Abstract A29: Advanced nano-proteomics based on ultrasensitive CE toquantitate protein expression/phosphorylation levels: Discovery of novel proteins involved in chemoresistance/invasion of lung cancer cells.

Abstract

Abstract Our laboratory has shown that resistance to chemotherapeutic agent cisplatin (chemoresistance) in non-small cell lung cancer (NSCLC) cells involves exaggerated phosphorylation of transcription factor CREB and high-level expression of pro-survival/anti-apoptotic proteins c-IAP1, livin, Mcl-1 and survivin, resulting from hyperactivation of a novel serine/threonine kinase, protein kinase G-Iα (PKG-Iα) (Wong et al. 2012). PKG-Iα also promotes cell proliferation/DNA synthesis and migration/invasion in both NSCLC and ovarian cancer cells (Fiscus et al., 2012; Wong et al. 2012). Because of low expression levels, these proteins are difficult to accurately quantify by conventional Western blotting. To solve this problem, we are developing new applications for NanoPro 100 and NanoPro 1000 (ProteinSimple), state-of-the-art instruments based on advanced “nano-proteomics” ultilizing ultrasensitive capillary electrophoresis (CE)-based technologies (Fiscus and Johlfs, 2012; Fiscus et al., 2012). The NanoPro 100/NanoPro 1000 are capable of accurately quantifying protein expression/phosphorylation levels with sensitivities >100-times and >500-times, respectively, that of conventional Western blots, thus allowing, for the first time, accurate measurements of lower-abundance proteins (e.g. PKG-Iα, survivin and other pro-survival proteins). Extremely small samples of <100 cells are needed. Because of exceptional sensitivity and superior separation of proteins of similar molecular weight (e.g. isoforms/splice variants), compared with Western blots, NanoPro 100/1000 are used in our laboratory to discover novel signaling proteins and their isoforms/splice variants involved in chemoresistance and migration/invasion of lung cancer and mesothelioma cells. We found that some lung cancer/mesothelioma cells express two splice variants of PKG-I, i.e. PKG-Iα and PKG-Iβ (difficult to detect by Western blot). This new technology shows a novel “signaling interdependence” between c-Src tyrosine kinase and PKG-Iα in lung cancer and mesothelioma cells, similar to c-Src/PKG-Iα interdependence that promotes cell proliferation and chemoresistance in ovarian cancer cells (Fiscus et al. 2012). This advanced “nano-proteomics” helps in discovery of many more novel proteins and phosphorylations, which can be used for developing new anti-cancer therapies (based on accurate molecular targeting) for treating lung cancer and mesothelioma. Wong JC, Bathina M and RR Fiscus (2012) Cyclic GMP/protein kinase G type-Iα (PKG-Iα signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells. J Cell Biochem. 113: 3587-3598. Fiscus RR, Leung EL, Wong JC and MG Johlfs (2012) Nitric Oxide/Protein Kinase G-IαPromotes c-Src Activation, Proliferation and Chemoresistance in Ovarian Cancer. In: Farghaly, S. (ed). Ovarian Cancer - Basic Science Perspective. Intech Open Access Publisher. pp 315-334. Fiscus RR and MG Johlfs (2012) Protein Kinase G (PKG): Involvement in Promoting Neural Cell Survival, Proliferation, Synaptogenesis and Synaptic Plasticity and the Use of New Ultrasensitive Capillary-Electrophoresis-based Methodologies for Measuring PKG Expression and Molecular Actions. In: Mukai H (ed). Protein Kinase Technologies, in NEUROMETHODS series, Springer. pp 319-348. Citation Format: Ronald R. Fiscus, Mary G. Johlfs, Janica C. Wong, Renee Coffman, Harry Rosenberg. Advanced nano-proteomics based on ultrasensitive CE toquantitate protein expression/phosphorylation levels: Discovery of novel proteins involved in chemoresistance/invasion of lung cancer cells. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A29.