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  • br Introduction br In solid

    2020-08-07


    1. Introduction
    In solid tumors, cytostatic drugs have been shown to effectively inhibit the growth of cancer cells but have not been efficacious on cancer metastasis [1]. Metastasis is the leading cause of cancer-related death [2,3] and cancer cell migration is an essential stage of metastasis [4,5]. Therefore, the unmet needs of anti-cancer drug discovery include identifying agents that prevent or block metastasis but that are also safe, selective, and effective. Oncogenic kinase inhibitors targeting signal transduction in cell migration lead to resistance due to highly redundant and bypassable signaling pathways [6]. To overcome drug resistance, migrastatics targeting the modulators or effectors of cancer cell migration and invasion have been proposed as a promising ther-apeutic strategy for treatment of solid tumors [6,7]. Target mechanisms for migrastatics include Thonzonium Bromide polymerization and actomyosin-mediated contractility [7]. Remodeling of the cytoskeleton plays a pivotal role in
    cell migration, invasion, and metastasis [8]. Drugs targeting actin fi-laments and microtubules have gained attention as anti-proliferative and anti-migratory agents [9]. Actin regulatory proteins are highly expressed in cancer cells and exhibit positive correlations with inva-siveness and the metastatic potential of cancer cells [10,11]. Com-pounds targeting tropomyosin preferentially disrupt the actin cytoske-leton of tumor cells and display low toxicity [12]. Therefore, a novel class of compounds targeting actin regulatory proteins have garnered attention as potential drug candidates; however, there are currently no FDA-approved drugs.
    The actin-related protein 2/3 (Arp2/3) complex consists of seven subunits (Arp2, Arp3, and ARPC1-5) and participates in migration, proliferation, endocytosis, phagocytosis, and pathogen infection [10]. Within lamellipodia at the leading edges of migrating cells, the Arp2/3 complex forms Y-branched actin networks that can nucleate new fila-ments [13,14]. The Arp2/3 complex interacts with the WA/VCA
    Corresponding authors at: Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahakro, Daejeon 34141, Republic of Korea. E-mail addresses: [email protected] (D.C. Han), [email protected] (B.-M. Kwon).
    1 These authors contributed equally to this work.
    domain of a nucleation promoting factor to enhance actin poly-merization [14]. It was reported that a positive correlation between the expression of Arp2/3 and the malignancy of glioma specimens, in many cases, a deregulation of the Arp2/3 system promotes cancer progression and directly impacts on patient survival [15,16].
    Small molecule inhibitors of the Arp2/3 complex have been re-ported to inhibit actin filament nucleation [17,18]. According to the crystal structure of the Arp2/3 complex with CK666, it binds in a pocket between Arp2 and Arp3 and blocks its movement into active short-pitch conformation [19]. CK869 contacts the hydrophobic pocket of Arp3 and allosterically destabilize the short-pitch Arp2-Arp3 interface [19]. These Arp2/3 inhibitors suppress cell migration by targeting Arp2 and Arp3 but also affect the migration of normal cells [18,20,21]. ARPC2 promotes cancer cell proliferation and invasion by regulating the ex-pression of oncogenes and tumor suppressor genes [22]. In gastric cancer tissues, ARPC2 expression is higher than that of normal gastric tissues and is associated with high tumor stage, lymph node invasion, and poor prognosis of ARPC2-positive patients [22]. Thus, ARPC2 has been suggested to be a potential target for cancer diagnosis and therapy.
    Due to the lethality of Arp2, Arp3, or ARPC3 null cells and animals, functional studies of the Arp2/3 complex have been conducted using RNA interference and a conditional Arp2/3 complex subunit-deficient mice, as well as chemical inhibitors for the Arp2/3 complex. Depletion of one of the Arp2/3 complex subunits by RNA interference results in disrupted lamellipodia formation and reduces directional cell migration in fibroblasts, epithelial cells, and cancer cells [23]. The functional roles of ARPC2 in vivo have been extensively studied in lnk4a/Arf-deficient mice with conditional knockout of ARPC2 [20,24]. Recently, a report showed that knockout of ARPC1B in megakaryocytic cells via CRIPSR-Cas9 knockout system led platelet abnormalities [25].
    In this study, we sought anti-migratory compounds from a library of 719 FDA-approved drugs or clinically tested compounds using pheno-type-based screening. We identified Benp as a potent anti-migratory drug and confirmed the anti-metastatic activity of Benp using an in vivo animal model. ARPC2 was identified as a direct target of Benp, which was further validated by computational docking study and label-free biochemical and biophysical assays. Benp showed anti-migratory ac-tivity at a lower dose (2 μM) compared with Arp2/3 inhibitors, in-cluding CK666 (100 μM) and CK869 (20 μM). In addition, Benp selec-tively suppressed cancer cell migration and invasion but not normal cells. Using ARPC2-depleted cancer cells, we elucidated the functional roles of ARPC2 in lamellipodia formation of cancer cells and metastasis in animal model and further validated ARPC2 as a target protein of Benp. Taken together, our data suggest that Benp is the first identified ARPC2 inhibitor with anti-metastatic activity, therefore, Benp is also the first small molecule inhibitor of Arp2/3 complex subunits. The se-lective inhibition of cancer cell migration and invasion by this ARPC2 inhibitor provides a new paradigm for discovery of anti-cancer and anti-metastatic drugs with reduced general toxicity.