Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas

Abstract

The majority of anaplastic large cell lymphomas (ALCLs) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is oncogenic due to its constitutive tyrosine kinase activity. Transformation by NPM-ALK not only increases proliferation, but also modifies cell shape and motility in both lymphoid and fibroblastic cells. We report that the Rac1 GTPase, a known cytoskeletal regulator, is activated by NPM-ALK in ALCL cell lines (Karpas 299 and Cost) and transfected cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We have identified Vav3 as one of the exchange factors involved in Rac1 activation. Stimulation of Vav3 and Rac1 by NPM-ALK is under the control of Src kinases. It involves formation of a signaling complex between NPM-ALK, pp60c-src, Lyn and Vav3, in which Vav3 associates with tyrosine 343 of NPM-ALK via its SH2 domain. Moreover, Vav3 is phosphorylated in NPM-ALK positive biopsies from patients suffering from ALCL, demonstrating the pathological relevance of this observation. The use of Vav3-specific shRNA and a dominant negative Rac1 mutant demonstrates the central role of GTPases in NPM-ALK elicited motility and invasion.

Introduction

Anaplastic large cell lymphomas (ALCLs), a subtype of high-grade non-Hodgkin's lymphomas of T or null phenotype, are characterized by the aberrant expression of the oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) in 75% of cases (Lamant et al., 1996; Duyster et al., 2001; Falini, 2001). The constitutive tyrosine kinase activity of NPM-ALK is responsible for malignant transformation of fibroblasts and lymphoid cells and was shown to induce B- and T-cell lymphomas in transgenic mice (Chiarle et al., 2003).

NPM-ALK-induced transformation depends on the activation of signaling pathways shared by many oncogenic tyrosine kinases. Pro-mitogenic functions include binding of adaptors, such as Shc, Grb2 and IRS1, to regulators of the Erk pathway, phospholipase Cγ (PLCγ), tyrosine phosphatases and the proto-oncogene pp60c-src (Fujimoto et al., 1996; Bai et al., 1998; Cussac et al., 2004; Honorat et al., 2006; Marzec et al., 2007). Antiapoptotic functions are related to the activation of the survival phosphatidylinositol 3-kinase (PI3K)/AKT pathway and of the Jak/STAT3–5 module (Bai et al., 2000; Amin et al., 2003; Chiarle et al., 2005).

However, a hierarchy of downstream signaling, in terms of importance for transformation, diagnosis and prognosis of the disease, remains to be established. With this goal in mind, several large or medium scale proteomic and transcriptomic studies were undertaken by several groups (Lamant et al., 2006; Lim and Elenitoba-Johnson, 2006). New partners have been found that account for different functions of NPM-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 ‘cytoskeleton and motility regulators’ in the context of ALCLs (Crockett et al., 2004; Cussac et al., 2006). Ambrogio et al. (2005) reported the association of NPM-ALK with p130Crk associated substrate (p130Cas) and described its role in actin depolymerization and transformation. Along these lines, NPM-ALK expression alters fibroblasts shape dramatically. They display an elongated phenotype with extensions similar to what is observed in PC12 cells transformed by the native ALK receptor, indicating that the kinase activity is responsible for the change in morphology.

