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Molecular analysis and biological implications of STAT3 signal transduction
Schuringa, Jan Jacob
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Chapter 9
SUMMARY, DISCUSSION and FUTURE PERSPECTIVES
Jan-Jacob Schuringa1,2, Edo Vellenga2, and Wiebe Kruijer1
1
2
Biological Center, Department of Genetics, Haren, and
University Hospital Groningen, Department of Hematology, Groningen, The Netherlands.
124
Chapter 9
Introduction
Since the discovery of the STAT family of transcription factors in the early 90’s, research
has focussed on the roles of STATs in a variety of biological processes as well as on the
molecular mechanisms that are involved in STAT activation. Although a detailed
description on how cytokines and growth factors utilize STAT signaling pathways to elicit
biological responses is now available, many questions remain concerning the specificity
and fine tuning of signals that challenge cells. Also, the causal relationship between
disturbed STAT signaling and the malignant progression of human cancers is only
beginning to become unraveled.
The work described in this thesis focussed both on the molecular mechanisms that are
involved in IL-6-induced STAT3 signal transduction, as well as on some of the biological
functions of STAT3, including STAT3 signaling in early embryonic carinoma cells.
Closely related issues concern the role of STAT3 in oncogenesis, which was studied in
detail in acute myeloid leukemia as well as in cells that express the MEN2A oncogene.
1. The signal transduction cascade involved in IL-6-induced STAT3
ser727 phosphorylation: kinetics and specificity
Although STAT tyrosine phosphorylation is the first critical event in ligand-induced
STAT activation, which allows STAT dimerization, translocation the nucleus and binding
of response elements in target gene promoters, STAT3 serine phosphorylation is a second
event that critically regulates STAT activity (reviewed in [63]). Except for STAT2 and
STAT6, all other STATs contain serine residues that become phosphorylated in a
stimulus-regulated manner [1,61-63]. This second phosphorylation event provides the
possibility to modulate and fine-tune STAT signal transduction and might introduce
specificity in the effects that cytokines and growth factors have on cells.
Figure 1 describes a model for IL-6-induced STAT3 tyr705 and ser727 phosphorylation in
HepG2 cells (Chapters 2 and 3). IL-6 initiates signaling by associating with its ligandbinding receptor (IL-6R), which allows dimerization of the gp130 receptor components.
The gp130 associated JAK kinases transphosphorylate tyrosine residues 767, 814, 905 and
915 that form docking sites for STAT3 once phosphorylated. Association of STAT3 with
the gp130 receptor enables JAK-mediated STAT3 tyr705 phosphorylation. In HepG2
cells, this occurs with rather quick kinetics: within 2 min upon IL-6 stimulation maximal
levels of STAT3 tyr705 phosphorylation were observed (Chapter 3). Once phosphorylated
on tyr705, STAT3 dimerizes via reciprocal interactions between the SH2 domains that
enable nuclear translocation. This fast nuclear import process was also demonstrated by
immunofluorescence microscopy studies revealing that STAT3 cytoplasmic-nuclear
translocation occurs within 5 min upon IL-6 stimulation (Fig.2, see also Chapter 3).
IL-6-induced STAT3 ser727 phosphorylation involves the sequential activation of Vav,
Rac-1, MEKK, SEK-1/MKK-4 and PKCδ. The guanine nucleotide exchange factor Vav is
associated with the membrane-distal region of the gp130 receptor and becomes
phosphorylated on tyrosine residue(s) upon IL-6 stimulation (Chapter 2, [34]). Within 5
min, Vav associates with the small GTPase Rac-1, and probably regulates GDP-GTP
exchange on Rac-1 [223], thus leaving it in the activated conformation. The kinetics of IL6-induced Vav tyrosine phosphorylation correlate with the kinetics of dissociation of the
Summary, Discussion and Future perspectives
125
Vav-Rac-1 complex, suggesting that tyrosine phosphorylation of Vav is not a prerequisite
for Vav-mediated Rac-1 activation but rather plays a role in the dissociation process,
although mechanistic explanations for this model are still lacking. Upon activation, Rac-1
initiates a signal transduction cascade that is comprised of the MAP kinase kinase kinase
MEKK-1, the MAP kinase kinase SEK-1/MKK-4 and PKCδ. IL-6 induces a transient
activation of SEK-1/MKK-4 as determined by phosphorylation of the residue Thr223
within 5 min, which reached maximal levels at 10 min upon IL-6 stimulation (Chapter 2).
