Large-scale proteomics differentiates cholesteatoma from surrounding tissues and identifies novel proteins related to the pathogenesis.

Cholesteatoma is the growth of keratinizing squamous epithelium in the middle ear. It is associated with severe complications and has a poorly understood etiopathogenesis. Here, we present the results from extensive bioinformatics analyses of the first large-scale proteomic investigation of cholesteatoma. The purpose of this study was to take an unbiased approach to identifying alterations in protein expression and in biological processes, in order to explain the characteristic phenotype of this skin-derived tumor. Five different human tissue types (cholesteatoma, neck of cholesteatoma, tympanic membrane, external auditory canal skin, and middle ear mucosa) were analyzed. More than 2,400 unique proteins were identified using nanoLC-MS/MS based proteomics (data deposited to the ProteomeXchange), and 295 proteins were found to be differentially regulated in cholesteatoma. Validation analyses were performed by SRM mass spectrometry. Proteins found to be up- or down-regulated in cholesteatoma were analyzed using Ingenuity Pathway Analysis and clustered into functional groups, for which activation state and associations to disease processes were predicted. Cholesteatoma contained high levels of pro-inflammatory S100 proteins, such as S100A7A and S100A7. Several proteases, such as ELANE, were up-regulated, whereas extracellular matrix proteins, such as COL18A1 and NID2, were under-represented. This may lead to alterations in integrity and differentiation of the tissue (as suggested by the up-regulation of KRT4 in the cholesteatoma). The presented data on the differential protein composition in cholesteatoma corroborate previous studies, highlight novel protein functionalities involved in the pathogenesis, and identify new areas for targeted research that hold therapeutic potential for the disease

Minimum fold changes of proteins meeting the group A and B criteria for fold change.

<p>Only comparisons including cholesteatoma are shown. Neck: Neck of cholesteatoma; Tymp: Tympanic membrane; EACS: external auditory canal skin; Mucosa: Middle ear mucosa.</p

Cytokeratins.

<p>All identified cytokeratins (Hair- and hair follicle keratins excluded) were grouped according to Moll <i>et al</i>.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104103#pone.0104103-Moll1" target="_blank">[38]</a>. Letters above the standard deviation bars indicate the tissues compared to which differences were found in the pairwise comparisons between the four keratinizing tissues (results from comparisons including the middle ear mucosa are not shown). a: Group A proteins; b: Group B proteins; b(t): Triplicate values in group B. Gene names shown below the columns. Tymp Membr: Tympanic membrane; Chol: Cholesteatoma.</p

Network of differential level proteins in cholesteatoma with associations to connective tissue.

<p>A. The top scoring, automatically-synthesized network of related proteins in IPA: Connective Tissue Development and Function, Embryonic Development, Organ Developmentive tissue.d proteins: Group B proteins.d EACS.holesteatoma a STRING. All proteins, except for MMP9, showed the same expression direction comparing cholesteatoma with tympanic membrane and EACS, respectively. Protein level differences meeting the group A or B criteria were detected in at least one of the two comparisons. 41 interactions (7.95 expected) were identified between the 23 proteins, network <i>p</i> value = 1.11e-16. B. Some of the top scoring significant associations of the network with: GO Biological Processes, GO Cellular Components, and KEGG Pathways; ordered by <i>p</i> value.</p

Identified proteins.

<p>Number and overlap of unique proteins identified in the three separate mass spectrometry experiments. In each experiment, a sample set of the five tissue types (pooled samples from three patients) were analyzed.</p

Top 20 Up-regulated proteins in cholesteatoma.

<p>Up-regulated proteins in cholesteatoma sorted by 1: The number of tissues compared to which cholesteatoma showed higher levels of the protein; 2: Protein group (fold change criteria A then B); 3: Fold change. C: Cholesteatoma; N: Neck of cholesteatoma; T: Tympanic membrane; S: External auditory canal skin; a: Protein group A; b: Protein group B; b(t): Triplicates in protein group B.</p

Synthesized network of immune response-related up-regulated proteins in cholesteatoma and neck of cholesteatoma.

<p>A. All proteins showed higher protein levels in cholesteatoma and neck of cholesteatoma compared to the external auditory canal skin. The proteins met the fold change criteria in at least one of the two tissue comparisons. B. Examples of significant associations of the network with: GO Biological Processes, GO Cellular Components and KEGG Pathways; ordered by <i>p</i> value. The networks were generated in STRING.</p

Simple overview of workflow.

<p>EACS: external auditory canal skin; Tymp: tympanic membrane; Neck: neck of cholesteatoma; Chol: cholesteatoma; Muc: middle ear mucosa; ME: Middle ear; LC: Liquid Chromatography; MS: Mass Spectrometry.</p

Overview of levels of 11 related proteins involved in inflammation, response to bacteria, and/or protein degradation.

<p>Letters above the standard deviation bars indicate the tissues compared to which differences were found in the pairwise comparisons between the four keratinizing tissues. Left: Leukocyte-associated proteins. Middle: Inhibitor of enzyme activity. Right other protein degrading and/or immune-response related proteins. a: Group A proteins, b: Group B proteins, b(t): Triplicate values in group B. Gene names below the columns. Tymp Membr: Tympanic membrane; Chol: Cholesteatoma.</p

Activation direction of biological functions in cholesteatoma.

<p>Statistical predictions on the direction of activation of biological functions associated with cholesteatoma (see experimental procedures). Biological functions with z - scores of the predictions above the numerical value 1.645 (90% significance level) are shown. In a first step, biological functions were statistically associated with cholesteatoma based on the up- and down-regulated proteins that could be assigned to these functions. Subsequently, predictions on the activation direction were calculated from the composition of up- and down-regulations among these proteins. The listed results were found in comparisons between cholesteatoma sack (C), neck of cholesteatoma (N), tympanic membrane (T), and external auditory canal skin (S). *The proteins had to show the same direction of expression in both tissues and meet the fold change criteria in at least one of the tissues.</p