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Tumor Biology |
Departments of Pathology and Pediatrics, BC Research Institute for Children’s and Women’s Health, Vancouver, British Columbia, V6H 3V4 [C. T., M. G., E. K., K. M., P. H. B. S.], and Department of Medical Genetics, Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, V5Z 4E6 [R. K.], Canada
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ETV6-NTRK3 chimeric transcripts encode the helix-loop-helix or pointed domain of ETV6 fused to the PTK domain of NTRK3 (1) . ETV6 (also known as TEL) is an ETS family transcription factor thought to play a major role in early hematopoiesis and angiogenesis (8 , 9) . The ETV6 gene has also been identified as a fusion partner in leukemia-associated chimeric proteins, such as ETV6-PDGFR (10) , ETV6-AML1 (11 , 12) , ETV6-JAK2 (13) , ETV6-ARG (14) , and others (15) . Moreover, an ETV6-NTRK3 variant fusion lacking ETV6 exon 5 has been reported recently in a case of AML occurring in an adult patient (16) . The NTRK3 gene (also known as TRKC) encodes the transmembrane surface receptor for neurotrophin-3 involved in growth, development, and cell survival in the central nervous system (reviewed in Ref. 17 ). Other reports highlight potential roles for NTRK receptors in oncogenesis. NTRK1 (TRKA) sequences were originally isolated from a colon carcinoma biopsy as part of an oncogene encoding the NH2-terminal portion of tropomyosin (TPM3) fused to a truncated tyrosine kinase receptor (18) . TPM3-NTRK1 fusions were subsequently detected in papillary thyroid carcinomas (19) . Altered NTRK signaling has been implicated in other neoplasms (reviewed in Ref. 20 ), including pancreatic adenocarcinoma (21) , AML (22) , and prostate cancer (23) . We showed that ETV6-NTRK3 functions as a chimeric PTK with potent transforming activity in NIH3T3 cells (24) . In addition, the ETV6-NTRK3 protein associated with AML induced a rapidly fatal myeloproliferative disease in a murine bone marrow transplant model system (25) . Therefore, ETV6-NTRK3 appears to have oncogenic activity in both mesenchymal and hematological cells, and elucidation of pathways activated by this molecule may provide novel insights into how NTRK signaling contributes to oncogenesis.
The mechanism of ETV6-NTRK3-mediated oncogenesis remains unknown. As in other ETV6 fusion proteins, ETV6-NTRK3 is capable of homodimerization or heterodimerization with endogenous ETV6 via the helix-loop-helix domain (24)
. Therefore, this domain likely mediates ligand-independent dimerization and PTK activation (24
, 25)
. This led us to hypothesize that ETV6-NTRK3 transformation is mediated by constitutive NTRK3 signaling. However, ETV6-NTRK3 fails to interact with adapter molecules known to associate with wild-type NTRK3 (24
, 25)
. These include Shc and Grb2, which link NTRK3 to the Ras-Erk1/2 MAP kinase pathway involved in mitogenesis or differentiation (17
, 26)
or the p85 subunit of PI3K linking NTRK3 with the PI3K-Akt neuronal survival pathway (17
, 26
, 27)
. This is actually predicted from the position of the ETV6-NTRK3 breakpoint, as NTRK3 tyrosine residue 516, the site of both Shc and p85 binding, is not present in ETV6-NTRK3 (1)
. ETV6-NTRK3 does bind PLC
, another known NTRK3 interactor which activates PKC, but PTK-active mutants unable to bind PLC did not show defects in transformation activity (24)
. Therefore, it remains unclear whether ETV6-NTRK3-mediated oncogenesis involves unknown links to Ras-Erk1/2 MAP kinase and/or PI3K-Akt cascades or whether novel mechanisms are activated by this chimeric oncoprotein.