Our group and others have described modifications of the expression of regulators of the Rho GTPases (Crockett et al., 2004; Cussac et al., 2006). We observed the extinction of the GTPase inhibitor RhoGDI2 in the proteome of an NPM-ALK(+) cell line, an effect shown to correlate with higher metastatic activity and poor prognosis in bladder cancers (Theodorescu et al., 2004; Cussac et al., 2006). These findings suggested that upregulation of Rho GTPases might be of importance during the progression of the disease. Recently, about 45 proteins were reported to interact with NPM-ALK, including various Rho GTPase activating proteins whose pattern of expression was also altered in a study of the transcriptome of NPM-ALK(+) cells (Crockett et al., 2004; Lamant et al., 2006). Rho GTPases mediate many aspects of cell biology including proliferation, regulation of the cell survival, polarity, adhesion, membrane trafficking and motility (Hall, 2005). The high incidence of overexpression of some GTPases (RhoA, RhoC, Rac1, Rac3 and Cdc42) or their regulators in human tumors suggests that GTPases play a role in carcinogenesis (Sahai and Marshall, 2002). The most studied members of the family are RhoA, Rac1 and Cdc42, which exert their transformant effects by regulating cell cycle progression via the cyclin-dependent kinases, and promoting migration and metastasis through regulation of cytoskeleton dynamics (Sahai and Marshall, 2002). Rac1 and Cdc42 regulate actin polymerization through the Arp2/3 complex with Rac1 involved in the generation of motile structures and Cdc42 in the establishment of polarity. RhoA organizes stress fibers predominantly through its effector Rho kinase (Hall, 1998). In addition, Rho GTPase signaling was demonstrated to be necessary for the oncogenicity of other proteins, especially for oncogenes derived from receptor tyrosine kinases, such as EGFR, IGFR, MET or RET (Aznar et al., 2004; Titus et al., 2005).

In this study, we demonstrate that Rac1 is activated in NPM-ALK expressing cells and is regulated by PI3K and Src family kinases. The Vav3 proto-oncogene is involved in bridging NPM-ALK and Rac1. The NPM-ALK chimera forms a multiprotein complex containing pp60c-src, Lyn and phosphorylated Vav3. Importantly, we observed activation of Vav3 in tumors from patients developing ALCL. Altering either Vav3 or the Rac pathway, by RNA interference or with dominant negative mutants and toxins, blocked invasion by NPM-ALK(+) cells. Altogether, our data demonstrate a critical role for Rho GTPases in ALCL.

Results

NPM-ALK activates the GTPase Rac1 via PI3K and Src

Activation of the Rho GTPases was first studied in two NPM-ALK positive ALCL cell lines, Karpas 299 (common type) and Cost (small cells, aggressive variant) (Falini et al., 1998; Lamant et al., 2004). Pull-down assays demonstrated that Rac1 was strongly activated in both cell lines (Figure 1a). NPM-ALK activation can be monitored by its autophosphorylation on tyrosine 664. We used two complementary approaches to determine whether NPM-ALK was responsible for Rac1 activation. First, we took advantage of the small molecule inhibitor WHI-154 that inhibits the ALK kinase (Marzec et al., 2005). Inactivation of NPM-ALK resulted in a marked decrease in Rac1 activation (Figure 1a). Second, we examined Rac1 in cell lines of independent origin (NIH-3T3 fibroblasts and Ba/F3 lymphoid cells) that were demonstrated to become transformed by stable expression of ALK oncogenic fusions (Armstrong et al., 2004). NPM-ALK-dependent Rac1 activation was again observed (Figures 1b and c).

Figure 1
figure1

Rac1 is activated downstream of NPM-ALK. Levels of active Rac1 were evaluated by pull-down experiments with GST-PBD as described in Materials and methods in (a) Karpas 299 and Cost cells treated for 1 h with 15 μM of the NPM-ALK inhibitor WHI-154, and (b) Control or NPM-ALK expressing NIH-3T3 cells, (c) Rac1 activation is dependent on PI3K and Src. Control or NPM-ALK expressing Ba/F3 cells were treated for 30 min with 25 μM of the PI3K inhibitor LY294002 or 2 μM of the Src inhibitor SU6656. Active Rac1-GTP and total Rac1 in the extracts were assessed by western blotting with antibodies to Rac1. The activation of NPM-ALK was followed with antibodies to the phosphorylated Y664 (pNPM-ALK(Y664)) and NPM-ALK level with ALK antibody. Phosphorylation of Rac effectors PAK1/2 was followed with anti-phospho-PAK1/2 (S199–204/S192–197) antibodies. α-Tubulin was used as loading control. (d) and (e) Karpas 299, Cost, Ba/F3 and NIH-3T3 cells were treated as above then levels of active Cdc42 and active RhoA were evaluated by pull-down experiments with GST-PBD and GST-RBD, respectively. Active Cdc42-GTP, RhoA-GTP, total Cdc42 and RhoA in the extracts were assessed by western blotting with antibodies to Cdc42 and RhoA. Data are representative of three to five experiments.

