Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK+ ALCL) is characterized by constitutive activation of the Janus kinase (JAK)3/signal transducers and activators of transcription 3 (STAT3) signaling pathway. SHP1, a tyrosine phosphatase that negatively regulates JAK/STAT, is frequently absent in ALK+ ALCL owing to gene methylation. To test the hypothesis that loss of SHP1 contributes to JAK3/STAT3 activation in ALK+ ALCL cells, we induced SHP1 expression using 5-aza-2′-deoxycytidine (5-AZA), an inhibitor of DNA methyltransferase, in ALK+ ALCL cell lines, and correlated with changes in the JAK3/STAT3 pathway. 5-AZA gradually restored SHP1 expression in Karpas 299 and SU-DHL-1 cells over 5 days. The initially low level of SHP1 expression did not result in significant changes to the expression or tyrosine phosphorylation of JAK3 and STAT3. However, higher levels of SHP1 seen subsequently correlated with substantial decreases in JAK3 and pJAK3, followed by pSTAT3 (but not STAT3). Importantly, the decrease in JAK3 was abrogated by MG132, a proteasome inhibitor. 5-AZA induced no significant increase in apoptosis but it sensitized ALCL cells to doxorubicin-induced apoptosis. Our findings support the concept that loss of SHP1 contributes to the constitutive activation of JAK3/STAT3 in ALK+ ALCL cells. SHP1 appears to downregulate JAK3 by two mechanisms: tyrosine dephosphorylation and increased degradation via the proteasome pathway.
Anaplastic large cell lymphoma (ALCL) is a distinct type of aggressive non-Hodgkin's lymphoma characterized by CD30 expression, a sinusoidal infiltrative pattern and a T/null-cell immunophenotype.1 Approximately 60% of these tumors have the chromosomal translocation, t(2;5)(p23;q35), that juxtaposes the nucleophosmin (NPM) gene at 5q35 with the anaplastic lymphoma kinase (ALK) gene at 2p23, leading to the expression of the aberrant fusion gene protein, NPM-ALK.2 NPM-ALK is oncogenic; previous data suggest that it directly contributes to lymphomagenesis through deregulating multiple signaling pathways by virtue of its tyrosine kinase activity.3, 4, 5, 6, 7 More recent studies have also implicated the signal transducer and activator of transcription 3 (STAT3) signaling pathway in the pathogenesis of ALK+ ALCL.8, 9, 10 STAT3 itself is oncogenic,11 and many types of human cancer demonstrate constitutive activation of STAT3.12, 13, 14, 15 It is believed that STAT3 promotes oncogenesis by upregulating a number of proteins that promote anti-apoptotic effects or cell-cycle progression, such as Bcl-XL, Mcl-1, survivin and cyclin D3. It has been previously shown that ALK+ ALCL tumors consistently show relatively high levels of STAT3 activation,8, 9, 16 and we have previously reported that specific blockade of STAT3 activation in ALK+ ALCL cell lines using a dominant-negative construct effectively triggers apoptosis and cell-cycle arrest, which correlates with downregulation of these STAT3 downstream targets.10 More recently, Chiarile et al.17 have provided further evidence to support the role of STAT3 in NPM-ALK-mediated tumorigenesis.
Although it is clear that NPM-ALK activates STAT3 in ALK+ ALCL cells, accumulating evidence suggests that other biochemical abnormalities also contribute to the high level of STAT3 activation seen in this cell type. Tyrosine kinases other than NPM-ALK have been shown to activate STAT3, such as src18 and JAK3,9, 19 with the latter being one of the normal physiologic activators of STAT3. Another group of defects is related to the loss of the negative regulators for the JAK/STAT signaling pathway such as SHP1.20 SHP1, a non-transmembrane protein tyrosine phosphatase, is expressed primarily in hematopoietic cells.21, 22, 23 It is known to function as an important negative regulator in various signal-transduction pathways including those of cytokine receptors (e.g. Epo-R, IL-3R, IL-2R), growth factors with an intrinsic tyrosine kinase activity (e.g. CSF-1, EGF, c-Kit) and the antigen receptors of B and T cells.24, 25 SHP1 has been shown to silence the JAK/STAT pathway by dephosphorylating and inactivating JAK.26, 27, 28, 29 SHP1 has been reported to have tumor suppressor function.30 In a previous study, we demonstrated that loss of SHP1 expression related to gene methylation is found in ALK+ ALCL cell lines and is detectable in the majority of ALK+ ALCL tumors.31 Nevertheless, the role of SHP1 in ALK+ ALCL cells has not been extensively studied. The possible relationship between loss of SHP1 and the high levels of JAK3/STAT3 activation in this tumor type has been largely speculative.
