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*
Department of Pathology and Laboratory Medicine and
Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104;
Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105;
Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark; and
¶ Center for Biotechnology, Temple University, Philadelphia, PA 19122
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of ALK, which is physiologically confined to cells of the nervous system (23), results in lymphomas from at least five different translocations involving ALK gene locus on the short arm of chromosome 2 (24, 25, 26, 27, 28, 29, 30, 31, 32, 33). The most frequent is a translocation t(2;5) (p23;q35) that occurs in up to 80% of cases of the ALK+ T/null-cell lymphoma (TCL). It fuses the ALK gene with a gene encoding nucleophosmin (NPM), which is a ubiquitously expressed protein involved in shuttling of ribosomal components between the cytoplasm and the nucleus (34, 35, 36, 37). The resulting 80-kDa NPM/ALK chimeric protein contains the oligomerization motif of NPM fused to the cytoplasmic portion of ALK that includes an intact kinase catalytic domain (38, 39, 40). NPM/ALK is constitutively activated through tyrosine autophosphorylation (41, 42). It has cell-transforming properties as demonstrated in in vitro (41, 42, 43) and in vivo (44, 45) experimental systems. However, the mechanisms of NPM/ALK-mediated oncogenesis remain poorly defined (41, 45, 46).
STATs are members of the ubiquitously expressed family of transcription
factors activated in response to growth factors and cytokines (reviewed
in Refs. 47, 48, 49). Activation of STATs requires
phosphorylation of their tyrosine residues either by the receptors that
often display an intrinsic tyrosine kinase activity or by
receptor-associated Jak/Tyk kinases. The phosphorylated STATs form
dimers that translocate into the nucleus and initiate transcription of
the growth factor/cytokine-responsive genes. STATs play a critical role
in promoting cell proliferation and cell protection from apoptosis.
Several members of the STAT family have been identified and designated
STAT1 to STAT6. STAT5 comprises two closely related but distinct STATs
labeled STAT5a and STAT5b. STAT1 and STAT3 have two isoforms (STAT1 and
STAT3 
) resulting from an alternative gene splicing.
Here we show that NPM/ALK induces constitutive activation of STAT3 in ALK+ TCL cells. This activation was present in all ALK+ TCL cell lines (4 of 4) and tissues (10 of 10) tested. STAT3 activation was selective because the other six STATs were inconsistently activated in the ALK+ TCL cell lines. In addition, STAT3 became phosphorylated in BaF3 lymphoid cells after transfection with NPM/ALK, but not with an empty vector. Furthermore, STAT3 was constitutively associated with ALK. We also showed that the ALK+ TCL cell lines expressed PP2A phosphatase (50), activity of which was critical for maintenance of the constitutive tyrosine phosphorylation of STAT3. Finally, the ALK+ T cell lines failed to express a negative regulator of STAT3 activation, PIAS3 (49, 51). Biological and clinical implications of these findings are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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JB6, SU-DHL1, Karpas 299, and SUP-M2 cell lines are derived from four different CD30+ anaplastic large TCLs and carry the t(2;5) chromosomal translocation involving ALK and NPM genes as documented by cytogenetic, molecular, and/or protein expression analysis (38, 52). PB-1, 2A, and 2B T cell lines were established from a patient with a progressive, cutaneous ALK- TCL (53, 54). Sez-4 cell line was derived from a patient with cutaneous T cell lymphoma in a leukemic phase (Sezary syndrome) (54). YT is a human NK cell line. MOLT4, MOLT13, and Jurkat were derived from patients with T cell lymphoblastic lymphoma/leukemia. The murine pro-B cell lymphoid cell line BaF3 was transfected with a vector containing NPM/ALK construct or a control empty vector (44). PBMC and PHA-stimulated blasts from healthy adults served as normal controls and were obtained by centrifugation on Ficoll/Paque gradient (54). Cell lines and PHA blasts were cultured at 37°C with 5% CO2 in standard RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin/fungizone mixture, 2 mM L-glutamine, and, in the case of BaF3 cells, 10% IL-3 containing WEHI-conditioned medium (55).
