoelectric focusing protocol on strips with an immobilized pH gradient to separate peptides labeled with iTRAQ (isobaric tags for relative and absolute quantitation) tags followed by liquid chromatography-tandem mass spectrometry analysis. Gene expression measurements were done using Affymetrix GeneChip HG-U133plus2 microarrays and quantitative reverse transcriptase PCR (q-RT-PCR). We identified 3,886 proteins with at least two peptides. Of them, 1,061 proteins were differentially expressed [FC ≥ 1.5; FDR ≤ 0.01] in two pair-wise comparisons: AD vs. NC and AC vs. AD while 15 and 23 proteins were progressively up-regulated and down-regulated in the NC/AD/AC sequence, respectively. The quantitative proteomic information was subsequently correlated with microarray data. For a collection of genes with the same direction of changes of both mRNA and protein levels, we obtained 785/853/795 genes in AD vs. NC/AC vs. NC/AC vs. AD comparison, respectively. Further evaluation of sequentially altered gene expression by q-RT-PCR on individual samples of 24 NCs, 42 ADs, and 26 ACs confirmed progressive expression of six genes: biglycan, calumenin, collagen type XII, alpha 1 (COL12A1), monoamine oxidase A (MAOA), ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), and MOCO sulphurase C-terminal domain-containing 2 (MOSC2). Among them, three continuously down-regulated (MAOA, ENTPD5, and MOSC2) and one continuously overexpressed (COL12A1) are reported, to our best knowledge, for the first time in a connection to colon cancer onset.

Modeling oncogenic signaling in colon tumors by multidirectional analyses of microarray data directed for maximization of analytical reliability.

BACKGROUND: Clinical progression of colorectal cancers (CRC) may occur in parallel with distinctive signaling alterations. We designed multidirectional analyses integrating microarray-based data with biostatistics and bioinformatics to elucidate the signaling and metabolic alterations underlying CRC development in the adenoma-carcinoma sequence. METHODOLOGY/PRINCIPAL FINDINGS: Studies were performed on normal mucosa, adenoma, and carcinoma samples obtained during surgery or colonoscopy. Collections of cryostat sections prepared from the tissue samples were evaluated by a pathologist to control the relative cell type content. The measurements were done using Affymetrix GeneChip HG-U133plus2, and probe set data was generated using two normalization algorithms: MAS5.0 and GCRMA with least-variant set (LVS). The data was evaluated using pair-wise comparisons and data decomposition into singular value decomposition (SVD) modes. The method selected for the functional analysis used the Kolmogorov-Smirnov test. Expressional profiles obtained in 105 samples of whole tissue sections were used to establish oncogenic signaling alterations in progression of CRC, while those representing 40 microdissected specimens were used to select differences in KEGG pathways between epithelium and mucosa. Based on a consensus of the results obtained by two normalization algorithms, and two probe set sorting criteria, we identified 14 and 17 KEGG signaling and metabolic pathways that are significantly altered between normal and tumor samples and between benign and malignant tumors, respectively. Several of them were also selected from the raw microarray data of 2 recently published studies (GSE4183 and GSE8671). CONCLUSION/SIGNIFICANCE: Although the proposed strategy is computationally complex and labor-intensive, it may reduce the number of false results.