Summary Beginning of metastasis, cancer cells detach from the primary tumor and they can survive even under loss of anchorage; however, the detachment-elicited mechanisms have remained unknown. Here, we found that Na + ,K + -ATPase α3-isoform (α3NaK) in human cancer cells is dynamically translocated from intracellular vesicles to the plasma membrane when the attached cells are detached and that this mechanism contributes to the survival of the detached (floating) cancer cells. α3NaK was detected in the plasma membrane of floating cancer cells in peritoneal fluids of patients, while it was in the cytoplasm of the cells in primary tumor tissues. On cancer cell detachment, we also found the focal-adhesion-kinase-dependent Ca 2+ response that induces the α3NaK translocation via nicotinic acid adenine dinucleotide phosphate pathway. Activation of AMP-activated protein kinase was associated with the translocated α3NaK in the plasma membrane. Collectively, our study identifies a unique mechanism for survival of detached cancer cells, opening up new opportunities for development of cancer medicines.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+,K+‐ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT‐29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+,K+‐ATPase α1‐isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3H]2‐deoxy‐ d‐glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+,K+‐ATPase α3‐isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain‐decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh‐84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh‐84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+‐releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs‐decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+‐ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol‐3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP‐mediated Ca2+ mobilization followed by the dynamin‐dependent GLUT1 endocytosis in human cancer cells.

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