In unstimulated cells, SEK-1/MKK-4 is present as a complex with PKCδ, and upon IL-6
stimulation PKCδ dissociates from SEK-1/MKK-4 within 10-15 min. Presumably, SEK1/MKK-4 phosphorylates PKCδ on Thr505, which then translocates to the nucleus.
Figure 1. Proposed model for IL-6-induced STAT3 ser727 phosphorylation.
Phosphorylated PKCδ was only found in the nucleus reaching maximal phosphorylation
levels at 5-10 min upon IL-6 stimulation, suggesting that PKCδ nuclear translocation is a
rather quick event. At timepoint 15 min, PKCδ associates with STAT3 and phosphorylates
STAT3 on ser727. STAT3 ser727 phosphorylation reaches maximal levels after 15 min.
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Importantly, STAT3 ser727 phosphorylation was was only detected in the nuclear
fractions and was absent from the cytoplasmic fractions. Taken together, these results
suggest that IL-6-induced STAT3 ser727 phosphorylation is a nuclear event, particularly
since STAT3 nuclear translocation occurs within 5 min upon IL-6 stimulation and
precedes STAT3 ser727 phosphorylation.
In agreement with our data, Jain et al. have described that PKCδ is directly involved in IL6-induced STAT3 ser727 phosphorylation [243]. In contrast, they report that STAT3PKCδ associations occur mainly in the cytoplasm. In immunoprecipitation studies from
IL-6-stimulated nuclear fractions of HepG2 cells, no nuclear PKCδ was detected, whereas
we find a significant amount of PKCδ in total nuclear fractions of HepG2 cells (chapter 3).
Possibly, due to a lack of detectable immunoprecipitated PKCδ from nuclear fractions
they were not able to observe a nuclear PKCδ-STAT3 association. Furthermore, they
indicate that STAT3 tyr705 phosphorylation appears to be a prerequisite for PKCδmediated STAT3 ser727 phosphorylation, particularly since stimulation with PMA, which
is a very potent activator of PKCδ and does not induce STAT3 tyr705 phosphorylation,
does not result in association of PKCδ with STAT3 [243]. Since we find that practically
all tyrosine phosphorylated STAT3 is present in the nucleus within 5 min upon IL-6
stimulation, these data suggest that IL-6-induced STAT3 ser727 phosphorylation is a
nuclear event.
We can exclude the possibility that the ERK signal transduction cascade is involved in IL6-induced STAT3 ser727 phosphorylation in HepG2 cells since inhibition at various levels
of this signaling cascade did not interfere with IL-6-induced STAT3 ser727
phosphorylation (Chapter 2). Also, PI-3K or Src activity is not required since inhibition of
the kinase activity of these molecules by treating cells with the chemical inhibitors
wortmannin and PP2A, respectively did not reduce IL-6-induced STAT3 ser727
phosphorylation. Inhibition of p38 kinase activity by using the inhibitor SB203580 also
did not interfere with IL-6-induced STAT3 ser727 phosphorylation, but rather enhanced
both ser727 phosphorylation as well as STAT3 transactivation (Chapter 2). Since LPS and
TNFα induce SOCS-3 expression via the p38 pathway [136], we speculated that a similar
mechanism might be involved in IL-6 signaling as well. Inhibition of p38 kinase activity
would then prevent the IL-6-induced upregulation of SOCS-3, which would allow an
increase in STAT3-mediated gene transcription. However, IL-6 still induced SOCS-1 and
SOCS-3 RNA in the presence of SB203580 to similar levels and with similar kinetics as
compared to untreated cells (data not shown), indicating the p38 kinase activity is not
required for the IL-6-induced upregulation of SOCS proteins. Possibly, p38 is required to
activate a (nuclear) phosphatase in order to downregulate STAT3 signal transduction.