Tissue sections were obtained from a total of 10 patients. The patients varied in age from 1 to 16 years (median, 8.5), nine were males, and one was a female. All but one presented with lymphadenopathy, the remaining one with a s.c. mass in the thigh. All lymphomas showed anaplastic large cell morphology, usually a monomorphic variant type, and expressed CD30 and epithelial membrane Ags, but not CD15 Ag. In seven patients, T cell origin of the lymphoma was determined by a positive immunohistochemical staining for at least one T cell-associated marker (CD3, CD43, and/or CD45R0). None of the lymphomas expressed B cell-associated marker CD20. Expression of ALK protein was confirmed in all 10 cases by the immunohistochemistry. Formalin-fixed, paraffin-embedded tissue was available from eight patients, and cryopreserved, snap-frozen tissue was available from four patients, in two of whom the frozen tissue was the only specimen analyzed.
In vitro tyrosine kinase activity of NPM/ALK
NPM/ALK tyrosine kinase activity was examined using an ELISA-based protein tyrosine kinase assay kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s manual. Briefly, cells were lysed with lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 20 mM Tris (pH 7.4)) containing protease and phosphatase inhibitors (1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 mM sodium vanadate, 20 mM NaF). Immunoprecipitation with 4 µl of ALK goat polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and 20 µl of protein A-agarose (Life Technologies, Grand Island, NY) was performed. Immunoprecipitates were added to poly-Glu-Tyr substrate-coated wells. The plate was incubated for 30 min at room temperature in the presence of tyrosine kinase buffer (50 mM HEPES (pH 7.4), 20 mM MgCl2, 0.1 mM MnCl2, 0.2 mM Na3VO4, 0.3 mM ATP), washed, and incubated with a monoclonal anti-phosphotyrosine/peroxidase conjugate. After washing, solution containing peroxidase substrate was applied to the plate. The reaction was stopped by adding 2.5 N H2SO4, and activity was measured in a microplate ELISA reader. NPM/ALK tyrosine kinase activity was compared with activity of two different concentrations (2 U and 4 U) of a purified human epidermal growth factor receptor supplied by the kit’s manufacturer.
Protein expression and phosphorylation
These experiments were performed as described previously (54, 55, 56). In brief, the cells (5–10 x 106) under study were washed and lysed in ice-cold lysis buffer. In some experiments, the cells were exposed for 5 min to 500 U of IL-2 or medium alone before lysis. The cell lysates were centrifuged, and supernatants were precleared with protein A-Sepharose (Sigma-Ald-rich) and incubated with a primary Ab specific for ALK, Jak3, STAT 1–6, PP2A, or PIAS1/3 (all Abs from Santa Cruz Biotechnology) and protein A-Sepharose. In some experiments, Abs were preincubated with a specific inhibitory peptide (54). The immunoprecipitates were washed, boiled, separated on a 10% polyacrylamide-SDS gel, and transferred electrophoretically to hybridization transfer membranes. In some experiments, cell extracts were directly separated by electrophoresis without the preceding immunoprecipitation. The membranes were blocked with 2% BSA in the TBST buffer. To detect protein expression, the membranes were incubated with the same Abs that were used for immunoprecipitation. To detect protein association, combinations of the appropriate Abs were used (see Results). To detect protein phosphorylation, the membranes were incubated with the antiphosphotyrosine 4G10 Ab (Upstate Biotechnology, Lake Placid, NY) or Ab specific for STAT3 phosphorylated on tyrosine 705 (phospho-STAT3; Santa Cruz Biotechnology) or serine 727 (New England Biolabs, Beverly, MA). Next, the membranes were incubated with appropriate secondary, peroxidase-conjugated Abs. Blots were developed using the ECL chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
EMSA
Nuclei were isolated and extracted in lysis buffer comprised of 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1 µM antipain, 1 µM chymostatin, 1 µM leupeptin, 1 µM pepstatin, 1 mM PMSF, 0.1 µM aprotinin, 200 mM DTT, 20 mM MgCl2, and 5 mM EDTA. The oligonucleotide sequence of the STAT3 consensus binding site was as follows: 5'-GAT CCT TCT GGG AAT TCC TAG ATC-3' (Santa Cruz Biotechnology). In addition, an oligonucleotide containing mutation in the STAT3 binding site, 5'-GAT CCT TCT GGG CCG TCC TAG ATC-3', was used as a negative control. Binding reaction was performed in a total volume of 20 µl in 20 mM HEPES (pH 7.6), 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 30 mM KCl, and 0.2% Tween 20. Ten micrograms of nuclear protein were incubated for 15 min at room temperature with 1 µg of poly(dI-dC) and 30 fmol of digoxigenin (Boehringer Mannheim, Indianapolis, IN)-labeled oligo-DNA probe. The protein-DNA complexes were separated in the 6% nondenaturing acrylamide gels in 0.5x Tris-borate-EDTA buffer and were transferred electrophoretically to positively charged nylon membrane. The membrane was preincubated with blocking solution, incubated with Fab of antidigoxigenin Ab, and washed. Blots were developed using the CSPD system (Boehringer Mannheim).
