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Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3498
Abstract
Previously, we reported that whereas both signal transducers and activators of transcription (STAT) 5A and STAT5B can be activated with respect to tyrosine phosphorylation and DNA binding potential by Src kinase, only STAT5B was translocated to the nucleus, where it presumably activates unique downstream responses. To help elucidate the functional consequences of STAT5B activation by v-src, the properties of stably transfected NIH-3T3 cells containing both an intact and a dominant negative, COOH-terminal-truncated isoform of STAT5B were investigated. STAT5B enhanced the transforming potential of v-Src as reflected by both an increase in focus formation and growth in soft agar. STAT5B also enhanced v-Src-induced cell cycle progression and cell motility in NIH-3T3 cells. Furthermore, the dominant negative, COOH-terminal-truncated isoform of STAT5B was able to partially suppress v-Src-mediated cell transformation. These results support the hypothesis that STAT5B may enhance Src/Abl-induced tumorigenesis. Accordingly, the equilibrium between STAT5B and STAT5A and their naturally occurring truncated forms may therefore play a key role in the etiology of certain cancers.
Introduction
Protein tyrosine kinases have been implicated in the regulation of both proliferation and differentiation of mammary cells. Deregulation of many non-receptor tyrosine kinases results in a variety of pathological conditions including cancer (1, 2, 3) . In fact, a number of primary tumors and tumor-derived cell lines including breast cancer, colon cancer, melanoma, and sarcoma have been shown to possess elevated Src tyrosine kinase activity (4, 5, 6, 7) . Src and its family members are required for mitogenesis initiated by several growth factor receptors, including epidermal growth factor receptor (8 , 9) , platelet-derived growth factor receptor (10 , 11) , basic fibroblast growth factor receptor (12 , 13) , and colony-stimulating factor 1 receptor (8 , 14) . It is now well established that Src kinases play an important role in cell cycle control, cell proliferation and differentiation, and cell adhesion and movement (15) . Additional roles for Src kinases have been reported recently in the control of cell survival (16) and angiogenesis (17) .
In mammalian cells, seven STAT2 proteins have been described (18) . This family of transcription factors becomes activated by tyrosine and serine phosphorylation. After tyrosine phosphorylation on a single tyrosine residue (located at approximately amino acid 700), STATs dimerize and translocate into the nucleus, where they bind to specific DNA elements and regulate transcription of the respective target genes.
It has been demonstrated that in addition to their signaling functions in normal cells, STATs can also participate in oncogenesis (19) . Originally, cytokine receptor-mediated activation of the JAK family of tyrosine kinases was identified as the primary mechanism of STAT protein activation. More recently, a number of reports have correlated STAT activation with the activity of non-receptor proto-oncogenic tyrosine kinases such as v-Src (20, 21, 22) , v-Abl (23) , Lyn (24) , Lsk (25) , Bcr/Abl (26) , and Fes (27) . In particular, STAT3 activation has been shown to be required for transformation of mammalian fibroblasts (19 , 28) , and it was suggested that STAT3 can function as an oncogene (29) . There is some evidence that v-Src and Bcr/Abl do not activate JAK kinases but instead may be directly associated with STATs (30 , 31) . Furthermore, activation of Src kinases by cytokines may not always be dependent on JAK kinase activation. For example, stimulation of c-Src by prolactin is independent of JAK2 (32) .
In previous studies (33) we observed that STAT5B contains a unique sequence that may be a potential Src/Abl kinase recognition site. Immediately proximal to this site was a putative, additional nuclear localization domain. We demonstrated that whereas both STAT5A and STAT5B could be activated with respect to their tyrosine phosphorylation and DNA binding potential by Src kinase, only STAT5B was translocated to the nucleus, where it presumably activates downstream responses.
In the present study, we demonstrate that STAT5B accelerates v-Src oncogenic activity and, to a limited extent, cell growth. More importantly, STAT5B dramatically increases v-Src-induced tumorigenicity and cell motility. Furthermore, STAT5B significantly enhances Src-induced cell cycle progression in NIH-3T3 cells. Finally, a COOH-terminal-truncated, potentially dominant negative form of STAT5B is able to partially suppress these effects of v-Src. These results support the hypothesis that STAT5B may enhance Src/Abl-induced tumorigenesis. Accordingly, the equilibrium between STAT5B and STAT5A and their naturally occurring truncated forms may, therefore, play a key role in the etiology of certain cancers.
Results
Generation of Stably Cotransfected Cell Lines.
To test the effect of STAT5 on v-Src-inducible transformation, we used
bicistronic vectors pEFIRES-N or pEFIRES-P (34)
. In these
vectors, both the recombinant cDNA and the antibiotic resistance gene
(to neomycin or to puromycin) are transcribed as a single transcript
driven by the human polypeptide chain elongation factor 1
promoter.
The presence of an internal ribosome entry site ensures that any clones
that are resistant to the selected antibiotic also express high levels
of recombinant protein encoded by the cDNA cloned upstream from the
antibiotic resistance gene. STAT5 cDNAs for STAT5B, STAT5A, and
STAT5B
40 [the naturally occurring COOH-terminal-truncated form of
STAT5B lacking the transactivation domain (35)
] were
cloned into the vector containing the neomycin resistance gene.
v-src cDNA was inserted into a vector encoding the puromycin
resistance gene. NIH-3T3 cells were then transfected with the v-Src-
and STAT5-containing plasmids, and clones resistant to puromycin and to
neomycin were selected. Expression of the corresponding proteins was
confirmed by Western blot analysis (data not shown).
STAT5B Accelerates v-src-inducible Focus
Formation.
