Anaplastic Lymphoma Kinase Activates the Small Gtpase Rap1 Via the Rap1-Speciﬁc GEF C3G in Both Neuroblastoma and PC12 Cells

Oncogene (2010) 29, 2817–2830 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Anaplastic lymphoma kinase activates the small GTPase Rap1 via the Rap1-speciﬁc GEF C3G in both neuroblastoma and PC12 cells

C Scho¨nherr1, H-L Yang2, M Vigny3, RH Palmer1 and B Hallberg1

1Department of Molecular Biology, Umea˚ University, Umea˚, Sweden; 2College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, China and 3U839 INSERM/UPMC IFM, Paris, France

Many different types of cancer originate from aberrant the result of a chromosomal translocation event that signaling from the anaplastic lymphoma kinase (ALK) places the C-terminal portion of ALK together with the receptor tyrosine kinase (RTK), arising through different N-terminal part of NPM (Morris et al., 1994; Shiota translocation events and overexpression. Further, activa- et al., 1994, 1995). A variety of ALK fusion proteins ting point mutations in the ALK domain have been recently have since been described in inflammatory myofibro- reported in neuroblastoma. To characterize signaling in the blastic tumors, non-small cell lung cancer, diffuse B-cell context of the full-length receptor, we have examined lymphomas and squamous cell carcinoma of the whether ALK is able to activate Rap1 and contribute esophagus (reviewed by Palmer et al., 2009). Together to differentiation/proliferation processes. We show that with the reported expression of ALK in a number of cell ALK activates Rap1 via the Rap1-specific guanine- lines and tumors, these findings suggest a function for nucleotide exchange factor C3G, which binds in a inappropriately activated ALK in oncogenic progres- constitutive complex with CrkL to activated ALK. The sion (reviewed by Palmer et al., 2009). Neuroblastoma, activation of the C3G/Rap1 pathway results in neurite which is derived from neural crest cells and can occur in outgrowth of PC12 cells, which is inhibited by either the entire peripheral sympathetic nervous system, overexpression of Rap1GAP or siRNA-mediated knock- accounts for approximately 15% of all pediatric cancer down of Rap1 itself or the guanine nucleotide exchange deaths (Maris et al., 2007). Amplification of the ALK factor C3G. Significantly, this pathway also appears to locus has been observed in neuroblastoma cell lines as function in the regulation of proliferation of neuroblastoma well as patient samples (Lamant et al., 2000; Miyake cells such as SK-N-SH and SH-SY5Y, because abroga- et al., 2002; Osajima-Hakomori et al., 2005) and recent tion of Rap1 activity by Rap1-specific siRNA or over- studies have reported activating ALK point mutations expression of Rap1GAP reduces cellular growth. These in both familial and sporadic cases of neuroblastoma, in results suggest that ALK activation of Rap1 may contri- addition to a number of neuroblastoma cell lines (Caren bute to cell proliferation and oncogenesis of neuroblasto- et al., 2008; Chen et al., 2008; George et al., 2008; ma driven by gain-of-function mutant ALK receptors. Janoueix-Lerosey et al., 2008; Mosse et al., 2008). Taken Oncogene (2010) 29, 2817–2830; doi:10.1038/onc.2010.27; together, aberrant ALK activation appears to be published online 1 March 2010 involved in tumor progression in a growing number of tumor types, consequently making the understanding of Keywords: ALK; PC12 cells; neurite outgrowth; small signaling emanating from this RTK an important aim. GTPase; Rap1; neuroblastoma Although many studies have addressed the mechanisms of action used by oncogenic ALK fusion proteins (primarily NPM-ALK), the physiological functions of ALK and its downstream targets in mouse and humans are far from clear. In mammals, ALK has been Introduction suggested to be involved in the normal development and function of the nervous system (Iwahara et al., 1997; Anaplastic lymphoma kinase (ALK) belongs to the Morris et al., 1997; Vernersson et al., 2006; Bilsland insulin receptor superfamily of receptor tyrosine kinases et al., 2008). However, the reported presence of (RTKs) (Iwahara et al., 1997; Morris et al., 1997), and differently sized ALK mRNA, suggests that differential was initially characterized as one of the fusion partners splicing and additional functions of ALK may exist in the nucleophosmin (NPM)-ALK oncogene found in a (Morris et al., 1994). subset of anaplastic large cell lymphomas. NPM-ALK is Studies of ALK-like RTKs in other species have expanded the knowledge of ALK in normal physiology. In Drosophila melanogaster ALK has a major function Correspondence: B Hallberg, Department of Molecular Biology, in the development of the visceral musculature of the Umea˚University, Buil. 6L, Umea˚, Norrland S-901 87, Sweden. E-mail: [email protected] midgut in vivo (Loren et al., 2001, 2003; Lee et al., 2003; Received 4 June 2009; revised 11 December 2009; accepted 28 December Stute et al., 2004), a process during which Jelly belly 2009; published online 1 March 2010 (Jeb) ligand-mediated activation of ALK forces the Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2818 extracellular signal-regulated kinase (ERK)/mitogen- thyroid-stimulating hormone-stimulated thyroid tumori- activated protein kinase (MAPK) pathway to induce genesis (Lou et al., 2002; Hong et al., 2008). However, in muscle cell specification (Englund et al., 2003; Lee et al., contrast to these findings, Rap1 has been shown in other 2003; Stute et al., 2004). In agreement, the Caenorhab- studies to suppress oncogenesis (Lin et al., 2000). To ditis elegans Jeb homologue Hen-1 was recently iden- date a function for Rap1 in the development of tified as a ligand for SCD-2, the