Proteomic and molecular analysis in colorectal cancer: validation of the biomarkers desmin, SET and CK8.

There is potential for significant improvements to be made in the diagnosis, staging, treatment and monitoring of colorectal cancer (CRC). The elucidation of proteins and pathways involved in CRC would aid in the development of biomarkers for detection, identify patients at risk of relapse and potentially give rise to new molecular targets of treatment. Laser microdissectio (LMD) was used with minimal labelling two-dimensional gel electrophoresis (2D DIGE) and mass spectrometry to identify proteins significantly increased in eight early stage paired tumour-normal tissues. Seventeen individual proteins were identified as upregulated by >2 fold (P<0.05). The role of the proteins cytokeratin 8 (CK8) and SET in CRC were chosen for further analysis. A third protein, desmin, was chosen from a pilot study performed using LMD and saturation labelling 2D DIGE for further analysis. The protein desmin was identified as significantly upregulated in a pilot study that aimed to identify proteins with altered expression in the cancerous epithelium and surrounding microenvironment. Using immunofluorescence (IF), desmin expression levels in the tissue stroma of late stage tumours compared to early stage tumours was significantly increased, P<0.0001. The desmin expressing cells were identified to be pericytes, formed around mature vasculature as a result of angiogenic stimulation. From this work desmin appears to have potential use as a histopathology marker for the identification of late stage patients and may help identify patients who would not benefit from the current anti-angiogenic therapies due to the presence of mature tumour microvasculature. Three isoforms of CK8 were found to be upregulated in the DIGE study, with each isoform containing the phosphoserine (PS) residues 23, 431 and 73. Western blotting showed significantly increased phosphorylation levels at these sites in tumour compared to normal tissues. The MAP kinase ERK is known to phosphorylate the 73 and 431 residues and 50% of CRC patients have mutations in the EGFR/Ras/Raf/MEK/ERK signalling pathway (KRAS or BRAF mutations) resulting in constitutive ERK activation. Inhibition of EGFR activation in Caco2 cells showed a significant decrease (P<0.0001) in PS73 and PS431 levels by 59% and 65% respectively, indicating that patients with KRAS or BRAF mutations may have significantly increased PS73 and PS431 levels. Previously it has been shown that high levels of CK8 phosphorylation may help to protect cells against caspase degradation and evasion of apoptosis. This is the first report of the differential expression of phospho-CK8 isoforms in CRC. SET is a known inhibitor of the tumour suppressor PP2A, a component of the GSK3 β complex that targets β catenin for degradation in the Wnt signalling pathway. Ninety percent of CRC patients have Wnt signalling pathway mutations resulting in constitutive pathway activation. The effect of knocking down SET via siRNA on β catenin levels was analysed in SW480 with constitutive Wnt signalling and HEK293 with low levels of Wnt signalling. No changes in β catenin levels were observed in the SW480 cells, however a 24.5% reduction was detected in the HEK293 cells. The role of SET in other cancer associated pathways was analysed in the SET knock down cells using the RT² Profiler PCR Array ‘Human Cancer Pathway Finder’ plates with the expression of genes involved in apoptosis, angiogenesis and adhesion found to be altered.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 201

An immunodominant La/SSB autoantibody proteome derives from public clonotypes

This item is under embargo for a period of 12 months from the date of publication, in accordance with the publisher's policy.The La/SSB autoantigen is a major target of long-term humoral autoimmunity in primary Sjögren’s Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti-Ro60/Ro52/La responses target an NH2-terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high-resolution Orbitrap mass spectrometry to determine the clonality, isotype and V-region sequences of LaA-specific autoantibodies in seven patients with primary SS. Anti-LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope-specific affinity chromatography were analysed by combined database and de-novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa-restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3-30 heavy chain paired with IGKV3-15 light chain and the second by an IGHV3- 43/IGKV3-20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity-determining regions, consistent with a common breach of B cell tolerance followed by antigendriven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein–RNA complexes are mediated by public sets of autoreactive B cell clonotypes.This work was supported by an Australian National Health and Medical Research Council grant 1041900 to T. P. Gordon and T. K. Chataway

Secreted human Ro52 autoantibody proteomes express a restricted set of public clonotypes

This article appeared in a journal published by Elsevier Ltd. Under Elsevier's copyright, mandated authors are not permitted to make work available in an institutional repository.Long-lived secreted autoantibody responses in systemic autoimmunity are generally regarded to be polyclonal and to express a diverse B-cell repertoire. Here, we have used a proteomic approach based on de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against a prototypic systemic autoantigen, Ro52 (TRIM21). Remarkably, anti-Ro52 autoantibodies from patients with primary Sjögren’s syndrome, systemic lupus erythematosus, systemic sclerosis or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain paired with one of two closely-related heavy chains; and were public in unrelated patients. Targeted mass spectrometry using these uniquely mutated V region peptides as surrogates detected anti-Ro52 autoantibodies in human sera with high sensitivity and specificity compared with traditional ELISA. Mass spectrometry-based detection of specific autoantibody motifs provides a powerful new tool for analysis of humoral autoimmunity.Australian National Health & Medical Research Counci

