Platelet-Derived Extracellular Vesicles Stimulate Migration through Partial Remodelling of the Ca2+ Handling Machinery in MDA-MB-231 Breast Cancer Cells

Abstract

Background: Platelets can support cancer progression via the release of microparticles and microvesicles that enhance the migratory behaviour of recipient cancer cells. We recently showed that platelet-derived extracellular vesicles (PEVs) stimulate migration and invasiveness in highly metastatic MDA-MB-231 cells by stimulating the phosphorylation of p38 MAPK and the myosin light chain 2 (MLC2). Herein, we assessed whether the pro-migratory effect of PEVs involves the remodelling of the Ca2+ handling machinery, which drives MDA-MB-231 cell motility. Methods: PEVs were isolated from human blood platelets, and Fura-2/AM Ca2+ imaging, RT-qPCR, and immunoblotting were exploited to assess their effect on intracellular Ca2+ dynamics and Ca2+-dependent migratory processes in MDA-MB-231 cells. Results: Pretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in Ca2+ release from the endoplasmic reticulum (ER) due to the up-regulation of SERCA2B and InsP3R1/InsP3R2 mRNAs and proteins. The consequent enhancement of ER Ca2+ depletion led to a significant increase in store-operated Ca2+ entry. The larger Ca2+ mobilization from the ER was required to potentiate serum-induced migration by recruiting p38 MAPK and MLC2. Conclusions: PEVs stimulate migration in the highly metastatic MDA-MB-231 breast cancer cell line by inducing a partial remodelling of the Ca2+ handling machinery.

Proteomic and functional profiling of platelet-derived extracellular vesicles released under physiological or tumor-associated conditions

“…The procedure for PEVs isolation (Fig. 1 ) was based on the strategy adopted in our previous investigations [ 6 , 7 , 12 ], and is detailed in the materials and methods section. The two populations of PEVs have been previously analyzed for their ability to stimulate the intrinsic aggressiveness of MDA-MB-231 cells [ 6 , 7 , 12 ] but they have not been characterized for their distinctive molecular signature.…”

“…The ability of PEVs to regulate specific processes in target cells has been widely documented [ 6 – 9 , 12 , 17 – 19 ]. It is reasonable to hypothesize that the capacity of PEVs to control biological responses may depend on the delivery of proteins involved in the regulation of specific pathways.…”

“…The majority of previous studies aimed to understand the functional effects of PEVs on target cells in the context of cancer, focused their attention on medium/large PEVs released in response to stimulation with common platelet agonists, such as thrombin and collagen [ 6 , 8 , 9 , 12 , 17 , 18 ]. We have previously demonstrated that cancer cells themselves can activate platelets and induce a massive release of PEVs [ 7 ].…”

“…A growing body of evidence suggests that PEVs are also important regulators of cancer progression and modulate key steps of the metastatic cascade [ 4 , 5 ]. Specifically, PEVs display the remarkable ability to bind tumor cells and to regulate their survival and intrinsic aggressiveness [ 6 – 12 ]. In previous studies, we have characterized the mechanism of platelet activation induced by breast cancer cells [ 13 ], and we have demonstrated that breast cancer cells can also induce the release of PEVs [ 7 ].…”

The C-Type Lectin Receptor CD93 Regulates Platelet Activation and Surface Expression of the Protease Activated Receptor 4

“…Platelet samples were lysed by addition of half a volume of SDS-sample buffer 3X (37.5 mM Tris, 288 mM glycine, 6% SDS, 1.5% dithiothreitol, 30% glycerol, 0.03% bromophenol blue, 3% β-mercaptoethanol, pH 8.3), and then heated at 95°C for 3 minutes. Equal amount of total platelet proteins from different samples were separated by SDS-PAGE and transferred to PVDF membranes as described 28 . Membranes were probed with the antibodies reported in the text and in the figure legends.…”