homologue of neuroblastoma has not been investigated. mammalian ALK (Ishihara et al., 2002; Liao et al., Rap1 is activated by GEFs such as C3G, the Epac 2004; Reiner et al., 2008). In both these species, ALK family proteins, CD-GEFs and PDZ-GEFs (reviewed by has furthermore been shown to be important for Bos et al., 2001). Interestingly, during mouse embryo- neuronal functions. In the fruit fly ALK and Jeb are genesis, C3G has essential functions in vascular matura- required for neuronal circuit assembly in the visual tion and the development of the nervous system (Voss system, mediating correct targeting of photoreceptor et al., 2003, 2006, 2008). Further, small-interfering axons (Bazigou et al., 2007), whereas ALK in C. elegans RNAs (siRNA)-mediated knockdown of C3G results is expressed in motor neurons, where it is involved in the in decreased neurite outgrowth in PC12 cells and C3G neuronal control of entry into dauer as well as synapse has been suggested to regulate differentiation and stabilization (Liao et al., 2004; Reiner et al., 2008). survival of human neuroblastoma cells (Hisata et al., Furthermore, in the zebrafish model system it has been 2007; Radha et al., 2008). Besides C3G, the atypical shown that LTK, which belongs to the LTK/ALK RapGEF DOCK4, which displays dual activity toward family, is expressed in developing neural crest cells and both Rap1 and Rac, also appears to exhibit oncogenic is important for the specification of iridophores from the capacity (Yajnik et al., 2003). neural crest linage (Lopes et al., 2008). Due to its very low intrinsic GTPase activity, To date, there are no reports of Jeb-like ligands for inactivation of Rap1 relies on GTP hydrolysis by GAPs, mammalian ALK. Instead, the small heparin binding including Rap1GAP and members of the Spa1 family peptide ligands, midkine and pleiotrophin, have been (reviewed by Bos et al., 2001). Importantly, tuberin, a described as activating ligands for vertebrate ALK. product of a familial tuberous sclerosis gene, that also However, the functional significance of this ligand/ displays in vitro Rap1GAP activity, induces benign receptor interaction in vivo is not fully understood tumors when deleted (Wienecke et al., 1995). Enhanced (Stoica et al., 2001, 2002; Muramatsu, 2002). Rap1 activity due to decreased Rap1GAP expression In the mouse, ALK is strongly expressed in the has been recently reported in prostate cancer cell lines, nervous system during embryogenesis, suggesting a leading to increased cell migration and invasive behavior function for mammalian ALK in this tissue. Supporting (Bailey et al., 2009). Thus, deregulated Rap1 signaling is this hypothesis, in vitro studies have shown that ALK implicated in the pathogenesis of a variety of malig- induces neuronal differentiation of PC12 and SK-N-SH nancy types, including leukemia, neuroblastoma and cells in a MAPK-dependent manner (Souttou et al., prostate cancer (reviewed by Minato and Hattori, 2009). 2001; Motegi et al., 2004; Gouzi et al., 2005; Moog-Lutz Here we observe that stimulation of mALK triggers et al., 2005; Yang et al., 2007). Moreover, a recent study activation of Rap1 and that this activity is indeed of ALK knockout mice has implicated a function for important for efficient ALK-mediated neurite out- ALK in the adult brain, where it appears to regulate the growth in PC12 cells. In part, ALK-induced Rap1 function of the frontal cortex and hippocampus, and activation appears to be mediated by C3G, which in may be involved in dopaminergic signaling (Bilsland complex with CrkL is recruited to the activated mALK et al., 2008). receptor. Furthermore, we have observed that the Rap1 is a small GTPase involved in the regulation of activation of Rap1 by ALK contributes to cell growth cell adhesion, polarity, secretion and cell–cell junction and tumorigenesis of the neuroblastoma cell lines SK-N- formation, processes in which it is controlled by an SH and SH-SY5Y, emphasizing a significant function of extensive set of activating guanine nucleotide exchange Rap1 in ALK-induced cell transformation. factors (GEFs) and inhibitory GTPase-activating proteins (GAPs) (Raaijmakers and Bos, 2009). Interest- ingly, Rap1 appears to have a function in neurite outgrowth and neuronal polarization by mediating Results sustained MAPK pathway activation in addition to regulating the cytoskeleton (York et al., 1998; Kao Rap1 is activated in response to ALK stimulation et al., 2001; Wu et al., 2001; Schwamborn and Puschel, We wished to investigate whether mouse ALK is able to 2004). Further, Rap1 is also involved in the regulation activate Rap1. To do this, we used a PC12 cell system in of cell proliferation. When overexpressed, Rap1 induces which expression of ALK can be induced by tetra- oncogenic transformation in Swiss-3T3 cells (Altschuler cycline, together with an agonist monoclonal antibody and Ribeiro-Neto, 1998), and has been reported to be (mAb46) that has been previously reported to bind and activated in metastatic mammary carcinomas, during activate mALK in vivo (Yang et al., 2007). Indeed, melanoma tumorigenesis as well as metastasis (Hem- activation of mALK with mAb46 resulted in an meryckx et al., 2001; Gao et al., 2006). Rap1 also extensive Rap1 activation, as shown by pull down with appears to be involved in the process of thyroid GST-RalGDS (Figure 1). Active Rap1 (that is, GTP mitogenesis and has been suggested to contribute to bound) could be detected as early as 5 min after

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2819 PC12 cells (Figures 3a and b). Similarly, mAb46- induced neurite outgrowth in mALK-expressing PC12 cells was also robustly inhibited by the presence of HA- Rap1GAP (Figures 3a and b).

