Inhibition of Cell Migration by Pitenins: the Role of ARF6
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Oncogene (2012) 31, 4317–4332 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Inhibition of cell migration by PITENINs: the role of ARF6 B Miao1, I Skidan2, J Yang3, Z You1,XFu4, M Famulok5, B Schaffhausen1, V Torchilin2, J Yuan6 and A Degterev1 1Department of Biochemistry, Tufts University School of Medicine, Boston, MA, USA; 2Department of Pharmaceutical Sciences, Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, USA; 3Neuroscience Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA; 4Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA; 5LIMES Institute, Program Unit Chemical Biology and Medicinal Chemistry, University of Bonn, Bonn, Germany and 6Department of Cell Biology, Harvard Medical School, Boston, MA, USA We have reported previously the development of small- Introduction molecule phosphatidylinositol-3,4,5-trisphosphate (PIP3) antagonists (PITs) that block pleckstrin homology (PH) Phosphatidylinositol-3,4,5-trisphosphate (PIP3), a lipid domain interaction, including activation of Akt, and show product of phosphatidylinositol-3-kinase (PI3K), con- anti-tumor potential. Here we show that the same trols a complex cellular signaling network regulating cell molecules inhibit growth factor-induced actin remodeling, survival and motility (Park et al., 2008). This makes lamellipodia formation and, ultimately, cell migration and PIP3 one of the most important second messengers invasion, consistent with an important role of PIP3 downstream from growth factor and oncogene signals. in these processes. In vivo, a PIT-1 analog displays PIP3 exerts its effect through binding to the plekstrin significant inhibition on tumor angiogenesis and metas- homology (PH) domains of multiple downstream tasis. ADP ribosylation factor 6 (ARF6) was recently effector proteins (Park et al., 2008). Dysregulation of identified as an important mediator of cytoskeleton and PI3K signaling, which is very frequently observed in cell motility, which is regulated by PIP3-dependent human tumors (Cantley, 2002; Vivanco and Sawyers, membrane translocation of the guanine nucleotide ex- 2002), has made this pathway a very important target change factors (GEFs), such as ADP-ribosylation factor for drug discovery. In many cases, regulation of PIP3 nucleotide binding site opener (ARNO) and general targets, such as PDK1 and Akt, and their downstream receptor for 3-phosphoinositides (GRP1). We demon- signaling pathway has been a primary focus in drug strate that PITs inhibit PIP3/ARNO or GRP1 PH discovery, because of the role of these pathways in the domain binding and membrane localization, resulting in regulation of cancer cell survival and metabolism. the inhibition of ARF6 activation. Importantly, we show Regulation of cell motility is another very important that expression of the constitutively active mutant of function of PIP3. This effect is exerted through multiple ARF6 attenuates inhibition of lamellipodia formation and mechanisms, including regulation of Akt and PDK1, as cell migration by PITs, confirming that inhibition of well as a large number of PH domain-containing ARF6 contributes to inhibition of these processes by PITs. proteins, such as ADP-ribosylation factor nucleotide Overall, our studies demonstrate the feasibility of binding site opener (ARNO) and general receptor for developing specific small-molecule targeting PIP3 binding 3-phosphoinositides (GRP1), controlling activation of by PH domains as potential anticancer agents that can Rho and ADP ribosylation factor (ARF) families of simultaneously interfere with cancer development at GTPases. Thus, dysregulation of PI3K signaling plays a multiple points. profound, yet complex, role in the control of the cancer Oncogene (2012) 31, 4317–4332; doi:10.1038/onc.2011.593; cell invasiveness (Cantley, 2002; Vivanco and Sawyers, published online 19 December 2011 2002). We have recently described two new classes of antagonists of PIP3/PH domain binding, termed PITE- Keywords: ARF6 guanine nucleotide exchange factor; NINs (PITs) (Miao et al., 2010). In this work, we have ARNO/GRP1 membrane translocation; lamellipodia investigated the effects of these molecules on the suppression; anti-angiogenesis; anti-metastasis regulation of actin cytoskeleton and cell motility. In particular, our initial results suggested that PITs target PH domains of GRP1 and ARNO, in addition to those of Akt and PDK1. GRP1 and ARNO are guanine exchange factors (GEFs) of ARF GTPases (Frank et al., 1998a, b; Langille et al., 1999; Caumont et al., 2000; Santy and Casanova, 2001). Therefore, we have Correspondence: Dr A Degterev, Department of Biochemistry, Tufts explored the