RAS-induced transformation of mammary epithelial cells relies on ZEB1-dependent cellular reprogramming through a paracrine process

The distinct frequency of activation of the RAS/MAPK signaling pathway in human cancers suggests a context-dependent cellular state of vulnerability to RAS transformation. While uncommon in breast cancers, oncogenic activation of this pathway is frequent in claudin-low (CL) tumors, a subtype of breast malignancies enriched in features of epithelial-mesenchymal transition (EMT), suggesting an interplay between RAS activation and EMT. Using inducible models of human mammary epithelial cells, we show that RAS-mediated transformation relies upon cellular reprogramming governed by the EMT-inducing transcription factor ZEB1. The path to ZEB1 induction involves a paracrine process: cells entering a senescent state following RAS induction release proinflammatory cytokines, notably IL-6 and IL1 which promote ZEB1 expression and activity in neighboring cells, thereby fostering their malignant transformation. Collectively, our findings unveil a previously unprecedented role for senescence in bridging RAS activation and EMT over the course of malignant transformation of human mammary epithelial cells. Overall design: We performed single-cell RNA sequencing analysis in a RAS inducible cellular model, HME-RASER, generated by introducing a 4-hydroxytamoxifen (4-OHT)-inducible form of HRASG12V into human mammary epithelial (HME) cells. We analyzed induced HME-RAS ER cells at different time points (D0, D3, D7, D14 and D20). Expression profiles were obtained for 12,357 genes in 445 cells (117 cells at D0, 83 cells at D3, 74 cells at D7, 82 cells at D14 and 89 cells at D20, Samples with less than 50000 reads were excluded).