Immunohistochemical staining
The staining was performed as described previously (56, 57) on formalin-fixed, paraffin-embedded, and cryopreserved tissue sections using a standard streptavidin-biotin complex technique (Research Genetics, Huntsville, AL) and Abs against the following Ags: ALK, CD3, CD15, CD20, CD43, CD45R0, CD30, and epithelial membrane Ag (all Abs from DAKO, Carpinteria, CA) and phosphotyrosine (705)-STAT3 (Cell Signaling Technology, Beverly, MA). To detect ALK expression, heat-induced Ag retrieval was performed using pH 6 citrate buffer and 10-min slide immersion into 95°C waterbath.
RNA isolation and RT-PCR
Total cellular RNA was extracted by RNeasy kit (Qiagen, Valencia, CA). RT-PCR (56) was performed with 1 µg of total RNA in RT buffer mixture containing 200 U of SuperScript II reverse transcriptase (Life Technologies). RNase H (Promega, Madison, WI) was added and cDNA was purified on Centri-Sep columns (Princeton Separations, Princeton, NJ). PCR was performed with 2 µl of cDNA in a PCR buffer mixture containing 2 U of Taq polymerase (Life Technologies). The PIAS3-specific primers were designed based on the PIAS3 genomic DNA sequence from the GenBank database using an Oligo 4.0 computer program (National Biosciences, Plymouth, MN). The PCRwas performed at 94°C for 3', followed by 30 cycles: 45 s at 94°C, 30 s at 57°C, and 1.5 min at 72°C. The amplified products were visualized in UV light by staining with ethidium bromide after electrophoresis in the 1.5% agarose gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several T cell lines carrying the t(2;5) translocation were
established from patients with ALK+ TCL
(38, 52). To investigate the expression and functional
status of NPM/ALK fusion oncoprotein in such cell lines, we performed
immunoprecipitation of the cell lysates with an anti-ALK Ab
followed by Western blotting with, respectively, the anti-ALK Ab
and an antiphosphotyrosine Ab. As shown in Fig. 1
A, the
ALK+ T cell lymphoma cell lines JB6, SU-DHL1, and
Karpas 299 expressed the NPM/ALK (80 kDa) protein, whereas the control
PB-1 cell line derived from a t(2;5)-negative cutaneous T cell lymphoma
(53, 54) did not express NPM/ALK or any other form of ALK.
The expressed NPM/ALK was strongly phosphorylated on the tyrosine
residues in all four ALK+ TCLs tested (Fig. 1B), presumably due to autophosphorylation (41, 42). To show directly that the constitutively activated NPM/ALK
displays tyrosine kinase activity, we examined the cell lysate
immunoprecipitates obtained with the ALK Ab for in vitro tyrosine
kinase activity, using a synthetic substrate rich in tyrosine residues.
As shown in Fig. 1C, NPM/ALK was constitutively
enzymatically active in the ALK+ TCL cell
lines.
