A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation

Angela Burleigh, Steven McKinney, Jazmine Brimhall, Damian Yap, Peter Eirew, Steven Poon, Viola Ng, Adrian Wan, Leah Prentice, Lois Annab, J Carl Barrett, Carlos Caldas, Connie Eaves, Samuel Aparicio

IntroductionThe extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. Here we applied an unbiased approach to understanding the functional role of signaling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes.MethodsWe developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide siRNA screen evaluating the functional effect of silencing each gene. Our selection endpoint was inhibition of growth. Rigorous post-screen validation processes including RT-QPCR to ensure on-target silencing, deconvolution of pooled siRNAs, and independent confirmation of effects with lentiviral shRNA constructs, identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional (3D) Matrigel growth/differentiation assays and primary human mammary epithelial cell colony assays, we confirm that these growth effects are not limited to the 184hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance.ResultsWe identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and GPCRs, as well as the endothelin receptor, PROCR. The majority (43 out of 47) of genes identified in 2D were also required for 3D growth, with HSD17B2, SNN, and PROCR showing greater than 10-fold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation/polarization in 3D cultures. The 47 genes identified show a significant non-random enrichment for differential expression among 10 molecular subtypes of breast cancer, sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5, and TMEM14C exhibit breast cancer subtype-independent overall survival differences.ConclusionDiverse transmembrane signals are required for mammary epithelial cell growth in 2D and 3D conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.