|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on October 3, 2002; DOI 10.1182/blood-2002-07-2130.
NEOPLASIA
From the Experimental Transplantation and Immunology
Branch, Center for Cancer Research, National Cancer Institute, National
Institutes of Health, Bethesda, MD; and Division of Monoclonal
Antibodies, Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD.
Despite some exciting new leads in molecular pathogenesis,
AIDS-defining primary effusion lymphoma (PEL) remains a fatal
malignancy. The lack of substantial progress in the management of PEL
demands innovative treatment approaches. Targeting intracellular
molecules critical to cell survival is one unexplored strategy for
treating PEL. Here we show that inhibition of signal transducer and
activator of transcription-3 (STAT3) leads to apoptosis in PEL cells.
STAT3 is constitutively phosphorylated in PEL cell lines BC-1, BCBL-1, and VG-1. Transduction of dominant-negative STAT3 and pharmacological STAT3 inhibition caused caspase-dependent cell death. Although STAT3
activation is known to induce expression of Bcl-2 family proteins, PEL
cell apoptosis was independent of Bcl-2,
Bcl-XL, or Mcl-1 protein expression.
Instead, STAT3 inhibition induced transcriptional repression of
survivin, a recently identified inhibitor of apoptosis. Forced
overexpression of survivin rescued VG-1 cells from apoptosis induced by
STAT3 inhibition. Our findings suggest that activated STAT3 signaling
directly contributes to malignant progression of PEL by preventing
apoptosis, acting through the prosurvival protein survivin. Since
constitutive STAT3 activation and survivin expression have been widely
documented in different types of cancers, their linkage may extend
to many malignancies and be critical to their pathogenesis.
(Blood. 2003;101:1535-1542) Since the advent of highly active
antiretroviral therapy for HIV infection, life expectancy has improved
substantially for HIV-infected individuals in whom virus replication
can be successfully suppressed.1 Despite significant
efforts to improve its outcome, non-Hodgkin lymphoma (NHL) remains a
lethal complication of AIDS.2,3 AIDS-related NHLs present
a predilection for widespread, extranodal disease; involvement of the
central nerve system; frequent Epstein-Barr virus (EBV) infection; and
poor responses to dose-intensive chemotherapy with high relapse
rates.4-7 Among AIDS-related NHLs, primary effusion
lymphoma (PEL) generally has an extremely aggressive clinical course
with a median survival of only 3 months.8 PEL usually
presents as a lymphomatous effusion in body cavities, and most cases
exhibit dual infection with EBV and Kaposi sarcoma-associated herpesvirus (KSHV).9,10 Pleural and abdominal effusions
from patients with AIDS-PEL contain various inflammatory
cytokines.11,12 Among them, interleukin 10 (IL-10)
and, to a lesser degree, viral IL-6 (vIL-6), the KSHV homolog
of human IL-6,13 serve as autocrine growth factors for PEL
cells.14 Vascular endothelial growth factor (VEGF),
although not known to be an autocrine growth factor, is produced at a
high level by PEL cells that also express VEGF receptor-1.15 Noteworthy, IL-10, vIL-6, and VEGF all
induce phosphorylation of signal transducer and activator of
transcription-3 (STAT3) for intracellular
signaling.16-18 Ligand-dependent activation of STAT
proteins is associated with organ development, cell proliferation, and
differentiation, while constitutive activation of STATs often results
in growth deregulation.19,20 STAT3 protein is
constitutively activated in many human cancer cells, and aberrant STAT3
signaling is implicated as an important process in malignant
progression.20 However, the mechanisms by which STAT3
contributes to oncogenesis have not been identified. Here we show that
constitutive phosphorylation of STAT3 plays a critical role in
promoting survival of KSHV+ PEL cells and that
inhibition of STAT3 activity results in reduced protein expression of
survivin, a member of the inhibitor of apoptosis protein (IAP)
family.21 Our findings provide evidence that survivin directly contributes to malignant progression of PEL and that targeting
survivin by STAT3 inhibition may provide a novel therapeutic approach
for this aggressive lymphoma.