Rap1 GEF C3G is activated on stimulation of mALK Figure 1 mALK stimulation leads to activation of Rap1. Rap1 The Rap1-specific GEF C3G is expressed in PC12 cells pull downs of cell lysates from PC12 Tet-on clone PCmALK and has been reported to have a function in neurite transfected with inducible mouse anaplastic lymphoma kinase outgrowth (York et al., 1998; Kao et al., 2001). We (ALK) expression. Cells were induced for ALK expression and therefore investigated whether activation of mALK had serum starved for 24 h before stimulation with 1 mg/ml mAb46 for the indicated times. Cell lysates were precleared (see Materials and any effect on C3G activation. Initially, we performed methods section), and incubated with GST-RalGDS beads. Bound immunoprecipitation experiments to examine whether proteins were subsequently analyzed by immunoblotting with anti- C3G is recruited to mALK in response to receptor Rap1 antibodies. Rap1 in whole-cell lysates was used as loading stimulation. As previously reported, a constitutive control. Detection of phosphorylated ERK (p-ERK) was used as a control for mALK stimulation. interaction between C3G and CrkL could be clearly detected by immunoprecipitation with antibodies against either C3G or CrkL, independent of mALK stimulation (Figures 4a and b) (Feller et al., 1995). stimulation, was sustained for at least 60 min and On stimulation with mAb46, we could observe tyro- decreased only after 120 min. Interestingly, the pattern sine phosphorylation of mALK, indicating successful of Rap1 activation is highly similar to that of ERK activation of the receptor (Figure 4a). Activation of phosphorylation and activation, which was monitored mALK-induced signaling was moreover confirmed by as a control for ALK activation. phosphorylation of ERK in cell lysates as a result of stimulation by the ligand mAb46. In addition, after Rap1 knockdown inhibits ALK-stimulated mAb46 stimulation, we found mALK protein to be neurite outgrowth present in coimmunoprecipitates, using either anti-C3G Next we wanted to investigate whether Rap1 is involved or anti-CrkL antibodies, indicating the formation of a in ALK-mediated stimulation of neurite outgrowth. multiprotein complex comprising C3G, CrkL and Differentiation and consequently neurite outgrowth of mALK (Figures 4a and b). Taken together, these results PC12 cells can be efficiently achieved by stimulating suggest that activated mALK recruits the constitutive mALK with mAb46 (Yang et al., 2007). To experimen- C3G/CrkL complex to the site of receptor stimulation. tally clarify a putative function for Rap1 in ALK- To further address the mechanism of activation of induced neurite outgrowth, we first used RNA inter- C3G, we analyzed the tyrosine-phosphorylation status ference (RNAi) to knockdown Rap1 expression in PC12 of C3G in response to mALK stimulation. To do this, cells. Importantly, the reduction of Rap1 protein levels we used commercially available beads coupled to anti- in PC12 cells was substantial and independent of mALK phosphotyrosine antibodies (4G10) to immunoprecipi- expression status (Figure 2a, compare lane 4 with 3). tate proteins specifically modified by phosphorylation When knocking down Rap1, we observed a distinct on tyrosine. Indeed, after 30 min of stimulation with decrease in nerve growth factor (NGF)-induced neurite mAb46, we could observe a clear tyrosine phosphoryla- outgrowth of approximately 50% as compared to tion of C3G (Figure 4c). Taken together, we conclude nontreated PC12 cells (Figure 2b). Moreover, this result that activated mALK recruits C3G/CrkL and that this was confirmed with a second independent siRNA complex formation results in tyrosine phosphorylation targeting Rap1 (Supplementary Figure 2). Similar of C3G. results were also obtained in mALK-expressing PC12 cells on stimulation with mAb46, in which transfection C3G knockdown inhibits ALK-stimulated of siRNA against Rap1 caused a 40% reduction of neurite outgrowth neurite outgrowth as compared with control cells. To address a function of C3G in ALK-induced neurite outgrowth, we once more used RNAi to suppress Rap1GAP expression blocks mAb46-induced neurite endogenous C3G expression levels in PC12 cells. C3G outgrowth in mALK-expressing PC12 cells protein levels were decreased to a similar extent in both In a second approach to block Rap1 activity in the cell, mALK-expressing and mALK-nonexpressing PC12 cells PC12 cells were transfected with a plasmid encoding the (Figure 5a, compare lane 4 with 3 and lane 2 with 1), Rap1-specific GAP Rap1GAP (HA-Rap1GAP), which although the knockdown was not as efficient as down- is known to abrogate Rap1 activity. Subsequent to regulation of endogenous Rap1 levels by siRNA transfection of mALK-expressing PC12 cells with (compare Figure 5a with Figure 2a). Interestingly, in HA-tagged Rap1GAP, we monitored mAb46-induced cells expressing mALK, which were not treated with neurite outgrowth. In agreement with previous C3G-siRNA, we observed increased C3G protein levels results, inhibition of Rap1 activity by Rap1GAP over- on stimulation with mAb46 (Figure 5a, compare lane 3 expression caused a dramatic reduction of NGF- with 1 and Figure 5d, compare lane 2 with 1). This induced neurite outgrowth in mALK-nonexpressing increase is in agreement with recently reported observa-

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2820

Figure 2 Knockdown of Rap1 by small-interfering RNA (siRNA) leads to reduced neurite outgrowth in PCmALK cells. PCmALK cells either expressing mALK or not were transfected with siRNAs against Rap1 (150 pmol) or control siRNA (150 pmol) together with 0.5 mg GFP with Amaxa Nucleofection Kit V. (a) Immunoblot Rap1 expression levels after treatment of mALK-negative and mALK-positive PCmALK cells with Rap1 siRNA. ALK induction was visualized by immunoblotting with anti-ALK antibody. Total ERK (pan-ERK) levels were monitored as loading control. (b) Quantiﬁcation of neurite outgrowth assays of mALK-negative and mALK-positive PCmALK cells transfected with either control siRNA or siRap1 together with pmaxGFP. Cells were stimulated with 100 ng/ml nerve growth factor (NGF) or 1 mg/ml mAb46, respectively. After 48 h 100 GFP-positive cells were analyzed and cells extending neurites with a length twice as long as the cell body were counted as neurite bearing cells. The experiment was carried out in triplicate. Each sample within an experiment was performed in duplicate.