Small GTPase activation requires guanosine exchange factors (GEFs), which can be regulated by PI3K products and kinases of the Src family (Hall, 2005). The p85 regulatory subunit of PI3K and pp60c-src were identified as downstream targets of NPM-ALK. Treatment of serum and IL3-deprived Ba/F3 cells with 25 μM of LY294002 (PI3K inhibitor) or 2 μM of SU6656 (indolinone inhibitor of Src kinases) abolished Rac1 activation (Figure 1c), showing that PI3K and Src kinases are important for NPM-ALK to signal to the GTPase. We previously demonstrated that NPM-ALK is a substrate for pp60c-src (Cussac et al., 2004). Hence, SU6656 treatment reduced NPM-ALK phosphorylation, making it difficult to conclude on a direct role of Src on Rac1 activation pathway (Figure 1c). To overcome this, we used RNA silencing to target pp60c-src and Lyn, two Src kinases involved in hematological malignancies (Cussac et al., 2004; Contri et al., 2005; Thompson et al., 2005), and found that both kinases could regulate NPM-ALK Y664 autophosphorylation (not shown). Activation of PAK1 (p21-activated kinase), a downstream target of Rac1, demonstrated the same pattern of regulation as the GTPase, as shown with antibodies to the active phosphorylated form of the kinase, indicating the functional relevance of Rac1 activation in terms of downstream signaling (Figure 1c).

Finally, evaluation of the activation status of RhoA and Cdc42 failed to demonstrate significant modifications in ALCLs and transfected cells (Figures 1d and e).

The proto-oncogene Vav3 is activated downstream of NPM-ALK

The Vav proto-oncogenes are the only GEFs with a structural hallmark of signal transducer proteins represented 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 kinase receptors (Bustelo, 2000; Hornstein et al., 2004). They are activated by phosphorylation by members of the Src family, and Vav1 and Vav3 were found to be associated with NPM-ALK partners, such as Grb2, Shc, the p85 regulatory unit of PI3K, pp60c-src and PLCγ (Bustelo, 2001). We therefore checked whether they could be targets of NPM-ALK by studying the activating phosphorylation on Y174 of Vav1 and Y173 of Vav3 with specific antibodies. Although some phosphorylation could be detected, inhibition of ALK did not affect the level of Vav1 Y174 phosphorylation, suggesting that NPM-ALK does not regulate Vav1 in ALCLs (Figure 2). Conversely, Figure 3a shows that NPM-ALK expression resulted in a robust phosphorylation of Vav3 in both Ba/F3 and NIH-3T3 cells. The same observation was made in Karpas 299 and Cost cells where ALK inhibition decreased Vav3 phosphorylation significantly (Figure 3b). We evaluated the status of Vav3 in protein extracts from four frozen lymph nodes from patients suffering from NPM-ALK(+) ALCLs after immunohistochemistry analysis of the biopsies (not shown). Again, Vav3 phosphorylation increased in tumor samples compared with control lymph nodes or peripheral blood lymphocytes, demonstrating the pathophysiological relevance of this observation (Figure 3c).

Figure 2
figure2

Modification of NPM-ALK activity does not affect phosphorylation of Vav1. The activating phosphorylation on Y174 of Vav1 was measured on Vav1 immunoprecipitates from Karpas 299 and Cost cells treated for 1 h with 15 μM WHI-154. Proteins present in the immunoprecipitates were analysed by western blotting with polyclonal anti-Vav1 and anti-pVav1 (Y174) antibodies. The activation of NPM-ALK was followed with the anti-pNPM-ALK(Y664) antibody, loading was assessed with the ALK antibody. Data are representative of two experiments.

