Identification of proteomic differences between squamous cell carcinoma of the lung and bronchial epithelium

Abstract

Proteins that exhibit different expression levels in normal and malignant lung cells are good candidate biomarkers to improve early diagnosis and intervention. We used a quantitative approach and compared the proteome of microdissected cells from normal human bronchial epithelium and squamous cell carcinoma tumors of histopathological grades G2 and G3. DIGE analysis and subsequent MS-based protein identification revealed that 32 non-redundant proteins were differentially regulated between the respective tissue types. These proteins are mainly involved in energy pathways, cell growth or maintenance mechanisms, protein metabolism, and the regulation of DNA and RNA metabolism. The expression of some of these proteins was analyzed by immunohistochemistry using tissue microarrays containing tissue specimen of 55 patients, including normal bronchial epithelium, squamous cell carcinomas, adenocarcinomas, and large cell carcinomas. The results of the immunohistochemical studies correlated with the proteome study data and revealed that particularly HSP47 and a group of cytokeratins (i.e. cytokeratins 6a, 16, and 17) are significantly co-regulated in squamous cell carcinoma. Furthermore cytokeratin 17 showed significantly higher abundance in G2 grade compared with G3 grade squamous cell carcinomas in both the gel-based and the immunohistochemical analysis. Therefore this protein might be used as a marker for stratification between different tumor grades.

Fig. 1.

Microdissection of squamous cell carcinoma tumors and normal bronchial epithelium. Tissue sections were stained with hematoxylin and eosin. Neighboring sections stained only with hematoxylin were used for needle microdissection. Tumor (Tu) tissue (A, before and B, after needle micro-dissection) was specifically enriched and separated from surrounding stroma (C, before and D, after micro-dissection) just like bronchial epithelium was specifically enriched and separated from surrounding connective tissue and smooth muscle cells.

Fig. 2.

Representative 2D DIGE images showing expression profiles from 5000 microdissected cells. Protein samples of bronchial epithelium and G2 and G3 grade squamous cell carcinoma were labeled with Cy5 in a saturation labeling procedure. In each gel an internal Cy3-labeled protein standard was co-separated to improve spot matching and quantification of spot volumes. For each sample 2500 spots were revealed.

Fig. 3.

Classes and functions of identified proteins. The human proteome reference database was screened for classes and function of the proteins identified as differentially regulated in SCC compared with normal bronchial epithelium.

Fig. 4.

Hierarchical clustering of 2D DIGE spot data. Normalized and standardized spot volumes were subjected to a hierarchical complete linkage clustering algorithm. 85 protein spots are included showing a differential regulation (t test p < 0.05, -fold change >3.4 or <−3.4) in at least one of the comparisons of normal bronchial epithelium and G2 or G3 grade tumors. The identities of differential regulated spots are given on the right side of the heat map as well as the gene symbols of proteins identified using mass spectrometry (supplemental Table 3). Red squares in the heat map represent high spot intensities, whereas green squares represent low spot intensities. The normal bronchial epithelium samples are clearly separated from the SCC (G2 and G3) samples. VIM, vimentin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HNRP, heterogeneous nuclear ribonucleoprotein; LMNA, lamin A; ALDOA, aldolase A; TAGLN, transgelin; PC, pyruvate carboxylase; FH, fumarate hydratase.

Fig. 5.

Immunohistochemistry on tissue arrays revealing the expression of proteins found to be overexpressed in squamous carcinoma. A representative picture of bronchial epithelium, squamous cell carcinoma, adenocarcinoma, and large cell carcinoma staining is shown for each antibody.

Fig. 6.

Receiver operation characteristic curve describing the discrimination of G2 and G3 grade SCC tumors using anti-cytokeratin 17 immunostaining. 15 SCC tissues (10 G2 and five G3 grade tumors) were stained, and the staining was scored considering the staining intensity and number of stained tumor cells.