Abstract 3201: Identification of the type 2 cystatins (SA/SN) as a novel biomarkers for stress-induced senescence in human non-small cell lung cancer lines

Abstract

Abstract Purpose: Stress-induced cellular senescence (SIS) is becoming increasingly well recognized as an important process in premalignant neoplastic lesions and the response to chemotherapeutic drugs and radiation. In addition, senescent cells may release growth factors, cytokines, proteases, and other factors that affect the tumor microenvironment. The standard maker of SIS (staining for β-galactoside [β-gal] at pH 6) cannot be used in fixed tissues. New and more consistent biomarkers of SIS that could be used in tissue sections are thus needed to improve early detection of premalignant lesison, to design and monitor cancer therapeutic approaches, and to gain additional mechanistic understanding. Experimental Procedures: Doxorubicin (Dox) was employed to induce senescence in A549 and L55 human non-small cell lung cancer lines. Gene expression profiles were compared in control vs Dox-treated A549 cells. Validation was carried out by real time quantitative PCR (qPCR). Type 2 Cystatin (SA/SN) protein expression levels were evaluated by immunoblotting and immunohistochemistry (IHC) using an antibody (Santa Cruz) that recognized both proteins. Results: Dox induced senescence of A549 and L55 cells as evidenced by cellular enlargement, cessation of proliferation, and positive staining for ß-gal at pH 6.0. Microarray screening revealed marked upregulation of the genes for Cystatin SA and SN, that was confirmed by qRT-PCR. High protein expression levels of type 2 cystatin (SA/SN) was demonstrated in Dox-treated cells by immunoblotting and IHC. IHC was successful in ethanol-fixed and formalin-fixed paraffin embedded slides. Conclusions: The cystatin superfamily is a well-established family of cysteine protease inhibitors that have been suggested to affect cell differentiation, growth, and migration through protease inhibition and by direct receptor mediated effects. The role of cystatin SA/SN in SIS is under study. However, our results identify two Type 2 Cystatins (SA/SN) that appear to be promising biomarkers for SIS in human non-small cell lung cancer. Cystatin staining could be utilized in fixed tissue sections to screen for oncogene-induced premalignant lesions and for studying the effects of chemotherapeutic drugs and radiation in lung cancer or bronchial biopsy samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3201.