Abstract A50: Nonredundant functions of splicing factors in breast-cancer initiation and metastasis

Abstract

Abstract Alternative splicing is a key control point in gene expression, whose misregulation contributes to cancer malignancy. Although certain splicing factors (SFs) and their targets are altered in human tumors, the functional significance of these alterations remains unclear. We previously demonstrated that the splicing factor SRSF1 is upregulated in human breast tumors and promotes transformation in vivo and in vitro. SRSF1 is a prototypical member of the SR protein family, composed of 12 structurally related proteins. However, little is known about differences and redundancies in their splicing targets and biological functions. Here, we investigated whether additional SFs also promoted breast cancer, using transformation models that mimic the relevant biological context. In parallel, we used RNA sequencing (RNA-seq) to systematically identify their oncogenic splicing targets. By mining a large collection of human tumors from the TCGA project, we defined the molecular portraits of SFs alterations in breast tumors. We identified five SFs amplified and/or overexpressed in at least 10% of breast tumors. We then used SF-overexpressing human mammary epithelial MCF-10A cells grown in organotypic 3-D culture; these cells form polarized growth-arrested acinar structures, similar to the terminal units of mammary ducts. Various breast-cancer oncogenes are known to disrupt acinar growth and/or architecture. Interestingly, only certain SFs were oncogenic in this context, differentially affecting cell proliferation, apoptosis, or acinar organization, suggesting non-redundant functions. We then characterized the splicing targets relevant for SF-mediated transformation. We developed a bioinformatics pipeline to identify and quantify splicing variation in RNA-seq data. We defined the global repertoire of SF-regulated splicing events in 3-D culture and compared the target specificities of various SR proteins. In addition, we identified splicing targets regulated both in 3-D culture as well as in human breast tumors. Strikingly, SFs that promoted similar phenotypic changes shared a significant number of splicing targets, suggesting that they regulate common genes to promote tumor initiation. Furthermore, specific SFs affected targets previously associated with epithelial to mesenchymal transition, and increased cell migration or invasion. Finally, we uncovered that the splicing regulator TRA2β is required for the maintenance of metastatic properties of human breast-cancer cells in 3-D culture and in mouse orthotopic models. Furthermore, TRA2β levels correlate with increased metastatic incidence in breast cancer patients. Thus TRA2β represent a potential target for therapeutics development. In summary, we gained new insights into the biological functions of SR proteins and identified novel oncogenic SF-regulated splicing events involved in tumor initiation and metastasis. Citation Format: Olga Anczuków, Shipra Das, Kuan-Ting Lin, Jie Wu, Martin Akerman, Senthil K. Muthuswamy, Adrian R. Krainer. Nonredundant functions of splicing factors in breast-cancer initiation and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A50.