Abstract 1965: Inhibiting SIAH2 E3 ligase function disrupts focal adhesion and cell junction, inhibits cell mobility and attachment, and blocks tumor invasion and metastasis in oncogenic K-Ras-driven tumors

Abstract

Abstract Oncogenic EGFR/HER2/K-RAS activation is known to reduce focal adhesion and cell attachment, alter cell junction, and increase cell motility during normal tissue development as well as tumor invasion and cancer metastasis. Deciphering the mechanisms that promote cancer cell dissemination and metastasis in response to oncogenic ERBB/K-RAS “pathway” activation and finding novel targets for inhibiting invasion and metastasis remains an important goal in cancer biology. SIAH2, the human homologue of a highly conserved RING-domain E3 ubiquitin ligase SINA that is the most downstream signal modulator identified in Drosophila RAS signaling pathway, is critically required for proper mammalian ERBB/K-RAS signal transduction. SIAH2 insufficiency was shown to impede oncogenic K-RAS signal transduction, thereby obstructing K-RAS-driven tumorigenesis and metastasis in the preclinical models. However, the exact molecular mechanism of SIAH2 function downstream of the oncogenic ERBB/K-RAS activation remains to be characterized in human cancer cells. In this study, we use immunoprecipitation to isolate novel SIAH2-interacting proteins from multiple highly aggressive human cancer cells with oncogenic K-RAS hyperactivation. Three LIM-domain focal adhesion proteins (Trip6/FHL2/LPXN) were identified by Mass Spectrometry analysis, and their interactions with SIAH2 were verified biochemically by bi-directional co-immunoprecipitation (co-IP). Trip6/FHL2/LPXN has been reported to play important roles in cell focal adhesion formation and cell migration. We demonstrated that SIAH2 biochemically interacts with, ubiquitinates and thereby causes the degradation of these LIM domain proteins, demonstrating that TRIP6, FHL2 and LPXN are bona fide SIAH2 substrates in vivo. Our immunofluorescence (IF) staining results showed that SIAH2-deficiency disrupts Trip6/FHL2/LPXN localization at the focal adhesion site, results in defects in focal adhesion, cell junction, cell attachment and cell motility. Conversely, ectopic expression of TRIP6, FHL2 and LPXN can partially rescue SIAH2-deficient cancer cells from the pronounced defects in cell motility, cell death and tumor growth. The discovery that SIAH2 directly modulates focal adhesion may provide a novel molecular mechanism to explain the well-documented increases in cancer cell dissemination, tumor invasion and metastasis in response to ERBB/K-RAS pathway activation in human cancer cells. We may have uncovered a novel function of SIAH in modulating focal adhesion and cell junction in human cancer cells. By circumventing EGFR/HER2/K-RAS signaling pathway at its most downstream signaling module, we hope to validate and develop novel anti-SIAH-based anti-K-RAS anticancer therapies against oncogenic K-RAS-driven tumors in the future. Citation Format: Ming Bian, Yang Liao, Rebecca L. Schmidt, Monicah M. Njogu, Rie Takahashi, Zandra E. Walton, Amy H. Tang. Inhibiting SIAH2 E3 ligase function disrupts focal adhesion and cell junction, inhibits cell mobility and attachment, and blocks tumor invasion and metastasis in oncogenic K-Ras-driven tumors. [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 1965. doi:10.1158/1538-7445.AM2014-1965