ABSTRACT
Protein microarrays are of increasing importance for high-throughput screening of fresh tissues. In our study, protein microarrays were generated by printing antibodies onto membranes to characterize protein profiles expressed by head and neck squamous cell carcinomas (HNSCCs). Cellular proteomes of 30 matched normal squamous epithelial cells and carcinoma specimens were analyzed after tissue microdissection using microarrays composed of 83 different antibodies. As controls, Western blot analysis and tissue microarrays (TMAs) containing 98 HNSCC specimens were used. Of the 83 proteins examined, 14 showed differential expression between HNSCCs and normal epithelium. The protein microarray approach revealed an upregulation of 8 proteins and a downregulation of 6 proteins. Bag-1, Cox-2, Hsp-70, Stat3, pescadillo, MMP-7 (matrilysin), IGF-2, and cyclin D1 were identified to be significantly upregulated, whereas suppressor of cytokine signaling 1, thrombospondin, TGF-beta1, Jun, Fos, and Fra-2 were downregulated. The differential expression of these proteins was confirmed using Western blot and TMA. Upon correlation of differentially regulated proteins with the clinicopathologic data of our patients, MMP-7 (matrilysin) was found to be associated with survival in univariate, but not multivariate, analysis. These data indicate that our protein arrays provide protein information in a systematic, reproducible, and also high-throughput fashion.
Subject(s)
Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell/pathology , Head and Neck Neoplasms/pathology , Protein Array Analysis/methods , Blotting, Western , Carcinoma, Squamous Cell/metabolism , Cyclin D1/analysis , Head and Neck Neoplasms/metabolism , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Matrix Metalloproteinase 7/analysis , Multivariate Analysis , Proteome/analysis , Reproducibility of Results , STAT3 Transcription Factor/analysis , Tissue Array AnalysisABSTRACT
Characterization of the protein profiles expressed by hepatocellular carcinomas (HCCs) may identify the genes involved in hepatocellular carcinogenesis and offers the possibility of elucidating clinical biomarkers. In an effort to discover such proteins and pathways that are deregulated in hepatocellular carcinogenesis, cellular proteomes of matched normal liver cells and carcinoma were analysed by tissue microdissection and protein microarrays. Using protein microarrays made up of 83 different antibodies, it was possible to monitor alterations of the protein levels in HCC and non-neoplastic liver tissue. Further analysis of altered proteins was performed using western blot analysis and tissue microarrays (TMAs) containing 210 HCC specimens and corresponding liver tissue. The protein microarray approach revealed differential expression between HCC and normal liver of 32 of the 83 proteins examined: 21 of these were up-regulated and 11 down-regulated. IGF (insulin growth factor) II, ADAM (a disintegrin and metalloproteases) 9, STAT (signal transducers and activators of transcription) 3, SOCS (suppressors of cytokine signalling) 3, and cyclin D1 were significantly up-regulated and collagen I, SMAD 4, FHIT (fragile histidine triad), and SOCS1 were down-regulated. The differential expression of these proteins was confirmed using western blot analysis and TMAs. Correlation of differentially regulated proteins with clinico-pathological data showed that cyclin D1 and SOCS1 were associated with tumour prognosis in univariate analysis, but not multivariate analysis. These data indicate that the development of an array-based approach for the determination of protein profiles in HCC may facilitate the identification of new proteins associated with carcinogenesis or prognosis.