Figure 3
figure3

The exchange factor Vav3 is phosphorylated in NPM-ALK expressing cells and in tumors from patients. The activating phosphorylation on Y173 of Vav3 was measured in total lysates from (a) control or NPM-ALK expressing Ba/F3 cells or NIH-3T3 cells, and (b) Karpas 299 and Cost cells treated for 1 h with 15 μM WHI-154. Lysates were immunoblotted with anti-pVav3 (Y173), anti-pNPM-ALK (Y664), anti-ALK or anti-Vav3 antibodies as loading control. Data are representative of three experiments. (c) Total lysates from two frozen control (LN) or four NPM-ALK positive lymph nodes and peripheral blood lymphocytes (PBL) from two healthy donors were immunoblotted with anti-pVav3 (Y173) or anti-human Vav3 antibodies.

In addition, we investigated whether Src kinases were involved in Vav3 phosphorylation. Treatment of Ba/F3 cells with SU6656 resulted in a decreased signal (Figure 4a, left panel). We then used RNA interference to target pp60c-src and Lyn. RNA silencing of pp60c-src demonstrated that it was responsible for Vav3 phosphorylation in this model (Figure 4a, right panel). Finally, we transfected cells with the L211Q inactive mutant of Vav3 containing a point mutation in the exchange DH (Dbl homology) domain (Figure 4b). In addition, we depleted Vav3 with specific short hairpin RNAs (shRNAs) (Figure 4c). In both cases, Rac1GTP levels were reduced, showing that functional Vav3 is required for Rac1 activation downstream of NPM-ALK.

Figure 4
figure4

Vav3 participates in Rac1 activation downstream of NPM-ALK. (a) NPM-ALK expressing Ba/F3 cells were treated for 30 min with 2 μM SU6656 or 1 h with 15 μM WHI-154 (left panel) or nucleofected with nothing (control) or 4 μM of siRNA targeting pp60c-src or Lyn (right panel). Total cell lysates were immunoblotted with the indicated antibodies. Tubulin was used as a loading control. (b) Control or NPM-ALK expressing NIH-3T3 cells were nontransfected (NT) or transfected with a plasmid expressing the GEF inactive L211Q human Vav3 mutant. Active Rac1-GTP was measured by pull-down experiments. Expression of the Vav3 mutant was assessed in total lysates with antibodies to human Vav3. (c) NPM-ALK expressing NIH-3T3 cells were not transfected (NT) or transfected with control vector (control) or a vector expressing short hairpin RNA (shRNA) to Vav3. After 40 h, levels of Rac1-GTP were assessed by pull-downs. The level of Vav3 was measured with anti-mouse Vav3.

The SH2 domain of Vav3 drives its association with phosphorylated NPM-ALK

Immunoprecipitations with an antibody directed against ALK demonstrated that a complex containing phosphorylated Vav3, pp60c-src and Lyn co-precipitated with the active oncogene (Figure 5a). In all cases, a decrease (but not abrogation) in the association is observed upon WHI-154 treatment suggesting that activation of NPM-ALK is important in the stabilization of the complex (Figure 5a). Figure 5b shows that the reciprocal immunoprecipitation of Vav3 also led to the same results. Overexpression of Vav3 mutants in NPM-ALK transfected cells confirmed the association of NPM-ALK, Vav3 and active Src kinases in this model (Figure 5c). Indeed, the Y173F mutant that was shown to adopt an open conformation facilitating access to partners and to GTPases (Llorca et al., 2005), binds to NPM-ALK strongly (Figure 5c). Interestingly, when we expressed the R697A mutant with a disabled phosphotyrosine binding SH2 domain, no binding to NPM-ALK was observed, demonstrating that the SH2 domain is crucial for the interaction (Figure 5c).