In this study, we focused on the potential roles of the Ras-Erk1/2 and PI3K-Akt pathways in ETV6-NTRK3 transformation and assessed whether their pharmacological inhibition could block ETV6-NTRK3-mediated transformation. We found that expression of the fusion protein was associated with constitutively high levels of phosphorylated Mek1 and Akt, even in the absence of serum. Moreover, ETV6-NTRK3-expressing cells showed serum-independent elevation of cyclin D1 protein. When we tested a panel of specific signaling inhibitors for their ability to reverse the transformed phenotype of ETV6-NTRK3-expressing cells, we found that inhibition of either the Ras-Erk1/2 MAP kinase or the PI3K-Akt pathway alone completely blocked colony formation of ETV6-NTRK3-expressing cells in soft agar assays. Furthermore, the constitutive expression of cyclin D1 protein in ETV6-NTRK3-expressing cells was down-regulated by MEK1 or PI3K inhibition, suggesting a link between activation of both pathways and aberrant cell cycle progression in ETV6-NTRK3 transformation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retroviral Constructs.
cDNAs encoding ETV6-NTRK3 or the kinase dead mutant (K380N) were inserted into the retroviral vector MSCVpac (28)
at the EcoRI site. The pWZL RKIP Blast construct was a generous gift from Dr. Kam C. Yeung (Brown University, Providence, RI). The DN-Ras construct contained a 17N mutation.
Generation of Retrovirally Transduced Cell Lines.
Retroviral vector plasmid DNA was transfected into the BOSC23 ecotropic retroviral packaging cell line using calcium phosphate precipitation as described by Pear et al. (29)
. Supplemental Gag/Pol (pGP1) and Env plasmids were used during the transfection procedure to increase viral titers. Retrovirus-containing supernatants were collected from the BOSC cells 48 h after transfection and used to infect NIH3T3 cells. Infected cells were selected for using the appropriate antibiotic [2 µg/ml Puromyocin for 48 h (Sigma Chemical Co.), 900 µg/ml Geneticin for 7–9 days (Life Technologies, Inc.), and 5 µg/ml Blasticidin S hydrochloride for 3 days (ICN)]. Protein expression was determined by Western blotting. Cells coexpressing two different constructs were made by transfecting and selecting for the control (MSCV) or ETV6-NTRK3 constructs into already established cell lines (DNRas or RKIP). Expression of both proteins was confirmed by Western blot analysis.
Kinase Inhibitor Studies.
MSCV and ETV6-NTRK3 NIH 3T3 cells were plated in six-well dishes at 
70% confluence. Cells were grown for 
48 h in media containing the following kinase inhibitors: U0126 (25 µM; Calbiochem), PD098059 (75 µM; Sigma Chemical Co.), Wortmannin (25 nM; Calbiochem), and LY294002 (25 µM; Calbiochem). Pictures of the cells were taken at various time points. For U0126 On/Off experiments, cells were plated as above and incubated in media containing 10 µl/ml DMSO (Vehicle), 2.5, or 25 µM U0126 for 24 h. At 24 h, fresh media containing the inhibitor were placed on the cells and incubated for an additional 24 h (On 24 h + 24 h). Pictures of the cells were taken, and cell lysates were prepared at this time point. Additional well cells treated for 24 h with U0126 were rinsed three times with PBS and were incubated in fresh media lacking the inhibitor for an additional 24 h (On 24 h/Off 24 h). Pictures were taken at this time point, and cell lysates were also prepared from these cells.
Lysate Preparation and Immunoblotting.
Cells were rinsed once with PBS and lysed with 500 µl of phosphorylation solubilization buffer (50 mM HEPES, 100 mM NaF, 10 mM Na4P2O7, 2 mM Na3VO4, 2 mM EDTA, 2 mM NaMoO4 and 0.5% NP40) containing phosphatase inhibitors (10 µg/ml Leupeptin, 10 µg/ml Apoprotinin, and 250 µM phenylmethylsulfonyl fluoride). The cells were solubilized for 30 min at 4°C on a shaking platform. Lysates were cleared by centrifugation at 12,000 x g for 10 min at 4°C. Protein quantification of the lysates was performed using a detergent-compatible protein assay kit from Bio-Rad. Total cell lysate (30 µg) was mixed with Laemmli buffer and electrophoresed overnight on 10–12% SDS-polyacrylamide gels according to standard methods. Electrophoresed proteins were transferred to Immobilon-P (Millipore) before immunoblot analysis with the indicated antibodies. Proteins were visualized with enhanced chemiluminescence (Amersham) according to the manufacturer’s protocols.