Further experiments are required to resolve this issue.
It is somewhat peculiar that the MAP kinase JNK is not involved in IL-6-induced STAT3
ser727 phosphorylation in HepG2 cells. In many cases, JNK-1 is the end-point kinase of
the Rac-MEKK-SEK-1 signal transduction cascade. Although it has been demonstrated
that JNK-1 is the kinase that phosphorylates STAT3 on ser727 in response to stress stimuli
such as UV and Anisomycin [83], we can exclude the possibility that JNK-1
phosphorylates STAT3 in response to IL-6 based on the following observations: (i) JNK-1
is not activated in response to IL-6 stimulation (Chapter 2 and 3); (ii) JNK-1 is not
nuclear-translocated in response to IL-6 (Chapter 2); (iii) no association between SEK1/MKK-4 and JNK-1 was detected in mammalian two hybrid assays (Chapter 3); (iv) no
IL-6-induced JNK-1 association with STAT3 was observed in immuno-precipitation
Summary, Discussion and Future perspectives
127
Figure 2. Kinetics of IL-6-induced STAT3 nuclear translocation. In unstimulated cells (0 min), STAT3 is
distributed over the cytoplasm and the nucleus. Upon stimulation, STAT3 translocates to the nucleus within 5
min. After 60 min of IL-6 stimulation, STAT3 is relocated to the cytoplasm.
128
Chapter 9
studies (data not shown). Thus, we conclude that JNK-1 is not involved in IL-6-induced
STAT3 ser727 phosphorylation. PKCδ is the end-point kinase of the Rac-MEKK-SEK-1
signal transduction cascade and phosphorylates STAT3 on the ser727 residue in response
to IL-6. It is plausible that JNK-1 and PKCδ are anchored in different signal transduction
protein complexes and that these complexes are activated in a strictly ligand-dependent
manner. Recently, two groups of proteins have been identified which might function as
scaffold-proteins for the JNK-1 signal transduction cascade, the JNK-1 Interacting
Proteins (JIP) and JNK/Stress-activated protein kinase-Associated Proteins (JSAP)
[343,344]. These putative scaffold proteins interact with specific members of the JNK
signal transduction cascade, including isoforms of the MAP kinase kinase kinases MEKK1, -2, -3 and -4, the MAP kinase kinases SEK-1/MKK-4 and MKK-7, and the MAP
kinases JNK-1, -2 and –3. It appears that these scaffold proteins only interact with a
specific subset of isoforms of the JNK signal transduction cascade, and that these proteins
selectively enhance the activation of signaling pathways by forming an anchor for the
specific proteins. Thus, scaffold proteins will contribute to the specificity of numerous
distinct signaling pathways in cells. The fact that these scaffold proteins can homo- or
hetero-dimerize either via leucine-zipper or SH3 domains and contain multiple regulatory
phosphorylation sites might introduce further specificity. It will be challenging to study
whether scaffold proteins are also involved in IL-6-induced activation of the Vav-RacMEKK-SEK/MKK-4-PKCδ signal transduction cascade and determine their role in IL-6induced STAT3 ser727 phosphorylation.