Cell lines
Western blotting and reverse transcriptase-polymerase chain
reaction (RT-PCR)
RT-PCR was performed as previously described,15 with the use of the following primer pairs: (1) vIL-6 forward, 5'-ATGTGCTGGTTCAAGTTGTGGTCTC-3'; reverse, 5'-TCACGTCGCTCTTTACTTATCGTG-3'; (2) viral FLICE-inhibitory protein forward, 5'-ATACAAGCCGGCACCATGGCC-3'; reverse, 5'-CTATGGTGTATGGCGATAGTGCC-3'; (3) viral cyclin forward, 5'-CACCCTGAAACTCCAGGC-3'; reverse, 5'-GATCCGATCCTCACATAGCG-3'; (4) latency-associated nuclear antigen (LANA) forward, 5'-GCAGTCTCCAGAGGTCTTCTC-3'; reverse, 5'-CGGAGCTAAAGAGTCTGGTG-3'; and (5) viral survivin forward, 5'-CAGCTTGAGTCAGTTTAGCACTGG-3'; reverse, 5'-GTACTACAGCTCTCAGGCCAATGT-3'. The quality of RNA was confirmed in all samples by parallel RT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).15 Construction and transfection of a dominant-negative STAT3-expressing vector The cDNA of STAT3D, a dominant-negative form of murine STAT3 in which Phe424 and Phe435 of STAT3 are replaced with alanines26 (a kind gift from Dr Koichi Nakajima, Osaka City University, Japan), was inserted into the EcoRI site of the pEGFP-C2 vector (BD Biosciences Clontech, Palo Alto, CA). This construct expresses STAT3D as a fusion protein to the C-terminus of enhanced green fluorescence protein (EGFP). Plasmid vectors were transfected into cells with the use of Lipofectamine 2000 (Invitrogen) and transferrin (Sigma Chemical). Cells were harvested 2 and 3 days after transfection for the performance of Western blotting and apoptosis assays. Sorting of EGFP+ population was performed by means of the FACSVantage SE cell sorter (BD Biosciences, Sunnyvale, CA).Apoptosis assay Cells were washed with ice-cold PBS, resuspended in annexin V binding buffer (BD Pharmingen, San Diego, CA), and subsequently stained with annexin V-fluorescein isothiocyanate (FITC) or annexin V-phycoerythrin (PE) (BD Pharmingen) according to the manufacturer's recommendation. Propidium iodide (PI) was used as an indicator of cell viability. To examine the effect of tyrphostin AG490, dead cells were removed with the use of LSM lymphocyte separation medium (ICN, Aurora, OH), and the remaining cells were incubated at 5 × 105/mL in 12-well plates (2 mL per well) with AG490 or 0.1% DMSO for 48 hours. In the assays in which the caspase pathway was examined, the cell-permeable caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl( -methyl ester-fluoromethy
ketone (Z-VAD-fmk) (Calbiochem) was added at 100 µM
to the cell suspension before the induction of apoptosis. Data
acquisition and analysis were performed by means of a fluorescence microscope (Eclipse E600; Nikon, Melville, NY) or a FACScalibur cytofluorimeter with CELLQuest software (BD Biosciences).
DNA synthesis assay Cells were cultured in 96-well microwell plates at 104 cells per well in RPMI 1640 with 10% fetal bovine serum (FBS) and graded concentrations of AG490 in an equal volume of drug solvent DMSO for 24 hours. DNA synthesis was measured by [3H]-thymidine incorporation (0.5 µCi [18.5 kBq] per well) (Perkin-Elmer) during the last 6 hours of culture. All assays were performed in triplicate cultures.Electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared as described previously.27 Protein concentrations were determined and normalized by the addition of extraction buffer. Electrophoretic mobility shift assays were performed as previously described.28 An oligonucleotide probe (5'-AGCTTAGGTTTCCGGGAAAGCAC-3') corresponding to the IL-6 response element of the intercellular adhesion molecule-1 (ICAM-1) promoter29 was end-labeled with the use of polynucleotide kinase and 32P- -adenosine triphosphate (ATP).
Competitive inhibition experiments were performed with the use of a
50-fold molar excess of unlabeled oligonucleotide. For supershift
assays, prepared extracts were incubated with rabbit polyclonal Abs
against STAT1 and/or STAT3 (Santa Cruz Biotechnology) for 60 minutes
prior to addition of labeled probe. Samples were then electrophoresed
through a 6% nondenaturing polyacryamaide gel and dried, and STAT-DNA
complexes were detected by autoradiography.