Antibodies.
The following antibodies were used in the Western blotting experiments described in this paper: Phospho-Akt (both Ser 473 and Thr 308) and total Akt (Cell Signaling Technology; 1:1000), Phospho-Mek1/2 (Ser217/221; Cell Signaling Technology; 1:1000), phospho-Erk1/2 (Thr202/Tyr204) and total Erk1/2 (Cell Signaling Technology; 1:1000), cyclin D1 (Upstate Biotechnology; 1:2000), and Grb2 (Transduction Labs; 1:5000). Additional antibodies used but not described include TrkC (C-14, 1:1000 dilution; Santa Cruz Biotechnology), HA antibody (HA.11, 1:2000 dilution; BabCO), Phospho and total p38 (Cell Signaling Technology; 1:1000), and Phospho and total SAPK/JNK (Cell Signaling Technology; 1:1000).
Northern Blot Analysis.
Northern blotting was performed according to standard methods. Total RNA was extracted using one-step TRIzol extraction (Life Technologies, Inc.) from NIH 3T3 cells expressing various constructs (Fig. 1C)
. In some cases, ETV6-NTRK3 cells were incubated with the following kinase inhibitors: U0126 (25 µM), LY0294002 (25 µM), or K252a (20 nM) for 24 h before lysis. Denatured total RNA (20 µg) was blotted and probed with 100 ng of 
-32P dCTP (Amersham)-labeled cDNA probes. The murine Cyclin D1 probe was obtained from Invitrogen (accession no.: AI323180), and the B-actin probe was obtained from Clontech.
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8 x 104 cells/35-mm dish. Bottom layers were made up of 0.4% agar in 9% CS DMEM. Cells were resuspended in a top layer of 0.2% agar in 9% CS DMEM. Cells were fed every other day by placing two drops of media (+/- inhibitors) on the top layer. After 2 weeks at 37°C, the number of single cells and colonies per high power view were counted. Results were formulated as a percentage of colonies formed per total number of cells plated.
FACS Analysis.
Cell cycle analysis was performed by serum starving cells overnight in 0.5% CS/DMEM and then stimulating them with 9% CS/DMEM for 48 h. In the case of EN cells, stimulation was also performed in the presence of the following inhibitors: U0126 (25 µM), Wortmannin (2 nM), and LY294002 (25 µM). Cells were trypsinized and pelleted, rinsed 1 x with PBS, and fixed with 70% ethanol overnight at 4°C. The fixed cells were spun down, resuspended in 100 µg/ml RNase A, and incubated at 37°C for 30 min. Lastly, 40 µl of 20 µg/ml propidium iodide were added, and cells were analyzed by FACS. Doublet discrimination was performed, and the percentage of cycling cells was determined using the ModFit program.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ETV6-NTRK3 cells had constitutively high levels of Mek1 phosphorylation even in the absence of serum. Whereas control cells responded to serum stimulation by the expected initial increase in Mek1 phosphorylation followed by a return toward basal levels by 6 h, levels remained elevated in ETV6-NTRK3 cells even after extended serum stimulation. In contrast, cells expressing the nontransforming, kinase-inactive ETV6-NTRK3 mutant EN-K380N (24)
demonstrated identical Mek1 phosphorylation patterns as vector control cells (data not shown). Interestingly, Mek1 activation did not correlate with differences in Erk1/2 phosphorylation (see Fig. 1B
). In fact, we reproducibly observed lower Erk1/2 phosphorylation in ETV6-NTRK3 cells compared with controls, although total Erk1/2 levels were similar, and both cell types responded similarly to serum with an increase in Erk1/2 phosphorylation followed by a decay toward basal levels (Fig. 1B)
. We also assessed the phosphorylation status of SAPK/JNK and p38 MAP kinases in ETV6-NTRK3-expressing cells but found no evidence that these cascades were activated by the fusion oncoprotein (data not shown). These studies indicate that although the ETV6-NTRK3 oncoprotein appears to constitutively activate components of the Ras-Erk1/2 pathway, this activation is not apparent at the level of increased MAP kinase phosphorylation. The basis of this observation is currently under investigation (see "Discussion").