We hypothesized that loss of SHP1 contributes to the constitutive activation of JAK3 and STAT3 in ALK+ ALCL cells. To test this hypothesis, we treated two ALK+ ALCL cell lines with 5-aza-2′-deoxycytidine (5-AZA), a DNA methyltransferase inhibitor, and correlated changes in the JAK3/STAT3 signaling pathway.
Materials and methods
Cell lines and tissue culture
ALK-positive ALCL cell lines, Karpas 299 and SU-DHL1, have been previously characterized and described.32, 33 These two cell lines were maintained in RPMI 1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), penicillin (10 000 U/ml; Sigma, St Louis, MO, USA), streptomycin (10 mg/ml; Sigma), and L-glutamine (200 mM, Life Technologies). U266, a myeloma cell line, was also maintained in RPMI similar to that described for ALCL cell lines. GP293, an embryonic kidney cell line, was maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% FBS and antibiotics (10 mg/ml streptomycin and 10 000 U/ml penicillin). All cell cultures were maintained under an atmosphere of 95% O2 and 5% CO2 in 98% humidity at 37°C.
5-AZA was purchased from Sigma-Aldrich (Toronto, Ontario, Canada). 5-AZA was initially dissolved in acetic acid and diluted to 10 μg/μl. The aliquoted 5-AZA was frozen at −20°C until being used. The final concentrations of 5-AZA used throughout this study were 0, 5 and 10 μ M, and the use of these concentrations was based on the findings of two previous studies34, 35 as well as those of our preliminary studies. In all experiments, cells were seeded in six-well culture plates at a density of 106/ml, and 5-AZA in fresh medium was added to the cell culture daily in order to maintain the constant concentration. Cells were harvested daily for 5 days for analysis. MG132, purchased from Calbiochem (EMD Biosciences, San Diego, CA, USA), was initially dissolved in dimethyl sulfoxide (DMSO) and further diluted with sterile, de-ionized water to a final concentration of 1 mM. The aliquoted MG132 was then stored at −20°C. At the time of the experiment, MG132 was thawed and diluted with tissue culture media to a final concentration of 10 μ M. AG490 was purchased from Calbiochem (San Diego, CA, USA) and diluted with DMSO (final concentration, 1 mM).
Western blot analysis and antibodies
Western blot analysis was performed using standard techniques and details have been described previously.19 All antibodies used in this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) unless otherwise specified: Bcl-2 (Dako, Carpinteria, CA, USA), Bcl-xL (Zymed, San Francisco, CA, USA), survivin (Novus Biologicals, Littleton, CO, USA), and actin (Sigma, St. Louis, MO, USA). All primary antibodies were used in a 1:1000 dilution.
Cell cycle analysis using propidium iodide and flow cytometry
Karpas 299 and SU-DHL-1 cells treated with 5-AZA were incubated with propidium iodide (Molecular Probes, Eugene, OR, USA) following the manufacturer's protocol. Briefly, the cells were washed and fixed in cold ethanol for 15 min and incubated with propodium iodide for 30 min. Thereafter, cells were analyzed with a flow cytometer (Becton Dickinson, San Jose, CA, USA).