tions in the neuroblastoma cell line IMR-32, made by C3G in the activation of Rap1, we used Rap1 pulldown Radha et al. (2008). The molecular mechanisms behind assays to investigate mALK-expressing PC12 cells that the increase of C3G proteins are unclear and we have were transfected either with sicontrol or siC3G. Indeed, not investigated this further in this study. After NGF mALK-positive PC12 cells with downregulated C3G stimulation, neurite outgrowth in mALK-nonexpressing expression levels display reduced levels of GTP-bound PC12 cells with suppressed C3G levels was reduced Rap1 in comparison to control on stimulation by approximately 30% as compared to control cells (Figure 5d, compare lane 4 with 2). On the basis of (Figures 5b and c). This was confirmed with a second these results, we conclude that C3G can be activated by independent siRNA targeting C3G (Supplementary mALK and indeed appears to have a function in Figure 2). Neurite outgrowth induced by mAb46- mAb46-induced neurite outgrowth and the activation mediated mALK activation was reduced to a compar- of Rap1 in PC12 cells. able degree in cells transfected with siC3G. Given that To verify this further, we used YFP- or V5-tagged the decrease in neurite outgrowth after treatment with human C3G to analyze the effect of C3G overexpression siC3G was not as pronounced as in Rap1 knockdown on neurite outgrowth in mALK-expressing and mALK- cells, we hypothesize that this may reflect other possible nonexpressing PC12 cells (Supplementary Figure 1). mechanisms of Rap1 activation and/or the incomplete Transfection of either YFP- or V5-tagged human C3G knockdown of C3G. Further, to confirm a function for had no significant impact on basal neurite outgrowth

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2821

Figure 3 Inhibition of neurite outgrowth in PCmALK cells by overexpression of Rap1GAP. PCmALK cells either expressing mALK or not were transfected with 2.5 mg Rap1GAP and 0.5 mg GFP using the Amaxa Nucleofection Kit V. Transfected cells were stimulated with 100 ng/ml nerve growth factor (NGF) and 1 mg/ml mAb46, respectively. After 48 h, 100 GFP-positive cells were analyzed and neurite cells were scored as described above. (a) Number of neurites per 100 transfected cells. The experiment was carried out in triplicate. Each sample within an experiment was performed in duplicate. (b) Representative images of neurite outgrowth. levels in either mALK-expressing or mALK-nonexpres- stimulated mALK-nonexpressing PC12 cells, V5-tagged sing PC12 cells. However, whereas overexpression of C3G could indeed trigger a mild but signiﬁcant increase C3G had no effect on neurite outgrowth in NGF- (23%) in neurite outgrowth in ALK-expressing PC12

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2822 cells stimulated with mAb46. These results are in lower panels, compare lane 6 with 5, and Figure 1). agreement with a role for C3G downstream of mALK. Further, treatment with NVP-TAE684 reduced mALK- driven ERK phosphorylation (Figure 6a, top panels, compare lane 6 with 8). As control, we treated PC12 The small inhibitor NVP-TAE684 inhibits the activation cells with NVP-TAE684 inhibitor before stimu- of ALK as well as Rap1 lation with NGF, which mediates phosphorylation The ALK inhibitor, NVP-TAE684, has been shown to of ERK and differentiation of PC12 cells (Gotoh et al., be an inhibitor of NPM-ALK-driven cell proliferation 1990; Traverse et al., 1992; Hallberg et al., 1998; and blocks activation of downstream targets of NPM- Galkin et al., 2007). As predicted, NVP-TAE684 does ALK signaling, such as ERK (Galkin et al., 2007). We not inhibit TrkA receptor signaling in response to stimu- have therefore used NVP-TAE684 to examine signaling lation with NGF and a clear ERK activation was downstream of mouse ALK in this study. We observed a observed (Figure 6a, top panel, compare lanes 3 and 4 clear abrogation of Rap1-GTP loading and phospho- with 1 and 2). rylation of ERK on treatment of mouse ALK-expressing cells with 0.2 mM NVP-TAE684 before stimulation (Figure 6a, compare lane 8 with 5, 6 and 7). As shown Rap1 does not appear to be required for mALK activation previously, activation of mALK with mAb46 without of Erk inhibitor pretreatment results in a clear activation of We have also examined the requirement of Rap1 for Erk Rap1 in GST-RalGDS pulldown assays (Figure 6a, phosphorylation in response to activation of ALK.

Figure 4 mALK stimulation induces complex formation with C3G and CrkL. PCmALK cells expressing mALK were serum starved for 24 h before stimulation with 1 mg/ml mAb46 for 30 min. Precleared cell lysates were incubated with protein G-coupled anti-C3G or CrkL antibodies, respectively, for 1 h at 4 1C. Samples were analyzed on SDS–polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies as indicated. (a) C3G immunoprecipitation. Phosphorylation of mALK was detected by pY99 antibodies. (b) CrkL immunoprecipitation. (c) 4G10 anti-phosphotyrosine (4G10) immunoprecipitation. To detect the tyrosine phosphorylation status of C3G, we stimulated PCmALK cells either with 1 mg/ml mAb46 for 30 min or with 10% fetal calf serum for 10 min as positive control. PCmALK cell lysates were subsequently incubated with 4G10 beads, and p-C3G was detected by anti-C3G immunoblotting.