Cytokine measurements Cells were suspended at 1 × 106/mL in RPMI 1640 supplemented with 10% FBS, and cultured for 24 hours in the presence of AG490 or 0.1% DMSO. Conditioned media were tested by enzyme-linked immunosorbent assay (ELISA) with the use of human VEGF, IL-6, and IL-10 Quantikine kits (R&D Systems), following the manufacturer's instructions. The human IL-6 ELISA does not detect vIL-6.12 ELISA for KSHV vIL-6 was performed as described previously.12Assays of reporter gene activity The human survivin promoter activity was examined by 2 types of reporter gene systems: EGFP and heat-stable secreted alkaline phosphatase (SEAP). The human survivin promoter reporter constructs were derived from a 1.6-kb genomic fragment containing the 5' region of the survivin gene upstream of the ATG translational start codon. Specific gene amplification by PCR was performed on genomic DNA isolated from human peripheral blood mononuclear cells with the following primers: psurvivin S-AseI, 5'-GGTGATTAATATATGAGTATTCTTTGTACT-3'; psurvivin AS- HindIII, 5'-AGGGAAGCTTCTTGAATGTAGAGAT-3'. After digestion with AseI and HindIII, the 1.6-kb fragment corresponding to positions 1512 to +70 of the human survivin promoter was purified, and the AseI-HindIII site of pEGFP-N3 vector (BD
Biosciences Clontech), encoding the cytomegalovirus promoter, was
replaced with the human survivin promoter. The construct DNA
sequence was confirmed by cycle sequencing by means of the ABI PRISM
dye terminal cycle sequencing kit (Applied Biosystems, Foster City,
CA). To construct SEAP2-psurvivin, p-SEAP2-basic vector
(BD Biosciences Clontech) was digested with XhoI,
blunt-ended, and digested with HindIII. The human
survivin promoter fragment was excised from plasmid psurvivin-EGFP by AseI digestion, blunting, and
HindIII digestion, and was subsequently subcloned into the
p-SEAP2-basic vector. The structure of psurvivin-SEAP was
confirmed by restriction enzyme mapping. Transient transfection of
psurvivin-EGFP, SEAP2-psurvivin, p-SEAP2-basic, and p-SEAP2-promoter (BD Biosciences Clontech) was
performed with the use of Lipofectamine 2000 and transferrin. After 24 hours, cells were suspended at 5 × 105/mL in RPMI 1640 supplemented with 10% FBS, and cultured for additional 48 hours in the
presence of AG490 or 0.1% DMSO. EGFP levels were determined by flow
cytometry. SEAP assays were performed with the use of supernatants
heated at 65°C for 30 minutes to inactivated endogenous alkaline
phosphatase. The SEAP activity was measured by using a colorimetric
assay based on hydrolysis of the para-nitrophenyl phosphate.
Forced overexpression of survivin in PEL cells To construct a survivin-expressing vector, survivin cDNA was amplified by RT-PCR with the use of the following primers: survivin S-BamHI, 5'-GGCGGATCCATGGGTGCCCCGACGT-3'; survivin AS-XbaI, 5'-CCGTCTAGAGGCCTCAATCCATGGCA-3'. After digestion with BamHI and XbaI, this fragment was subcloned into the BamHI-XbaI site of pEF/Bsd vector (Invitrogen). The construct DNA sequence was confirmed by cycle sequencing. The vector was transfected into VG-1 cells by means of GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA). After selection with Blasticidin S (Invitrogen), stable survivin-transfected and control vector-transfected clones from each cell line were used for subsequent assays.
Dominant-negative STAT3 induces apoptosis in PEL cells Aberrant STAT activation is found in many malignancies, and constitutive activation of STAT3 is associated with cellular transformation, including B-cell transformation by EBV.19,20,30 To examine STAT3 activation in KSHV-infected cells, Western blot analysis was performed for PEL cell lines BC-1, BCBL-1, and VG-1. BAF130 cells, representing murine BAF-B03 cells stably transfected with human gp130, are known to proliferate in response to v-IL-6.31 Phosphorylation of STAT3 was detected in BAF130 cells stimulated with recombinant vIL-6 (Figure 1A). Without exogenous cytokine stimulation, STAT3 phosphorylation was documented in all 3 PEL cell lines. Consistent with a previous report,23 phosphorylated STAT3 was not detected in Bcr-Abl-transformed K562 cells although unphosphorylated STAT3 was expressed abundantly in these cells.