Reverse transcription-polymerase chain reaction
Total cellular RNA was extracted by the RNeasy MINI Handbook Kit (Qiagen, Valencia, CA, USA). Reverse transcriptase-PCR (RT-PCR) was performed using the one-step kit purchased from Qiagen following the manufacturer's protocol. Briefly, the reaction mixture, which was 50 μl in the final volume, consisted of 2 μg RNA template and 0.6 μ M of forward and reverse primers for SHP1. The RT reaction consisted of one cycle of incubation for 30 min at 50°C, and the PCR consisted of 40 cycles of the following conditions: 95°C for 1 min, then 60°C for 1 min, followed by 72°C for 2 min. The final extension time was 10 min at 72°C. The SHP1 PCR primer was described previously: forward primer, 5′-IndexTermCTCTCCGGAAGCCCCCAGGATG-3′, and reverse primer, 5′-IndexTermCCACCTGAGGACAGCACCGCT-3′.36 The expected product size of the SHP1 amplicon is of 1749 bp The sequences of the β-globin primers are as follows: (sense): 5′-IndexTermCCTGGCTCACCTGGACAACCTCAA-3′ and (antisense): 5′-IndexTermTAGCCACACCAGCCACCACTTTCT-3′. The expected product size of the β-globin amplicon is of 190 bp. The amplified PCR products were then separated on 1% agarose gel and visualized with ethidium bromide stain.
Measurement of ALK tyrosine kinase activity
The activity of NPM-ALK tyrosine kinase was measured using a commercially available kit (Sigma, St Louis, MO, USA) and details of this method have been described previously.19
Gene transfection of NPM-ALK
The NPM-ALK cDNA was initially amplified using an NPM-ALK plasmid (a kind gift by Dr S Morris, St Jude’ Children Research Hospital, Memphis, TN, USA) using a primer set: ALK (forward) 5′-IndexTermATGGAAGATTCGATGGACATG-3′ and NPM (reverse) 5′-IndexTermTCAGGGCCCAGGCTGGTT-3′. The NPM-ALK cDNA was blunt ligated with pCruz His™, which was initially digested with EcoRV. The resulted vector was transformed into Escherichia coli on ampicillin-resistant plates, and plasmids were isolated by the Mini-prep kit (Qiagen). The sequence and orientation of the insert were confirmed by DNA sequencing. Transfection experiments were carried out in six-well plates using LipofectAMINE (Invitrogen Life Technologies) according to the manufacturer's protocol.
Statistical analysis was performed by Student's t-test. A P-value of <0.05 was considered to be statistically significant.
5-AZA induced SHP1 expression in ALK+ ALCL cells
SHP1 was not detectable in either Karpas 299 or SU-DHL-1 at the steady state, as shown by Western blots and RT-PCR. As shown in Figure 1, SHP1 mRNA became detectable in Karpas 299 with addition of 5-AZA to the cell culture 24 h after the initiation of the experiment, with 10 μ M of 5-AZA inducing a higher level than 5 μ M. Western blot analysis showed that SHP1 protein was expressed in Karpas 299, first detectable 2 days after the initiation of the experiment (Figure 2a). The protein level of SHP1 increased gradually with time, reaching the highest level on day 5. On day 2, 10 μ M of 5-AZA induced a higher level of SHP1 than 5 μ M, but there were no appreciable differences in the SHP1 level between these two different dosages of 5-AZA from day 3 to day 5. Induction of SHP1 was similarly observed for SU-DHL-1, except that a higher level of SHP1 was induced with 10 μ M compared to 5 μ M, as illustrated in Figure 2b (day 5 results).