Figure 5 Knockdown of C3G by small-interfering RNA (siRNA) leads to reduced neurite outgrowth in mALK-stimulated PCmALK cells. PCmALK cells either induced to express mALK or not were transfected with siRNAs against C3G (150 pmol) or control siRNA (150 pmol) together with 0.5 mg GFP with Amaxa Nucleofection Kit V. (a) Immunoblot analysis of downregulated C3G expression levels by siC3G in mALK-negative and mALK-positive PCmALK cells. pan-ERK was monitored as loading control. (b) Quantiﬁcation of neurite outgrowth assays of mALK-negative and mALK-positive PCmALK cells transfected with either control siRNA or siC3G together with pmaxGFP. Cells were stimulated with 100 ng/ml nerve growth factor (NGF) or 1 mg/ml mAb46, respectively. After 48 h 100 GFP-positive cells were analyzed and neurite bearing cells were scored as above. The experiment was carried out in triplicate. Each sample within an experiment was performed in duplicate. (c) Representative images of neurite outgrowth. (d) Rap1 pull downs of cell lysates from mALK-expressing PC12 cells. Cells were transfected with siRNAs against C3G (150 pmol) or control siRNA (150 pmol) with Amaxa Nucleofection Kit V, induced for ALK expression and serum starved for 24 h before stimulation with 1 mg/ml mAb46 for the indicated times. Cell lysates were precleared (see Materials and methods section), and incubated with GST-RalGDS beads. Bound proteins were subsequently analyzed by immunoblotting with anti-Rap1 antibodies. Rap1 in whole-cell lysates was used as loading control. Detection of phosphorylated ERK (p-ERK) was used as a control for mALK stimulation.

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2823 Indeed, in our hands, we observe that both mALK and of Rap1 (Figures 6b and c, compare lanes 1–6 with TrkA are able to stimulate phosphorylation of ERK in a 7–12). In a second approach to evaluate the importance sustained manner regardless of the presence or absence of Rap1 activity for activation of ERK, ALK-expressing

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Figure 6 Knockdown of Rap1 by small-interfering RNA (siRNA) does not affect ERK phosphorylation both in ALK-nonexpressing and ALK-expressing PCmALK cells. (a) Rap1 pull downs of cell lysates from mALK-expressing PCmALK cells. Cells were induced for ALK expression and serum starved for 24 h. Before stimulation with 1 mg/ml mAb46 for the indicated times, cells were treated with 0.2 mM NVP-TAE684 for 1 h. Cell lysates were precleared (see Materials and methods section), and incubated with GST-RalGDS beads. Bound proteins were subsequently analyzed by immunoblotting with anti-Rap1 antibodies. Rap1 in whole-cell lysates was used as loading control. Detection of phosphorylated ERK (p-ERK) was used as a control for mALK stimulation and inhibition of mALK by NVP-TAE684. (b) Effect of Rap1 knockdown by siRNA on ERK phosphorylation in mALK-nonexpressing PC12 cells and (c) mALK-expressing PCmALK cells. Cells were transfected with siRNAs against Rap1 (150 pmol) or control siRNA (150 pmol) with Amaxa Nucleofection Kit V. Starved ALK-nonexpressing PC12 cells were stimulated with 100 ng/ml nerve growth factor (NGF) (b) and starved ALK-expressing PC12 cells were stimulated with 1 mg/ml mAb46 (c) for the indicated times. Precleared cell lysates were subjected to immunoblot analysis for ERK phosphorylation (d). Overexpression of Rap1GAP was detected by HA antibodies. pan- ERK was monitored as loading control. The experiment was carried out in triplicate.

