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Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-04-1204.
REVIEW ARTICLE
From the Leukemia Section, Department of Medicine, and
Department of Molecular and Cellular Biology, Roswell Park Cancer
Institute, Buffalo, NY.
Signal transducer and activator of transcription (STAT)
proteins are a 7-member family of cytoplasmic transcription factors that contribute to signal transduction by cytokines, hormones, and
growth factors. STAT proteins control fundamental cellular processes,
including survival, proliferation, and differentiation. Given the
critical roles of STAT proteins, it was hypothesized that inappropriate
or aberrant activation of STATs might contribute to cellular
transformation and, in particular, leukemogenesis. Constitutive
activation of mutated STAT3 has in fact been demonstrated to result in
transformation. STAT activation has been extensively studied in
leukemias, and mechanisms of STAT activation and the potential role of
STAT signaling in leukemogenesis are the focus of this review. A better
understanding of mechanisms of dysregulation of STAT signaling pathways
may serve as a basis for designing novel therapeutic strategies that
target these pathways in leukemia cells.
(Blood. 2003;101:2940-2954) Signal transducer and activator of transcription
(STAT) proteins are a family of latent cytoplasmic transcription
factors involved in cytokine, hormone, and growth factor signal
transduction.1-7 STAT proteins mediate broadly diverse
biologic processes, including cell growth, differentiation, apoptosis,
fetal development, transformation, inflammation, and immune response.
The intent of this review is to provide a brief synopsis of the role of
STAT activation in signal transduction, the structure of STAT proteins,
mechanisms of aberrant signal transduction, and the role of STAT
proteins in normal and malignant hematopoiesis. The review focuses in
particular on the role of STAT activation in leukemogenesis.
The interaction of a cytokine with its ligand-binding receptor
STAT proteins were originally discovered in interferon
(IFN)-regulated gene transcription in the early
1990s.10-12 Since then, a number of cytokines have been
recognized to activate various STAT proteins (Table
1). Seven members of the STAT family of transcription factors have been identified in mammalian cells: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6.
Convincing evidence from genetic mapping studies indicates a common
ancestral origin that gave rise to 3 chromosomal clusters of STAT genes
through a series of duplication processes (Table 2).37
Previous characterization of the crystal structure of STAT
molecules allowed a better understanding of the distinct functional domains within the STAT proteins.38,39 Several domains are conserved in all STAT family members (Figure 2; Table
3).
STAT isoforms lacking regions of the c-terminal domain have a
competitive dominant-negative (DN) effect on gene induction mediated by
the STAT pathway, counteracting the effects of the full-length isoform
STAT
c-Terminally truncated STAT isoforms can be generated by 2 different
mechanisms. The first mechanism is alternative mRNA
splicing.10,60-68 Splicing joins the coding sequences
(exons) by removing the intervening noncoding sequences (introns) from
primary transcripts. Alternative splicing generates an enormous
repertoire of functional diversity by producing multiple RNAs and
proteins from a single gene. In the case of STAT3
Studies of targeted deletion of STAT genes in mice have provided
insight into the roles of STAT proteins in response to various cytokines in vital biologic functions (Table
1).13-36,74-76 Models demonstrated the relevance of the
STAT isoforms as mediators of cytokine receptor-specific signaling
reactions, eg, STAT1 for IFN-
The G-CSF and IL-6 family of cytokines, which share gp130 as a common signal transducing subunit, and the GM-CSF and IL-3 family of cytokines are the main cytokines involved in myeloid differentiation. Other hematopoietic growth factors are also implicated to a lesser degree. STAT3 and STAT5 are the major STAT family members governing signal transduction in growth factor-regulated control of myelopoiesis.77,78 Studies with STAT null-mutant mice showed no role for STAT1, STAT4, or STAT6.74 The critical role of STAT3 in myeloid differentiation has been demonstrated with the use of DN mutants.79-81 STAT3 activation by the gp130 family of cytokines in M1 murine myeloid leukemia cells is associated with growth arrest and morphologic differentiation, and blocking IL-6- and LIF-induced activation of STAT3 in DN STAT3 mutants defective in either the tyrosine phosphorylation