A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections

The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify and quantify proteins of interest from a consecutive section of the same tissue. Here, we present data from concurrent precancerous lesions from the endometrium and fallopian tube of a single patient. Using this complementary approach, we monitored the abundance of hundreds of proteins within the precancerous and neighboring healthy regions. The method described here represents a useful tool to maximize the number of molecular data acquired from small sample sizes or even from a single case. Our initial data are indicative of a migratory phenotype in these lesions and warrant further research into their malignant capabilities

Desmin expression in colorectal cancer stroma correlates with advanced stage disease and marks angiogenic microvessels

Abstract Introduction Biomarkers that improve stratification of colorectal cancer patients for adjuvant therapy versus resection alone, or that are predictive of response to therapeutic agents, have the potential to greatly improve patient selection for such therapies. The aim was to determine proteins differentially expressed within the malignant epithelial glands and closely associated stromal elements compared to matched normal mucosa, and to characterise the over-expression of one such protein as a potential biomarker. Methods Protein from laser microdissected tumor and normal mucosa was analysed by two dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry to determine differentially over expressed tumor proteins. Tumor over-expression of one such protein, desmin, was quantified using immunofluorescence staining in a larger cohort. Dual staining for desmin and vimentin, or desmin and von Willebrand factor, was performed to determine the cell type of interest. Results Desmin expression was significantly increased between stage I and III tumors, (P P Conclusion Pericyte coverage of vasculature is a marker of vessel maturation, hence desmin expression may have use as a marker for microvessel maturation. Clinical trials will be needed to determine its use in identifying tumors that will be less responsive to anti-angiogenic therapy.</p

Long-term Ro60 humoral autoimmunity in primary Sjogren's syndrome is maintained by rapid clonal turnover

This article appeared in a journal published by Elsevier Ltd. Under Elsevier's copyright, mandated authors are not permitted to make work available in an institutional repository.Long-term humoral autoimmunity to RNA–protein autoantigens is considered a hallmark of systemic autoimmune diseases. We use high resolution Orbitrap mass spectrometric autoantibody sequencing to track the evolution of a Ro60-specific public clonotypic autoantibody in 4 patients with primary Sjögren's syndrome. This clonotype is specified by a VH3–23/VK3–20 heavy and light chain pairing. Despite apparent stability by conventional immunoassay, analysis of V-region molecular signatures of clonotypes purified from serum samples collected retrospectively over 7 years revealed sequential clonal replacement. Prospective longitudinal studies confirmed clonotype loss and replacement at approximately three-monthly intervals. Levels of secreted anti-Ro60 clonotypes fluctuated markedly over time, despite minimal changes in clonal affinity. Our novel findings indicate a relentless turnover of short-lived clonotypic variants, masquerading as long-lived Ro60 humoral autoimmunity.Australian National Health & Medical Research Counci

Label-Free Quantification Mass Spectrometry Identifies Protein Markers of Chemotherapy Response in High-Grade Serous Ovarian Cancer

Eighty percent of ovarian cancer patients initially respond to chemotherapy, but the majority eventually experience a relapse and die from the disease with acquired chemoresistance. In addition, 20% of patients do not respond to treatment at all, as their disease is intrinsically chemotherapy resistant. Data-independent acquisition nano-flow liquid chromatography–mass spectrometry (DIA LC-MS) identified the three protein markers: gelsolin (GSN), calmodulin (CALM1), and thioredoxin (TXN), to be elevated in high-grade serous ovarian cancer (HGSOC) tissues from patients that responded to chemotherapy compared to those who did not; the differential expression of the three protein markers was confirmed by immunohistochemistry. Analysis of the online GENT2 database showed that mRNA levels of GSN, CALM1, and TXN were decreased in HGSOC compared to fallopian tube epithelium. Elevated levels of GSN and TXN mRNA expression correlated with increased overall and progression-free survival, respectively, in a Kaplan–Meier analysis of a large online repository of HGSOC patient data. Importantly, differential expression of the three protein markers was further confirmed when comparing parental OVCAR-5 cells to carboplatin-resistant OVCAR-5 cells using DIA LC-MS analysis. Our findings suggest that GSN, CALM1, and TXN may be useful biomarkers for predicting chemotherapy response and understanding the mechanisms of chemotherapy resistance. Proteomic data are available via ProteomeXchange with identifier PXD033785