Discovery of new membrane-associated proteins overexpressed in small-cell lung cancer

Abstract

Introduction: Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with no early detection strategy or targeted therapy currently available. We hypothesized that difference gel electrophoresis (DIGE) may identify membrane-associated proteins (MAPs) specific to SCLC, advance our understanding of SCLC biology, and discover new biomarkers of SCLC.

Methods: MAP lysates were prepared from three SCLCs, three non-small-cell lung cancers, and three immortalized normal bronchial epithelial cell lines and coanalyzed by DIGE. Subsequent protein identification was performed by mass spectrometry. Proteins were submitted to Ingenuity Pathway Analysis. Candidate biomarkers were validated by Western blotting (WB) and immunohistochemistry (IHC).

Results: Principal component analysis on the global DIGE data set demonstrated that the four replicates derived from each of the nine cell lines clustered closely, as did samples within the same histological group. One hundred thirty-seven proteins were differentially expressed in SCLC compared with non-small-cell lung cancer and immortalized normal bronchial epithelial cells. These proteins were overrepresented in cellular/tissue morphology networks. Dihydropyrimidinase-related protein 2, guanine nucleotide-binding protein alpha-q, laminin receptor 1, pontin, and stathmin 1 were selected as candidate biomarkers among MAPs overexpressed in SCLC. Overexpression of all candidates but RSSA in SCLC was verified by WB and/or IHC on tissue microarrays. These proteins were significantly associated with SCLC histology and survival in univariables analyses.

Conclusion: DIGE analysis of a membrane-associated subproteome discovered overexpression of dihydropyrimidinase-related protein 2, guanine nucleotide-binding protein alpha-q, RUVB1, and stathmin 1 in SCLC. Results were verified by WB and/or IHC in primary tumors, suggesting that investigating their functional relevance in SCLC progression is warranted. Association with survival requires further validation in larger clinical data sets.

FIGURE 1

Isolation of membrane-associated proteins. Membrane-associated (mbr) and cytosolic (cyto) extracts were resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis, and blots were incubated with anti-E-cadherin, NCAM, EGFR, AKT, or actin antibodies. E-cadherin, NCAM, EGFR, and AKT levels were normalized to actin. In each quadruplicate analysis, membrane proteins E-cadherin, NCAM, and EGFR were mainly expressed in membrane-associated extracts, whereas cytosolic protein AKT was mainly expressed in cytosolic extracts. Blots are representative of three independent experiments. AKT, v-Akt murine thymoma viral oncogene homolog 1; EGFR, epidermal growth factor receptor; NCAM, neural cell adhesion molecule.

FIGURE 2

DIGE results. A, PCA of the DIGE global data set. The axes correspond to the first three principal components. The apparent groups yielded by PCA are enclosed in circles: blue circle for immortalized normal bronchial epithelial cell lines (normal), green circle for NSCLC cell lines, and red circle for SCLC cell lines. The four replicates from each of the nine cell lines clustered very closely, as did samples within each histological subgroup. B, C, Quantitative analysis of protein expression in the nine cell lines analyzed by DIGE. Representative graphs of protein features significantly overexpressed (B) and underexpressed (C) in SCLC compared with immortalized normal bronchial epithelial and NSCLC cell lines (p < 10⁻³). The Y axis represents the standardized log protein abundance generated by the DeCyder analysis. DIGE, difference gel electrophoresis; NSCLC, non small-cell lung cancer; SCLC, small-cell lung cancer; PC, principle component; PCA, principal component analysis.

FIGURE 3

Verification of the candidate biomarkers’ expression by Western blotting. Membrane-associated protein lysates were resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis, and blots were incubated with an antibody against DPYL2, phospho-DPYL2 (Thr⁵¹⁴/Ser⁵¹⁸), GNAQ, RSSA, RUVB1, STMN1, or phospho-STMN1 (Ser¹⁶). Expression levels were normalized to actin. Overexpression of DPYL2, GNAQ, and STMN1 was observed in SCLC compared with immortalized normal bronchial epithelial (normal) and NSCLC cell lines, while RSSA and RUVB1 expression was similar in all the cell lines. Increased phosphorylation of DPYL2 and STMN1 was also observed in SCLC compared with normal and NSCLC cell lines, suggesting an activation of DPYL2 and STMN1 in SCLC. DPYL2, dihydropyrimidinase-related protein 2; GNAQ, guanine nucleotide binding protein alpha-q; NSCLC, non small-cell lung cancer; RSSA, laminin receptor 1; RUVB1, pontin; SCLC, small-cell lung cancer; STMN1, stathmin 1.

FIGURE 4

Verification of the candidate biomarkers’ expression by immunohistochemistry: representative cases of 130 studied NSCLC and 36 studied SCLC. Tissue sections were incubated with an antibody against DPYL2, GNAQ, RSSA, RUVB1, or STMN1. A J, Representative images of NSCLC and SCLC tissues with a staining score close to the average staining score for each biomarker in each histological subgroup. A, NSCLC tumor with a DPYL2 staining score of 200. B, SCLC tumor with a DPYL2 staining score of 300. C, NSCLC tumor with a GNAQ staining score of 100. D: SCLC tumor with a GNAQ staining score of 300. E, NSCLC tumor with an RSSA staining score of 100. F, SCLC tumor with an RSSA staining score of 100. G, NSCLC tumor with an RUVB1 staining score of 100. H, SCLC tumor with an RUVB1 staining score of 200. I, NSCLC tumor with an STMN1 staining score of 0. J, SCLC tumor with an STMN1 staining score of 300 (magnification: ×10 in the large windows and ×40 in the small windows; scale bars: 100 μm in the large windows and 20 μm in the small windows). DPYL2, dihydropyrimidinase-related protein 2; GNAQ, guanine nucleotide binding protein alpha-q; NSCLC, non small-cell lung cancer; RSSA, laminin receptor 1; RUVB1, pontin; SCLC, small-cell lung cancer; STMN1, stathmin 1.