site (Y705F; STAT3F) or the DNA binding site (EE434-435AA; STAT3D) results in maturation arrest.80-82 These data suggest that STAT3 is necessary in gp130-mediated differentiation of myeloid lineage cells. In contrast, the amount of STAT3 protein decreases during differentiation of embryonic stem (ES) cells,83 and STAT3F84 or specific STAT3 antisense oligodeoxynucleotides (ODNs)85 promote differentiation and block self-renewal of ES cells. These contradictory data suggest that cytokines transmit specific signals to direct lineage commitment of pluripotent hematopoietic stem cells and that specific target genes are stimulated in different cells. G-CSF-induced myeloid differentiation has been demonstrated to be
mediated by STAT3 activation.79,86,87 DN STAT3 mutants, STAT3F and STAT3D, prevented G-CSF-induced granulocytic
differentiation in murine myeloid LGM-1 cells, but cell proliferation
was not impaired.79 These data suggest that STAT3
activation is crucial for G-CSF-induced differentiation but not
growth. In a similar fashion, the introduction of DN STAT3 constructs,
STAT3F and STAT3D, into mouse myeloid cell lines suppressed
G-CSF-induced neutrophilic differentiation,87,88 most
probably because of STAT3-mediated up-regulation of the
cyclin-dependent kinase (cdk) inhibitor p27 (Kip1).88
Other evidence of STAT3 involvement in G-CSF-induced myeloid
differentiation is the identification of a novel STAT3 recruitment
motif within the G-CSF receptor (G-CSFR).89 The cytoplasmic phosphotyrosine residues Y704 and Y744 of G-CSFR were demonstrated to mediate G-CSF-induced differentiation of M1 leukemia cells.90 These residues have been reported to function as
docking sites for STAT3 STAT5 has also been implicated in myeloid differentiation induced by
IL-3, G-CSF, and GM-CSF.91-93 The detection of STAT5 mRNA by polymerase chain reaction (PCR) was suggested to represent an early marker of differentiation in ES cells.83 In
addition, STAT5 activation has been shown to be necessary for
G-CSF-induced myeloid differentiation.92 Ilaria et
al92 generated both an NH2-terminal mutant
STAT5a/WKR (W255KR The antiapoptotic activity of STAT5 was shown to be necessary during the terminal stages of myeloid differentiation.93 For example, primary chicken myeloblasts expressing DN STAT5 were not capable of generating mature neutrophils because of apoptosis, which was reversed by Bcl-2.93 Similarly, bone marrow myeloid cells from STAT5a/STAT5b-knock-out mice showed a differentiation defect and underwent apoptosis during GM-CSF-dependent maturation in vitro. The antiapoptotic protein Bcl-xL was induced in response to GM-CSF and IL-3 through a STAT5-dependent pathway, indicating that antiapoptotic effects of STAT5 are due to induction of the Bcl-x gene.93 These data suggest that STAT5 is required for granulocytic differentiation and has a permissive role in promoting survival and proliferation of differentiating myeloid progenitor cells. The transcriptional activation domain of STAT proteins provides
functional specificity, including commitment to myeloid
differentiation.50-53 Therefore, the distinct
transactivating capabilities of the
Dysregulation of STAT signaling pathways, particularly STAT3 and STAT5, has been demonstrated to contribute to malignant cellular transformation.98,99 STAT proteins are postulated to play important roles in oncogenesis by 2 distinct mechanisms: constitutive activity of the full-length molecule and expression of a c-terminally mutated one. Constitutive activation of STAT1, STAT3, and STAT5 has been
demonstrated to be associated with malignant transformation induced by
various oncoproteins.59,99-102 Full-length STAT3 is
constitutively activated in NIH3T3 fibroblast cell lines transformed by
the oncogenic v-Src tyrosine kinase, and the level of constitutive
STAT3 activity correlates directly with oncogenic transformation by
Src.59,100-102 The transforming ability was suppressed by
DN STAT3 mutants, including recombinantly generated STAT3F, STAT3D,
