Abstract 2696: Deciphering the interactome of the metastasis-suppressor protein RAI2

Abstract

Abstract We have identified low RAI2 expression as a novel metastasis-associated genetic alteration especially related to the presence of disseminated tumor cells in the bone marrow of breast cancer patients and poor outcome (Werner et al, Cancer Discovery, 2015). In hormone-dependent breast and prostate cancer cell lines RAI2 depletion induces down-regulation of hormone receptor expression. Furthermore, RAI2 depletion is associated with chromosomal instability. Nevertheless, to date the exact molecular mechanisms behind these observations remain largely elusive. To better define the RAI2 interactome we performed SILAC-immunoprecipitation quantitative proteomics. HEK293T cells were stably isotope labeled with amino acids in cell culture and transfected with a plasmid for expression of a HA-tagged RAI2 protein. Whole protein extracts were used for co-immunoprecipitation and possible interacting proteins of RAI2 were determined by mass spectrometry based on their SILAC ratios in enriched cell lysates from RAI2 over-expressing and control cells. Subsequently, the DAVID functional annotation tool was used for annotation of co-precipitated proteins. For validation of newly identified potential interactions, we applied combined co-immunoprecipitation, immunoblot and microscopic imaging analysis. The quantitative proteomics approach revealed high confidence interactions of the RAI2 protein with nine gene products (CTBP1, CLTC, HSPA8, NONO, HSPA9, HSPA1A, TNRC6B, UPF1, AP2B1). Additionally, eleven low confidence interactions (HSPA5, PARP1, SFPQ, NUDT21, KRT8, DDX24, FXR1, HSPB1, COL17A1, SLC25A11, FARSA) were identified. The highest SILAC ratio was obtained for CTBP1. Functional annotation showed significant enrichment for the GO terms mRNA processing (GO:0006397, p=0.018), paraspeckles (GO:0042382, p<0.001) and polyA RNA binding (GO:0044822, p<0.001). We validated molecular interactions of RAI2 with CTBP1 and the paraspeckles proteins NONO and SFPQ by combined co-immunoprecipitation and immunoblot analysis. Immunofluorescence staining showed that the RAI2 protein is predominantly co-localized with CTBP1 in nuclear speckles, which is dependent on an intact non-consensus bipartite CTBP-binding domain of the RAI2 protein. In conclusion, the RAI2 protein interacts with different scaffolding proteins and various RNA binding proteins that are involved in mRNA processing, i.e. post-transcriptional regulation. Based on these findings we conclude that the tumor suppressive function of the RAI2 protein is mainly associated with post-transcriptional regulation probably by orchestrating protein scaffolds and subnuclear compartmentalization. Citation Format: Stefan Werner, Kai Bartkowiak, Pascal Steffen, Katharina Besler, Lena Böttcher, Hartmut Schlüter, Klaus Pantel, Harriet Wikman. Deciphering the interactome of the metastasis-suppressor protein RAI2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2696.