We next assayed Akt activation and found a similar pattern of phosphorylation as for Mek1. There was constitutive serum-independent phosphorylation of Akt serine 473 (see Fig. 1A
) and threonine 308 (data not shown) in ETV6-NTRK3 cells compared with control cells or cells expressing EN-K380N. We could detect only a minimal additional increase in Akt phosphorylation when ETV6-NTRK3 cells were treated with serum, and levels remained high even after extended serum stimulation (Fig. 1A)
.
Because ETV6-NTRK3 does not interact directly with Shc, Grb2, or PI3K p85 (24)
, we screened other potential adapter molecules known or hypothesized to associate with wild-type NTRK3 or other NTRK PTKs. However, immunoprecipitation with -ETV6-NTRK3 antibodies followed by Western blotting using a series of specific antibodies failed to detect interactions between ETV6-NTRK3 and ABL (30)
, SH2Bß (31)
, SRC (32)
, or SHIP2 (Ref. 33
and data not shown). Therefore, ETV6-NTRK3 appears to be linking via as yet unknown mechanisms to both the Ras-Erk1/2 and the PI3K-Akt pathways.
Constitutive Expression of Cyclin D1 in NIH3T3 Cells Transformed by ETV6-NTRK3.
Activation of both the Ras-Erk1/2 and PI3K-Akt cascades is associated with increased expression of cyclin D1 and promotion of the G1 to S phase transition (reviewed in Refs. 34
and 35
). Therefore, we compared the levels of cyclin D1 proteins in ETV6-NTRK3 and vector control cells and found that cells expressing the fusion protein exhibited constitutively high levels of cyclin D1 even after overnight serum starvation (see Fig. 1A
). Northern analysis revealed a marked elevation of cyclin D1 transcripts in cells expressing either activated H-Ras, ETV6-NTRK3, or the EN-PLC-transforming mutant that fails to bind PLC (see Fig. 1C
) but not in cells expressing the nontransforming mutants EN-K380N or EN-Y x 3F (24)
. Therefore, cyclin D1 regulation by ETV6-NTRK3 appears to be, at least in part, transcriptional. Cyclin D1 elevation was also observed in ETV6-NTRK3-expressing NIH3T3 cells grown as spheroids in agar-coated dishes (data not shown). Even after 24 h of growth in suspension cultures, ETV6-NTRK3 cells exhibited high levels of cyclin D1, suggesting that elevation of these proteins was not dependent on signaling provided by anchorage to plastic dishes. These results suggest a link between Mek1 and/or Akt activation and the expression of cyclin D1 mRNA and protein in ETV6-NTRK3-transformed cells.
Mek1 Inhibitors Block ETV6-NTRK3-induced Morphological Transformation in a Reversible Manner.
To gain additional insights into signaling cascades activated by ETV6-NTRK3, we screened the Mek1 inhibitors U0126 and PD098059 (36)
and the PI3K inhibitors LY294002 and Wortmannin (37)
for their effects on morphological transformation of NIH3T3 cells. Equivalent numbers of ETV6-NTRK3 and vector control cells were synchronized by overnight serum starvation and then grown in 10% serum to near confluency. Cells were then treated with the above inhibitors and monitored microscopically over 48 h for changes in phenotype. Only the Mek1 inhibitors had any appreciable effect on ETV6-NTRK3 cell morphology. Cells treated with either U0125 or PD098059 for 6–24 h flattened out, demonstrated decreased nuclear-to-cytoplasmic ratios and reduced refractivity, and became contact inhibited (Fig. 2
, top panel). Cell viability was unaffected at the concentrations used for phenotypic reversion. Neither LY294002 nor Wortmannin had any appreciable effects on morphological transformation over a range of concentrations tested (data not shown). These results indicate that the Ras-Erk1/2 pathway is important for ETV6-NTRK3-induced phenotypic transformation of NIH3T3 cells but that the PI3K-Akt pathway is not involved in these morphological changes.