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2825 PC12 cells were transfected with a plasmid encoding the oncogenic translocation-ALK fusion proteins (Palmer Rap1-specific GAP, Rap1GAP (HA-Rap1GAP), which et al., 2009). In mammalian systems, it has been shown abrogates Rap1 activity and neurite outgrowth from that the full-length ALK RTK interacts with and these cells (Figure 3). mediates the activation of proximal signaling proteins, Subsequent to transfection of mALK-expressing including Shc, FRS, PI3-kinase and PLCg, which in PC12 cells with HA-tagged Rap1GAP, ALK-induced many cases leads to activation of ERK (Piccinini et al., ERK phosphorylation was monitored over time as 2002; Motegi et al., 2004; Degoutin et al., 2007). Along indicated in Figure 6d. In agreement with our data using with others, we have previously shown that activation of siRNA against Rap1 above, inhibition of Rap1 activity ALK triggers the differentiation of PC12 cells into by Rap1GAP overexpression did not reduce the ability sympathetic-like neurons, inducing a substantial neurite of activated mALK to mediate sustained phosphoryla- outgrowth (Souttou et al., 2001; Motegi et al., 2004; tion of ERK (Figure 6d). Thus, we conclude that Rap1 Gouzi et al., 2005; Moog-Lutz et al., 2005; Yang et al., activity is not absolutely required for ERK activation; 2007). In Drosophila, ALK activates an ERK-dependent however, it is necessary for the neurite outgrowth pathway leading to downstream transcription of mole- responses of ALK-expressing PC12 cells. cules such as Duf/Kirre, Org, Hand and the TGF-b ligand Dpp (Englund et al., 2003; Lee et al., 2003; Varshney and Palmer, 2006; Shirinian et al., 2007), Rap1 activity is required for efficient cell growth whereas in C. elegans ALK (SCD-2) has been suggested of neuroblastoma cell lines to modulate the TGF-b pathway (Reiner et al., 2008). Although the mALK-inducible PC12 cell line provides In response to treatment with NGF, PC12 cells an excellent and controlled model for investigating undergo differentiation from immature chromaffin cells signaling events downstream of ALK, the question of into cells that resemble sympathetic neurons. This how significant the function of Rap1 is in ALK signaling process is characterized by extension of neurites and under physiological conditions, remains. The recent development of electrical excitability (Greene and discovery of activating ALK mutations in human Kaplan, 1995). The molecular mechanism behind the neuroblastoma, together with reports implicating the differentiation process in PC12 cells relies on the ability C3G/Rap1 pathway in the survival of the human of NGF to induce sustained activation of the ERK neuroblastoma cell line IMR-32 (Caren et al., 2008; MAPK family of serine/threonine kinases, which is Chen et al., 2008; George et al., 2008; Janoueix-Lerosey mediated by Ras and Rap1 (Qiu and Green, 1991; et al., 2008; Mosse et al., 2008; Radha et al., 2008), Marshall, 1995; York et al., 1998). suggests that the involvement of the C3G/Rap1 pathway We show in this investigation that the stimulation of in the growth of neuroblastoma cell lines may occur in ALK, using agonist antibodies, results in activation of an ALK-dependent manner. To investigate this hypoth- Rap1. Further, neurite outgrowth after ALK activation esis, we used SK-N-SH and SH-SY5Y neuroblastoma is reduced by transfecting cells with Rap1GAP or with cell lines, which carry an activating F1174L point two independent siRNAs against either Rap1 or its mutation in the ALK receptor (Chen et al., 2008; GEF C3G, suggesting a function for Rap1 activation in et al. et al. George , 2008; Janoueix-Lerosey , 2008). ALK-stimulated neurite outgrowth. Furthermore, we Using RNAi or HA-Rap1GAP to downregulate Rap1 also observed a decrease in the ability of mALK to load activity, we analyzed Rap1-dependent cell growth of Rap1 proteins with GTP when cells are transfected with SK-N-SH and SH-SY5Y cells by monitoring the two different siRNA against C3G and Rap1 or treated amount of viable cells over a period of 5 days with a small selective ALK inhibitor, NVP-TAE684, on (McDermott et al., 2008). In both cell lines, reduction stimulation. of endogenous Rap1 expression levels by siRNA Although we clearly observe a function for Rap1 in resulted in a decrease of cell growth (Figures 7a–c). In the ALK-induced differentiation response, we have been addition, reduction of Rap1 protein was able to block unable to define a function for Rap1 in the activation the stimulation of cell growth by mAb46, which can be of ERK by ALK. Our investigations show that the observed in SH-SY5Y but not in SK-N-SH cells phosphorylation status of Erk on stimulation of mALK (Figures 7b and c). Furthermore, we observed that the is unaffected by either the knockdown of C3G and Rap1 inhibition of Rap1 activity by Rap1GAP had the same siRNA or by the transient overexpression of Rap1GAP effect on cell growth as depletion of Rap1 by RNAi (Figures 5 and 6). This would suggest that Rap1 (Figures 7a–c). Therefore, we suggest that Rap1 not activation is not a critical requirement in the activation only is important for PC12 cell differentiation in of ERK by ALK in our system. In addition, we response to ALK activation via C3G, but that it also also observe a prolonged phosphorylation of ERK on has a significant function in regulating the growth of stimulation with NGF when cells were treated with neuroblastoma cell lines. siRNA Rap1, which is in agreement with previous published results (Zwartkruis et al., 1998). Although activation of Rap1 and C3G appear to be Discussion essential for neurite outgrowth in ALK-expressing PC12 cells, our results imply another scenario leading to the Our understanding of the signaling events elicited by the prolonged ERK phosphorylation that does not invoke native ALK receptor has lagged behind studies of the activation of Rap1 and C3G. The question of the

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2826

Figure 7 Abrogation of Rap1 activity in the neuroblastoma cell lines SK-N-SH and SH-SY5Y leads to reduced cell growth independent of stimulation. For cell growth determination, 1 Â 104 cells of SK-N-SH (a) and SH-SY5Y (b) neuroblastoma cell lines were seeded per well in 24-well plates. Cells were transfected and treated in the following way: 0.4 mg pcDNA3 as negative control (~), as positive control 0.4 mg pcDNA3 and stimulation with 1 mg/ml mAb46 the following day (’), 20 pmol siRap1 (m), 20 pmol siRap1 and stimulation with mAb46 ( Â ) or 0.4 mg HA-Rap1GAP and stimulation with mAb46 (). The amount of viable cells was determined by Trypan blue staining over 5 days. Each sample was performed in triplicate. (c) Expression levels of HA-Rap1GAP and Rap1 in SK-N-SH and SH-SY5Y cells.

molecular mechanisms underpinning the phosphoryla- is sufﬁcient to propel the differentiation process. Rather, tion of Erk on stimulation of mouse ALK constitutes Rap1 and putative downstream target molecules of the basis for future investigations. Although not Rap1 are determinants for differentiation in PC12 cells examined in this study, the FRS2 adaptor has been stimulated with NGF and in stimulated ALK-expressing reported to be recruited by ALK after antibody-based PC12 cells. ALK activation in PC12 cells (Degoutin et al., 2007), It is important to note that difﬁculties arise in suggesting that an ALK/FRS2/CrkL/C3G-complex comparing results of similar nature, such as PC12 cells may be assembled on activation of the ALK RTK. and Rap1 activation, because the genetic composition Previous reports indicate that overexpression of a and variation of different clones of PC12 cells varies. constitutively active Rap1 mediates neurite outgrowth Our results regarding C3G are in line with those of in PC12 cells (Vossler et al., 1997), and it has been Hisata et al. (2007) and Radha et al. (2008), who have suggested that neurite outgrowth by NGF does not also observed decreased neurite outgrowth on knock- require sustained activation of Erk (York et al., 1998). down of endogenous Rap1 or C3G levels in PC12 cells. Here we show that sustained activation of Erk pathway The molecular mechanism used appears to involve both