In HepG2 cells, IL-6-induced STAT3 signaling is transient. Within 60 min, both STAT3
tyr705 and ser727 phosphorylation is reduced to basal levels and STAT3 is re-entered into
the cytoplasm. The kinetics of STAT3 dephosphorylation and cytoplasmic re-localization
occur with similar kinetics as the upregulation of SOCS proteins. SOCS proteins
downregulate STAT signal transduction by association with Jaks or the activated receptor
complex, thereby preventing STAT tyrosine phosphorylation. SOCS-1 and SOCS-3
mRNA was first detected upon 30 min of IL-6 stimulation and prolonged for several hours
(data not shown), suggesting that this negative feedback loop indeed downregulates
STAT3 signal transduction. However, SOCS proteins will only inhibit STAT3 signaling at
the receptor level and will prevent a re-activation of STAT3 when the IL-6-gp130 receptor
complex is still in its active conformation. Thus, phosphatases must play an important role
as well. Presumably, as is the case for STAT1 [115], nuclear tyrosine phosphatases will
de-phosphorylate STAT3 tyrosine residues in the nucleus, which allows dissociation of the
STAT3 dimer. STAT3 monomers will then be shuttled to the cytoplasm via a Nuclear
Export Signal (NES)-mediated mechanism as has also been demonstrated for STAT1 [71].
In the cytoplasm, SOCS proteins will now prevent a re-activation of STAT3. Further
experiments are required to identify the tyrosine phosphatase(s) involved, as well as the
roles that potential serine phosphatases, receptor internalization and protein degradation
might fulfill in the downregulation of IL-6-induced STAT3 signal transduction.
An important point concerns the ligand-dependent and cell-type specific conditions that
determine which signal transduction cascade is utilized to phosphorylate STAT3 on ser727
(reviewed in [63]). For instance, IL-2 induces STAT3 ser727 phosphorylation via the
MEK/ERK pathway in T lymphocytes, but in combination with IL-12 it utilizes the p38
pathway [88]. EGF induces ser727 phosphorylation via the MEK/ERK pathway in NIH-
Summary, Discussion and Future perspectives
129
3T3 cells [82], similar as insulin, OnM and LIF in adipocytes [345] and GM-CSF and GCSF in human neutrophils [346]. Stress stimuli such as UV and TNFα utilize the JNK
pathway to phosphorylate STAT3 on ser727 in COS-7 cells [83], whereas v-Src-induced
STAT3 ser727 phosphorylation is mediated by both JNK-1 and p38 in v-Src-transformed
NIH-3T3 cells [89]. In HepG2 cells, IL-6-induced STAT3 ser727 phosphorylation is
independent of ERK activation (Chapter 2). Jain et al demonstrated that PKCδ directly
phosphorylates STAT3 on ser727 [243], in agreement with our data. So, although IL-6 is
capable of activating ERK-1 (Chapter 2), ERK is not involved in the IL-6-induced ser727
phosphorylation of STAT3, while other ligands such as EGF or insulin do utilize this
signal transduction cascade in some cells. Similar, in response to stress stimuli, JNK-1 is
the end-point kinase of the JNK signal transduction cascade, whereas in the case of IL-6,
JNK-1 is not involved. The kinetics and the level of activation of the specific signal
transduction cascades might account for these observations, as well as the involvement of
scaffold proteins, which activation patterns might depend on the cytokine or growth factor
that a cell is challenged to.
2. The role of STAT3 ser727 phosphorylation in the regulation of gene
transcription
Based on the experiments described in Chapters 2-4, we conclude that ser727
phosphorylation is required for maximal STAT3 transactivation in response to IL-6
stimulation. STAT3β, which lacks the C-terminal transactivation domain and the mutant
STAT3 ser727ala display strongly reduced transcriptional activities as compared to
STAT3α. The STAT3 ser727asp mutant, in which the serine residue is replaced by the
negatively charged aspartate residue, displays similar transcriptional activities as STAT3α.
However, this mutant has become independent of activation of the Vav-Rac-MEKK-SEK1/MKK-4-PKCδ signal transduction cascade, indicating that the role of this signaling
pathway is to provide STAT3 with a negative charge at position 727 (Chapter 4). These
observations are further underscored by experiments in which C-terminal fragments of
STAT3 were fused to the DNA-binding domain of GAL4 and were tested for
transcriptional activities in GAL-4-luciferase reporter assays. The 65 C-terminal amino
acids of STAT3 can serve as an independent Transactivation domain (TAD), particularly
when a negative charge is introduced at position 727 (Chapter 4).