STAT3S, and c-terminally truncated splice variant STAT3 Constitutively activated STAT may exert its transforming activity through the induction of an antiapoptotic pathway. Inhibition of the STAT3 pathway has been shown to induce apoptosis in breast cancer cell lines.103 Two members of the antiapoptotic Bcl-2 family, Bcl-xL and Mcl-1, were shown to be up-regulated in multiple myeloma cells in which constitutive STAT3 activity was induced by IL-6.104-107 In head and neck cancers, constitutive STAT3 activity with up-regulated epidermal growth factor receptor (EGFR) signaling plays an important role in malignant proliferation through a Bcl-x-induced antiapoptotic mechanism.108-110 These data suggest that constitutive STAT protein activity may induce antiapoptotic pathways in various malignancies. The candidate target genes regulated by the STAT pathways, such as c-myc, cyclin D1, and Bcl-x, appear to contribute to oncogenesis by inducing cell proliferation and survival through the control of cell cycle progression and/or prevention of apoptosis. A genetically mutated STAT3 (STAT3-C) with the substitution of 2 cysteine residues within the c-terminal loop of the SH2 domain has been demonstrated to possess intrinsic oncogenic potential in the absence of tyrosine phosphorylation and to act as a transforming agent.111 This molecule is constitutively active, forms homodimers spontaneously, independently of tyrosine phosphorylation, migrates to the nucleus, binds to DNA, and induces transcription. At the molecular level, this mutant molecule up-regulates the expression of cyclin D1, Bcl-x, and c-myc. Transfection of STAT3-C into rodent fibroblasts also induces the formation of transformed colonies in soft agar and produces tumors in nude mice. These data suggest that altering the c-terminal domain of STAT3 induces constitutive activation. This observation provides further evidence that STAT3 activation may be oncogenic by itself and is not just a consequence of tyrosine phosphorylation. Interaction of the STAT pathway with other signaling pathway(s) from the hematopoietic growth factor receptor, eg, the mitogen-activated protein (MAP) kinase pathway, may also play a role in oncogenic transformation.112,113 Activation of the MAP kinase has been demonstrated in AML and in multiple myeloma,112,113 and some direct "cross-talk" may exist between these pathways.114,115
Constitutive activation of STAT proteins has been reported in a number of malignant cell lines and human cancers.98 Although this review concentrates on leukemia, the large body of information on the role of STAT proteins in solid tumors will be briefly summarized. The role of STAT molecules in breast cancer has been extensively
studied.116 Constitutive activation of STAT3 and/or STAT1 has been detected in breast carcinoma cell
lines103,117-119 and human breast carcinoma nuclear
extracts,103,120 but not in cell lines derived from
nonmalignant mammary gland epithelium103,118,119 or in
cells from healthy human breast tissue.120 STAT3 activity correlates with elevated EGFR and Src expression and with their respective tyrosine kinase activities.98 Abrogation of
constitutive STAT3 activity by DN STAT3 mutants induces apoptosis and
growth arrest in breast cancer cell lines,103 suggesting a
pivotal role for constitutively active STAT3 in breast cancer
development, possibly via an aberrant EGFR pathway and/or Src kinase
activity. Similarly, human head and neck squamous cell carcinomas
(HNSCCs) also display constitutive STAT3 activity, which mediates
activation of EGFR tyrosine kinase induced by transforming growth
factor Src kinase-mediated activation of STAT3 has been shown to be essential
in prostate and ovarian carcinomas.117 Interestingly, enhanced expression of breast cancer susceptibility gene 1 (BRCA1) in prostate cancer cell lines was shown to induce
constitutive tyrosine and serine phosphorylation of STAT3 and upstream
activation of STAT3, JAK1, and JAK2.124 Additionally,
autocrine stimulation by IL-6 induces prostate cancer cell growth
accompanied by activation of STAT3.125 Likewise, IL-6
treatment of colorectal carcinoma cells induces activation of STAT1
and, to a lesser extent, STAT3.126 Finally, murine B16
melanoma cells display constitutive STAT3 activity with an unknown
activating cytokine, and DN STAT3
IL-6 signaling mediated by STAT3 transcriptional activity is the
major pathway involved in growth and differentiation of B cells into
malignant plasma cells.112,128 Indeed, STAT3 is
constitutively active in human bone marrow mononuclear cells from
patients with multiple myeloma and in the IL-6-dependent human myeloma
cell line U266, which expresses high levels of the antiapoptotic
protein Bcl-xL.104,105 IL-6-dependent
constitutive STAT3 activity signaling confers resistance to apoptosis
in U266 cells.104 Inhibition of STAT3 signaling by DN
STAT3 or by AG490, an inhibitor of the JAK2 kinase, has been shown to
block Bcl-xL expression, with subsequent induction of
apoptosis.104 The expression of Mcl-1, another
antiapoptotic protein, has been shown to be up-regulated by IL-6 in
human myeloma cells through the STAT3 pathway.106
