Abstract 5020: A genome-scale ORF screen reveals an alternative splicing program that regulates mesenchymal and stem-like cell states in breast cancer

Cells residing in mesenchymal state are often associated with stem cell properties. The phenotypic changes from epithelial to mesenchymal cell state, or from non-stem-like to stem-like cell state contribute to tumor heterogeneity and play important roles in tumor initiation, progression and metastasis. To systematically interrogate the modulators of epithelial-to-mesenchymal transition, we performed a genome-scale ORF screen to identify regulators of mesenchymal and stem-like cell states using a barcoded human ORFome expression library in human mammary epithelial cells. In the screen, we used flow cytometric analysis of the CD44 cell surface marker and identified 68 ORFs that can switch cells from CD44 low state to CD44 high state in 7 days. Among these genes, the RNA splicing factors were highly enriched as analyzed by GO terms and Gene set enrichment analysis (GSEA).We employed six different assays for candidate validation: 1) Induction of CD44 cell surface markers; 2) Evaluation of the expression of EMT markers; 3) Test of the ability to form mammospheres; 4) Investigation of the expression during EMT induction; 5) Test of the necessity of these splicing factors for EMT and stem-like states; 6) Examination of the ability to promote tumor formation in vivo. We discovered that QKI and RBFOX1 were both necessary and sufficient to promote EMT and stem-like states. MBNL1, MBNL2 and CELF4 were sufficient to induce some mesenchymal markers. We further investigated the downstream targets of these splicing factors by RNA-sequencing analysis. We found that QKI and RBFOX1 regulated the alternative splicing of genes in 5 functional modules: 1) Cell motility and ECM/cytoskeleton organization; 2) Stem cell fate determination; 3) Oncogenic signaling; 4) Epigenetic targets; 5) cell polarity. Strikingly, using molecular and biochemical assays, we found that QKI and RBFOX1/2 interacted and cooperatively regulated the alternative splicing of a large number of overlapping transcripts, including Filamin B (FLNB). QKI and RBFOX1 induced a shorter isoform via exon skipping. which plays a key functional role in the regulation of EMT. Importantly, the expression of QKI, RBFOX1/2 and the short isoform of FLNB are elevated in basal B type of breast cancer cell lines and in basal-like breast cancer patient samples, the subtype of breast cancer that displays higher degree of mesenchymal and stem-like traits. In conclusion, alternative RNA splicing plays a key role in the regulation of EMT and stem-like cell states. QKI and RBFOX1/2 are both necessary and sufficient to promote EMT and stem-like traits. Alternative splicing of FLNB controlled by QKI and RBFOX1/2 is one of the key downstream targets that regulates EMT. Thus, the molecular targets and mechanism identified in this study may aid in the development of new diagnostic and therapeutic approaches for breast tumors, especially for basal-like breast cancer. Citation Format: Ji Li, Peter Choi, Christine Chaffer, Katherine Labella, Jong Wook Kim, John Doench, Chao Dai, Andrew Giacomelli, Seav Huong Ly, Justin Hwang, Andrew Hong, Nina Ilic, Ole Gjoerup, Matthew Meyerson, Angela Brooks, Robert Weinberg, William Hahn. A genome-scale ORF screen reveals an alternative splicing program that regulates mesenchymal and stem-like cell states in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5020. doi:10.1158/1538-7445.AM2017-5020