Abstract 3794: The role of LMTK3 in breast cancer cell growth and invasion.

Abstract

Abstract Abstract Title: The role of LMTK3 in breast cancer cell growth and invasion Background: We have identified Lemur tyrosine kinase-3 (LMTK3) as a novel regulator of estrogen receptor α (ERα) that is associated with disease free and overall survival and predicted response to endocrine therapies in breast cancer. In addition, inhibition of LMTK3 in tamoxifen (tam)-sensitive and tam-resistant xenograft mouse models led to tumor volume reduction. Moreover, a genome microarray analysis revealed various genes modulated by LMTK3 implicated in different canonical pathways. Herein we present evidence that further elucidates the mechanism of LMTK3 involvement in breast cancer growth and invasion. Methods: Stable over-expressed and depleted LMTK3 breast cancer cell lines were generated to study the effects of LMTK3 on proliferation and invasion. Various molecular and cellular biology techniques (including RT-PCR, immunofluorescence, immunoprecipitation and invasion assays) were carried out to elucidate the participation of LMTK3 in various signalling pathways. Stable isotope labelling by amino acids in cell culture (SILAC) was undertaken to identify and analyse novel LMTK3-regulated proteins. Results: Transwell and collagen 1 invasion assays results demonstrated that over-expression of LMTK3 in non-invasive MCF7 cells promote invasion while conversely knockdown of LMTK3 in MDA-MB-231 cells significantly inhibited invasion. Moreover, stable LMTK3 over-expressing cells form larger and looser acini structures compared to parental cells in 3D matrigel overlay assays. Wound healing assays demonstrated an increased motility of cells that over-express LMTK3 compared to LMTK3 knock-out cell lines. Cell adhesion assays revealed preferential binding of LMTK3 over-expressing cell lines to different ECM ligands. RT-qPCR analysis indicated up-regulation of an invasive gene signature (including MMPs, vimentin, and others) associated with LMTK3 over-expression. SILAC analysis identified various novel LMTK3-regulated proteins and phospho-proteins. In addition, modulation of LMTK3 affected the expression of specific members of the integrin family, as well as proteins involved in the focal adhesion complex. Co-immunoprecipitation and immunofluorescence experiments further helped us identify interacting partners of LMTK3 pertaining to the pathways conferring the observed pro-invasive phenotypic results. Finally, the involvement of LMTK3 in promoting tumorigenicity was confirmed in an in vivo xenograft mouse model. Conclusion: We show that LMTK3 is important for mediating breast cancer cell growth and invasion and propose a model of the mechanism of LMTK3 action in these processes. These data position LMTK3 in the map of well-known oncogenic signaling cascades. Citation Format: Yichen Xu, Aleksandra Filipovic, Hua Zhang, Lei Cheng Lit, Ylenia Lombardo, Justin Stebbing, Georgios Giamas. The role of LMTK3 in breast cancer cell growth and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3794. doi:10.1158/1538-7445.AM2013-3794