In order to study the functional role of the negative charge at position 727, either provided
by a phosphate group or the ser727asp mutant, STAT3 associating proteins were studied
in relation to the negative charge at position 727. A model is presented in figure 3. The coactivator protein p300 associates with STAT3 in vivo upon IL-6 stimulation, and p300
strongly associates with the minimal TAD of STAT3 when serine 727 is mutated into
aspartate (Chapter 4). The function of the association of p300 with STAT3 is to allow high
levels of gene transcription. This is reflected by the fact that overexpression of p300
strongly enhanced the transcriptional activities of STAT3α and STAT3 ser727asp, but not
of STAT3β or STAT3 ser727ala (Chapter 4). P300 contains a histone acetyltransferase
(HAT) domain that is required for its cooperative transcriptional activity with other
signaling molecules, including c-Jun and p53 [258,259]. By acetylation of histones, p300
is involved in chromatin remodeling and can subsequently modulate gene transcription.
Furthermore, it is now being appreciated that besides phosphorylation, acetylation is also
an important trigger that affects the activity of a variety of signal transduction molecules,
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Chapter 9
including transcription factors. In the case of STAT3 however, we were not able to detect
any IL-6-induced actelyation of STAT3 (data not shown) suggesting that p300 does not
directly acetylate STAT3. Alternatively, we propose a model in which p300 enhances
STAT3 transactivation by functioning as a bridging factor coupling DNA-bound, ser727
phosphorylated STAT3 to the basal transcription machinery via association of p300 with
the RNA polymerase II complex of proteins (Figure 3).
In contrast to the positive contribution of STAT3 ser727 phosphorylation on the
intititation of gene transcription described in this thesis, negative effects of ser727
phosphorylation on STAT3 transactivation have been described as well. For example, Jain
et al. have described that overexpression of PKCδ strongly enhances STAT3 ser727
phosphorylation, but reduces STAT3 transactivation [243]. In contrast, we find that
overexpression of constitutive active RacV12, which strongly enhances STAT3 ser727
phosphorylation, also enhances STAT3 transactivation levels (chapter 3). Thus, these data
suggest that the inhibitory effects of overexpression of PKCδ on STAT3 transactivation
are not directly linked to enhanced STAT3 ser727 phosphorylation, but are due to
secondary effects like the activation of phosphatases or the upregulation of negative
feedback proteins like Suppressors of Cytokine Signaling. We find that inhibition of
PKCδ activity, either by treating cells with rottlerin or by overexpression of dominant
negative PKCδ, results in a reduced STAT3 ser727 phosphorylation that is linked to a
severely impaired STAT3 transactivation.
Figure 3. Proposed model for the role of STAT3 ser727 phosphorylation in the initiation of gene
transcription. P300 serves as a bridging factor coupling ser727 phosphorylated, DNA-bound STAT3 to the
basal transcription machinery (TBP, TATA Binding Protein; TAF, TBP Associated Factor).
3. STAT3 associating proteins/cross-talk
In contrast to many other cellular signal transduction cascades, the TK-STAT pathway is
rather direct: STATs function both as signal transducers transmitting signals from the
cytoplasmic receptors to the nucleus as well as transcription factors thus initiating gene
transcription. However, STATs do not act alone. Interactions of STATs with a variety of
other proteins have been described, including nuclear hormone receptors, minichromosome maintenance proteins, p300/CBP and members of the AP-1 family of
transcription factors. These STAT interacting proteins function to modulate STAT
signaling at various steps and mediate the crosstalk of STATs with other cellular signaling
pathways.