SHP1 expression correlated with decreased expression of JAK3, pJAK3 and pSTAT3
We then evaluated if the restoration of SHP1 expression is associated with any changes in the JAK3 tyrosine phosphorylation in both ALK+ ALCL cell lines. As illustrated in Figure 2a and c, no substantial changes in the expression of JAK3 and pJAK3 were identified shortly after treating Karpas 299 cells with 5-AZA. Nevertheless, both JAK3 and pJAK3 markedly decreased on day 3 when compared to cells treated with diluent only (negative controls). When the pJAK3 levels were assessed after correction of the total JAK3 was made, we identified that pJAK3 was downregulated more than that of the total JAK3 (Figure 2c). The decreased levels of JAK3 and pJAK3 inversely correlated with the marked increase in the SHP1 protein level on day 3. Whereas there were no appreciable changes in the total STAT3 protein level throughout the experiment, pSTAT3 decreased with 5-AZA. Compared to the decreases in JAK3 and pJAK3, the decrease in pSTAT3 was delayed, with >50% reduction identifiable on day 5 of treatment with 10 μ M of 5-AZA (Figure 2a and c). Similar results were obtained from SU-DHL-1 cells treated with 5-AZA (illustrated in Figure 2b). To ensure these changes were not owing to a nonspecific drug effect of 5-AZA, we performed similar experiments using U266 cells, a myeloma cell line that we have confirmed to have constitutive activation of STAT3 and SHP1 expression at the steady state. No detectable changes in SHP1, pSTAT3 and STAT3 were identified in U266 cells treated with 5-AZA for 5 days. These findings support a link between the restoration of SHP1 expression and changes in the JAK3/STAT3 pathway.
Decreased STAT3 activation was associated with modulation of some but not all STAT3 downstream targets
We have previously shown that specific blockade of STAT3 in ALK+ ALCL cells using a dominant-negative construct induced significant alterations in known downstream targets of STAT3 signaling, including Bcl-2, Mcl-1, SOCS3, survivin and cyclin D3.10 Thus, we assessed if the decrease in pSTAT3 associated with SHP1 expression is sufficient to induce similar changes in these STAT3 downstream targets. As illustrated in Figure 2d, the expression levels of cyclin D3 and survivin showed no detectable changes throughout the experiment, but Mcl-1, SOCS-3 and Bcl-2 showed concentration-dependent and time-dependent decreases in their protein levels after treatment with 5-AZA. The decrease in the protein level of Bcl-2 was first noted on day 3, whereas the decreases in SOCS-3 and Mcl-1 were first detectable on day 5. Changes in the STAT3 downstream targets for SU-DHL-1 cells are also illustrated in Figure 2d (right panel). The overall pattern was similar although more marked downregulation in cyclin D3 and Mcl-1 was noted. Similar experiments were performed in U266; no detectable changes were noted in these targets.
Proteasome inhibitor, MG132, inhibited 5-AZA-induced downregulation of JAK3, pJAK3 and STAT3
SHP1 has been reported to inhibit JAKs not only by tyrosine dephosphorylation, but also by decreasing the total protein level via the proteasome pathway.37 Thus, we tested if proteasome inhibitor, MG132, can inhibit the 5-AZA-induced downregulation of the total JAK3 protein level. Both Karpas 299 and SU-DHL-1 cells were incubated in the presence of 5 μ M of 5-AZA for 4 days, treated with MG132 (10 μ M) and harvested at 0, 2, 6 and 10 h after the addition of MG132. As illustrated in Figure 3, JAK3, which was present at a low level after 4 days of 5-AZA treatments, increased in expression in a time-dependent manner. SHP1, pJAK3 and pSTAT3 all increased in their levels with the addition of MG132.
5-AZA did not induce significant changes to the ALK tyrosine kinase activity
As we previously showed that JAK3 might potentiate the tyrosine kinase activity of NPM-ALK in ALK+ ALCL cell lines,19 we tested whether there are changes in the tyrosine kinase of NPM-ALK on day 4, during which both JAK3 and pJAK3 had been downregulated. As shown in Figure 4, in vitro tyrosine kinase assay showed that Karpas 299 cells treated with 5 μ M of 5-AZA for 4 days induced a slight increase in ALK enzymatic activity to 120% of its baseline level, although this difference is not statistically significant (P>0.05). In contrast, a selective JAK inhibitor, AG490, significantly reduced the ALK enzymatic activity to approximately 42% of the negative control cells (acetic acid only).
