Abstract 1: Role of the splicing factor SF2/ASF in mammary epithelial cell transformation (2010)

Abstract Cancer cells often display aberrant profiles of alternative splicing, leading to the production of isoforms that can stimulate cell proliferation and migration or improve resistance to apoptosis. Some of these alterations are caused by mutations at splice sites or in splicing-regulatory elements, but changes can also be caused by alterations in the expression of proteins involved in splice-site selection. Changes in the expression of splicing factors have been reported in different types of cancers, such as colon, ovarian, and breast. Our lab recently demonstrated that the splicing factor SF2/ASF can be oncogenic and is often upregulated in human cancers. Several studies have suggested a role for splicing factors in the development of breast tumors in both human and mice. Thus, our goal is to examine the role of splicing factors in transformation and tumor maintenance of mammary epithelial cells. We have analyzed the ability of the splicing factor SF2/ASF to promote transformation of MCF-10A human mammary epithelial cells, which form organized acinar structures in three-dimensional (3D) culture. Cells slightly overexpressing SF2/ASF form significantly larger acini than the control, but retain a proper acinar organization with a hollow lumen. They show increased proliferation and delayed apoptosis in 3D culture, and can also form colonies in soft agar, a measure of anchorage-independent growth. Interestingly, these features require the first RNA-recognition motif (RRM1) of SF2/ASF, as a deletion mutant lacking this motif has the same phenotype as the control. Furthermore, we find that SF2/ASF can cooperate with the myc oncogene but not with ErbB2 to transform mammary epithelial cells grown in 3-D, and also to promote anchorage-independent growth in soft agar. In contrast, when SF2/ASF is overexpressed together with the HPV16 E7 oncogene, MCF-10A acini are smaller than the controls, suggesting competitive pathways. We are currently investigating if epithelial cells overexpressing SF2/ASF alone or with the above oncogenes can form tumors in a xenograft model, after transplantation into epithelium-free fat pads of three-week-old mice. We are also investigating the signaling pathways involved in SF2/ASF-mediated transformation, focusing on mTOR signaling, as well as on the isoforms of the downstream kinase S6K1, which has been linked to SF2/ASF's oncogenic properties in murine fibroblasts. In addition, we are in the process of identifying the splicing targets of SF2/ASF in MCF-10A in 3D culture. This study will allow us to assess the role of SF2/ASF in breast cancer. By identifying oncogenic splicing factors and their specific targets, we hope in the future to be able to contribute to the development of alternative cancer therapies based on modulating the expression or activity of these factors or their targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1.