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2827 CrkL and C3G, because stimulation of ALK results in activating ALK point mutation F1174L, strengthens formation of a multiprotein complex, containing CrkL the potential importance of the C3G/Rap1 pathway for and C3G, together with the activated ALK receptor. cell proliferation in neuroblastoma. Indeed, activation of ALK also leads to tyrosine Interestingly, we were unable to observe an increase in phosphorylation of C3G, which is in agreement with cell proliferation of SK-N-SH cells on stimulation with previous reports showing C3G activation in response to anti-ALK agonist antibodies in conditions of normal various exogenous stimuli (de Jong et al., 1998; Ichiba serum levels. However, abrogation of the Rap1 signaling et al., 1999). cascade either by siRNA or by overexpression of Rap1- Rap1 is the first small GTPase that has been GAP clearly reduced cell proliferation in both cell lines. investigated downstream of mALK so far and it is clear Previous observations—before the identification of the that inhibition of mALK using the small ALK-inhibitor ALK F1174L mutation in SK-N-SH cells—of cells NVP-TAE684 abrogates the GTP-loading of Rap1, cultured in reduced serum conditions suggest that together with diminished ERK activation. A number of stimulation of ALK with agonist antibodies promotes reports suggest that C3G has an inhibitory function in cell growth and neurite outgrowth in SK-N-SH cells, cell proliferation (Schmitt and Stork, 2002; Stork, 2003; indicating a function for ALK in pathogenesis of Guerrero et al., 2004). Indeed, activation of C3G by neuroblastoma (Motegi et al., 2004). In contrast to NGF or overexpression of C3G has been shown to SK-N-SH cells, SH-SY5Y cells showed an increase in mediate cell-cycle arrest in the neuroblastoma cell line cell growth on stimulation with mAb46. In considera- IMR-32 (Radha et al., 2008), reinforcing a function for tion of potential treatments for patients with neuro- Rap1 in this process. blastoma, such observations highlight the importance of Although it is clear that activation of ALK in PC12 understanding the genetic disposition of individual cells triggers activation of the small GTPase Rap1 as tumors, because both cell lines contain the same well as neurite outgrowth, the significance of this mutation within the kinase domain of ALK, but only pathway in tumor development is unclear. It has been SH-SY-5Y cells can be further stimulated on ALK shown that Rap1 is activated by cAMP, which can activation. Although intriguing, the mechanisms behind inhibit or enhance cell proliferation, depending on this discrepancy are unknown and have not been the cell type. It has been further proposed that both investigated here. What is clear is that in both cell lines, the inhibitory and enhancing effects of cAMP on cell abrogation of the ALK-Rap1 connection reduced the proliferation are mediated by Rap1, which can act either cell proliferation rate dramatically. antagonistically or synergistically with Ras, depending Taken together, the results presented here show that on the cellular context (Bos et al., 2001; Schmitt ALK activation leads to formation of a complex and Stork, 2001; Ribeiro-Neto et al., 2002). Thus far, between the receptor and the CrkL/C3G protein no Rap1-activating mutations have been identified in complex, in which C3G becomes tyrosine phosphory- human tumors (Gyan et al., 2005; Zemojtel et al., 2006; lated in response to ALK stimulation. This activated Matsuse et al., 2009). However, this does not exclude the complex activates Rap1, leading to neurite outgrowth in possibility that Rap1 is a potential member in oncogen- PC12 cells. Further, we show that Rap1 has an esis via alternative mechanisms, such as overexpression important function in the proliferation of tumorigenic or downregulation of Rap1, or any of the regulatory neuroblastoma cells. Currently, there are no clinically proteins acting upstream and downstream of Rap1 approved treatments for oncogenic malignancies (Altschuler and Ribeiro-Neto, 1998; Ishida et al., 2003; caused by inappropriate ALK activation that directly Yajnik et al., 2003; Tsygankova et al., 2007; Bailey et al., target ALK or downstream ALK-driven signaling. 2009). However, disruption of the ALK/Rap1 pathway may A function for ALK in cancer development is well prove worthy of further investigation. established, and the recently reported ALK gain-of- function mutations observed in patients with neuroblastoma (Caren et al., 2008; Chen et al., 2008; George Materials and methods et al., 2008; Janoueix-Lerosey et al., 2008; Mosse et al., 2008; Palmer et al., 2009) suggest that a connection Antibodies between ALK and Rap1 may be of relevance in Mouse anti-Rap1 (1:1000) and mouse anti-pan-ERK (1:5000) neuroblastomas. The IMR-32 neuroblastoma cell line were purchased from BD Transduction Laboratories, Franklin in which C3G has been examined (Radha et al., 2008) Lakes, NJ, USA; rabbit anti-pERK (1:1000) from Cell is characterized as containing a wild-type ALK RTK. Signaling Technology, Danvers, MA, USA; and rabbit anti- However, the growth of this cell line is inhibited by PF- C3G (H-300 and C-19) (1:1000), rabbit anti-CrkL (C-20) 2341066, a c-MET and ALK inhibitor, suggesting that (1:1000) in addition to mouse anti-phosphotyrosine pY99 ALK signaling contributes to IMR-32 cell growth at (1:1000) from Santa Cruz Biotechnology, Santa Cruz, CA, some level (Janoueix-Lerosey et al., 2008). Furthermore, USA. Anti-phosphotyrosine 4G10 agarose conjugate was obtained from Upstate, Lake Placid, NY, USA (Cat. No. ALK-specific small-hairpin RNA treatment of IMR-32 16-101). The activating monoclonal antibody mAb46 and the cells leads to considerable inhibition of focus formation chicken anti-mALK antibody have been described previously and growth arrest (Janoueix-Lerosey et al., 2008). (Moog-Lutz et al., 2005; Yang et al., 2007). The horseradish Examination of two additional neuroblastoma cell peroxidase-coupled secondary antibodies goat anti-rabbit IgG lines—SK-N-SH and SH-SY5Y—which contain the and goat anti-mouse IgG were from Thermo Scientific, Waltham,

