Abstract 4439: Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation and oncogenesis
Authors: Njogu Monicah M. , Bian Ming Lei , Tang Amy H. 
Published: May 2015

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. Our aim was to use a proteomic approach to identify differentially expressed SIAH2-interacting proteins isolated from normal epithelial cells, benign tumor and malignant cancer cells, and delineate their roles in K-Ras-driven cellular transformation and oncogenesis. In this study, affinity purification was conducted in triplicates to isolate the SIAH2 protein complexes from normal bronchial epithelial cells (BEAS-2B), RAS-transformed BEAS-2B (BZR) and oncogenic K-RAS activated NSCLC (A549) cells. The extracted proteins were trypsin-digested and peptides subjected to high throughput liquid chromatography tandem mass spectrometry (LC-MS/MS). The data was processed and used in searches against the latest versions of the SwissProt and NCBI protein databases. Peptide and protein identifications were filtered at a False Discovery Rate (FDR) of 1% using the decoy database strategy. Dynamic changes, post-translational modifications and altered expression of individual protein components in the SIAH2-complexes were identified and quantified in a pairwise comparison. Out of the proteins identified as putative SIAH2 interacting partners, twelve top hit SIAH2-interacting proteins were chosen for validation and further analysis. Using Western blots, immunofluorescence staining and co-immunoprecipitation assays, we have verified and confirmed that TRIP6 (Thyroid Receptor Interacting Protein 6) is a newly identified SIAH2 substrate in six human cancer cell lines (pancreatic cancer (MiaPaCa), lung cancer (A549), cervical cancer (HeLa), triple negative breast cancer (MDA-MB-231), breast cancer (MDA-MB-468), melanoma (SK-MEL-28). 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, distinct changes of SIAH2 complexes were detected in normal epithelial, benign tumor and metastatic cancer cells. Our proteomic ID experiments are still ongoing. We have found 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, Amy H. Tang. Proteomic analysis of SIAH2 E3 ligase complex in oncogenic K-Ras-driven cell transformation and oncogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4439. doi:10.1158/1538-7445.AM2014-4439