In Chapter 5, a direct interaction between STAT3 and AP-1 transcription factors is
described. A model is presented in which STAT3 directly binds to its response element
Summary, Discussion and Future perspectives
131
and c-Jun or c-Fos associate with STAT3 without binding to the DNA. The consequence
of this interaction is an enhanced STAT3-driven transcription. The transactivation domain,
the DNA binding domain as well as the serine residues 63 and 73 of c-Jun appear to be
required for this cooperativity. Similar results have been described by Zhang et al, who
identified the interacting regions in STAT3 and c-Jun that participate in cooperative
transcriptional activation [68]. Two regions in STAT3 were found to associate with c-Jun,
a region within the coiled-coil domain and a portion of the DNA binding domain distant
from DNA contact sites.
Interestingly, we find that co-stimulation with IL-6 and TPA induces a synergistic
transactivation of the IRE reporter in HepG2 cells, which is coupled to a strong TPAinduced upregulation of c-Jun and c-Fos expression via the ERK pathway. Thus, crosstalk
exists between IL-6/STAT3 signal transduction and activation of the ERK pathway, and
the cooperativity between STAT3 and AP-1 proteins provides the possibility to fine-tune
the expression of downstream target genes.
A mechanistic explanation for the observed cooperation between STAT3 and AP-1
proteins in target gene expression can not be given yet. Two possibilities might be
considered. First, it is conceivable that association of c-Jun or c-Fos with STAT3 stabilizes
STAT3 DNA binding. Thus, the off-rate of STAT3 dissociation from the DNA might be
reduced which would finally result in higher levels of gene transcription. Alternatively,
association of c-Jun or c-Fos with STAT3 might allow or facilitate the recruitment of coactivator proteins. The co-activator CBP/p300 might be a possible candidate, since it has
been demonstrated that phosphorylation of the serine residues 63 and 73 of c-Jun enables
the recruitment of CBP/p300 which is a prerequisite for high levels of gene transcription
[258]. We find that the serine residues 63 and 73 of c-Jun are required for its cooperativity
with STAT3, indicating that such a model indeed might be valid. However, we can not
exclude the possibility that other co-activator proteins might be recruited by c-Jun or c-Fos
as well. Further studies are required to determine whether p300 indeed interacts with the
STAT3/AP-1 DNA-bound complex in vivo and what the exact sites of interaction are,
since we also find that p300 can interact with the C-terminus of STAT3.
4. STAT3 in oncogenesis
The consistent finding of disturbed STAT signaling in a variety of human tumors has led
to the hypothesis that a constitutive STAT activation might be causal in the malignant
progression of cancers [163,164,166,328]. Probably the most direct evidence for this
hypothesis stems from an article by Bromberg and colleagues entitled ‘STAT3 as an
oncogene’ [191]. They demonstrated that by introducing mutations in the C-terminal loop
of the SH2 domain, STAT3 dimerizes spontaneously and induces constitutive gene
expression. This constitutive active STAT3 molecule induces cellular transformation of
immortalized fibroblasts and tumor formation in nude mice. Target genes of STAT3 that
are induced include cyclin D1 and the anti-apoptosis protein Bcl-xL, although the exact
gene expression pattern that is involved in the transformation process still needs to be
elucidated. A direct link between cell-cycle progression and STAT3 activation was
described by Fukada and colleagues [154]. STAT3 plays a key role in G1 to S phase cellcycle transition through upregulation of cyclins D2, D3 and A and cdc25A, and the
concomitant downregulation of p21 and p27 in the BAF/B03 pro-B cell line. Thus, a
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constitutive STAT3 activation might contribute to a growth advantage of malignant cells
over the normal counterpart.
In acute leukemias, a constitutive activation of STAT3, STAT5 and in some cases STAT1
has been observed [164]. In acute myeloid leukemia (AML), we observed a constitutive
activation of STAT3 in approximately 25% of the investigated cases, as determined by
constitutive and non-IL-6 inducible STAT3 tyr705 and ser727 phosphorylation as well as
by constitutive DNA binding (Chapter 6). Although the majority of the investigated AML
cells secreted low to undetactable levels of IL-6 into the medium, the AML cells
characterized by constitutive STAT3 activation displayed strongly enhanced IL-6
secretion levels. Since interference with neutralizing anti-IL-6 antibodies reduced
constitutive STAT3 phosphorylation to low basal levels and restored the IL-6 inducibility
of STAT3 we conclude that the elevated IL-6 secretion levels cause the constitutive
STAT3 activation in the investigated AML cells. Previous studies have demonstrated that
the transcription factor NF-κB is an important mediator of IL-6 gene expression [77].