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, top panel). However, 24 h after inhibitor withdrawal, ETV6-NTRK3-expressing cells had reestablished a transformed phenotype, even when initially treated at the high U0126 concentration (Fig. 2
, top panel). This indicates that the block in phenotypic transformation by U0126 is reversible. Moreover, the reemergence of the transformed phenotype was associated with increased levels of phosphorylated Erk1/2 (Fig. 2
, bottom panels). Therefore, although overall levels of Erk1/2 phosphorylation are not elevated in ETV6-NTRK3 cells compared with controls (Fig. 1B)
, the ability of Mek1 to phosphorylate Erk1/2 remains intact in these cells.
Both Mek1 and PI3K Inhibitors Block Soft Agar Colony Formation of ETV6-NTRK3-expressing Cells.
One of the hallmarks of transformed cells is the ability to grow in an anchorage-independent manner. We demonstrated previously that ETV6-NTRK3-expressing NIH3T3 cells readily form anchorage-independent macroscopic colonies when plated in soft agar (24)
. Therefore, to further assess the role of signaling pathways in ETV6-NTRK3 transformation, we examined the effects of the above panel of signaling inhibitors on soft agar colony formation. The addition of U0126 to bottom and top soft agar layers completely blocked ETV6-NTRK3 colony formation (Fig. 3A)
, although cells remained alive as single cells. To confirm that ETV6-NTRK3 requires a functional Ras-Erk1/2 pathway to confer anchorage-independent growth properties to cells, we coexpressed either a DN form of Ras (H-Ras 17N; Ref. 38
) or the Raf kinase inhibitor protein RKIP (39)
, along with ETV6-NTRK3 in NIH3T3 cells. Expression of DN-Ras or RKIP both markedly reduced soft agar colony formation of ETV6-NTRK3-expressing cells, although RKIP was more potent (Fig. 3A)
. Interestingly, the effects of DN-Ras and RKIP were associated with only a partial reduction of Mek1 and Erk1/2 phosphorylation (Fig. 3B)
, indicating that the interplay between components of the Ras-Erk1/2 pathway in ETV6-NTRK3 transformation is likely complex.
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, the addition of LY294002 completely inhibited Akt phosphorylation at serine 473; phosphorylation at threonine 308 was also blocked (data not shown). Moreover, the addition of LY294002 to the bottom and top soft agar layers completely blocked ETV6-NTRK3 colony formation in soft agar assays (Fig. 3D)
. Similar effects were observed with Wortmannin, although it was slightly less effective compared with LY294002 (Fig. 3D)
. Because the PI3K-Akt pathway is known to function in cell survival (40
, 41)
, it was important to confirm that the observed effect on colony formation by blockade of this pathway is not only a function of cell loss attributable to increased apoptosis. In fact, cells remained alive as single cells in the presence of the PI3K inhibitors, and there was no apparent decrease in viability compared with nontreated cells (data not shown). We also tested the NTRK tyrosine kinase inhibitor K252a (42)
. However, this reagent did not have any appreciable effect on ETV6-NTRK3-mediated soft agar colony formation at the range of concentrations tested (data not shown). Therefore, our data indicate that both the Ras-Erk1/2 and PI3K-Akt pathways are essential for ETV6-NTRK3-mediated, anchorage-independent growth. Interestingly, although PI3K inhibitors did not noticeably affect Erk1/2 phosphorylation in serum-stimulated ETV6-NTRK3 cells and Mek1 inhibitors had no effect on Akt phosphorylation (data not shown), LY294002 treatment of these cells appeared to enhance Mek1 phosphorylation (Fig. 3C)
. More studies are required to rigorously evaluate potential signaling cross-talk between these pathways in ETV6-NTRK3-expressing cells.
