The lysophosphatidic acid-regulated signal transduction network in ovarian cancer cells and its role in actomyosin dynamics, cell migration and entosis

Abstract

Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived and OVCAR8 cells, RNA sequencing, measurements of intracellular Ca 2+ and cAMP as well as cell imaging. The function of LPARs and downstream signaling components in migration and entosis were analyzed by selective pharmacological inhibitors and RNA interference. Results: Phosphoproteomic analyses identified > 1100 LPA-regulated sites in > 800 proteins and revealed interconnected LPAR1, ROCK/RAC, PKC/D and ERK pathways to play a prominent role within a comprehensive signaling network. These pathways regulate essential processes, including transcriptional responses, actomyosin dynamics, cell migration and entosis. A critical component of this signaling network is MYPT1, a stimulatory subunit of protein phosphatase 1 (PP1), which in turn is a negative regulator of myosin light chain 2 (MLC2). LPA induces phosphorylation of MYPT1 through ROCK (T853) and PKC/ERK (S507), which is majorly driven by LPAR1. Inhibition of MYPT1, PKC or ERK impedes both LPA-induced cell migration and entosis, while interference with ROCK activity and MLC2 phosphorylation selectively blocks entosis, suggesting that MYPT1 figures in both ROCK/MLC2-dependent and -independent pathways. We finally show a novel pathway governed by LPAR2 and the RAC-GEF DOCK7 to be indispensable for the induction of entosis. Conclusion: We have identified a comprehensive LPA-induced signal transduction network controlling LPA-triggered cytoskeletal changes, cell migration and entosis in HGSC cells. Due to its pivotal role in this network, MYPT1 may represent a promising target for interfering with specific functions of PP1 essential for HGSC progression.

Phosphoproteomics Reveals Selective Regulation of Signaling Pathways by Lysophosphatidic Acid Species in Macrophages

“…We were, however, unable to detect significant LPArelated effects in a macropinocytosis assay, which monitored the uptake of dextran by MDMs (see Figure S3). Nonetheless, LPA may influence other forms of phagocytosis that operate through mechanisms distinct from macropinocytosis [17][18][19]52,53].…”

“…The impact of LPA on cancer cell signal transduction, as well as its role in enhancing tumor cell migration and invasion are relatively well-established [4,9,[16][17][18]. This is in marked contrast to its effect on macrophages, which, despite their crucial role in the OC TME, remains surprisingly understudied in a recent review [19].…”

Entosis: the core mechanism and crosstalk with other cell death programs

“…Additional cytoskeletal or cortical regulators of entosis that affect cell tension and/or RhoA–ROCK pathway activity include interactions with microtubules 18 , 22 , cell polarity proteins 23 , calcium signaling and interaction with Septins and myosin light chain kinase (MLCK) 24 , myosin light chain phosphatase (MYPT1) 25 , membrane composition and cholesterol 26 , and small GTPase proteins, including cell division cycle 42 (Cdc42) 27 , 28 and, intriguingly, ras-related C3 botulinum toxin substrate 1 (Rac1) 15 , 25 . Rac1 is a key driver of phagocytosis, during which it regulates the remodeling of the actin cytoskeleton into dendritic networks within engulfing cells, such as macrophages.…”

The Emerging Role of LPA as an Oncometabolite

“…In high-grade serous ovarian cancer, increased LPA levels activate LPAR1 to promote Rho-associated protein kinase (ROCK)-and PKC/ERK-induced myosin phosphatase target subunit 1 (MYPT1) phosphorylation, leading to cytoskeletal changes followed by enhanced migration and entosis [77]. The treatment of ovarian cancer cells with LPA also promotes O-GlcNAcylation of ezrin-radixin-moesin (ERM) cytoskeletal proteins [78], and although the exact mechanism is not entirely understood, LPA-induced activation of ERM suggests the role of LPA in the adhesion and migration of ovarian cancer cells.…”