Moreover, constitutive NF-κB DNA binding has been observed in many acute myeloid
leukemia cells [75]. Further studies are required to determine whether there exists a
correlation between constitutive NK-κB activity and enhanced IL-6 secretion in AML
cells, which would finally result in a constitutive activation of STAT3. Figure 4 describes
a model, in which the constitutive secretion of cytokines induces signal transduction in an
autocrine or paracrine manner. Whether this model reflects a general mechanism that
results in the malignant progression of leukemias remains to be elucidated but elevated
levels of cytokine production have indeed been described in a variety of leukemias and
lymphomas.
Figure 4. Elevated levels of IL-6 production induce constitutive activation of STAT3 in acute myeloid
leukemia cells.
Besides disturbed cytokine production, numerous oncogenes have been described which
initiate STAT signaling in the absence of ligand stimulation. For example, v-Src, v-Eyk, vRos, and v-Fps have been describe to activate STAT3 (see also Table 4, reviewed in
Summary, Discussion and Future perspectives
133
[163]). Furthermore, it has been demonstrated that constitutive activation of the tyrosine
kinase receptor Flt-3 induces STATs, which results in cellular transformation [347,348].
Often, these oncogenes contain a constitutive tyrosine kinase activity and activate STATs
directly or via the persistent activation of signal transduction cascades. In Chapter 7, we
describe that the MEN2A oncogene induces constitutive STAT3 activation. The Ret protooncogene encodes for a member of the receptor family of tyrosine kinase (RTK)
superfamily that plays a crucial role during the development of the enteric nervous system
[306,309,349]. Germline mutations at cysteine residues in the extracellular domain
(Cys609, -611, -620, -630, and –634) are responsible for the majority of multiple
endocrine neoplasia type 2A (MEN2A) [310,311]. MEN2A mutations induce a ligandindependent constitutive activation of RET which leads to abnormal cell growth,
differentiation defects and cellular transformation. We find that NIH-3T3 cells stably
expressing MEN2A and STAT3α, but not STAT3β, are characterized by enhanced
proliferation in soft agar and cyclin D1 promoter activity, indicating that malignant cell
growth induced by MEN2A involves its activation of STAT3. Interestingly, deletion of the
C-terminal TAD of STAT3 severely reduced the MEN2A-induced cellular transformation,
strongly suggesting that besides STAT3 tyr705 phosphorylation, ser727 phosphorylation
is required for its full cell-biological activity. Similar observations have been published for
v-Src-induced cellular transformation [184,185]. STAT3 enhances the transformation of vSrc while STAT3β inhibits v-Src-induced transformation of NIH-3T3 cells [184,185].
These data strongly underline the importance of the C-terminal transactivation domain for
STAT3 signaling in vivo. The data presented in Chapter 7 provide a novel example of how
STAT3 might contribute to the malignant progression of cancers in endocrine tissues that
express the MEN2A oncogene and it will be challenging to determine whether there is
indeed a causal relationship between a persistent activation of STAT3 and the
development of multiple endocrine neoplasia type 2A in patients.
5. The role of STAT3 in embryonic carcinoma cells
Recent advances in mouse embryonic stem cell biology have elucidated the critical roles
of LIF and STAT3 for stem cell self-renewal and the maintenance of pluripotency
[156,159,335]. As a consequence, mouse ES cells can now be cultured in the absence of
embryonic fibroblast feeder-cell layers when LIF is administered to the medium.