5-AZA induced cell-cycle arrest and sensitized ALCL cells to doxorubicin-induced apoptosis
Compared to untreated Karpas 299 and SU-DHL-1 cells, treatment with 5-AZA induced a significant decrease in the cell viability as assessed by Trypan blue staining. As shown in Figure 5a, 5-AZA induced a significant decrease in the number of viable cells in both cell lines compared to the untreated cells. As illustrated in Figure 5b, cell cycle analysis revealed that there was a decrease in the proportion of Karpas 299 cells in the S phase as well as the G2/M phase. The S-phase fraction decreased from 31 (untreated) to 22 and 21%, at 5 and 10 μ M of 5-AZA, respectively. There was no detectable increase in the sub-G0/G1 fraction. The lack of an increase in apoptosis was further supported by the absence of a detectable increase in the cleaved caspase-3 level shown on Western blots (not shown). Despite the absence of increased apoptosis, 5-AZA potentiated apoptosis induced by doxorubicin, a chemotherapeutic drug used commonly to treat ALCL patients. As shown in Figure 5c, Karpas 299 cells were treated with a relatively low dose (0.1 μ M) of doxorubicin. Compared with the use of doxorubicin alone, the combination of doxorubicin and 5-AZA was more effective in reducing the number of viable cells, although the difference between the two experimental conditions is statistically significant only on day 4.
In view of a recent publication that active STAT3 binds to DNA methyltransferase 1 (DNMT1) and promotes methylation/silencing of SHP1,38 we tested if transfection of NPM-ALK, which activates STAT3 in ALK+ ALCL cells, leads to downregulation of SHP1. After gene transfection into GP293 cells, STAT3 became tyrosine phosphorylated and SHP1 was downregulated (Figure 6), and the decrease was estimated to be 32% using densitometry analysis (after correction of a slight difference between the actin bands). Interestingly, the total STAT3 band was consistently decreased slightly (averaging 88% of the negative control).
Loss of SHP1 associated with gene hypermethylation is a relatively common abnormality in hematologic cancers;19, 39, 40 the exact biological significance of this abnormality has not been extensively studied. The main objective of this study is to correlate the restoration of SHP1 expression using 5-AZA and changes in the JAK3/STAT3 signaling pathway in ALK+ ALCL cells, which are often characterized by the absence of SHP1 and constitutive activation of the JAK3/STAT3 pathway.
We found that 5-AZA induced a time-dependent increase in the SHP1 mRNA and protein level in both ALK+ ALCL cell lines, and this change coincided with the downregulation of pJAK3 and JAK3, followed by a decrease in pSTAT3. Substantial (i.e. >50% of the negative controls) downregulation of both JAK3 and pJAK3 was detectable. The finding that pJAK3 did not decrease in their levels until day 3 of the experiment is rather unexpected, as the current concept is that SHP1 is a rapid acting tyrosine phosphatase. This unexpected result suggests that the initially low level of SHP1 is not sufficient in balancing the overactivated JAK3. We also found that reduction in the total JAK3 protein level was dependent on the proteasome degradation pathway, as a proteasome inhibitor, MG132, efficiently blocked the decrease of JAK3. A parallel observation was made by Wu et al.,37 who found that expression of SHP1 in HTB26 cells negatively regulates JAK1 by dephosphorylation and promotion of proteasome degradation. Thus, SHP1 negatively regulates the JAK/STAT pathway through at least two different mechanisms. Additional studies are required to delineate how SHP1 promotes proteasome degradation of JAK3. Interestingly, as shown in Figure 3, both pSTAT3 and SHP1 increased in their levels after addition of MG132, and these findings support the concept that STAT3 activation is more related to the expression/activation level of JAK3 than the SHP1 expression. Another interesting observation is that a marked reduction of the pSTAT3 level was relatively delayed compared to that of JAK3 or pJAK3. This finding suggests that SHP1 downregulated STAT3 activation indirectly, likely via decreasing JAK3 expression and activation. This is in keeping with our previous finding that JAK3 contributes to STAT3 activation in ALK+ ALCL cells.18
Zhang et al.38 recently reported that active STAT3 forms a complex with DNMT1, which binds to the SHP1 promoter and facilitates gene methylation of SHP1. As NPM-ALK is an important activator of STAT3 in ALK+ ALCL, we tested if ectopic NPM-ALK expression in GP293 cells results in downregulation of SHP1. Results from our gene transfection experiments (Figure 6) are entirely in keeping with this concept. However, our findings suggest that STAT3 activation alone is not sufficient to induce complete SHP1 silencing; additional mechanisms likely exist to achieve complete SHP1 gene silencing. A mild decrease in the total STAT3 level was identified after NPM-ALK; the mechanism underlying this finding is unclear.