To investigate the role of STAT3 in PEL cell survival, we constructed a vector that expresses STAT3D, a dominant-negative form of STAT3,26 as a fusion protein of EGFP. Tyrosine-phosphorylated STAT dimers translocate to the nucleus and bind specific DNA elements leading to transcriptional activation.19 Since the DNA-binding site is mutated, STAT3D can act as a specific competitive inhibitor of wild-type STAT3.26 When PEL cells were transfected with this construct, the expression of dominant-negative STAT3 was reflected by the intensity of EGFP. Expression of EGFP-STAT3D induced significant apoptotic cell death in PEL cells. The occurrence of apoptosis was measured by fluorescence-activated cell sorter (FACS) analysis of plasma membrane annexin V binding.32 At 3 days after transfection, up to 89% of STAT3D-expressing PEL cells underwent apoptosis while STAT3D transfection minimally increased the annexin V+ population in K562 cells (Figure 1B). EGFP itself was not toxic to PEL cells since fewer than 5% of cells expressing EGFP alone were apoptotic. The vast majority of VG-1 cells expressing EGFP-STAT3D (green) were positive for annexin V (red) (Figure 1C). This observation was further supported by FACS analysis demonstrating that most of the cells in the EGFP-STAT3D+ population shifted to the small-sized/PI+ fraction (Figure 1D). Similar results were obtained in BC-1 and BCBL-1 cells (data not shown). By contrast, K562 cells expressing EGFP-STAT3D continued to grow for more than 2 months in the presence of G418 (data not shown). These results suggest that constitutive STAT3 activation is critical to PEL cell survival. Pharmacological STAT3 inhibition induces apoptosis in PEL cells We examined the effect of pharmacological STAT3 inhibition on PEL cell growth. Tyrphostin AG490 is described as a Janus kinase 2 (JAK2) inhibitor leading to suppression of constitutively activated STAT3 molecules in acute leukemias and multiple myelomas.32,33 We looked for this effect of AG490 in PEL. An EMSA using an oligonucleotide probe corresponding to the IL-6 response element of the ICAM-1 promoter documented constitutive DNA binding activity in BC-1 cells (Figure 2A). Supershift assays identified these DNA-binding complexes as homodimers and heterodimers of STAT1 and STAT3 (Figure 2B). AG490 treatment caused disappearance of the bands corresponding to STAT3/3 and STAT1/3, but not STAT1/1 (Figure 2A), suggesting that AG490 elicits a selective inhibitory effect on STAT3 activation.
We then examined the effects of AG490 using 2 cell lines, K562 and TF-1, in which JAK2 plays a primary role for cell growth. K562 cells display constitutive phosphorylation of JAK2 that is critical to Bcr-Abl malignant transformation.34 TF-1 is a multifactor-dependent erythroleukemia cell line that exhibits rapid JAK2 phosphorylation in response to GM-CSF or IL-6, followed by STAT1/3/5 activation.24 When incubated with AG490 for 24 hours, K562 cells displayed decreased DNA synthesis in a dose-dependent manner (Figure 2C). TF-1 cells stimulated with exogenous GM-CSF or IL-6 showed a sensitivity to AG490 inhibition similar to that of K562 cells (Figure 2C). Of note, PEL cell lines exhibited a much greater sensitivity to AG490 than K562 cells or TF-1 cells (Figure 2C). This reduction in DNA synthesis correlated directly with the inhibitory effects of AG490 on DNA-binding activity (Figure 2A). We next examined whether AG490 can promote apoptotic cell death. Following 48-hour exposure to AG490, PEL cells displayed a marked increase in annexin V binding measured by flow cytometry, with up to 78% of cells undergoing apoptosis (Figure 2C). By contrast, only a modest increase in annexin V+ cells was documented in K562 cells, which harbor constitutively activated JAK2,34 in spite of the fact that AG490 caused 70% reduction in [3H]-thymidine incorporation in these cells. These results suggest that AG490 induces mainly a cytostatic effect in K562 cells and a cytotoxic effect in PEL cells. KSHV-derived proteins and autocrine growth factors PEL is characterized by its consistent infection with KSHV. An important issue is whether STAT3 inhibition suppresses presumed oncogenic functions of KSHV, leading to apoptosis of PEL cells. KSHV encodes several proteins that regulate the proliferation and survival of infected cells. Some viral proteins are expressed only in the lytic phase, such as the viral homolog of Bcl-2 (open reading frame 16 [orf 16]), whereas others are expressed in latently infected PEL cells.35 Viral FLICE-inhibitory protein (orf K13/orf 71) protects cells from Fas-mediated apoptosis, and viral cyclin (orf 72) inactivates the tumor-suppressor function of retinoblastoma protein.36,37 Latency-associated nuclear antigen (orf 73) deregulates the retinoblastoma and p53 proteins.38 Recently, the viral homolog of a spliced variant of survivin has been identified within KSHV orf K7.39 The orf K7 (viral survivin) is expressed in latently infected PEL cells,35 and its protein product inhibits Fas-mediated apoptosis.39 By RT-PCR, no significant reduction of message from these viral genes was detected after incubation with AG490 (Figure 3A). In addition, expression of vIL-6 (orf K2) and viral survivin was not increased by AG490. Both vIL-6 and viral survivin are expressed constitutively in KSHV-infected PEL cells, but their expression is further induced in the lytic phase.12,35 These results suggest that AG490 does not induce KSHV lytic replication or regulate latent genes that can modulate cell cycle and survival.
