Oncogene (2008) 27, 2728–2736 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALKsignaling in anaplastic large cell lymphomas A Colomba1,2, D Courilleau1,2, D Ramel1,2, DD Billadeau3,4, E Espinos1,2, G Delsol1,2, B Payrastre1,2 and F Gaits-Iacovoni1,2 1INSERM, U563, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France; 2Universite´ Toulouse III Paul-Sabatier, IFR30, Toulouse, France; 3Division of Developmental Oncology Research, Mayo Clinic College of Medicine, Rochester, MN, USA and 4Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN, USA The majority of anaplastic large cell lymphomas (ALCLs) (Lamant et al., 1996; Duyster et al., 2001; Falini, 2001). express the nucleophosmin-anaplastic lymphoma kinase The constitutive tyrosine kinase activity of NPM-ALK (NPM-ALK) fusion protein, which is oncogenic due to its is responsible for malignant transformation of fibro- constitutive tyrosine kinase activity. Transformation by blasts and lymphoid cells and was shown to induce B- NPM-ALKnot only increases proliferation, but also and T-cell lymphomas in transgenic mice (Chiarle et al., modifies cell shape and motility in both lymphoid and 2003). fibroblastic cells. We report that the Rac1 GTPase, a NPM-ALK-induced transformation depends on the known cytoskeletal regulator, is activated by NPM-ALK activation of signaling pathways shared by many in ALCL cell lines (Karpas 299 and Cost) and transfected oncogenic tyrosine kinases. Pro-mitogenic functions cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We include binding of adaptors, such as Shc, Grb2 and have identified Vav3 as one of the exchange factors IRS1, to regulators of the Erk pathway, phospholipase involved in Rac1 activation. Stimulation of Vav3 and Cg (PLCg), tyrosine phosphatases and the proto- Rac1 by NPM-ALKis under the control of Src kinases. It oncogene pp60c-src (Fujimoto et al., 1996; Bai et al., involves formation of a signaling complex between NPM- 1998; Cussac et al., 2004; Honorat et al., 2006; Marzec ALK, pp60c-src, Lyn and Vav3, in which Vav3 associates et al., 2007). Antiapoptoticfunctionsare related to the with tyrosine 343 of NPM-ALKvia its SH2 domain. activation of the survival phosphatidylinositol 3-kinase Moreover, Vav3 is phosphorylated in NPM-ALKpositive (PI3K)/AKT pathway and of the Jak/STAT3–5 module biopsies from patients suffering from ALCL, demonstrat- (Bai et al., 2000; Amin et al., 2003; Chiarle et al., 2005). ing the pathological relevance of this observation. The use However, a hierarchy of downstream signaling, in of Vav3-specific shRNA and a dominant negative Rac1 terms of importance for transformation, diagnosis and mutant demonstrates the central role of GTPases in prognosis of the disease, remains to be established. With NPM-ALKelicited motility and invasion. this goal in mind, several large or medium scale Oncogene (2008) 27, 2728–2736; doi:10.1038/sj.onc.1210921; proteomicand transcriptomicstudies were undertaken published online 12 November 2007 by several groups (Lamant et al., 2006; Lim and Elenitoba-Johnson, 2006). New partners have been Keywords: anaplasticlarge celllymphomas; NPM- found that account for different functions of NPM- ALK; Rho GTPases; Vav3 ALK, such as the regulation of mRNA turnover (Fawal et al., 2006). Among others, proteins regulating cell shape and cytoskeleton plasticity, two important features altered in transformed cells, were identified, leading to investigations of molecules classified as Introduction ‘cytoskeleton and motility regulators’ in the context of ALCLs (Crockett et al., 2004; Cussac et al., 2006). Anaplasticlarge celllymphomas (ALCLs), a subtype of Ambrogio et al. (2005) reported the association of high-grade non-Hodgkin’s lymphomas of T or null NPM-ALK with p130Crk associated substrate phenotype, are characterized by the aberrant expression (p130Cas) and described its role in actin depolymeriza- of the oncogenic fusion protein nucleophosmin-anaplas- tion and transformation. Along these lines, NPM-ALK ticlymphoma kinase (NPM-ALK) in 75% of cases expression alters fibroblasts shape dramatically. They display an elongated phenotype with extensions similar to what is observed in PC12 cells transformed by the Correspondence: Dr F Gaits-Iacovoni, INSERM, U563, Dpt d’Onco- native ALK receptor, indicating that the kinase activity gene` se, Signalisation et Innovation the´ rapeutique, CHU Purpan, BP is responsible for the change in morphology. 