Furthermore, the presence of IFN- Constitutive activity of STAT3 and STAT5, but not STAT1, was demonstrated in the mouse T-cell lymphoma cell line, LSTRA, with overexpression of the Lck protein, a Src family tyrosine kinase.130 In addition, constitutive activity of STAT1 and STAT3 was reported to be related to the presence of Epstein-Barr virus (EBV) DNA in Cherry lymphoblastoid cells (LCL) and Burkitt lymphoma cells131; this activity was associated with IL-10 and Bcl-2 protein expression. Cells with no EBV or IL-10 expression did not have constitutive STAT activity.131 Consistent with these results, endogenous IL-10 was shown to induce STAT3 activation in an acquired immunodeficiency syndrome (AIDS)-related Burkitt lymphoma cell line, 2F7, leading to the overexpression of the antiapoptotic protein Bcl-2.132 Treatment with anti-CD20 monoclonal antibody, Rituximab, decreased transcription and production of IL-10, which disrupted IL-10 autocrine/paracrine loops, with consequent down-regulation of STAT3 binding activity and, in turn, decreased Bcl-2 expression.132 The significance of STAT3 activation in the apoptotic pathway has been further demonstrated in T-cell large granular lymphocyte (LGL) leukemia associated with antiapoptotic Mcl-1 overexpression.133 Inhibition of STAT3 signaling causes apoptosis of leukemic LGLs and reduced Mcl-1 expression. These results demonstrate that activated STAT3 has an antiapoptotic effect in tumor cells. STAT3 and/or STAT5 are constitutively activated in human T-cell lymphotrophic virus type I (HTLV-I)-related adult T-cell leukemia/lymphoma134 and HTLV-I-transformed T cells in association with the acquisition of IL-2-independent growth.135,136 Constitutive STAT3 phosphorylation on Tyr705 was detected in self-renewing CD5+ murine B-1 lymphocytes.137 Similar to primary B-1 cells, nuclear extracts of CD5+ B-cell lymphoma cells have been shown to contain a constitutively active STAT3 that is phosphorylated on Tyr705 and Ser727.137 Suppression of STAT3 expression in these cells was associated with a block in the G1 phase of the cell cycle, indicating a role for STAT3 in growth and immunoglobulin production of B-cell lymphoma through control of cell cycle progression. STAT3 and STAT5 were also shown to be constitutively activated in cutaneous lymphomas, including cutaneous anaplastic large T-cell lymphoma,138 Sézary syndrome,138,139 and mycosis fungoides.140,141 The abrogation of STAT3 signaling resulted in a decrease in antiapoptotic Bcl-2 protein and an increase in proapoptotic Bax protein, with subsequent induction of apoptosis in mycosis fungoides cells.141 Recently, constitutively activated STAT3 was identified in Hodgkin disease (HD) cell lines.142 Additionally, constitutive phosphorylation of STAT3 and STAT6 was demonstrated in Reed-Sternberg cells from patients with HD.143 STAT6 activation was due in part to IL-13 signaling in these cells, and abrogation of IL-13 signaling resulted in the inhibition of STAT6 phosphorylation and cellular proliferation, suggesting a vital role for STAT6-mediated IL-13 signaling in the development of HD.
The role of aberrant STAT signaling and constitutive STAT activation in leukemias has been a recent focus of intensive research. A growing body of evidence indicates a fundamental causative role for dysregulated STAT signaling mechanisms in both acute and chronic leukemias. The initial hypothesis implicating STAT activation in leukemogenesis stemmed from studies of the fruit fly Drosophila.144-146 The hopscotch (hop) locus encodes a Drosophila JAK homolog.147 A single Drosophila STAT gene, called D-STAT, STAT92E, or Marelle, has been identified, which functions in the embryonic development of the Drosophila.148-150 In Drosophila melanogaster, the dominant temperature-sensitive gain-of-function hop JAK kinase mutations hopTum-1 and hopT42 increase tyrosine kinase activity and cause clonal proliferation of plasmatocytes, similar to the clonal proliferation of leukemia cells.151-154 These mutations hyperphosphorylate and hyperactivate D-STAT when overexpressed in Drosophila melanogaster cells and lead to a leukemia-like phenotype. However, introducing a lack-of-function D-STAT mutation into these cells did not totally reverse the overproliferation and leukemia-like abnormalities,154 suggesting that hyperactivation of STAT alone is most probably not sufficient for proliferation and survival. Acute leukemias Leukemia cells and normal hematopoietic progenitors proliferate in the bone marrow stroma. Long-term bone marrow culture provides a means to examine the interplay between hematopoietic cells and bone marrow stroma.155 The adherent layer in such a system (equivalent to stromal cells) modulates hematopoiesis in vitro. Several groups, including ours, have shown that cultured stroma from a subset of patients with AML produces multiple cytokines.156,157 What needs to be determined is why