Ca2+ mediates extracellular vesicle biogenesis through alternate pathways in malignancy

Abstract

Extracellular vesicles (EVs) are small extracellular membrane vesicles that serve as important intercellular signalling intermediaries in both malignant and non-malignant cells. For EVs formed by the plasma membrane, their biogenesis is characterized by an increase in intracellular calcium followed by successive membrane and cytoskeletal changes. EV production is significantly higher in malignant cells relative to non-malignant cells and previous work suggests this is dependent on increased calcium mobilization and activity of calpain. However, differences in calcium-signalling pathways in the context of malignant and non-malignant EV biogenesis remain unexplored. Here, we demonstrate vesiculation is greater in malignant MCF-7 cells relative to non-malignant hCMEC-D3 cells, increases in free cytosolic Ca²⁺ via endoplasmic reticulum (ER) Ca²⁺ store depletion with thapsigargin increases EV biogenesis in both cell types, and vesicular induction is abolished by the intracellular Ca²⁺ chelator BAPTA-AM. Store-operated calcium entry (SOCE) plays an essential role in the maintenance of EV biogenesis after store depletion. These findings contribute to furthering our understanding of extracellular vesicle biogenesis. Furthermore, since EVs are key mediators in the intercellular transfer of deleterious cancer traits such as cancer multidrug resistance (MDR), understanding the molecular mechanisms governing their biogenesis in cancer is the crucial first step in finding novel therapeutic targets that circumvent EV-mediated MDR.

Keywords: Biogenesis; SERCA; calcium; cancer; endoplasmic reticulum; extracellular vesicles; microvesicles; multidrug resistance; sarco/endoplasmic reticulum calcium ATPase; store operated calcium entry.

Figure 1.

AFM topographical images of vesiculation following increased intracellular calcium in malignant and non-malignant cells.

Figure 2.

Increasing intracellular calcium with thapsigargin (TG) induces plasma membrane EV biogenesis in malignant MCF-7 cells.

Figure 3.

Effects of Thapsigargin (TG)-induced microvesicle (MV) biogenesis on cell viability over 24 h.

Figure 4.

Vesiculation in malignant and non-malignant cells following manipulation of the calcium-calpain plasma membrane EV biogenic pathway.

Figure 5.

Proposed model for the role of the endoplasmic reticulum and SOCE in MV biogenesis.