Abstract
The mammalian Diaphanous-related (mDia) formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin), as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2) inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf) in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ≥5–10 μM induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10–20 and 50 μM, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.
Highlights
Dynamic regulation of the cytoskeleton impacts cell motility in a variety of in vitro and in vivo tumor models
We demonstrated in glioblastoma multiforme (GBM), IMM agonists, and their pegylated- (PEG) Dia auto-regulatory domain (DAD) domain peptide counterparts are superior to small molecule inhibitor of Formin Homology-2 (FH2) domain (SMIFH2)-mediated antagonism in blocking directional GBM cell migration and invasion in vitro in multicellular 3D spheroids, and in an ex vivo rat brain slice model of spheroid invasion (Arden et al, 2015)
We found that while IMM and PEG-DAD compounds were well tolerated in zebrafish embryos, SMIFH2 induced significant toxicity to embryos at concentrations below those shown to be functionally antagonistic to filamentous actin (F-actin) dynamics in vitro
Summary
Dynamic regulation of the cytoskeleton impacts cell motility in a variety of in vitro and in vivo tumor models. Formin family proteins regulate both actin and microtubule network dynamics. MDia formins stabilize microtubules (Gundersen and Bulinski, 1988; Tran et al, 2007; Bartolini et al, 2008; Thurston et al, 2012). Forminregulated microtubules organize tracks for vesicle transport, underlie microtentacle formation, and facilitate pro-migratory Golgi re-orientation to aid in polarized cell movement (Nobes and Hall, 1999; Ridley et al, 2003; Yamana et al, 2006; Whipple et al, 2007; Andres-Delgado et al, 2012; Morris et al, 2014; Boggs et al, 2015; Bartolini et al, 2016). Targeting formins with small molecules may be an effective therapeutic intervention with potential to impact both the microtubule and actin dynamics during cell division and migration
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