Both Mek1 and PI3K Inhibitors Block Cyclin D1 Elevation in ETV6-NTRK3-expressing Cells.
Previous reports have correlated the elevation of cyclin D1 expression with Ras activation (reviewed in Ref. 34
) or with activation of the PI3K-Akt pathway (43)
. Therefore, to better understand how the Ras-Erk1/2 and PI3K-Akt cascades might contribute to ETV6-NTRK3 transformation, we next tested whether the observed elevation in cyclin D1 in ETV6-NTRK3-expressing NIH3T3 cells was mediated by one or both of these pathways. ETV6-NTRK3 and control cells were serum starved overnight and then incubated with serum +/- Mek1 or PI3K inhibitors. U0126 markedly decreased levels of cyclin D1 in ETV6-NTRK3 monolayer cells, which became most evident by 24 h after the addition of the inhibitor (Fig. 4A)
. We next tested the effects of PI3K inhibition on cyclin D1 expression. We observed that LY294002 and Wortmannin caused a reduction of cyclin D1 levels in ETV6-NTRK3-expressing cells (see Fig. 4A
for LY294002 results). However, this effect was transient with cyclin D1 protein levels returning to preinhibitor levels by 24 h. Therefore, only inhibition of the Ras-Erk1/2 pathway led to persistent down-regulation of cyclin D1 expression. To confirm the U0126 results, we compared cyclin D1 levels in cells expressing ETV6-NTRK3 to those coexpressing ETV6-NTRK3, along with either DN-Ras or RKIP. RKIP markedly decreased cyclin D1 expression, whereas DN-Ras had only minimal effects on cyclin D1 protein levels (Fig. 4B)
, consistent with our finding that RKIP is more effective than DN-Ras at blocking soft agar colony formation in ETV6-NTRK3-expressing cells (Fig. 3A)
. Similar results were obtained when cells cultured in suspension were tested; the addition of U0126 to the media also greatly reduced levels of cyclin D1 in ETV6-NTRK3-expressing cells (data not shown). To independently test the effects of signaling blockade on cell cycle progression, we performed FACS analysis on NIH3T3 cells expressing various constructs, which were grown in the presence or absence of the above inhibitors. As shown in Fig. 5
, U0126, Wortmannin, and LY294001 all reduced the percentage of cycling cells expressing ETV6-NTRK3, as did coexpression of RKIP, along with ETV6-NTRK3. These results indicate that both the Ras-Erk1/2 and PI3K-Akt pathways contribute to regulation of cyclin D1 protein expression and cell cycle progression in ETV6-NTRK3 cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The unique ability of ETV6-NTRK3 to activate both the Ras-Erk1/2 and PI3K-Akt pathways may be key to its oncogenic activity. An association between aberrant activation of the Ras-Erk1/2 pathway and cellular transformation is well described in the literature (45, 46, 47, 48, 49) . Alterations in the PI3K-AKT pathway have also been observed in humans tumors, including amplification of the AKT ß isoform in breast and ovarian carcinomas (50) , amplification of the PI3K regulatory subunit in ovarian carcinoma (51 , 52) , and mutations of the PTEN tumor suppressor gene leading to constitutive AKT activation (reviewed in Ref. 53 ). Although these and other studies (reviewed in Ref. 54 ) illustrate the oncogenic potential of each individual pathway, it is becoming increasingly clear that a synergistic effect exists between Ras-Erk1/2 and PI3K-Akt cascades in transformation. Activated Ras mutants that are incapable of activating PI3K are unable to transform NIH3T3 cells unless an activated (nontransforming) viral form of Akt is coexpressed (55) . Moreover, transformation of rat intestinal epithelial cells by oncogenic Ha-Ras not only leads to coactivation of PI3K-Akt but is blocked by PI3K inhibitors (56) .