We examined whether STAT3 inhibition modified the secretion of growth factors that play a pathogenetic role in PEL (Figure 3B). Secretion of vIL-6, an autocrine growth factor for some PEL cells, was not modified by AG490 in BC-1 or BCBL-1 cells but was reduced by 50% in VG-1 cells. Since vIL-6 expression is up-regulated during KSHV replication, this result further suggests that AG490 does not promote KSHV lytic replication in PEL cells. Production of IL-10, another autocrine growth factor for some PEL cells, was increased by 3-fold in BCBL-1 cells, perhaps reflective of an autocrine IL-10 feedback response,16 but did not change in BC-1 cells. In VG-1 cells, IL-10 was not detectable, but vIL-6 levels were much higher than those of BC-1 and BCBL-1, suggesting that autocrine growth stimulation in VG-1 cells may rely on different cytokines from those in BC-1 and BCBL-1 cell lines. However, we have no evidence that a 50% reduction in vIL-6 production is detrimental to the survival of VG-1 cells. AG490 did not change VEGF production in PEL cells even though STAT3 plays a role in transcriptional regulation of the VEGF gene in other cells.40 Secretion of IL-6 was reduced by 75% in BC-1 cells treated with AG490, but IL-6 does not affect BC-1 cell proliferation14 so it is unlikely that this reduction mediates AG490-induced apoptosis. Together, these results suggest that growth factor modulation is not essential to AG490-induced apoptosis. STAT3 inhibition reduces survivin expression By regulating expression of the Bcl-2 family of antiapoptotic proteins in many cell types, STAT3 activation can inhibit apoptosis and promote cell proliferation. BCL-2 family members can protect cancer cells from apoptosis induced by a variety of stimuli, including anticancer agents.32,33,41 In PEL cells, however, we did not find diminished Mcl-1 and Bcl-XL expression following treatment with AG490 (Figure 4A). Bcl-2 was detectable only in BC-1 cells, and expression of this protein was not diminished by AG490 treatment. A novel modulator of the cell death/viability balance in cancer was recently identified as survivin, a member of the IAP family.21 It is noteworthy that expression of survivin was significantly reduced in AG490-treated PEL cells. By contrast, minimal reduction of survivin expression was observed in K562 cells. In PEL cells, inhibition of survivin expression was noted within 12 hours after AG490 treatment (Figure 4B). Suppression of survivin expression was also induced by dominant-negative STAT3 (Figure 4C). Transduction of EGFP-STAT3D was associated with reduced survivin expression in PEL cells but not in K562 cells. These results suggest that STAT3 inhibition induces a selective down-regulation of survivin expression in PEL cells.
Recent studies suggest that survivin protects cells from apoptosis by
inhibiting caspase proteases directly or indirectly.42 Cleavage and activation of caspases plays a central role in the initiation and execution of apoptosis.42 To examine if
AG490-induced apoptosis is a caspase-dependent phenomenon, PEL cells
were incubated with the broad-spectrum caspase inhibitor Z-VAD-fmk.
