Piezo1 activation attenuates thrombin-induced blebbing in breast cancer cells

Abstract

Cancer cells exploit a variety of migration modes to leave primary tumors and establish metastases, including amoeboid cell migration facilitated by bleb formation. Here we demonstrate that thrombin induces dynamic blebbing in the MDA-MB-231 breast cancer cell line, and confirm that PAR2 activation is sufficient to induce this effect. Cell confinement has been implicated as a driving force in bleb-based migration. Unexpectedly, we find that gentle contact compression, exerted using a "Cell Press" to mechanically stimulate cells, attenuated thrombin-induced blebbing with an associated increase in cytosolic calcium. Thrombin-induced blebbing was similarly attenuated using Yoda1, an agonist of the mechanosensitive calcium channel Piezo1, and its capacity to attenuate blebbing was impaired in Piezo1 depleted cells. Additionally, Piezo1 activation suppressed and reversed the thrombininduced phosphorylation of ERM proteins, which are implicated in the blebbing process, and this activity was in part mediated through activation of the PP1A/PP2A family of serine/threonine phosphatases. Our results provide mechanistic insights into Piezo1 activation as a suppressor of dynamic blebbing, specifically that which is induced by thrombin.2 increase hydrostatic pressure within the cell 21 ; in parallel, actomyosin contractility is essential for the retraction of expanded PM blebs 5 .Piezo1 is a mechanosensitive cation channel 22,23 implicated in diverse areas of cell biology including shear stress sensing in endothelial cells 24 ; regulation of red blood cell volume 25 ; cell division in epithelial cells 26 ; and as a confinement sensor that suppresses PKA activity to optimize cell motility 27 . Piezo1 is found in the plasma membrane as a homotrimer and in closed conformation induces a bowlshaped indentation in the PM. In response to increased membrane tension Piezo1 transitions to a more planar arrangement and gates Ca 2+ influx 28,29 . Furthermore, loss of cortical actin, as seen during the initial expansion of PM blebs, reduces the activation threshold of Piezo1 30 .Here, we identify thrombin as a bleb induction stimulus, and Piezo1 activation, through contact compression or stimulation with the Piezo1 agonist Yoda1, as an effective mechanism to suppress dynamic blebbing in breast cancer cells. Thrombin induced blebbing was associated with increased ERM phosphorylation that was suppressed or reversed through Piezo1 activation, which in turn was dependent on the downstream activity of PP1/PP2A serine/threonine phosphatases. We propose that Ca 2+ influx via Piezo1 activates PP1/PP2A phosphatases to dephosphorylate ERMs, which in turn reduces the actomyosin contraction forces applied to the PM; thereby reducing the likelihood of dynamic blebbing. Materials and Methods Reagents and chemicalsSiR-Actin (Spirochrome, Tebu-bio, Roskilde, Denmark) was diluted to 1 mM in dimethyl sulfoxide (DMSO; ThermoFisher Scientific, Uppsala, Sweden) and stored at -20 o C. Fluo-4 and Fura Red calcium indicators (ThermoFisher Scientific) were resuspe...

Channeling the Force: Piezo1 Mechanotransduction in Cancer Metastasis

“…Piezo1 downregulation can accelerate cell migration in lung cancer by promoting ameboid-type migration [ 61 , 62 ]. In MDA-MB-231 cells, Piezo1 inhibited blebbing, providing more evidence that Piezo1 prevents ameboid migration [ 64 ]. In studies of non-small cell lung cancer (NSCLC), Piezo1 knockdown enhanced migration and tumor growth [ 63 ].…”

Reduction of breast cancer extravasation via vibration activated osteocyte regulation

“…For example, Piezo1 activation inhibited blebbing of MDA-MB-231 breast cancer, reducing its ameboid migration. 48 Conversely, blockage of Piezo1 using GsMTx-4 reduced migration velocity and motility of MCF-7 breast cancer cells, suggesting that Piezo1 promoted cancer migration and invasion. 49 Due to the unavoidable diffusion in devices in our study, Yoda1 may directly affect cancer and endothelial cell activities; hence follow-up studies are needed.…”

Melanoma cells adopt features of both mesenchymal and amoeboid migration within confining channels

“…Additionally, it has been shown that the tumor microvasculature is under a "pathological state of coagulation." In fact, the introduction of thrombin, which activates PAR-1 signaling, was recently shown to promote blebbing in breast cancer cells (O'Callaghan et al 2020). Thus, in terms of how they may regulate the motile behavior of melanoma cells, several aspects of 3D confinement within microchannels remain to be addressed.…”

“…Although here we have simulated channel-like environments in general, it remains possible that the introduction of channel coatings, such as VCAM-1 (which decorates micro-vessel walls), may also regulate the motile behavior of melanoma cells. Additionally, it has been shown that the tumor micro-vasculature is under a “pathological state of coagulation.” In fact, the introduction of thrombin, which activates PAR-1 signaling, was recently shown to promote blebbing in breast cancer cells 20 . Thus, in terms of how they may regulate the motile behavior of melanoma cells, several aspects of 3D confinement within microchannels remain to be addressed.…”