Figure 5
figure5

Vav3 forms a complex with NPM-ALK and Src kinases. (a) NPM-ALK was immunoprecipitated with ALK1 antibodies from Karpas 299 and Cost cells treated for 1 h with 15 μM WHI-154. Proteins present in the immunoprecipitates were analysed by western blotting with the indicated antibodies. (b) Vav3 was immunoprecipitated from Karpas 299 and Cost cells with anti-Vav3 antibodies. Proteins in the complexes were analysed as described above. (c) Control or NPM-ALK expressing NIH-3T3 cells were nontransfected (NT), or transfected with wild-type human Vav3 (WT), active open Y173F Vav3, GEF inactive L211Q Vav3 or SH2 defective R697A mutants. Twenty-four hours after transfection, proteins in NPM-ALK immunoprecipitates were analysed by immunoblotting. Expression of the mutants in total lysates was assessed with anti-human Vav3 antibody. (d) HEK293 cells were transfected with vectors expressing wild-type Vav3 and NPM-ALK mutated on the indicated tyrosines. Twenty-four hours after transfection, proteins in NPM-ALK immunoprecipitates were analysed with the indicated antibodies. Data are representative of two to four experiments.

To determine which NPM-ALK tyrosine residue was required to bind Vav3, we co-transfected HEK293 cells with wild-type Vav3 and different mutants of NPM-ALK, in which targeted tyrosines were replaced by phenylalanines (Y338F, Y342F, Y343F, Y418F (docking for pp60c-src) and Y664F (docking for PLCγ) mutants). Our data indicate that among the tested mutants, only Y343F had an impact on Vav3 binding (Figure 5d). pp60c-src is not a physical intermediate of Vav3 and NPM-ALK association since mutation of its binding site (Y418F) had no effect on the association. Interestingly, assessment of the intrinsic tyrosine kinase activity of NPM-ALK with the anti-pNPM-ALK(Y664) antibody confirmed that three of the mutants, namely, Y338F, Y342F and Y343F, had reduced kinase activity (Duyster et al., 2001).The fact that two of them are still capable of binding Vav3 indicates that Y343 is probably phosphorylated by another kinase still to identify.

Small GTPases drive NPM-ALK-induced migration

NPM-ALK was reported to affect cell morphology, adhesion and migration in various cell types. Those functions have clearly been attributed to GTPases of the Rho family in many normal cell types and in tumoral cells (Sahai and Marshall, 2002; Hall, 2005). In invasion assays through 3D Matrigel chambers, transfection of adherent fibroblasts with the dominant negative mutant Rac1T17N completely blocked cellular migration of NIH-3T3 NPM-ALK cells (Figure 6a). Similarly, depletion of Vav3 by RNA interference also reduced migration, confirming that Vav3/Rac1 signaling plays a major role in mediating NPM-ALK effects on cellular migration and invasion in vitro (Figure 6b).

Figure 6
figure6

Small GTPases are required for NPM-ALK-dependent migration in vitro. (a). Control or NPM-ALK expressing NIH-3T3 cells were transfected with pcDNA3-EGFP-Rac1T17N or the control vector. Thirty hours after transfection, cells were plated on Matrigel-coated inserts and the number of migrating GFP positive cells was evaluated as percentage of control cells. Lysates were immunoblotted with anti-GFP to control EGFP-Rac1T17N expression. (b) Cells were transfected with the short hairpin RNAs (shRNA) Vav3 targeting vector or with the control vector and analysed as above. Lysates were immunoblotted with anti-mouse Vav3 to control Vav3 silencing. Control, cells transfected with the control vector; shRNA Vav3, cells transfected with the Vav3 targeting vector. The levels and the activation of NPM-ALK were assessed with anti-ALK and anti-pNPM-ALK(Y664) antibodies. Data are expressed as mean±s.e.m.