Unfortunately however, human ES cells do not respond to LIF in a similar fashion and still
require feeder-cell layers for support [338]. Embryonic carcinoma (EC) cells provide a
tool for analyzing the mechanisms that control differentiation during embryonic
development [336]. They express markers that are also expressed in ES cells, including
Oct-4, alkaline phosphatase and stage-specific embryonic antigens, and can be
differentiated into cell-types of all the germ layers. In Chapter 8, we studied the signal
transduction cascades that are initiated by LIF in mouse versus human embryonic
carcinoma cells (P19 EC versus Ntera/D1 EC cells). In P19 EC cells, LIF induces the
activation of both ERK as well as STAT3. Also, LIF significantly enhances the
proliferation of P19 EC cells, which is completely dependent on the LIF-induced ERK
activation and does not involve STAT3. In contrast, LIF neither activates STAT3 nor
ERK-1 in Ntera/D1 EC cells, although receptor components are properly expressed.
Interestingly, the negative feedback protein SOCS-1 is highly expressed in human
Ntera/D1 EC cells, which might prevent LIF-induced STAT3 signal transduction.
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Although further experiments are required to determine whether SOCS-1 expression is
indeed also elevated in human ES cells, it is tempting to speculate that LIF-STAT3
signaling might be disturbed in ES cells due to a constitutive activation of this negative
feedback loop. When this is indeed the case, it might become feasible to culture human ES
cells in the absence of feeder cells in the near future by using anti-sense oligonucleotidebased strategies to inhibit endogenous SOCS-1 expression.
Future perspectives
One of the most challenging goals in the near future will be the determination of STAT3
target genes in relation to cell-type specific events using micro-array technologies. Next, it
will be important to elucidate the relationship between the expression of a certain gene and
the observed phenotype(s). In the case of STAT3, the knockout mice are embryonic lethal
and thus the role of STAT3 in adult tissues could not be assessed. Conditional-knockout
approaches did yield considerable insight in the biological functions of STAT3 and it will
be of importance in the near future to identify the STAT3 target genes that account for the
specific cell-biological responses. This should yield important information on how normal
STAT3 signal transduction results in the appropriate phenotype, which can then be
correlated to disturbed gene expression patterns in human malignancies.
Also, it will be rewarding to study gene expression patterns when a cell is facing multiple
cytokines, for instance IL-6 in combination with ligands that activate the ERK, p38 or PI3K pathways. Thus, it will be possible to start elucidating the effects of cytokine-crosstalk
on the expression of genes. Since the completion of the sequencing of the human genome
it appears that remarkably few genes exist in humans as compared to for instance fruit flies
or nematodes (approximately 35.000 in humans versus approximately 13.600 in
Drosophila and approximately 17.300 in C. elegans). Without that many new genes
waiting to be discovered, it appears likely that the interplay amongst genes will be a new
focus of research. Possibly, the fate of cells might be considerably different when two
genes are expressed simultaneously as compared to a setting in which the individual genes
are expressed. Also, the levels of proteins that are translated in response to ligand
stimulation might be of much more importance then currently acknowledged, which
presumably will be crucial in the determination of cellular decisions.
Candidate genes regulated by STATs that may contribute to oncogenesis are now rapidly
being identified. While constitutive STAT activation is a common characteristic of
leukemias and other human cancers, the specific pattern of activated STATs and the
manner by which STAT activation occurs vary with each disease. Thus, there is increasing
interest in the development of treatments directed against specific steps in the signal
transduction cascades that lead to STAT activation. Human cancers continue to cause
significant mortality in adults and children and the use of standard cytotoxic chemotherapy
has reached its maximal applicability in the clinic. The STAT signaling pathway is an
attractive target for therapeutic intervention, and strategies designed to inhibit STAT
mediated gene transcription should be a focus of future research. An increasing number of
therapeutics that mimic or modulate the activity of cytokines or growth factors are
currently being generated using cell-based or biochemical high-throughput screens, which
will hopefully be applicable in the clinic.