Caspase-3-dependent activation of calcium-independent phospholipase A ₂ enhances cell migration in non-apoptotic ovarian cancer cells

Calcium-independent phospholipase A₂ (iPLA₂) plays a pivotal role in phospholipid remodeling and many other biological processes, including inflammation and cancer development. iPLA₂ can be activated by caspase-3 via a proteolytic process in apoptotic cells. In this study we identify novel signaling and functional loops of iPLA₂ activation leading to migration of non-apoptotic human ovarian cancer cells. The extracellular matrix protein, laminin-10/11, but not collagen I, induces integrin- and caspase-3-dependent cleavage and activation of overexpressed and endogenous iPLA₂. The truncated iPLA₂ (amino acids 514-806) generates lysophosphatidic acid and arachidonic acid. Arachidonic acid is important for enhancing cell migration toward laminin-10/11. Lysophosphatidic acid activates Akt that in turn acts in a feedback loop to block the cleavage of poly-(ADP-ribose) polymerase and DNA fragmentation factor as well as prevent apoptosis. By using pharmacological inhibitors, blocking antibodies, and genetic approaches (such as point mutations, dominant negative forms of genes, and siRNAs against specific targets), we show that β₁, but not β₄, integrin is involved in iPLA₂ activation and cell migration to laminin-10/11. The role of caspase-3 in iPLA₂ activation and cell migration are supported by several lines of evidence. 1) Point mutation of Asp⁵¹³ (a cleavage site of caspase-3 in iPLA ₂) to Ala blocks laminin-10/11-induced cleavage and activation of overexpressed iPLA₂, whereas mutation of Asp⁷³³ to Ala has no such effect, 2) treatment of inhibitors or a small interfering RNA against caspase-3 results in decreased cell migration toward laminin-10/11, and 3) selective caspase-3 inhibitor blocks cleavage of endogenous iPLA₂ induced by laminin-10/11. Importantly, small interfering RNA-mediated down-regulation of endogenous iPLA₂ expression in ovarian carcinoma HEY cells results in decreased migration toward laminin, suggesting that our findings are pathophysiologically important.