Pattern recognition receptors (PRRs) mediate the innate immune responses and play a crucial role in host defense against pathogen infections. Apextrin C-terminal (ApeC)-containing proteins (ACPs), a newly discovered class of PRRs specific to invertebrates, recognize pathogens through their ApeC domain as intracellular or extracellular effectors. However, the other immunological functions of ACPs remain unclear. In this study, a membrane-localized ACP receptor was identified in the sea cucumber Apostichopus japonicus (denoted as AjACP1). The ApeC domain of AjACP1, which was located outside of its cell membrane, exhibited the capability to recognize and aggregate Vibrio splendidus. AjACP1 was upregulated upon V. splendidus infection, internalizing into the cytoplasm of coelomocytes. AjACP1 overexpression enhanced the phagocytic activity of coelomocytes against V. splendidus, while knockdown of AjACP1 by RNA interfere inhibited coelomocyte endocytosis. Inhibitor experiments indicated that AjACP1 regulated coelomocyte phagocytosis through the actin-dependent endocytic signaling pathway. Further investigation revealed that AjACP1 interacted with the subunit of the actin-related protein 2/3 complex ARPC2, promoting F-actin polymerization and cytoskeletal rearrangement and thereby affecting the coelomocyte phagocytosis of V. splendidus via the actin-dependent endocytic signaling pathway. As a novel membrane PRR, AjACP1 mediates the recognition and phagocytic activity of coelomocytes against V. splendidus through the AjACP1-ARPC2-F-actin polymerization and cytoskeletal rearrangement pathway.

Metastasis, in which cancer cells migrate to other tissues and form new tumors, is a major cause of both cancer death and treatment failure. In a previous study, benproperine (Benp) was identified as a cancer cell migration inhibitor and an inhibitor of actin-related protein 2/3 complex subunit 2 (ARPC2). However, Benp is a racemic mixture, and which stereoisomer is the active isomer remains unclear. In this study, we found that S-Benp is an active isomer and inhibits the migration and invasion of cancer cells much more strongly than R-Benp, with no effect on normal cells. The metastasis inhibitory effect of S-Benp was also verified in an animal model. Validating that inhibitors bind to their targets in cells and tissues has been a very challenging task in drug discovery. The direct interactions between ARPC2 and S-Benp were verified by surface plasmon resonance analysis (SPR), a cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS). In the mutant study with ARPC2F225A cells, S-Benp did not bind to ARPC2F225A according to CETSA and DARTS. Furthermore, we validated that S-Benp colocalized with ARPC2 in cancer cells and directly bound to ARPC2 in tumor tissues using Cy3-conjugated S-Benp according to CETSA. Finally, actin polymerization assays and immunocytochemistry showed that S-Benp suppressed actin remodeling such as lamellipodium formation. Taken together, our data suggest that S-Benp is an active stereoisomer of Benp and a potential metastasis inhibitor via ARPC2 binding.

ARPC2 is a subunit of the Arp2/3 complex, which is essential for lamellipodia, invadopodia and filopodia, and ARPC2 has been identified as a migrastatic target molecule. To identify ARPC2 inhibitors, we generated an ARPC2 knockout DLD-1 human colon cancer cell line using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system and explored gene signature-based strategies, such as a connectivity map (CMap) using the gene expression profiling data of ARPC2 knockout and knockdown cells. From the CMap-based drug discovery strategy, we identified pimozide (a clinically used antipsychotic drug) as a migrastatic drug and ARPC2 inhibitor. Pimozide inhibited the migration and invasion of various cancer cells. Through drug affinity responsive target stability (DARTS) analysis and cellular thermal shift assay (CETSA), it was confirmed that pimozide directly binds to ARPC2. Pimozide increased the lag phase of Arp2/3 complex-dependent actin polymerization and inhibited the vinculin-mediated recruitment of ARPC2 to focal adhesions in cancer cells. To validate the likely binding of pimozide to ARPC2, mutant cells, including ARPC2F225A , ARPC2F247A and ARPC2Y250F cells, were prepared using ARPC2 knockout cells prepared by gene-editing technology. Pimozide strongly inhibited the migration of mutant cells because the mutated ARPC2 likely has a larger binding pocket than the wild-type ARPC2. Therefore, pimozide is a potential ARPC2 inhibitor, and ARPC2 is a new molecular target. Taken together, the results of the present study provide new insights into the molecular mechanism and target that are responsible for the antitumor and antimetastatic activity of pimozide.