The partial but incomplete downregulation of pSTAT3 after 5-AZA treatment is expected, as the other activators of STAT3 such as NPM-ALK and src are probably functional. With the partial decrease in the pSTAT3 level, a number of STAT3 downstream targets showed detectable decreases in their expression (such as Mcl-1, SOCS3 and Bcl-2). The relative lack of changes in survivin and cyclin D3 in this study is probably owing to the persistence of STAT3 activation, albeit at a relatively low level. These findings suggest that the expression of various STAT3 downstream targets may be triggered at different levels of STAT3 activation.
Despite the decrease in Bcl-2 and Mcl-1 levels, we did not detect significant apoptotic cell death with 5-AZA. Lack of apoptotic cell death in our experiments can at least be partially explained by the persistent expression of survivin. Survivin is an important anti-apoptotic protein.41 Previous studies have shown that STAT3 induces its anti-apoptoic effects via induction of survivin,42 and enforced expression of survivin can suppress cell death induced by STAT3 inhibition.43 Nevertheless, we found that 5-AZA sensitized Karpas 299 cells to doxorubicin-induced apoptosis, and this may be due to decreases in other anti-apoptotic proteins such as Mcl-1 and Bcl-2. In support of this concept, the difference in apoptosis between Karpas 299 cells treated with combined 5-AZA and low-dose doxorubicin and cells treated with low-dose doxorubicin alone was not statistically significant until day 4 of the experiment, and this correlates with the findings that Bcl-2 and Mcl-1 did not show substantial decreases in their expression level until day 4 or 5, respectively.
We found that 5-AZA induced cell-cycle arrest in Karpas 299 cells, predominantly in the G2/M phase. This is almost certainly not related to cyclin D3, a G1 promoter that did not show significant changes in its expression level after 5-AZA. From the literature, it has been reported that the p38/MAPK pathway is modulated by 5-AZA,44 and downregulation of this pathway may be responsible for the G2/M arrest observed in this study. As 5-AZA can modulate the expression of many genes, depending on the gene methylation pattern of specific cell types, it is highly likely that other genes that may have been upregulated by this agent in ALK+ ALCL cells are responsible for these biological effects.
Lastly, after we submitted this manuscript for publication, Honorat et al.45 have published that NPM-ALK is a substrate for SHP1, and SHP1 negatively regulates NPM-ALK and decreases tumorigenecity. Thus, these findings are in keeping with the concept that loss of SHP1 contributes to the pathogenesis of ALK+ ALCL. As mentioned in discussion of this paper, there is a discrepancy related to the SHP1 expression in Karpas 299 cells between the two studies. We believe that this discrepancy is likely owing to the existence of different subclones of this cell line.
We found that restoration of SHP1 expression induced by 5-AZA correlates with a significant downregulation of the JAK3/STAT3 signaling in both ALK+ ALCL cells. Our data support the concept that loss of SHP1 contributes to the pathogenesis of ALK+ ALCL. In addition to its role as a tyrosine phosphatase, SHP1 silences JAK3 by virtue of promoting JAK3 degradation via the proteasome pathway.
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This study is supported by research grants from the Alberta Cancer Board and the University of Alberta Hospital Research Foundation awarded to RL.
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Han, Y., Amin, H., Frantz, C. et al. Restoration of shp1 expression by 5-AZA-2′-deoxycytidine is associated with downregulation of JAK3/STAT3 signaling in ALK-positive anaplastic large cell lymphoma. Leukemia 20, 1602–1609 (2006). https://doi.org/10.1038/sj.leu.2404323
- anaplastic large cell lymphoma
- JAK3/STAT3 signaling
- DNA methylation
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