AG490-mediated killing of VG-1 cells was significantly reduced in the
presence of Z-VAD-fmk (Figure 5). A
similar effect was noted in BC-1 and BCBL-1 cells (data not shown).
Together, these results indicate that AG490-induced PEL cell death is
mediated mainly by a caspase-dependent pathway, and raise the
possibility that STAT3 signaling may contribute to the prevention
of programmed cell death by inducing survivin expression.
Transcriptional suppression of survivin gene expression by AG490 To determine if the AG490-induced decrease in survivin protein expression is due to repression of survivin promoter activity, a SEAP reporter construct was made. The 1.6-kb survivin promoter fragment (positions1512 to +70) was cloned into a SEAP
reporter plasmid, designated SEAP2-psurvivin (Figure
6A). The pSEAP2-promoter is a positive
control reporter vector that contains the SV40 early promoter upstream
of the SEAP gene. After transfection (72 hours), alkaline phosphatase
activity was detected in culture supernatants of both K562 and BC-1
cells transfected with pSEAP2-promoter. Equivalent or higher levels of
alkaline phosphatase activities were detected in cells transfected with
SEAP2-psurvivin, whereas minimal activity was detected in
cells transfected with the vector lacking a promoter. AG490 suppressed
survivin promoter activity significantly in BC-1
cells but only slightly in K562 cells. The alkaline phosphatase
activity was suppressed more than 90% in BC-1 cells transfected with
SEAP2-psurvivin. By contrast, AG490 reduced alkaline
phosphatase activity by approximately 50% in BC-1 cells transfected
with SEAP2-promoter even though a large proportion of BC-1 cells
underwent apoptosis.
To confirm the effect of AG490 on the live cell population, we used another reporter plasmid that contains the survivin promoter upstream of the EGFP gene. BC-1 cells were transfected with this reporter plasmid, and EGFP expression in the live cell population was examined after 48-hour incubation with AG490 or DMSO. Flow cytometry analysis revealed a marked reduction of EGFP expression in live PEL cells by AG490 (Figure 6B), and the vast majority of the live cells underwent apoptosis in the following 24 hours (data not shown). We conclude from these results that AG490 induces repression of the human survivin promoter in PEL cells, and suggest that constitutively activated STAT3 promotes transcriptional activation of the human survivin gene, providing PEL cells with a survival advantage. Forced expression of survivin rescues PEL cells from AG490-induced apoptosis To examine whether survivin repression is responsible for PEL cell apoptosis induced by STAT3 inhibition, we established stable clones of VG-1 cells transfected with a survivin construct driven by the human elongation factor-1 subunit. After selection of stable
transfectants, survivin-transfected as well as control vector-transfected cells were incubated with AG490. Expression of
survivin protein was retained after AG490 treatment in
survivin-transfected VG-1 cells, but was markedly reduced in control
vector-transfected cells (Figure 7A). In
DNA synthesis assays, enforced expression of survivin rendered VG-1
cells significantly less sensitive to AG490 (Figure 7B). After 48-hour
incubation with AG490, annexin V+ cells increased
significantly in control vector-transfected but not in
survivin-transfected cells (Figure 7C). Similar results were obtained
in BCBL-1 cells transfected with a survivin expression vector (data not
shown). These results show that survivin can counteract apoptosis
induced by AG490 in PEL cells, and provide evidence that constitutive
activation of STAT3 sustains growth and survival of PEL cells, at least
in part, through stimulation of survivin expression.