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To determine whether the constitutively active NPM/ALK activates
STATs, we examined the ALK+ TCL cell lines for
tyrosine phosphorylation of these signaling proteins. At first, we
concentrated on STAT3 and STAT5, which are perpetually activated as a
consequence of constitutive activation of the IL-2R complex-associated
Jak/STAT signaling pathway in the ALK-, human T
cell leukemia virus-I-positive, and human T cell leukemia
virus-I-negative T cell lines derived from cutaneous TCL involving skin
(54, 56, 58, 59, 60, 61). As shown in Fig. 2, A–C, STAT3, but
not STAT5 a or b, was constitutively phosphorylated in all three
ALK+ TCL cell lines tested. This activation
appeared maximal because it could not be enhanced by cell treatment
with IL-2. Interestingly, one of the lines, JB6, but not the other two
lines, displayed constitutive phosphorylation of Jak3, suggesting that
this tyrosine kinase may contribute to the constitutive activation of
STAT3 in the JB6 line. To determine whether STATs other than STAT3 and
STAT5 are activated in the ALK+ TCL cells, we
examined the tyrosine phosphorylation status of an additional four
known STATs (1, 2, 4, 6) in such cells. However, STAT3 was the only
STAT that was consistently tyrosine phosphorylated in all four
ALK+ TCL cell lines tested (Fig. 2D
and data not shown). From the other STATs, only STAT1 was
phosphorylated in two ALK+ TCL cell lines, JB6
and SUP-M2. These findings indicate that the STAT3 activation is rather
selective in the ALK+ TCL cells.
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) contains the
full-length molecule (91 kDa), whereas the fast migrating form
(STAT3) is slightly smaller due to truncation of the carboxyl
terminus domain. STAT3 is considered biologically more active
(60). Because the standard anti-STAT3 Ab that we used
in the experiments described in Fig. 2
, A–D,
recognized only STAT3, we used in this experiment a novel Ab
specific for phospho-STAT3 and reactive with both STAT3 forms to
determine which of them is preferentially phosphorylated in the
ALK+ TCL cells. This anti-phospho-STAT3 Ab
recognizes specifically STAT3 phosphorylated on tyrosine 705,
phosphorylation of which is critical for an effective STAT3 activation.
As shown in Fig. 2E, both and forms of STAT3 were
strongly tyrosine phosphorylated. This contrasted with the
ALK- cutaneous TCL cell lines, which displayed
preferential phosphorylation of STAT3.
To provide further evidence that STAT3 is continuously activated in
ALK+ TCL cells, we tested the ability of STAT3
derived from such cells to specifically bind DNA. As shown in Fig. 2F, STAT3 derived from four different
ALK+ TCL cell lines bound the STAT3 target
oligo-DNA sequence. The binding was, indeed, sequence specific, as
demonstrated by lack of STAT3 binding to a mutated form of the target
oligo-DNA that did not contain the consensus binding site for STAT3 due
to a three-base substitution.
Continuous activation of STAT3 in ALK+ TCL tissues
To determine whether STAT3 activation is present not only in the
cultured ALK+ TCL cell lines, but also in
ALK+ TCL cells derived directly from patients, we
examined by Western blotting and immunohistochemistry activation status
of STAT3 in lymphoma tissue samples from 10 patients with
ALK+ TCL, using the anti-phospho-STAT3 Ab. As
shown in Fig. 3, cells from all four
ALK+ TCL patients tested in Western blot
displayed tyrosine phosphorylation of STAT3. Interestingly, in contrast
to the ALK+ TCL cell lines, the lymphoma tissues
from most (3 of 4) patients displayed preferential phosphorylation of
the form of STAT3 (60). Whether this discrepancy
reflects a true difference between the cultured and tissue-derived
lymphoma cells or is related to some degree of contamination of the
lymphoma cell by reactive, nonmalignant cells, which may be expected to
preferentially activate STAT3 (62), is at present
unclear. To confirm that STAT3 is indeed continuously activated in
ALK+ TCL cells in vivo, we examined by
immunohistochemistry tissue sections from eight
ALK+ TCL patients for in situ expression of
phospho-STAT3.