Discussion

We report the PI3K- and Src-dependent activation of Rac1 downstream of NPM-ALK in two ALCL cell lines (Karpas 299 and Cost), Ba/F3 and NIH-3T3 transfected cells. To find the link between NPM-ALK and Rac, we focused on the Vav family of GEFs (Vav1, Vav2 and Vav3). In addition to their GEF activity, Vavs have the unique feature of acting as docking proteins, which makes them excellent candidates for relaying NPM-ALK functions (Bustelo, 2001). Indeed, these GEFs are major regulators of lymphocyte function, and Vav1 and Rac were involved in BCR-ABL signaling in leukemia (Bassermann et al., 2002; Cho et al., 2005). Overexpression of active Vav1 and Vav3 mutants have phenotypes reminiscent of NPM-ALK transformed cells (Movilla and Bustelo, 1999; Zeng et al., 2000; Hornstein et al., 2004). Although Vav2 was shown to act downstream of tyrosine kinases such as the EGF and PDGF receptors, there are major differences between Vav2 and NPM-ALK signaling. Specifically, overexpression of Vav2 generates strong stress fibers while NPM-ALK cells are characterized by a disappearance of actin cables (Liu and Burridge, 2000; Ambrogio et al., 2005). Moreover, Vav2 was described as being more prone to activate RhoA than Vav1 and Vav3, which fits with its action on the actin cytoskeleton but does not match the pattern of NPM-ALK-induced GTPase activation since RhoA activity was fairly weak and not affected by the oncogene. These observations do not favor a role of Vav2 in NPM-ALK transformation. Moreover, we demonstrated that no difference in Vav1 activating phosphorylation was observed when NPM-ALK activity was challenged. Conversely, Vav3 is phosphorylated on the activating Y173 downstream of NPM-ALK in ALCL and transfected cells as well as in lymph node biopsies originating from patients suffering from NPM-ALK(+) lymphomas, suggesting that this pathway could be a valid target in the human pathology. Because we demonstrated that it is ALK kinase activity that drives Rac1 activation, it is likely that other translocations involving the ALK kinase domain would also activate Rac1. A broader study will determine if activation of Vav3/Rac1 is a common feature of neoplasia resulting from deregulated ALK activity.

It is now well accepted that pathways regulated by Rho GTPases are very important in human cancers. Their role in tumorigenesis was first demonstrated in fibroblasts overexpressing dominant positive forms of RhoA, Rac1 or Cdc42. Not only was their proliferation deregulated, but they also induced lung metastasis when subcutaneously grafted in mice. Since then, they were shown to be necessary for transformation evoked by oncogenes such as Ras or receptor tyrosine kinases (Sahai and Marshall, 2002; Titus et al., 2005). Beside their effects on cell proliferation, GTPases are central modulators of the actin and microtubule cytoskeleton, cell adhesion and motility (Hall, 1998). Using an EGFP tagged version of the dominant negative mutant Rac1T17N, we demonstrated that small GTPases drove NPM-ALK elicited migration. In fact, the balance between Rac, Cdc42 and RhoA activities determines cell morphology and migration. In ALCLs and NPM-ALK transfected cells, Rac1 activity was strong while RhoA-GTP was difficult to detect. Indeed, it was demonstrated that Rac activation can downregulate RhoA, leading to the dissolution of focal adhesions and increasing cell motility (Rottner et al., 1999). We observed a 10-fold decrease in the number of focal adhesions associated with the expression of NPM-ALK in NIH-3T3 fibroblasts (not shown), the biological impact of a cross-regulation between Rac1 and RhoA in ALCL is currently under investigation. Interestingly, when we depleted cells for Vav3 using RNA interference, we also observed a blockade of migration through 3D Matrigel that was less intense than Rac1T17N (66.7% inhibition for shVav3 versus 84.25% for Rac1T17N). This Rac1 mutant acts by sequestering the upstream Rho GEFs and thereby inhibiting endogenous Rho GTPases. It then affects more than one GEF/GTPase couple and its stronger effect on migration suggests several GEFs act downstream of NPM-ALK. Accordingly, the use of the Rac GEF inhibitor NSC23766 (Gao et al., 2004), reported not to act on Vav, resulted in a blockade of migration similar to Vav3 depletion (61.28% of inhibition). We are currently investigating the synergy between different GEFs and their regulatory pathways in ALCLs.