Via autocrine production of growth factors or constitutive activation of intracellular signaling pathways, tumor cells can gain independence of exogenous growth factors.43 Unlike normal cells and tissues, constitutively activated STATs are detected in a wide variety of human cancer cells, and aberrant activation of STAT proteins is often associated with cellular transformation by various oncoproteins.19,20 Particularly in lymphoid cells, a link between STAT3 activation and regulation of Bcl-2 family protein expression has been demonstrated.32,33,41 STAT3 was required for Bcl-2 induction in pro-B cells,44 and for bcl-x gene expression in U266 myeloma cells.32 Inhibition of STAT3 signaling resulted in apoptosis and decreased Mcl-1 expression in large granular lymphocyte leukemias.33 More recently, inhibition of STAT3 signaling in B-NHL cells by endogenous IL-10 neutralization resulted in reduced Bcl-2 expression and sensitization to cytotoxic drugs.41 Here we show that STAT3 inhibition in PEL cells induced apoptosis without down-regulation of Bcl-2, Mcl-1, or Bcl-XL expression. Instead, expression of the IAP family protein survivin was significantly reduced by STAT3 inhibition, and forced expression of survivin rescued PEL cells from AG490-induced apoptosis. In contrast to other lymphoid malignancies in which STAT3 inhibition sensitized cells to Fas-mediated apoptosis and to cytotoxic agents,32,33,41 STAT3 inhibition by itself resulted in PEL cell death, suggesting that STAT3 is a desirable therapeutic target for PEL. Survivin has been implicated in both cell cycle control and
apoptosis resistance.42 Survivin promoter exhibits typical
M phase inducible transactivation,45 and interference with
the expression of survivin causes caspase-dependent cell death in the
G2/M phase of the cell cycle.46 The half-life
of survivin protein is only 30 minutes, and the ubiquitin-proteasome
pathway is believed to mediate degradation of this
protein.47 Here, we demonstrate that inhibition of STAT3
phosphorylation by AG490 results in transcriptional repression of the
human survivin promoter activity. STAT3 plays a key role in
G1-to-S phase cell cycle transition,44 and
survivin displays cell cycle-regulated expression that peaks in the
G2/M phase.45 Hence, reduced expression of
survivin might simply be the result of inhibited cell cycle progression
when STAT3 is inactivated. In a recent report, fibroblasts transformed by a persistently activated form of STAT3 presented increased survivin
expression, which was down-regulated by
interferon- Among molecules of the IAP family, survivin exhibits the most restricted expression in adult tissues.42 Only occasional normal adult human or mouse tissues express survivin, whereas most of the common human cancers do.21,42 Selectivity of expression and biological importance render survivin an attractive target for treatment, but the 3-dimensional structure of survivin reveals no obvious opportunity for drug design.42,52 Disruption of STAT3 activation by tyrphostin AG490 may provide a new therapeutic approach for targeting survivin and treating AIDS patients with PEL. Since constitutive STAT3 activation and survivin expression are both commonly detected in cancers, their linkage may extend far beyond PEL.
We thank Drs Robert Yarchoan, Koichi Akashi, and Masashi Narazaki for helpful comments; Mike Klutch and George Poy for their help in DNA sequencing; Dr William Telford for his technical advice in FACS analysis; and Dr Veena Kapoor for cell sorting.
Submitted July 18, 2002; accepted September 24, 2002.
Prepublished online as Blood First Edition Paper, October 3, 2002; DOI 10.1182/blood- 2002-07-2130.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yoshiyasu Aoki, Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Dr 12N226, Bethesda, MD 20892-1907; e-mail: aokiy{at}mail.nih.gov.
1.
Levine AM.
Evaluation and management of HIV-infected women.
Ann Intern Med.
2002;136:228-242
2.
Levine AM, Seneviratne L, Espina BM, et al.
Evolving characteristics of AIDS-related lymphoma.
Blood.
2000;96:4084-4090 3. Levine AM, Scadden DT, Zaia JA, Krishnan A. Hematologic aspects of HIV/AIDS. Hematology (Am Soc Hematol Educ Program). 2001;463-478.
4.
Shibata D, Weiss LM, Hernandez AM, Nathwani BN, Bernstein L, Levine AM.
Epstein-Barr virus-associated non-Hodgkin's lymphoma in patients infected with the human immunodeficiency virus.
Blood.
1993;81:2102-2109
5.
Kaplan LD, Straus DJ, Testa MA, et al.
Low-dose compared with standard-dose m-BACOD chemotherapy for non-Hodgkin's lymphoma associated with human immunodeficiency virus infection. National Institute of Allergy and Infectious Diseases AIDS Clinical Trials Group.
N Engl J Med.
1997;336:1641-1648 6. Kieff E. Current perspectives on the molecular pathogenesis of virus-induced cancers in human immunodeficiency virus infection and acquired immunodeficiency syndrome. J Natl Cancer Inst Monogr. 1998;23:7-14[Medline] [Order article via Infotrieve].
7.
Carbone A, Gloghini A, Larocca LM, et al.
Expression profile of MUM1/IRF4, BCL-6, and CD138/syndecan-1 defines novel histogenetic subsets of human immunodeficiency virus-related lymphomas.
Blood.
2001;97:744-751
8.
