Mobilization of Intracellular Calcium Stores and ER‐Mitochondrial Coupling in High‐grade Serous Ovarian Cancer (HGSOC) Cells

Abstract

Ca2+ signaling plays crucial roles in cancer metastasis by participating in proliferation, migration, invasion and transformation. However, specific involvements of various intracellular Ca2+ stores in these processes are not clearly defined, and the characteristics of the Ca2+ release mechanisms have not been determined in high‐grade ovarian cancer (HGSOC) cells. In the present study, we sought to determine the functional properties of the IP3‐receptor (IP3R), ryanodine receptor (RyR), and nicotinic acid adenine dinucleotide phosphate (NAADP) gated Ca2+ release pathways in the HGSOC cell line OVCA433. Subcellular Ca2+ signals in OVCA433 cells were monitored using confocal fluorescence microscopy with the cytoplasmic Ca2+ fluorescent dye Fluo‐4 AM. Activation of IP3R by photorelease of cell‐permeant caged‐IP3 using a 405 laser elicited a dramatic increase in cytosolic [Ca2+] indicating robust IP3R‐gated Ca2+ stores. Activation of RyRs using the common agonist caffeine or 4‐chloro‐m‐cresol (4CmC) activated rapid Ca2+ release, indicating the presence of functional RyR‐gated Ca2+ stores in these non‐excitable cells. Moreover, application of the cell permeant NAADP‐AM also caused intracellular Ca2+ release. These results demonstrated the presence of functional IP3R‐, RyR‐, and NAADP‐gated Ca2+ stores in OVCA433 cells. Super‐resolution confocal live cell imaging were preformed in OVCA433 cells loaded with Bodipy‐FI‐X ryanodine, CellLight ER‐RFP, Lysotracker Deep Red, and/or Mitotracker Orange, to further examined the subcellullar organization of the intracellular Ca2+ stores. We found that RyRs are expressed in a subpopulation of ER occupying special locations in the central and peripheral regions, and the ERs in the central region are closely associated with mitochondria. The lysosomal acidic stores are distributed in a random manner. The close association of central ERs and mitochondria suggests that ER‐mitochondrial coupling may occur in OVCA433 cells. This possibility was confirmed by photorelease of caged‐IP3 in a small region inside the cells by an UV laser which elicited significant increase in [Ca2+]mito measured by Rhod‐2. Application of caffeine to OVCA433 cells also elicited a rapid increase in cytosolic [Ca2+], which was followed by sustained increase in [Ca2+]mito. Moreover, caffeine‐induced Ca2+ release activated superoxide bursts in mitochondria of OVCA433 cells loaded with the mitochondrial ROS indicator dye MitoSOX Red. Our results, hence, for the first time, characterized the IP3R‐, RyR‐, and NAADP‐mediated Ca2+ release, and demonstrated IP3R and RyR‐dependent ER‐mitochondrial Ca2+ transfer and coupling in OVCA433 cells. The results provide the fundamental information on intracellular Ca2+ signaling in HGSOC cells and could be important for the study of Ca2+ signaling in ovarian cancer metastasis.Support or Funding InformationSupported by DOD W81XWH‐17‐1‐0146This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.