Different mechanisms can be hypothesized as to how synergism between the Ras-Erk1/2 and PI3K-Akt pathways may enhance oncogenic activity. First, it is well established that constitutive expression of Ras can induce apoptosis or cell cycle arrest through mechanisms involving p19ARF, p21CIP1, and p53 (reviewed in Ref. 34
). Several recent studies have demonstrated negative regulation of Raf1 through phosphorylation by Akt (57
, 58)
. There is also recent evidence that suppression of apoptosis by Raf1 and Mek1 requires a PI3K-dependent signal (59)
. One emerging model from these data is that unless the PI3K-Akt pathway is activated in parallel, continuously high Ras activity would otherwise lead to apoptosis or cell cycle arrest (35)
. In this way, suppression of excessive Raf-1 signaling by PI3K-Akt may be required for sustained proliferation of Ras-transformed cells. This scenario may explain at least in part why ETV6-NTRK3-expressing cells do not show elevated Erk1/2 phosphorylation compared with controls despite the Mek1 activation observed in the former (Fig. 1C)
. It is possible that by inducing the PI3K-Akt pathway in conjunction with Ras-Erk1/2, ETV6-NTRK3 may somehow maintain Ras-Erk1/2 activation at a level that favors sustained proliferation over apoptosis or cell cycle arrest. Consistent with this hypothesis, we observed that phosphorylated Mek1 was elevated in ETV6-NTRK3-expressing cells after treatment with the LY294002 PI3K inhibitor (Fig. 3C)
. Yip-Schneider et al. (60)
reported recently that pancreatic carcinoma cell lines with activating K-RAS mutations and high levels of functional MEK have attenuated ERK1/2 phosphorylation. Moreover, this correlated with up-regulation of a phosphatase, MKP-2, that is capable of dephosphorylating ERK1/2. It will be important to determine whether MAP kinase phosphatase-2 or other ERK1/2 phosphatases are active in cells expressing ETV6-NTRK3.
A second potential consequence of ETV6-NTRK3’s ability to activate both the Ras-Erk1/2 and PI3K-Akt pathways is suggested by reports of synergism between these cascades in cell cycle progression. Ras-Erk1/2 activation is thought to induce cyclin D1 expression predominantly via transcriptional regulation of the cyclin D1 promoter (reviewed in Refs. 34 and 61, 62, 63, 64 ). Cyclin D1 induction and cell cycle progression appear to require sustained Mek1 activation but only moderate Erk1/2 activation, whereas strong Erk activation leads to induction of cell cycle arrest or apoptosis as discussed above (34) . Therefore, the extent and duration of Erk1/2 activation may determine whether cells undergo proliferation versus cell cycle arrest or apoptosis. Moreover, Treinies et al. (65) demonstrated that whereas an activated form of Mek can induce DNA synthesis in NIH3T3 cells, PI3K signals are required for this effect. They found that PI3K activation was necessary in order for Mek activation to induce cyclin D1 expression and cell cycle entry. Similarly, it has been reported that LY294002 blocks Ha-Ras-induced cyclin D1 expression in transformed rat intestinal epithelial cells (56) and that Raf1 stimulates cyclin D1 transcription in synergy with activation of the PI3K-Akt pathway (66) . The ability of ETV6-NTRK3 to activate Akt, which then regulates Raf1 and Mek1 activity (57 , 58) , may be key to determining the balance between cell cycle progression and arrest or apoptosis in ETV6-NTRK3-transformed cells. On the other hand, the PI3K-Akt pathway also directly regulates cyclin D1 protein levels. Muise-Helmericks et al. (43) provide evidence for a posttranscriptional PI3K-dependent pathway that regulates cyclin D1 protein by increasing cyclin D1 mRNA translation. Furthermore, Akt phosphorylation of glycogen synthase kinase-3ß blocks its ability to phosphorylate and thereby enhance degradation of the cyclin D1 protein (67 , 68) . Therefore, more than one mechanism related to potential synergism between Ras-Erk1/2 and PI3K-Akt pathways may underlie the serum-independent elevation of cyclin D1 observed in ETV6-NTRK3-expressing cells.