Cesarman E, Chang Y, Moore PS, Said JW, Knowles DM.
Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.
N Engl J Med.
1995;332:1186-1191
9.
Nador RG, Cesarman E, Chadburn A, et al.
Primary effusion lymphoma: a distinct clinicopathologic entity associated with the Kaposi's sarcoma-associated herpes virus.
Blood.
1996;88:645-656 10. Tirelli U, Spina M, Gaidano G, Vaccher E, Franceschi S, Carbone A. Epidemiological, biological and clinical features of HIV-related lymphomas in the era of highly active antiretroviral therapy. AIDS. 2000;14:1675-1688[CrossRef][Medline] [Order article via Infotrieve].
11.
Aoki Y, Tosato G, Nambu Y, Iwamoto A, Yarchoan R.
Detection of vascular endothelial growth factor in AIDS-related primary effusion lymphomas.
Blood.
2000;95:1109-1110
12.
Aoki Y, Yarchoan R, Braun J, Iwamoto A, Tosato G.
Viral and cellular cytokines in AIDS-related malignant lymphomatous effusions.
Blood.
2000;96:1599-1601
13.
Aoki Y, Jaffe ES, Chang Y, et al.
Angiogenesis and hematopoiesis induced by Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6.
Blood.
1999;93:4034-4043
14.
Jones KD, Aoki Y, Chang Y, Moore PS, Yarchoan R, Tosato G.
Involvement of interleukin-10 (IL-10) and viral IL-6 in the spontaneous growth of Kaposi's sarcoma herpesvirus-associated infected primary effusion lymphoma cells.
Blood.
1999;94:2871-2879
15.
Aoki Y, Tosato G.
Role of vascular endothelial growth factor/vascular permeability factor in the pathogenesis of Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphomas.
Blood.
1999;94:4247-4254 16. Takeda K, Clausen BE, Kaisho T, et al. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity. 1999;10:39-49[CrossRef][Medline] [Order article via Infotrieve].
17.
Mullberg J, Geib T, Jostock T, et al.
IL-6 receptor independent stimulation of human gp130 by viral IL-6.
J Immunol.
2000;164:4672-4677
18.
Bartoli M, Gu X, Tsai NT, et al.
Vascular endothelial growth factor activates STAT proteins in aortic endothelial cells.
J Biol Chem.
2000;275:33189-33192 19. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell. 2002;109(suppl):S121-S131[CrossRef][Medline] [Order article via Infotrieve]. 20. Bromberg J. Stat proteins and oncogenesis. J Clin Invest. 2002;109:1139-1142[CrossRef][Medline] [Order article via Infotrieve]. 21. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997;3:917-921[CrossRef][Medline] [Order article via Infotrieve].
22.
Brander C, Suscovich T, Lee Y, et al.
Impaired CTL recognition of cells latently infected with Kaposi's sarcoma-associated herpes virus.
J Immunol.
2000;165:2077-2083
23.
de Groot RP, Raaijmakers JA, Lammers JW, Jove R, Koenderman L.
STAT5 activation by BCR-Abl contributes to transformation of K562 leukemia cells.
Blood.
1999;94:1108-1112
24.
Rajotte D, Sadowski HB, Haman A, et al.
Contribution of both STAT and SRF/TCF to c-fos promoter activation by granulocyte-macrophage colony-stimulating factor.
Blood.
1996;88:2906-2916
25.
Narazaki M, Yasukawa K, Saito T, et al.
Soluble forms of the interleukin-6 signal-transducing receptor component gp130 in human serum possessing a potential to inhibit signals through membrane-anchored gp130.
Blood.
1993;82:1120-1126 26. Nakajima K, Yamanaka Y, Nakae K, et al. A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells. EMBO J. 1996;15:3651-3658[Medline] [Order article via Infotrieve]. 27. Ueda A, Takeshita F, Yamashiro S, Yoshimura T. Positive regulation of the human macrophage stimulating protein gene transcription: identification of a new hepatocyte nuclear factor-4 (HNF-4) binding element and evidence that indicates direct associat | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
Burkitt lymphoma cell line. BAF130
cell line is a murine BAF-B03-derived cell line expressing transfected
human gp130 (a generous gift from Dr Masashi Narazaki, National Cancer
Institute, Bethesda, MD). These cells were maintained as
described previously.