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depicts results of such analysis
in a patient representative for the entire cohort. H&E-stained sections
(left two panels) show a diffuse infiltrate of large,
anaplastic lymphoma cells involving the lymph node parenchyma and
packing the marginal sinus. ALK staining highlights the large lymphoma
cells and shows lack of staining in the small, reactive lymphocytes.
Staining with the phospho-STAT 3 Ab shows an architectural pattern very
similar to the ALK staining, with the most intense staining of the
lymphoma cells in the marginal sinus and lack of staining of the
scattered reactive lymphocytes. The same results were obtained in all
eight patients examined, including two also tested by Western blot
(Fig. 3). These findings indicate that ALK expression is invariably
associated with activation of STAT3, not only in cultured but also in
primary lymphoma cells, and strongly suggest that ALK mediates STAT3
activation.
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To provide a direct evidence that NPM/ALK mediates activation of
STAT3, we evaluated STAT3 tyrosine phosphorylation in a lymphoid BaF3
cell line transfected with NPM/ALK (44) or an empty
vector. As shown in Fig. 5, constitutive
expression of NPM/ALK did, indeed, result in phosphorylation of STAT3.
The STAT3 phosphorylation was not related to an increase in the STAT3
expression because the NPM/ALK and vector-transfected cells expressed
similar amounts of STAT3 (Fig. 5C). Interestingly, the
but not the form of STAT3 was phosphorylated in the BaF3 cells
(Fig. 5B). The reason for the difference in the STAT3
phosphorylation pattern seen between the ALK+ TCL
(Figs. 2 and 3) and BaF3 cells is unclear. However, it may be
related to the ontogenetic differences between these two cell
populations (human mature T cell lymphoma cells vs murine immature,
pro-B cell leukemic cells).
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To determine whether NPM/ALK associates with STAT3 we performed
coprecipitation experiments using ALK and STAT3 Abs (Fig. 6). Western blotting of the ALK
immunoprecipitates with the STAT3-reactive Ab yielded a strong STAT3
band (91 kDa). Pretreatment of the ALK Ab with a specific blocking
peptide greatly diminished intensity of the band (Fig. 6A,
lane 4), whereas pretreatment of the ALK Ab with the STAT3
Ab-specific blocking peptide had no effect on the band intensity (Fig. 6A, lane 5), confirming specificity of the
reaction. The reciprocal immunoprecipitation with the STAT3 Ab followed
by immunoblotting with the ALK Ab yielded a dominant, NPM/ALK (80 kDa)
band, the intensity of which was not affected by preincubation of the
STAT3 Ab with the ALK inhibitory peptide, but was eliminated by
preincubation with the STAT3 peptide (Fig. 6B). As expected,
precipitation with the anti- ALK Ab of cell lysate from the
ALK- TCL cell line 2B (53, 54) did
not coprecipitate STAT3 (Fig. 6A), and the converse
experiment with the anti-STAT3 Ab precipitation followed by
anti-ALK Ab blotting also did not yield an ALK-specific band (Fig. 6B). These data strongly suggest that STAT3 is activated
directly by NPM/ALK.
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Recent findings indicate that PP2A phosphatase is involved in the
maintenance of perpetual STAT3 tyrosine phosphorylation in
ALK- cutaneous TCL cell lines that are mediated
by constitutive activation of the Jak3 kinase (50). The
exact mechanism of this enhancement is unclear, but may be related to
the PP2A-induced serine/theonine dephosphorylation of STAT3
(50). To determine whether PP2A also plays a role in the
continuous tyrosine phosphorylation of STAT3 mediated by NPM/ALK, we
examined the ALK+ TCL cell lines for expression
and functional status of this phosphatase. As shown in Fig. 7A, all four
ALK+ TCL cell lines expressed PP2A. Moreover, we
found that PP2A was associated with STAT3 (Fig. 7B). To
evaluate whether the expressed PP2A participates in the perpetual STAT3
tyrosine phosphorylation, we treated the ALK+ TCL
cell lines with the PP2A inhibitor calyculin A (50). This
treatment resulted in an almost complete eradication of the STAT3
phosphorylation in all cell lines (Fig. 7C). In only one
cell line, SUP-M2, phosphorylation of the fast migrating form of STAT3
remained unaffected; the slow migrating form in this cell line and the
fast and slow migrating forms in the remaining three cell lines became
completely tyrosine dephosphorylated. As expected, the expression of
STAT3 was not affected by the treatment with calyculin A (Fig. 7D). These findings indicate that PP2A plays a critical role
in maintaining constitutive tyrosine phosphorylation of STAT3 in the
NPM/ALK+ TCL cells.
