Abstract A36: Genome-scale ORF screening to identify modulators of the epithelial-to-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is an essential developmental program and is often reactivated during tumor initiation and progression. EMT is a reversible reprogramming of the cells. It not only promotes cell morphology alterations, but also provokes a profound cell state change in multiple regulatory circuits in global cell signaling, transcriptional and post-transcriptional modifications. Among all breast cancer subtypes, basal-like breast cancer displayed a high degree of mesenchymal and stem-like cell properties. To systematically interrogate the modulators of epithelial-to-mesenchymal transition, we performed a genome-scale ORF screen in human mammary epithelial cells. We used CD44 cell surface protein as a marker for cells reside in mesenchymal and stem-like cell state. CD44 low cells were presorted and induced with a barcoded genome-scale ORF library. After seven days of propagation, cells converted to CD44 high state were sorted by flow cytometry, and the barcodes enriched in the CD44 high population compared to the cells in the CD44 low population were evaluated with next-generation sequencing. Interestingly, 68 genes scored 3 standard deviations above the mean, including transcriptional factors, RNA splicing factors, epigenetic regulators, kinases/phosphatases, spermatogenesis regulators and amide metabolic modifiers. Strikingly, gene ontology and gene set enrichment analysis showed that RNA splicing process was the most significantly enriched biological process. 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. After performing these assays, we discovered the splicing factors/RNA binding proteins, QKI and RBFOX1, are both necessary and sufficient to promote EMT and stem-like states. We subsequently investigated the downstream targets of these splicing factors by RNA-sequencing analysis. Further, using biochemical and genetic approaches, we characterized the functional roles of these splicing factors and their downstream targets. In summary, alternative pre-mRNA splicing plays a key role in the regulation of EMT. 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. Genome-scale ORF screening to identify modulators of the epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A36.