leukemic cells have a different response to growth factors than normal hematopoietic cells residing in the same microenvironment. Reasons may include differences in the expression of receptor subunits, signal communicating proteins, or downstream target genes.AML is characterized by maturation arrest of a malignant clone of myeloid cells. Growth factors and growth factor signaling pathways are likely to determine the proliferation and differentiation state of the leukemic blasts in vivo.158 Receptors for growth factors that signal through STAT proteins are present on AML blasts and most of them are known to have intact function.158 Because multipotential nonleukemic hematopoietic cells undergo differentiation, whereas leukemic cells maintain proliferation rather than differentiation, in response to growth factors, aberration of signaling pathways is suggested to contribute to leukemogenesis. Constitutive activation of STATs has been demonstrated in leukemia cell lines159-163 and blasts from 22% to 100% of patients with AML by various groups.73,113,131,163-167 Gouileux-Gruart et al164 found constitutive activation of STAT3 in peripheral blood (PB) cells from 5 patients with AML; constitutive activity of STAT5 was also present in 2 of the 5 patients, and STAT1 was activated in 1 patient. The same group also reported a study of 14 patients with AML; 10 patients (71%) exhibited constitutive STAT1 and STAT3 activity, and 1 patient had STAT5 activity in addition to STAT1 and STAT3.131 In another study, constitutive STAT1 activity was associated with IL-3-independent proliferation in 10 of 20 patients (50%) with AML.165 In a recent study, 18 of 26 (69%) patients with AML exhibited constitutive STAT5 activity.166 Interestingly, this activity was associated with Flt3 phosphorylation in 70% of the cases.166 Hayakawa et al113 found constitutive STAT3 activity in 17 of 23 (74%) and STAT5 activity in 40 of 50 (80%) bone marrow samples from patients with AML. Approximately half of the samples tested revealed activation of the MAP kinase pathway; however MAP kinase activation did not correlate with constitutive STAT3/STAT5 phosphorylation. In an analysis of 36 pretreatment bone marrow samples from newly diagnosed adult patients with AML, we detected constitutive activation of STAT3 and STAT5 in 10 (28%) and 8 (22%) samples, respectively.73 Activation of both STAT3 and STAT5 was seen in 4 patients (11%). There was no STAT6 activation. In another study, we showed that constitutive STAT3 activity correlated with unfavorable treatment outcome.167 Disease-free survival was significantly shorter in patients with, as compared to without, constitutive STAT3 activity. This was the first demonstration of clinical significance of STAT proteins in any malignancy. It is yet unclear whether this adverse treatment outcome is associated with the presence of constitutive STAT activity itself or with a process that leads to constitutive STAT activity. Production of hematopoietic cytokines by leukemic blasts, with autocrine/paracrine stimulation of the JAK/STAT pathway, might be a possible mechanism for constitutive STAT activity in AML in some cases.168 IL-6 secretion from the leukemic blasts has been shown to cause constitutive STAT3 activity,168 as was also seen in multiple myeloma.104 However, IL-6 has antiproliferative effects in AML.169 Therefore, the role of IL-6-induced STAT3 activation in leukemogenesis remains controversial. Aberrant STAT activation may be associated with leukemic transformation by various oncoproteins.170 Several phosphotyrosine kinases, including Bcr-Abl and TEL-JAK2, have been shown to activate the STAT pathway in leukemias, without the need for receptor activation.171-174 However, involvement of these kinases in AML is rare. Aberrant regulation of apoptotic pathways may be another cause of leukemogenesis. Members of the antiapoptotic Bcl-2 family are up-regulated in various malignancies, including AML.105-107,110,175,176 However, there are no data suggesting a relationship between these antiapoptotic molecules and STAT activity in AML. Direct cross-talk between the STAT and MAP kinase pathways in AML has also been suggested to play a role in leukemogenesis.113,114 c-Terminally truncated STAT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
subunit is the first step in the formation of a signaling-competent receptor complex. This process involves the oligomerization of the
ligand-bound subunit with either another subunit or a separate, signal-transducing 
subunit.
,
AAA) and c-terminally truncated STAT5a/<53C, lacking the last 53 amino acids of murine STAT5a, which is similar to a naturally occurring isoform of rat
STAT5b.