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Recently, an inhibitor of STAT3 activation called PIAS3 has been
described (51). PIAS3 belongs to a novel family of
inhibitors, members of which are ubiquitously expressed and act by
direct binding to STATs, preventing them from binding to DNA (49, 51). It appears that PIAS proteins are highly specific in their
interaction with STATs because PIAS3 binds to STAT3 but not STAT1
(51), and, in turn, STAT1 comprises a target for PIAS1 but
not for PIAS3 (63). To determine whether dysregulation of
PIAS expression may contribute to the constitutive STAT3 activation
induced by NPM/ALK, we evaluated ALK+ TCL cell
lines for expression of PIAS3 and PIAS1 on both RNA and protein levels.
In contrast to the control normal T cell-rich PBMC- and PHA-stimulated
T cell blasts, three of four ALK+ T cell lines
tested failed to express mRNA coding for PIAS3. This lack of PIAS3 mRNA
expression was not limited to the ALK+ T cell
lymphoma lines, because most (two of three) ALK-
cutaneous TCL lymphoma lines, which display constitutive STAT3
activation mediated by Jak3 (Fig. 2) (54), also failed to
express PIAS3 transcript. It is noteworthy that three other
ALK- T cell lines derived from T cell lymphoblastic
lymphoma/leukemia, in which STAT3 is in the quiescent
tyrosine-nonphosphorylated state (59, 61), expressed the
transcript. To examine whether the TCL cell lines that expressed PIAS3
mRNA contained PIAS3 protein, we performed a Western blot analysis on
some of these T cell lines using an Ab that recognizes both PIAS3 and
PIAS1. As can be seen in Fig. 8C, no PIAS3 protein
(molecular mass, 68 kDa) could be detected in the
phospho-STAT3+ TCL lines, including the two PIAS3
mRNA. PIAS3 expression was identified in the control PBMC,
PHA-blasts and the T cell lymphoblastic lymphoma/leukemia cell
line MOLT4 which, as stated above, does not display constitutive
activation of STAT3 (54, 61). In contrast to PIAS3, all
normal and malignant cell populations, including the
ALK+ TCL cell lines, expressed PIAS1 (molecular
mass, 78 kDa). These findings indicate that loss of PIAS3 expression in
the ALK+ and other TCL cells is frequent,
apparently selective, and may be due to disruption of PIAS3 protein
synthesis on both pre- and posttranscriptional levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Little is known so far about the cell signaling pathways activated by
ALK in normal and malignant cells. Previous studies have shown a direct
interaction of NPM/ALK with Shc and insulin receptor substrate-1
(41, 42), raising a possibility that these adapter
proteins may play a role in ALK-mediated lymphomagenesis. However,
interaction of both Shc and insulin receptor substrate-1 may not be
essential for NPM/ALK-mediated cell transformation (41, 76). The potential role of another adapter molecule,
GRB2, in such a process is also controversial (41, 46). Furthermore, phospholipase C-
has been identified as a
major downstream target of NPM/ALK (76). Activation
of phospholipase C-, which in normal cells leads to the generation
of diacylglycerol and inositol triphosphate, activation of protein
kinase C, and calcium mobilization may play a role in the
NPM/ALK-mediated oncogenesis by transducing mitogenic signals
(76). Recently, we (77) and others
(78) have found that NPM/ALK also activates the
phosphatidylinositol 3-kinase/AKT signaling pathway.
