Proteomic Analysis of Laser-Captured Paraffin-Embedded Tissues: A Molecular Portrait of Head and Neck Cancer Progression

Abstract

Purpose: Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. Experimental Design: Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis. Results: A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. Conclusions: The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

Enhanced Interferon Signaling Pathway in Oral Cancer Revealed by Quantitative Proteome Analysis of Microdissected Specimens Using 16O/18O Labeling and Integrated Two-dimensional LC-ESI-MALDI Tandem MS

“…In addition, among the 53 up-regulated candidates identified from the three pairs of OSCC samples used here, more than 20 candidates have been confirmed and/or rediscovered to be overexpressed in OSCC tissues using a larger number of samples in the present study or by other groups (Ref. 15 and Table I and references cited therein). This observation indicates that, although potential bias may result from using specimens from a limited number of male patients, the quantitative proteome data set generated here can be useful for searching potential OSCC biomarkers.…”

“…This study explored the relative distribution of identified proteins in tissue samples by counting the peptide numbers of each protein detected. In addition, the authors validated the expression of cytokeratins 4 and 16, desmoplakin, desmoglein 3, and vimentin proteins using immunohistochemistry (15). Although these studies identified and confirmed several OSCC/HNSCC-associated proteins, a more global view of the changes in protein expression in microdissected OSCC cells and adjacent non-tumor epithelial cells can be achieved using a precise quantification approach.…”

“…However, relatively few studies have used these techniques to identify OSCC/HNSCC-associated proteins in tissue specimens from patients with OSCC/HNSCC (15,17,29). For example, Melle et al (29) used ProteinChip technology and 2D gel electrophoresis to examine the up-regulation of annexin V in microdissected HNSCC tissues.…”

“…Another recent study used LC-MS/MS to analyze the proteins extracted from microdissected formalin-fixed, paraffin-embedded tissue sections of normal oral epithelium as well as well, moderately, and poorly differentiated oral cancers. The authors identified 391 and 866 total proteins in the normal oral epithelia and in well differentiated oral cancer tumors, respectively (15). This study explored the relative distribution of identified proteins in tissue samples by counting the peptide numbers of each protein detected.…”

“…Recently state-of-the-art proteomics technologies have revealed alterations in protein abundance, posttranslational modification and turnover, and spatial and temporal distribution within tumor specimens. Using proteomics approaches, aberrantly expressed proteins have been identified in body fluids (12)(13)(14), frozen or paraffin-embedded tissues (15)(16)(17)(18), and cultured cell lines (19 -22). The fold changes in protein expression in samples from healthy and cancerous states as well as the roles of each protein in disease progression must be determined to identify potential candidates for biomarkers and therapeutic targets.…”