Abstract
Epithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on fluorescence ubiquitination cell cycle indicator, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates noncanonical NF-κB signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin-dependent kinase inhibitors, by blocking entry into quiescence.
Highlights
An important characteristic of epithelial cells is that, unlike fibroblasts, they do not undergo contact inhibition but continue to proliferate at confluence
With the goal of discovering novel factors that regulate epithelial homeostasis, we developed a powerful new assay based on a fluorescence-activated cell sorting (FACS) approach that we integrated with a genome-wide CRISPR/Cas9 sgRNA knockout (KO) screen to select for genes that are essential for cell cycle arrest at high density, but which do not impact proliferation of cells below the threshold for arrest
We chose the murine EpH4 mammary epithelial cell line for this screen, because EpH4 cells are highly polarized, form confluent epithelial sheets, and, most importantly, we confirmed that they efficiently arrest at high density
Summary
An important characteristic of epithelial cells is that, unlike fibroblasts, they do not undergo contact inhibition but continue to proliferate at confluence. This behavior enables the expansion of epithelial tissues during organismal growth without compromising the barrier function created by intercellular junctions. Compression, which increases density, can result in extrusion and apoptosis of cells so as to bring the epithelial layer back to its homeostatic state (Eisenhoffer et al, 2012). This control mechanism that prevents tissue overgrowth is essential for normal development, and it is lost in hyperplasia and in cancer. The mechanisms that underlie homeostatic cell density maintenance remain incompletely understood
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