3028, Toulouse cedex 3, Midi pyrenees 31024, France. E-mail: [email protected] Our group and others have described modifications of Received 11 May 2007; revised 27 September 2007; accepted 16 October the expression of regulators of the Rho GTPases 2007; published online 12 November 2007 (Crockett et al., 2004; Cussac et al., 2006). We observed Role of GTPases in NPM-ALK( þ ) ALCLs A Colomba et al 2729 the extinction of the GTPase inhibitor RhoGDI2 in the First, we took advantage of the small molecule inhibitor proteome of an NPM-ALK( þ ) cell line, an effect WHI-154 that inhibits the ALK kinase (Marzec et al., shown to correlate with higher metastatic activity and 2005). Inactivation of NPM-ALK resulted in a marked poor prognosis in bladder cancers (Theodorescu et al., decrease in Rac1 activation (Figure 1a). Second, we 2004; Cussac et al., 2006). These findings suggested that examined Rac1 in cell lines of independent origin (NIH- upregulation of Rho GTPases might be of importance 3T3 fibroblasts and Ba/F3 lymphoid cells) that were during the progression of the disease. Recently, about 45 demonstrated to become transformed by stable expres- proteins were reported to interact with NPM-ALK, sion of ALK oncogenic fusions (Armstrong et al., 2004). including various Rho GTPase activating proteins NPM-ALK-dependent Rac1 activation was again ob- whose pattern of expression was also altered in a study served (Figures 1b and c). of the transcriptome of NPM-ALK( þ ) cells (Crockett Small GTPase activation requires guanosine exchange et al., 2004; Lamant et al., 2006). Rho GTPases mediate factors (GEFs), which can be regulated by PI3K many aspects of cell biology including proliferation, products and kinases of the Src family (Hall, 2005). regulation of the cell survival, polarity, adhesion, The p85 regulatory subunit of PI3K and pp60c-src were membrane trafficking and motility (Hall, 2005). The identified as downstream targets of NPM-ALK. Treat- high incidence of overexpression of some GTPases ment of serum and IL3-deprived Ba/F3 cells with 25 mM (RhoA, RhoC, Rac1, Rac3 and Cdc42) or their of LY294002 (PI3K inhibitor) or 2 mM of SU6656 regulators in human tumors suggests that GTPases play (indolinone inhibitor of Srckinases) abolished Rac1 a role in carcinogenesis (Sahai and Marshall, 2002). The activation (Figure 1c), showing that PI3K and Src most studied members of the family are RhoA, Rac1 kinases are important for NPM-ALK to signal to the and Cdc42, which exert their transformant effects by GTPase. We previously demonstrated that NPM-ALK regulating cell cycle progression via the cyclin-depen- is a substrate for pp60c-src (Cussac et al., 2004). Hence, dent kinases, and promoting migration and metastasis SU6656 treatment reduced NPM-ALK phosphoryla- through regulation of cytoskeleton dynamics (Sahai and tion, making it difficult to conclude on a direct role of Marshall, 2002). Rac1 and Cdc42 regulate actin poly- Src on Rac1 activation pathway (Figure 1c). To merization through the Arp2/3 complex with Rac1 overcome this, we used RNA silencing to target pp60c-src involved in the generation of motile structures and and Lyn, two Srckinases involved in hematological Cdc42 in the establishment of polarity. RhoA organizes malignancies (Cussac et al., 2004; Contri et al., 2005; stress fibers predominantly through its effector Rho Thompson et al., 2005), and found that both kinases kinase (Hall, 1998). In addition, Rho GTPase signaling could regulate NPM-ALK Y664 autophosphorylation was demonstrated to be necessary for the oncogenicity (not shown). Activation of PAK1 (p21-activated kinase), of other proteins, especially for oncogenes derived from a downstream target of Rac1, demonstrated the same receptor tyrosine kinases, such as EGFR, IGFR, MET pattern of regulation as the GTPase, as shown with or RET (Aznar et al., 2004; Titus et al., 2005). antibodies to the active phosphorylated form of the In this study, we demonstrate that Rac1 is activated in kinase, indicating the functional relevance of Rac1 NPM-ALK expressing cells and is regulated by PI3K activation in terms of downstream signaling (Figure 1c). and Srcfamily kinases. The Vav3 proto-oncogeneis Finally, evaluation of the activation status of RhoA involved in bridging NPM-ALK and Rac1. The NPM- and Cdc42 failed to demonstrate significant modifica- ALK chimera forms a multiprotein complex containing tions in ALCLs and transfected cells (Figures 1d and e). pp60c-src, Lyn and phosphorylated Vav3. Importantly, we observed activation of Vav3 in tumors from patients developing ALCL. Altering either Vav3 or the Rac The proto-oncogene Vav3 is activated downstream of pathway, by RNA interference or with dominant NPM-ALK negative mutants and toxins, blocked invasion by The Vav proto-oncogenes are the only GEFs with a NPM-ALK( þ ) cells. Altogether, our data demonstrate structural hallmark of signal transducer proteins repre- a critical role for Rho GTPases in ALCL. sented by the SH3-SH2-SH3 (Src homology 2 or 3) module at their C terminus. This unique feature suggests that they could act as nucleation points for multiple signaling complexes after being recruited by tyrosine Results kinase receptors (Bustelo, 2000; Hornstein et al., 2004). They are activated by phosphorylation by members of NPM-ALK activates the GTPase Rac1 via PI3K and Src the Srcfamily, and Vav1 and Vav3 were found to be Activation of the Rho GTPases was first studied in two associated with NPM-ALK partners, such as Grb2, Shc, NPM-ALK positive ALCL cell lines, Karpas 299 the p85 regulatory unit of PI3K, pp60c-src and PLCg (common type) and Cost (small cells, aggressive variant) (Bustelo, 2001).