Physical confinement promotes mesenchymal trans-differentiation of invading transformed cells in vivo.

Metastasis is tightly linked with poor cancer prognosis, yet it is not clear how transformed cells become invasive carcinomas. We previously discovered that single KRasV12-transformed cells can invade directly from the epithelium by basal cell extrusion. During this process, cells de-differentiate by mechanically pinching off their epithelial determinants, but how they trans-differentiate into a migratory, mesenchymal phenotype is not known. Here, we demonstrate that basally extruded KRasV12-expressing cells become significantly deformed as they invade the zebrafish body. Decreasing the confinement that cells experience after they invade reduces the percentage of KRasV12 cells that trans-differentiate into mesenchymal cell types, while higher confinement increases this percentage. Additionally, increased confinement promotes accumulation of internal masses over time. Altogether, our results suggest that mechanical forces drive not only de-differentiation of KRasV12-transformed epithelial cells as they invade but also their re-differentiation into mesenchymal phenotypes that contribute to distant metastases.

KRas-transformed epithelia cells invade and partially dedifferentiate by basal cell extrusion.

Metastasis is the main cause of carcinoma-related death, yet we know little about how it initiates due to our inability to visualize stochastic invasion events. Classical models suggest that cells accumulate mutations that first drive formation of a primary mass, and then downregulate epithelia-specific genes to cause invasion and metastasis. Here, using transparent zebrafish epidermis to model simple epithelia, we can directly image invasion. We find that KRas-transformation, implicated in early carcinogenesis steps, directly drives cell invasion by hijacking a process epithelia normally use to promote death-cell extrusion. Cells invading by basal cell extrusion simultaneously pinch off their apical epithelial determinants, endowing new plasticity. Following invasion, cells divide, enter the bloodstream, and differentiate into stromal, neuronal-like, and other cell types. Yet, only invading KRasV12 cells deficient in p53 survive and form internal masses. Together, we demonstrate that KRas-transformation alone causes cell invasion and partial dedifferentiation, independently of mass formation.

The forces and fates of extruding cells.

Cell extrusion drives most epithelial cell death while maintaining a functional epithelial barrier. To extrude, a cell produces a lipid signal that triggers the neighboring cells to reorganize actin and myosin basally to squeeze the extruding cell out apically from the barrier. More studies continue to reveal other signals and mechanisms controlling apical extrusion. New developmental studies are uncovering mechanisms controlling basal extrusion, or ingression, which occurs when apical extrusion is defective or during de-differentiation in development. Here, we review recent advances in epithelial extrusion, focusing particularly on forces exerted upon extruding cells and their various later fates ranging from cell death, normal development, and cancer.

EWS/FLI utilizes NKX2-2 to repress mesenchymal features of Ewing sarcoma.

In Ewing sarcoma, NKX2-2 is a critical activated target of the oncogenic transcription factor EWS/FLI that is required for transformation. However, its biological function in this malignancy is unknown. Here we provide evidence that NKX2-2 mediates the EWS/FLI-controlled block of mesenchymal features. Transcriptome-wide RNA sequencing revealed that NKX2-2 represses cell adhesion and extracellular matrix organization genes. NKX2-2-depleted cells form more focal adhesions and organized actin stress fibers, and spread over a wider area-hallmarks of mesenchymally derived cells. Furthermore, NKX2-2 represses the actin-stabilizing protein zyxin, suggesting that these morphological changes are attributable to zyxin de-repression. In addition, NKX2-2-knockdown cells display marked increases in migration and substrate adhesion. However, only part of the EWS/FLI phenotype is NKX2-2-dependent; consequently, NKX2-2 is insufficient to rescue EWS/FLI repression of mesenchymalization. Strikingly, we found that EWS/FLI-and NKX22-repressed genes are activated by ZEB2, which was previously shown to block Ewing sarcoma epithelialization. Together, these data support an emerging theme wherein Ewing sarcoma cells highly express transcription factors that maintain an undifferentiated state. Importantly, co-opting epithelial and mesenchymal traits by Ewing sarcoma cells may explain how the primary tumor grows rapidly while also "passively" metastasizing, without the need for transitions toward differentiated states, as in carcinomas.