Recently, small GTPases have become potential candidates for anticancer therapy. Different types of natural molecules and synthetic drugs that inactivate GTPases or their effectors are available, some of them show striking antineoplastic or antimetastatic activity (Aznar et al., 2004; Fritz and Kaina, 2006). We have tested Clostridium toxins (Petit et al., 2003; Genth et al., 2006). Both Toxin B from Clostridium difficile (affects Rac, RhoA and Cdc42) and LT-IP82 from Clostridium sordellii (specific for Rac, Ras, Ral and Rap) affected NPM-ALK-induced migration but also blocked proliferation, demonstrating that the role of GTPases downstream of NPM-ALK is broader than migration and invasion (Colomba, A and Gaits-Iacovoni, F., unpublished). Altogether, our observations demonstrate a central role for Rac1 in NPM-ALK positive lymphomas. These data illustrate how an oncogene, here NPM-ALK, can potentiate its transforming activity by recruiting proto-oncogenes (such as pp60c-src and Vav3) in its vicinity. Interestingly, both the common type and a small cell variant of ALCL display the same pattern of activation of Rho GTPases, indicating that targeting GTPases could be a therapeutic avenue valid to treat not only the primary disease, but also prevent relapses of the most aggressive subtypes.

Materials and methods

Reagents and antibodies

Peripheral lymph nodes biopsies were obtained from patients diagnosed for NPM-ALK(+) ALCLs at the Department of Pathology of Toulouse University Medical Center (France), after informed consent. The study was approved by the institutional review board of the Purpan's Hospital, Toulouse, France. Cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). SU6656 and WHI-154 were from Calbiochem (San Diego, CA, USA). All other chemicals were from Sigma-Aldrich (St Louis, MO, USA). Antibodies used were ALK1 (DakoCytomation, Glostrup, Denmark); Rac1, human Vav3 and mouse Vav3 (Upstate Biotechnology, Lake Placid, NY); α-tubulin (Sigma-Aldrich); ALK, pPAK1/2 (S199–204/S192–197), pSrc (Y416), pALK (Y1604) that recognizes Y664 on NPM-ALK called pNPM-ALK (Y664) (Cell Signaling Technologies, Beverly, MA, USA); pVav3(Y173) (Biosource International); Vav1, pVav1(Y174), Cdc42, RhoA, c-src (clone H-12), Lyn and GFP (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin antisera were from Promega (Madison, WI, USA).

Cell culture, plasmids and transfection

HEK293 and NIH-3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium and Ba/F3 lymphoid cells in RPMI 1640 containing 2 ng ml−1 murine recombinant IL-3 (mrIL3) (R&D Systems Europe, Abingdon, UK). Media were supplemented with 10% fetal calf serum (FCS), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 0.5 mg ml−1 geneticin (G418) for cells stably expressing NPM-ALK. Human ALCLs cell lines Karpas 299 and Cost were cultured in Iscove's modified Dulbecco's medium supplemented with 15% FCS. Before immunoprecipitation, GTPases activation and western blot studies, Karpas 299, Cost and Ba/F3 cells were serum and IL3 starved overnight and NIH-3T3 cells were maintained in 2% serum overnight. NIH-3T3 were transfected using Lipofectamine (Invitrogen) and HEK293 with Effectene (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Ba/F3 NPM-ALK cells were transiently nucleofected using the Amaxa technology (Amaxa, Koeln, Germany). Briefly, 2 × 106 cells were nucleofected using the Amaxa solution kit V, 4 μM of siRNA smartpool pp60c-src or Lyn (Dharmacon Inc., Lafayette, CO, USA) and the program X-01 following the Amaxa guidelines. Cells were lysed 48 h post nucleofection. pcDNA3 containing wild-type NPM-ALK or the Y338F, Y342F, Y343F, Y418F and Y664F mutants were already described (Duyster et al., 2001; Cussac et al., 2004). pcDNA3-EGFP-Rac1T17N was a generous gift from Dr K Hahn (University of North Carolina, Chapel Hill, NC, USA). pCI2.F.hVav3.WT, pCI2.F.hVav3.Y173F, pCI2.F.hVav3.L211Q, pCDNA3.F.hVav3R697A, and pCMS3.H1P and pCMS3.H1P.shVav3 that contain a separate transcriptional cassette driving Green Fluorescent Protein (GFP) expression allowing easy identification of transfected cells were already described (Zakaria et al., 2004; Charvet et al., 2005).