In this report, we provide several lines of evidence that
constitutively active NPM/ALK mediates continuous activation of STAT3.
Strong tyrosine phosphorylation of STAT3 was present in
NPM/ALK+ cultured, tissue-based, and transfected
cells. In the ALK+ TCL cell lines, we also
confirmed STAT3 activation by the DNA binding assay. Activation of
STAT3 was selective because no other STAT protein was consistently
activated in any of the ALK+ TCL cell lines.
STAT3 seemed to be activated directly by NPM/ALK, as shown by
bidirectional NPM/ALK-STAT3 coprecipitation. It is well established
that activation of STAT3 plays a critical role in development and
function of normal hematopoietic and nonhematopoietic cells (reviewed
in Refs. 79 and 80). Although the final net
effect of STAT3 activation appears to depend on the contribution of
other costimulatory molecules and type and stage of differentiation of
the affected cell, STAT3 displays mitogenic and antiapoptotic
properties in at least some types of cells. Not surprisingly, there is
a growing body of evidence that STAT3 may play an important role in
pathogenesis of a broad spectrum of malignant tumors (79, 80). In regard to T cell malignancies, STAT3 is continuously
activated in T cell lines derived mainly from adult-type T cell
lymphoma/leukemia (58, 59, 61) and cutaneous T cell
lymphoma/Sezary syndrome (54, 60). STAT3 activation in
such cell lines results from a constitutive activation of the Jak/STAT
signaling pathway associated with the common -chain
(c) that is shared by receptors for IL-2,
IL-4, IL-7, IL-9, and IL-15 (48). It is noteworthy that in
a set of three cell lines from a patient with cutaneous T cell
lymphoma, activation of STAT3 was highly selective, although dependent
on the signaling mediated by the c-associated
Jak3 kinase (60). This finding, combined with the data
presented in this report, indicates that activation of STAT3 may be
important in the pathogenesis of several types of T cell lymphoma.
Furthermore, it suggests that NPM/ALK might use the critical part(s) of
the c-associated signaling pathways. In
addition to NPM/ALK, several other oncogenic kinases such as v-src,
v-fps, and v-sis activate STAT3 in a direct and indirect manner
(79, 80). This may explain the presence of activated STAT3
and other STATs in various types of human tumors, including carcinomas
that involve breast, head and neck, and lung. Activation of STAT3
appears critical for cell transformation mediated by v-src (81, 82); the exact role of STAT3 in tumorigenesis mediated by the
other oncogenic kinases including NPM/ALK remains to be elucidated. It
is noteworthy that activated STAT3 has recently been shown to be
directly oncogenic in vitro and in vivo in fibroblast transformation
models (83, 84). On the functional level, constitutive
activation of STAT3 in malignant cells mediates resistance to apoptosis
(85, 86, 87).
Under physiologic conditions, activation of STATs is only transient due
to inhibition of their activity by negative regulators, both direct and
indirect (49, 80). Our data indicate that dysregulation of
STAT3 activation in ALK+ TCL may be complex and
not dependent solely on the continuous phosphorylation mediated by
NPM/ALK, but also involving at least two other regulatory molecules:
PP2A phosphatase and PIAS3. PP2A, which binds to STAT3, is required for
the sustained tyrosine phosphorylation and activation of STAT3 in
cutaneous T cell lymphoma (50) and, as shown here,
ALK+ TCL (Fig. 7). However, the mechanism of
up-regulation of the STAT3 activity by PP2A is currently poorly
understood. It could be argued that the enhancing effect of PP2A
results from its suppression of STAT3 phosphorylation on serines and
threonines (50). However, the prevailing evidence
indicates that serine/threonine phosphorylation of STATs enhances
rather than decreases activation of STAT3 mediated by tyrosine
phosphorylation (79, 80). Furthermore, inhibition of PP2A
activity by calyculin A resulted in ALK+ TCL
cells in only a slight increase in the STAT3 serine phosphorylation
(data not shown) that was disproportionately low when compared with the
profound inhibition of the STAT3 tyrosine phosphorylation.