regulation of this family by PITs. ARF is University School of Medicine, Boston, MA 02111, USA. E-mail: [email protected] a member of the Ras superfamily of small GTPases. Received 3 June 2011; revised 21 September 2011; accepted 25 October Among the six known ARF isoforms, ARF6 has 2011; published online 19 December 2011 received significant attention because of its unique role Inhibition of cell migration by PITENINs B Miao et al 4318 in the regulation of actin remodeling at the plasma which in turn contributes to the inhibition of cancer membrane, formation of membrane ruffles and, ulti- cell migration. Several ARF GEF inhibitors have been mately, its contribution to the regulation of cell motility described previously. The activity of all known inhibi- and endosome recycling (Donaldson, 2003; Balana tors is based on the direct or indirect inhibition of et al., 2005). ARF6 has also been shown to play protein–protein interaction by Sec7 domains of GEFs important roles in tumor cell invasion in vitro and tumor (Renault et al., 2003; Hafner et al., 2006; Viaud et al., metastasis in vivo (Tague et al., 2004; Balana et al., 2005; 2007). Overall, our work presents a new strategy based Muralidharan-Chari et al., 2009). Screening of various on the inhibition of PH domain/phosphoinositide breast tumor cell lines has revealed a direct correlation binding and suppression of the plasma membrane between ARF6 protein expression and tumor invasive- translocation of GEFs. Conceptually, protein–lipid ness (Hashimoto et al., 2004). In contrast, downregula- interactions may be more readily targetable by chemical tion of ARF6 by small interfering RNA (siRNA) or inhibitors compared with that of protein–protein inter- expression of dominant-negative ARF6 mutants blocks actions, which frequently involve interactions of tumor cell migration and invasion, resulting in reduced extended flat protein surfaces that are difficult to disrupt metastasis in vivo (Hashimoto et al., 2004; Tague et al., by small molecules. 2004). ARF6 cycles occur between an inactive GDP-bound form and an active GTP-bound form. Activation of Results ARF6 is stimulated by GEFs, which promote the release of GDP and binding of GTP, whereas inactivation is PIT-1 inhibits PIP3 binding to PH domains of GRP1/ stimulated by GTPase-activating proteins (Nie et al., ARNO, and suppresses ARF6 activation 2003; Gillingham and Munro, 2007). Cytohesin family We have reported previously two distinct inhibitors of proteins ARNO and GRP1 are known phosphoinosi- PIP3/PH domain binding through a screen of B50 000 tide-dependent GEFs with preference for ARF6 in cells diverse small molecules, which were termed PIT-1 and and in vivo (Frank et al., 1998a, b; Langille et al., 1999; PIT-2 (Figure 1a) (Miao et al., 2010). They have been Caumont et al., 2000; Santy and Casanova, 2001). characterized as specific PIP3 antagonists with selectiv- ARNO and GRP1 possess similar domain architecture, ity towards a distinct sub-set of PIP3-specific PH consisting of an N-terminal coiled-coil domain, a domains (Miao et al., 2010). The finding that PITs are catalytic Sec7 domain (ARF-GEF activity), a phosphoi- able to inhibit PIP3 binding to PH domains of GRP1 nositide-binding PH domain and a short C-terminal and ARNO attracted our interest because both GRP1 basic amino-acid region (Chardin et al., 1996; Jackson and ARNO are GEFs of ARF6, an GTPase playing and Casanova, 2000). important roles in the regulation of lamellipodia ARF6 translocates to the plasma membrane in formation and cell migration. Importantly, membrane response to growth factor stimulation, where it is translocation of these GEFs mediated by their PH activated by GEFs and modulates actin remodeling domains’ binding to phosphoinositides (Chardin et al., and lamellipodia formation. Membrane translocation of 1996; Jackson and Casanova, 2000) is necessary for the both ARF6 and its GEFs is necessary for their efficient efficient ARF6 activation (Cavenagh et al., 1996; Yang interaction and ARF6 activation (Cavenagh et al., 1996; et al., 1998; Santy and Casanova, 2001; Macia et al., Yang et al., 1998; Santy and Casanova, 2001; Macia 2004). Consistent with our previous report (Miao et al., et al., 2004). The recruitment of ARNO and GRP1 to 2010), PIT-1 and PIT-2 effectively inhibited PIP3 the plasma membrane has been shown to be mediated binding to PH domains of GRP1 and ARNO in by the binding of their PH domains to phosphoinosi- fluorescence polarization (FP) assay using recombinant tides (Chardin et al., 1996; Jackson and Casanova, GRP1 or ARNO PH domains and a fluorescent 2000). Overexpression of ARNO causes cells to develop tetramethylrhodamine-labeled PIP3 molecule, whereas broad lamellipodia, separate from neighboring