Abstract 2008: Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation in human cancer

Abstract

Abstract Affinity purification-mass spectrometry (AP-MS) is a highly effective method of identifying and quantifying the individual constituents of molecular machines that are dynamically regulated by diverse cellular stimuli during tumor initiation, progression and metastasis. Seven In absentia homolog 2 (SIAH2) is an evolutionarily conserved E3 ubiquitin ligase and a key component of a molecular machine called “SIAH2-dependent proteolytic machinery” that serves as the most downstream signaling module critical for proper oncogenic ERBB/RAS signal transduction. As a newly identified promising anti-K-RAS and anticancer drug target, it is important to understand the dynamic regulation of SIAH function, substrate recognition and complex formation during tumor initiation, progression and metastasis. Our aim was to delineate the molecular regulation, substrate selection and target recognition of the SIAH proteolytic machinery in K-Ras-driven cellular transformation and oncogenesis; and to explore the SIAH-dependent K-RAS oncogene addiction and its future therapeutic application. In this study, affinity purification was conducted in triplicates to isolate the SIAH2 protein complexes from two panels of human epithelial cells: For the lung epithelial cells: we used the normal bronchial epithelial cells (BEAS-2B), RAS-transformed BEAS-2B (BZR) and non-small cell lung cancer (NSCLC) A549 cells with oncogenic K-RAS activation. For the mammary epithelial cells: we used normal mammary epithelial cells (HMEC and MCF-10A), benign breast tumor cells (MCF-7), MDA-MB-231 that carries both oncogenic K-RAS and B-RAF activation and MDA-MB-468. The extracted proteins were trypsin-digested and peptides subjected to high throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. SIAH2 was observed to propagate downstream RAS signals by affecting proteins involved in cellular junctions, focal adhesion, metabolism, mitochondrial function and kinase activities. Using Western blots, immunofluorescence staining and co-immunoprecipitation assays, we have verified and confirmed that focal adhesion proteins, TRIP6 (Thyroid Receptor Interacting Protein 6) and FHL2 (Four and Half Lim domain protein) are SIAH2 substrates in human cancer cell lines lung cancer (A549) and triple negative breast cancer (MDA-MB-231) and breast cancer (MDA-MB-468). We found that TRIP6 primarily localizes to the focal adhesions, cell junctions and the nucleus of the cells. Overexpression of SIAH2 induces the degradation of exogenous and endogenous TRIP6 while Inhibition of SIAH2 enzymatic function results in a stabilization of TRIP6 and mis-localization of TRIP6 in human cancer cells. In conclusion, we report that SIAH2 regulates focal adhesion, cell junction and cellular attachment by down regulating TRIP6 expression levels and may induce a more motile and aggressive phenotype in human cancer cells. Citation Format: Monicah M. Njogu, Ming Lei Bian, Jamie Eisner, Amy H. Tang. Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation in human cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2008. doi:10.1158/1538-7445.AM2015-2008