Oncogene Activation of the small GTPase Rap1 by ALK C Scho¨nherr et al 2828 MA, USA and rabbit anti-chicken IgY from Pierce, Rockford, Pull-down assay for GTP-Rap1 IL, USA (1:5000). PC12 cells expressing mALK were starved for 24 h and when indicated treated with 0.2 mM of the ALK inhibitor NVP- Plasmids TAE684 for 1 h before stimulation with 1 mg/ml mAb46 for the pMT2-HA-Rap1GAP, pcDNA3.1-YFP-humanC3G and indicated times (Moog-Lutz et al., 2005; Yang et al., 2007). pcDNA3.1-V5-humanC3G were generous gifts from JL Bos Whole-cell lysates were harvested and analyzed as described (Utrecht University, Utrecht, the Netherlands). For transfec- (Ren et al., 1999; Henriksson et al., 2002). tion control pmaxGFP from Amaxa Biosystems (Cologne, Germany) was used. For the GTP-Rap1 pull-down assay, Cell transfection and neurite outgrowth assay GST-RalGDS proteins were used (Franke et al., 1997). For the PCmALK cells were transfected using cell line nucleofector kit cell growth assay, cells were transfected with pcDNA3 V (Amaxa Biosystems) according to manufacturer’s protocol as control. with an efficiency of 60–80%. For the neurite outgrowth assay, The pTRE-Tight-puromycin (pTTP) plasmid was genera- cells were transfected with 0.5 mg pmaxGFP and 2.5 mg DNA ted from pTRE-Tight (BD Biosciences, San Jose, CA, USA, and 150 pmol siRNA, respectively. Cells were stimulated with Cat. No. 631059). The XhoI site at position 602 (two XhoI sites 100 ng/ml NGF 2.5S (Grade I) (Alomone Labs, Jerusalem, are present in pTRE-Tight) was eliminated with a fill-in Israel) or 1 mg/ml mAb46. Quantification of neurite outgrowth reaction. Thereafter the modified TRE-Tight was opened with in the transfected PCmALK cells was carried out as described PvuI/XhoI (position 1985/2), and this fragment was replaced (Moog-Lutz et al., 2005). Experiments were performed in with a PvuI/XhoI (position 3143/2) fragment from pTRE2pur triplicates, and each sample within an experiment was (BD Biosciences, Cat. No. 6254-1). A generated PCR NotI performed in duplicate. mALK fragment (position 428 to 5767) from pME-mALK (kind gift from Dr Tadashi Yamamoto, Tokyo, Japan) was inserted at the NotI site in the pTTP vector (creating Cell growth assay pTTPmALK) using standard plasmid manipulation proce- For cell growth determination, 1 Â 104 SK-N-SH and SH- dures and sequenced before used. SY5Y cells per well were seeded in 24-well plates. Transfection was performed with Lipofectamine 2000 (Invitrogen) accord- Cell culture ing to manufacturer’s protocol with an efficiency of 60–80%. The stable PC12 Tet-on clone PCmALK was generated by As control, cells were transfected with 0.4 mg pcDNA3. For transfecting (Lipofectamine; Invitrogen, Paisley, GB) PC12 knockdown of Rap1, cells were transfected with 20 pmol Tet-on cells with the pTTPmALK plasmid according to siRap1. Rap1 activity was inhibited by transfection with 0.4 mg manual (BD Biosciences, Cat. No. 630921). Stable clones were HA-Rap1GAP. The next day cells were stimulated with 1 mg/ selected in Dulbecco’s modified Eagle’s medium, containing ml mAb46. The amount of viable cells was determined by 10% horse serum (MP Biomedicals, Solon, OH, USA), 5% Trypan blue staining for 5 days. Each sample was performed tetracycline-screened fetal calf serum (HyClone, Logan, UT, in triplicate. USA), penicillin, streptomycin, L-glutamine, 100 mg/ml G418 and 2 mg/ml puromycin at 37 1C and 5% CO2. The cell clones were screened for mALK expression by induction with 1 mg/ml doxycycline for 24 h before analysis by immunoblotting. Neuroblastoma cell lines SK-N-SH and SH-SY5Y were Conflict of interest cultured in Dulbecco’s modified Eagle’s medium, containing 10% fetal calf serum, penicillin, streptomycin and L-glutamine. The authors declare no conflict of interest. Cell lysis, immunoprecipitation and immunoblotting were preformed as described (Yang et al., 2007).

RNA interference Acknowledgements For Rap1 and C3G knockdown, double-stranded 25-nucleotide RNA duplexes (Stealth RNA-mediated interference; We thank Caroline Grabbe for comments and critical reading Invitrogen) targeting Rap1 (using siRNA-Rap1 no. 2 in of the paper and Lovisa Olofsson for initial experiments. BH is Figures 2b, 6b and c and 7a and b and siRNA no. 3 in supported by a Grant (08-0597) from the Swedish Cancer Supplementary Figure 2, which does not recognize Rap1B) or Foundation, Lions Cancer Research Foundation, Umea˚, and C3G (using siRNA-C3G no. 1 in Figure 5 and siRNA-C3G Grant (08/084) from the Swedish Childhood Cancer Founda- no. 2 in Supplementary Figure 2) were transfected into tion. RHP is a Swedish Cancer Foundation Research Fellow PCmALK cells using cell line nucleofector kit V (Amaxa and is supported by grants from the Swedish Research Council Biosystems) with an efﬁciency of 60–80%. As a negative (621-2003-3399), the Swedish Childhood Cancer Foundation control Stealth RNAi negative control medium CG duplex (08/074) and the Association for International Cancer (Invitrogen) was used. Research (AICR 08-0177).

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