Of interest, given the difficulty in the clinical management of CFS and cellular CMN, is the possibility that MEK and PI3K inhibitors can be used alone or in combination for the treatment of human tumors expressing ETV6-NTRK3. This is in light of recent studies performed by Sebolt-Leopold et al. (49) showing marked inhibition of colon tumor growth by a p.o. active MEK inhibitor, PD184352, in vivo. In addition, another group has reported that PD98059 reduces the survival of AML cells in vitro (69) . Therefore, MEK inhibitors could represent a logical approach for controlling the growth of CFS or CMN tumors in vivo. Moreover, it is possible that blockade of the PI3K-AKT pathway in cells expressing ETV6-NTRK3 may lead to RAS-ERK1/2-mediated apoptosis, and this approach warrants additional study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Canadian Institutes of Health Research and from the National Cancer Institute, Director’s Challenge: Toward a Molecular Classification of Tumors (U01-CA88199). Dr. C. Tognon is the recipient of a postdoctoral fellowship from the Candlelighters Childhood Cancer Foundation/Canadian Institutes of Health Research. 
2 To whom requests for reprints should be addressed, at Departments of Pathology and Pediatrics, BC Research Institute for Children’s and Women’s Health, 950 West 28th Street, Vancouver, British Columbia, V6H 3V4 Canada. Phone: (604) 875-2936.
3 The abbreviations used are: CFS, congenital fibrosarcoma; AML, acute myeloid leukemia; CMN, congenital mesoblastic nephroma; PTK, protein tyrosine kinase; MAP, mitogen-activated protein; PI3k, phosphatidylinositol 3'-kinase; Ras-Erk1/2, Ras-Raf1-Mek1-Erk1/2; PLC, phospholipase-C; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; CS, calf serum; DN, dominant negative; FACS, fluorescence-activated cell sorter.
Received 7/ 6/01. Accepted 10/18/01.
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W. Jin, C. Yun, A. Hobbie, M. J. Martin, P. H.B. Sorensen, and S.-J. Kim Cellular Transformation and Activation of the Phosphoinositide-3-Kinase-Akt Cascade by the ETV6-NTRK3 Chimeric Tyrosine Kinase Requires c-Src Cancer Res., April 1, 2007; 67(7): 3192 - 3200. [Abstract] [Full Text] [PDF]
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 M. J. Martin, N. Melnyk, M. Pollard, M. Bowden, H. Leong, T. J. Podor, M. Gleave, and P. H. B. Sorensen The Insulin-Like Growth Factor I Receptor Is Required for Akt Activation and Suppression of Anoikis in Cells Transformed by the ETV6-NTRK3 Chimeric Tyrosine Kinase. Mol. Cell. Biol., March 1, 2006; 26(5): 1754 - 1769. [Abstract] [Full Text] [PDF]
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W. Jin, B.-C. Kim, C. Tognon, H.-J. Lee, S. Patel, C. L. Lannon, J. M. Maris, T. J. Triche, P. H. B. Sorensen, and S.-J. Kim The ETV6-NTRK3 chimeric tyrosine kinase suppresses TGF-{beta} signaling by inactivating the TGF-{beta} type II receptor PNAS, November 8, 2005; 102(45): 16239 - 16244. [Abstract] [Full Text] [PDF]
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C. E. Tognon, C. D. Mackereth, A. M. Somasiri, L. P. McIntosh, and P. H. B. Sorensen Mutations in the SAM Domain of the ETV6-NTRK3 Chimeric Tyrosine Kinase Block Polymerization and Transformation Activity Mol. Cell. Biol., June 1, 2004; 24(11): 4636 - 4650. [Abstract] [Full Text] [PDF]
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C. L. Lannon, M. J. Martin, C. E. Tognon, W. Jin, S.-J. Kim, and P. H. B. Sorensen A Highly Conserved NTRK3 C-terminal Sequence in the ETV6-NTRK3 Oncoprotein Binds the Phosphotyrosine Binding Domain of Insulin Receptor Substrate-1: AN ESSENTIAL INTERACTION FOR TRANSFORMATION J. Biol. Chem., February 20, 2004; 279(8): 6225 - 6234. [Abstract] [Full Text] [PDF]
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