Large-Scale Proteomics Differentiates Cholesteatoma from Surrounding Tissues and Identifies Novel Proteins Related to the Pathogenesis

Abstract

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.

Discovery of a Biological Mechanism of Active Transport through the Tympanic Membrane to the Middle Ear

“…Finally, competition by synthetic peptides that contain the targeted phage-displayed peptide sequence to compete with phage for binding implicates a receptor-mediated process for binding and translocation. While epithelia form protective barriers throughout the body, trans-epithelial transport of molecules and cells is common 54 , and is often required for normal epithelial function. For example, transport of antibodies and surveillance immune cells is part of the normal biology of all mucosae (e.g., gut and ME).…”

“…Leukocytes are also able to transit epithelial barriers via paracellular transport 56 , separating intercellular junctions in the process, to cross into mucosal lumens such as in the ME cavity during OM 57 . An alternative mechanism is trans-cellular transport, using cell surface receptors, which can occur in either a basolateral or laterobasal direction 54 . While molecules can move into and through a cell by passive diffusion, they are moved far more efficiently by active transport, including carrier- and receptor- mediated systems.…”

Investigation of genomic instability in cholesteatoma of patients

“…A recent study demonstrated that expression profile of cholesteatoma is similar to a metastatic tumor [4]. Britze, et al [5] demonstrated that several differentially expressed proteins related with cancer may cause cancer-like alterations and reorganization of the tissue. Taken together, these observations support that cholesteatoma has genetic instability.…”

“…In another recent study, several differentially expressed proteins related with cancer were shown in cholesteatoma comparing to corresponding skin lesions. It was reported that these proteins might cause cancer-like alterations and reorganization of the tissue that may explain the characteristic phenotype of the disease [5]. There are not any medical therapies for cholesteatoma and the only applied treatment now is surgical resection.…”

Downregulation of Calreticulin and Annexin A2 in Cholesteatoma by 2-DE Analysis

“…Down-regulation of several extracellular matrices and basement membrane proteins (such as COL18A1 and NID2) observed may have a major impact on tissue integrity. The up-regulation of ELANE and pro-inflammatory S100 proteins (eg S100A7A and S100A7) is mainly regulated biological areas in cholesteatoma (16).…”