Blebs promote cell survival by assembling oncogenic signalling hubs

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death(1). Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue(2,3). In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs(4-11). Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios(12). Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

Automated summary

Most human cells require anchorage for survival, and loss of cell-substrate adhesion can result in anoikis, a form of programmed cell death. Blebbing, the formation of small hemispherical plasma membrane protrusions, triggers the formation of signalling hubs that confer anoikis resistance by recruiting septin proteins as scaffolds for mutant NRAS and effectors. These signalling hubs activate pro-survival pathways, such as ERK and PI3K. Inhibition of blebs or septins in detached cells causes mislocalization of NRAS, reduced MAPK and PI3K activity, and cell death. Therefore, blebs play a crucial role in providing robust anoikis resistance.