Therefore, it is possible that PP2A may inhibit function of a currently
unknown tyrosine phosphatase normally involved in dephosphorylation of
STAT3 tyrosines.
In addition to expressing activated PP2A, ALK+
TCL cells failed to express PIAS3 that is a potent direct negative
regulator of STAT3 activity in normal cells by preventing its binding
to DNA (51). The aberrant lack of PIAS3 expression seemed
selective in ALK+ TCL because expression of PIAS1
that is a negative regulator of STAT1, but not STAT3 (63),
was not diminished in such cells. Based on these data, we propose a
model of a multifactoral control of STAT3 activation in
ALK+ TCL (Fig. 9).
According to this model, constitutively active NPM/ALK continuously
induces tyrosine phosphorylation of STAT3. This STAT3 phosphorylation
is sustained by PP2A, which inhibits the tyrosine dephophorylation. The
phosphorylated STAT3 can freely bind DNA without any interference by
PIAS3 that is not expressed in ALK+ TCL cells.
This multifactoral dysregulation of the STAT3 activation is, according
to our knowledge, quite novel, and its existence has a number of
important implications. First, it suggests that similar complex
dysregulation might govern activation of STAT3 and similar proteins in
other malignant tumors. Second, it raises a question of whether
aberrant function of PP2A and lack of expression of PIAS3 are mediated
by NPM/ALK or represent additional, NPM/ALK-independent oncogenic
mechanisms. Recent evidence, which indicates that NPM/ALK expression
alone may not be sufficient to mediate malignant cell transformation
(88), indirectly supports the latter possibility. However,
activation status of NPM/ALK, STAT3, and other NPM/ALK down-stream
effector molecules, not to mention expression of PIAS3 and activation
of PP2A, were not evaluated in that study. Therefore, further studies
are needed to better understand ALK-induced oncogenicity and the exact
role of STAT3 and its regulatory molecules in the pathogenesis of
ALK+ TCL. Finally, our findings provide argument
for combined therapy in ALK+ TCL possibly aimed
at direct inhibition of ALK activity and abrogation of its effects
indirectly by targeting downstream effectors and their regulatory
proteins.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mariusz A. Wasik, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 7.103 Founders, Philadelphia, PA 19104. E-mail address: wasik{at}mail.med.upenn.edu
3 Abbreviations used in this paper: ALK, anaplastic lymphoma kinase; TCL, T/null-cell lymphoma; NPM, nucleophosmin; PP2A, protein phosphatase A2; PIAS3, protein inhibitor of activated STAT3; c, common -chain.
Received for publication December 1, 2000. Accepted for publication October 22, 2001.
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References |
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in human T lymphocytes. Cytokine 10:735.[Medline]
to mediate its mitogenicity. Mol. Cell. Biol. 18:6951.This article has been cited by other articles:
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P. Lovato, C. Brender, J. Agnholt, J. Kelsen, K. Kaltoft, A. Svejgaard, K. W. Eriksen, A. Woetmann, and N. Odum Constitutive STAT3 Activation in Intestinal T Cells from Patients with Crohn's Disease J. Biol. Chem., May 2, 2003; 278(19): 16777 - 16781. [Abstract] [Full Text] [PDF]
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Z.-Y. Wang, Q. Zhang, J. Wilson, M. Z. Ratajczak, and M. A. Wasik Detection of Protein Tyrosine-Kinase (PTK) Gene Expression Pattern in Normal and Malignant T Lymphocytes by Combined PTK-Specific Polymerase Chain Reaction and Parallel Denaturing Gradient Gel Electrophoresis J. Mol. Diagn., May 1, 2003; 5(2): 113 - 120. [Abstract] [Full Text] [PDF]
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R. Chiarle, J. Z. Gong, I. Guasparri, A. Pesci, J. Cai, J. Liu, W. J. Simmons, G. Dhall, J. Howes, R. Piva, et al. NPM-ALK transgenic mice spontaneously develop T-cell lymphomas and plasma cell tumors Blood, March 1, 2003; 101(5): 1919 - 1927. [Abstract] [Full Text] [PDF]
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