Cell lysis, immunoprecipitation and immunoblotting

Total proteins were extracted with lysis buffer (50 mM Tris-base pH 8, 150 mM NaCl, 5 mM EGTA, 1% Nonidet P-40, 1 mM PMSF, 25 mM NaF, 2 mM Na3VO4, 10 μg ml−1 leupeptin and 2 μg ml−1 aprotinin). For lysates from NPM-ALK positive or negative lymph nodes from ALCLs patients, frozen tissues were sonicated in detergent-free lysis buffer, cleared by centrifugation and proteins in the supernatant quantitated with the Bio-Rad protein assay (Bio-Rad, Munich, Germany). For immunoprecipitations, clarified homogenates were incubated overnight at 4 °C with suited antibodies and a mix of protein A/G sepharose beads. After washes, proteins were eluted with Laemmli buffer and analysed by SDS–PAGE followed by western blotting on Immobilon-P membranes (Millipore, Billerica, MA, USA). Immunoreactive bands were detected by chemiluminescence with the SuperSignal detection system (Pierce Chemical Co, Rockford, IL, USA).

GTPases pull-down assays

The amounts of GTP-bound active Rac1, Cdc42 or RhoA were determinated by pull-down as previously described (Benard and Bokoch, 2002). Briefly, cells were lysed in ice-cold lysis buffer (50 mM Tris-base pH 7.4, 500 mM NaCl, 10 mM MgCl2, 2.5 mM EGTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 50 mM NaF, 1 mM Na3VO4, 10 μg ml−1 leupeptin and 2 μg ml−1 aprotinin) and clarified lysates were incubated with 30 μg GST-PBD (PAK1 Binding Domain for Rac1 and Cdc42) or GST-RBD (Rhotekin Binding Domain for RhoA) bound to glutathione sepharose at 4 °C for 30 min. Beads were washed with 50 mM Tris-base pH 7.4, 150 mM NaCl, 1 mM MgCl2, 5 mM EGTA, 1% triton X-100 and GTP-bound GTPases eluted with Laemmli buffer and subjected to SDS–PAGE followed by western blotting.

Cell invasion assay

To assess the role of Rac1 and Vav3 in migration, NIH-3T3 cells were transfected with pCMS3.H1P (expressing GFP), pCMS3.H1P.shVav3 (expressing GFP and shRNA targeting Vav3) or pcDNA3.EGFP.Rac1T17N. After 30 h, cells were seeded in 24-well plates on biocoated Matrigel Invasion Chambers that consisted in a 8 μm-size pore filter coated with a reconstituted basal membrane matrix (Becton Dickinson, Mountain View, CA, USA). Migration proceeded for 17 h at 37 °C. Then, cells on the upper side of the filters were scrapped with a cotton swab and cells positive for GFP were scored. A correction index was applied to the raw values, it corresponds to the growth of the various transfectants over 17 h. Results are expressed as percentages of control cells expressing GFP only.

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Acknowledgements

We thank Dr Popoff for Clostridium lethal toxins. We are grateful to Dr H Tronchère, Dr S Manenti, Dr MP Gratacap, Dr C Racaud-Sultan and Dr M Plantavid for helpful discussions. AC and DR were financed by the ‘Ministère de la Recherche et de la Technologie’ and the ‘Association pour la Recherche sur le Cancer’. This work was supported by grants from the INSERM, ARC, ARECA, La Ligue contre le Cancer, the ‘Cancéropôle Grand Sud-Ouest’ and the ‘Institut National du Cancer’ (INCa), the Région Midi-Pyrénées and the ‘Pôle de Compétitivité Cancer-Bio Santé’.

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Correspondence to F Gaits-Iacovoni.

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Colomba, A., Courilleau, D., Ramel, D. et al. Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas. Oncogene 27, 2728–2736 (2008). https://doi.org/10.1038/sj.onc.1210921

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Keywords

  • anaplastic large cell lymphomas
  • NPM-ALK
  • Rho GTPases
  • Vav3

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