Derek Murphy

Toward optimized antibody microarrays: a comparison of current microarray support materials

With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.

Widespread Dysregulation of MiRNAs by MYCN Amplification and Chromosomal Imbalances in Neuroblastoma: Association of miRNA Expression with Survival

MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.

MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-β signalling pathway

Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN.

MicroRNA Mediates DNA De-methylation Events Triggered By Retinoic Acid During Neuroblastoma Cell Differentiation

Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regimen for patients with high-risk disease, and a similar derivative, all-trans-retinoic acid (ATRA), causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA-induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. Four hundred and two gene promoters became demethylated, whereas 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were overexpressed by >2-fold, whereas 13 of the hypermethylated genes were underexpressed. Gene ontology analysis indicated that demethylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we show the downregulation of methyltransferases, DNMT1 and DNMT3B, along with the upregulation of endogenous microRNAs targeting them. Ectopic overexpression of miR-152, targeting DNMT1, also negatively affected cell invasiveness and anchorage-independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA demethylation changes contribute to the process of ATRA-induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation.

Protein arrays as tools for serum autoantibody marker discovery in cancer

Protein array technology has begun to play a significant role in the study of protein-protein interactions and in the identification of antigenic targets of serum autoantibodies in a variety of autoimmune disorders. More recently, this technology has been applied to the identification of autoantibody signatures in cancer. The identification of tumour-associated antigens (TAAs) recognised by the patients immune response represents an exciting approach to identify novel diagnostic cancer biomarkers and may contribute towards a better understanding of the molecular mechanisms involved. Circulating autoantibodies have not only been used to identify TAAs as diagnostic/prognostic markers and potential therapeutic targets, they also represent excellent biomarkers for the early detection of tumours and potential markers for monitoring the efficacy of treatment. Protein array technology offers the ability to screen the humoral immune response in cancer against thousands of proteins in a high throughput technique, thus readily identifying new panels of TAAs. Such an approach should not only aid in improved diagnostics, but has already contributed to the identification of complex autoantibody signatures that may represent disease subgroups, early diagnostics and facilitated the analysis of vaccine trials.

Global MYCN Transcription Factor Binding Analysis in Neuroblastoma Reveals Association with Distinct E-Box Motifs and Regions of DNA Hypermethylation

Background Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors. Methodology We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions. Conclusion Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the activity of individual genes, and that of a mediator of global chromatin structure.

Human IgG antibody profiles differentiate between symptomatic patients with and without colorectal cancer

Objective: Patients with cancer have antibodies against tumour antigens. Characterising the antibody repertoire may provide insights into aberrant cellular mechanisms in cancer development, ultimately leading to novel diagnostic or therapeutic targets. The aim of this study was to characterise the antibody profiles in patients whose symptoms warranted colonoscopy, to see if there was a difference in patients with and without colorectal cancer. Methods: Patients were recruited from a colonoscopy clinic. Individual serum samples from 43 patients with colorectal cancer and 40 patients with no cancer on colonoscopy were profiled on a 37 830 clone recombinant human protein array. Antigen expression was evaluated by quantitative reverse transcription-PCR and by immunohistochemistry on tissue microarrays. Results: Using a sex- and age-matched training set, 18 antigens associated with cancer and 4 associated with the absence of cancer (p<0.05) were identified and confirmed. To investigate the mechanisms triggering antibody responses to these antigens, antigen expression was examined in normal colorectal mucosa and colorectal carcinoma of the same patients. The identified antigens showed cellular accumulation (p53), aberrant cellular expression (high mobility group B1 (HMGB1)) and overexpression (tripartite motif-containing 28 (TRIM28), p53, HMGB1, transcription factor 3 (TCF3), longevity assurance gene homologue 5 (LASS5) and zinc finger protein 346 (ZNF346)) in colorectal cancer tissue compared with normal colorectal mucosa. Conclusions: It is demonstrated for the first time that screening high-density protein arrays identifies unique antibody profiles that discriminate between symptomatic patients with and without colorectal cancer. The differential expression of identified antigens suggests their involvement in aberrant cellular mechanisms in cancer.

Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays.

Antibodies are routinely used as research tools, in diagnostic assays and increasingly as therapeutics. Ideally, these applications require antibodies with high sensitivity and specificity; however, many commercially available antibodies are limited in their use as they cross-react with non-related proteins. Here we describe a novel method to characterize antibody specificity. Six commercially available monoclonal and polyclonal antibodies were screened on high-density protein arrays comprising of ~10,000 recombinant human proteins (Imagenes). Two of the six antibodies examined; anti-pICln and anti-GAPDH, bound exclusively to their target antigen and showed no cross-reactivity with non-related proteins. However, four of the antibodies, anti-HSP90, anti-HSA, anti-bFGF and anti-Ro52, showed strong cross-reactivity with other proteins on the array. Antibody-antigen interactions were readily confirmed using Western immunoblotting. In addition, the redundant nature of the protein array used, enabled us to define the epitopic region within HSP90 of the anti-HSP90 antibody, and identify possible shared epitopes in cross-reacting proteins. In conclusion, high-density protein array technology is a fast and effective means for determining the specificity of antibodies and can be used to further improve the accuracy of antibody applications.

Protein Macroarray Profiling of Serum Autoantibodies in Pseudoexfoliation Glaucoma

PURPOSE Complex repertoires of IgG autoantibodies have been detected against ocular antigens in patients with glaucoma. The goal was to identify and characterize the IgG autoantibody repertoires in sera of patients with pseudoexfoliation glaucoma (PXFG) with protein macroarrays. METHODS Serum samples of 21 patients with PXFG and 19 age- and sex-matched control subjects were profiled on high-density colony protein macroarrays expressing His-tagged recombinant human proteins derived from a human fetal brain cDNA library. Statistically prevalent expression clones in the PXFG group were sequenced. mRNA expression of identified antigens was examined in the rat ganglion cell line RGC-5 and in human brain and optic nerve cDNA. The IgG immunoreactivity of the sera of 20 control and 26 PXFG patients to purified C6orf129 was analyzed in a reverse enzyme-linked immunosorbent assay. RESULTS An increased prevalence was detected among the PXFG patients of serum antibodies to seven proteins: C6orf129; stathmin-like 4; transmembrane protein 9 domain family, member B; fibroblast growth factor receptor 3; cleft lip and palate transmembrane protein 1; EH-domain-containing protein 1; and eukaryotic translation elongation factor 2. All antigens were expressed in the RGC-5 cells and in cDNA from human brain and optic nerve, with the exception of stathmin-like 4, which was not expressed in the RGC-5 cells. The patients with PXFG had increased anti-C6orf129 IgG immunoreactivity compared with that in the control subjects (P < 0.05). CONCLUSIONS Screening high-density protein arrays identifies unique antibody profiles that may discriminate between patients with and without PXFG. Characterization of the autoantibody repertoire may provide new insights into the pathophysiology of PXFG.

Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

BackgroundProtein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown.ResultsAs a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKAs primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma.ConclusionsPTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.