Differential Protein Expression in Congenital and Acquired Cholesteatomas

Abstract

Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5-3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5-3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5-3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins may differ from those of acquired cholesteatomas, which originate from retraction pocket epithelia.

Fig 1. A representative case of congenital cholesteatomas examined in this study (Patient 2 in Table 1).

A. Otoscopic tympanic membrane findings. B. Axial computed tomography image of the temporal bone. C. Surgical findings. D. Congenital cholesteatoma specimen obtained in surgery (scale bar: 5mm).

Fig 2. Expression pattern of protein spots identified with 2-dimensional electrophoresis of congenital cholesteatoma.

Expression patterns in the 4 patients examined were similar (A-D).

Fig 3. Protein expression patterns in congenital cholesteatoma (left), acquired cholesteatomas (middle), and the retroauricular canal skin (right).

Fig 4. Venn diagram showing the number of identified protein spots in each tissue.

One hundred twenty seven spots were only expressed in congenital cholesteatoma.

Fig 5. Ten major spots only expressed in congenital cholesteatoma, as determined by image analysis.

Fig 6. Expression of forkhead transcriptional factor homolog (FKH 5–3, A) and titin (B) in congenital cholesteatoma, as detected with immunohistochemistry.

Both proteins were well-expressed in cholesteatoma matrix. Negative controls (C, D) are also shown for comparison.