To study the role of STAT5 in v-src-inducible cell
transformation, a focus formation assay was first used, which, in the
case of v-Src transformation, has been reported to correlate well with
growth in soft agar and tumorigenesis (36)
. NIH-3T3 cells
coexpressing STAT5B and v-Src began forming foci as early as 40 h
after plating (Fig. 1A)
and formed large colonies after 3 days (Fig. 1B)
. Cells expressing v-Src alone, v-Src in combination with
STAT5A, or v-Src in combination with the truncated form of STAT5B
(STAT5B) did not form foci during this time period (Fig. 1)
.
Normally, NIH-3T3 cells expressing v-Src began to form foci only
after 7–9 days, when they were plated at identical cell densities.
Expression of STAT5 proteins without v-Src did not induce focus
formation (data not shown). Thus, STAT5B appears to accelerate
v-Src-inducible transformation of NIH-3T3 cells.
|
B) resulted in >50%
suppression of v-Src-inducible colony formation. However, STAT5A in
combination with v-Src did not significantly change the transforming
potential compared with v-Src alone (Fig. 2)
.
|
. STAT5B in combination with v-Src also resulted in increased cell
growth, but these results were not significantly different as compared
with v-Src-transformed cells alone. Interestingly, however, the
COOH-terminal truncated form of STAT5B (STAT5B), when expressed
together with v-Src, decreased cell growth by 50% even when compared
with cells transformed with the control vectors (Fig. 3)
.
|
. After 1 month of serum withdrawal, many cells expressing both v-Src
and STAT5B were still alive, whereas those expressing v-Src alone were
dead. The truncated form of STAT5B had the opposite effect: after 3
days of serum withdrawal, all of the cells expressing v-Src and
STAT5B were nonviable (Fig. 4)
.
|
A, NP) arrested in the G1 phase of the
cell cycle. After 24 h of starvation, only 3.5–6.5% of the cells
were seen in the S + G2 phases of the cell cycle
(Fig. 5A)
. Transformation by v-Src increased cell cycle
progression (Fig. 5A)
, and coexpression of STAT5B and v-Src
significantly enhanced the number of cells in S +
G2 (Fig. 5A)
. After 5 days of serum
deprivation, expression of STAT5B resulted in a 3-fold greater number
of cells in S + G2 compared with cells expressing
v-Src alone (Fig. 5B)
.
|
. Interestingly, the COOH-terminal truncated form of STAT5B (STAT5B)
did not exert any detectable effect on v-Src-transformed
cells in this experiment.
|
The tumorigenic phenotype is characterized by the ability of cells to continuously proliferate in the absence of constant stimulation by growth factors. Tumor progression can be viewed as progression of tumorigenic cells toward an autonomous state, where they may no longer be dependent on paracrine interactions. It is not always apparent whether a specific gene is important in control of malignancy or is involved only in tumor initiation (reviewed in Ref. 39 ).
Recent studies suggest that activated STATs participate in oncogenesis (40) . Transformation by tyrosine kinase fusion genes associated with human leukemia has been persistently linked to activation of STAT5. For example, Bcr/Abl expression leads to STAT1 and STAT5 activation (26 , 30 , 41 , 42) . STAT5 is also activated by the Tel/platelet-derived growth factor ß receptor (PDGFßR), Tel/Abl, and Huntingtin Interacting Protein I/PDGFßR oncogenic fusion proteins (43 , 44) .
Although STAT5A and STAT5B share 93% identity at the amino acid level, in previous studies we observed that only STAT5B was able to translocate to the nucleus in response to Src and Abl kinase activation (33) . Studies of STAT5A and STAT5B knockout mice have also revealed significantly different phenotypes. STAT5A knockout mice showed impaired mammary gland development and lactation and a partial defect in T-cell growth. In contrast, STAT5B knockout mice are characterized by impaired sexual dimorphism and defects in T-cell growth and in natural killer cell development (45, 46, 47, 48) . However, there are differences in the phenotypes of mice with STAT5A or STAT5B mutations reported by different groups of investigators (49) that may be due to differences in the strain backgrounds used or the exact mutation constructed.
The experiments described above demonstrate that activation of
STAT5B accelerates focus formation of v-Src-transformed cells.
This acceleration may reflect the participation of STAT5B in tumor
progression. The assay measuring anchorage-independent growth of
cells revealed that STAT5B increases the transforming potential of
v-Src, demonstrating a possible role of STAT5B in enhancement of
tumorigenesis. Although endogenous STAT5B or STAT5A proteins were not
readily detectable in NIH-3T3 cells, the overexpression of the dominant
negative truncated form of STAT5B (STAT5B) suppressed
v-Src-inducible colony formation by more than 50%. We reported
previously (33)
that whereas constitutively active c-Src
bound to STAT5, it did not directly phosphorylate STAT5. Thus, the
inhibition of the transforming potential of v-Src by STAT5B might be
due to a competitive inhibition of Src action on other potential
substrates. STAT5B in combination with v-Src induced cell growth, but
not to an extent sufficient to explain the observed acceleration of
focus formation. This presumably also reflects the additional
effects of STAT5B and v-Src on cell survival and cell motility and
possibly reflects effects on cell-cell interactions and the actin
cytoskeleton.
A number of different studies have also demonstrated the direct participation and requirement of STAT3 in transformation in response to Src kinase activation in NIH-3T3 cells (19 , 28 , 29) . Therefore, there is a possibility that STAT5B and STAT3 may cooperate in the process of transformation. Thus, the functional importance of STAT3/STAT5 heterodimers in Src transformation needs to be investigated further.
Src kinase is known to influence cell survival (16)
. In
this study, we observed that STAT5B dramatically increased the survival
of v-Src-transformed cells grown in serum-free media, whereas STAT5B
exerted the opposite effect. This property of STAT5B suggests that it
may be an important player in the process of tumor progression in
certain cell types. Studies using STAT5A knockout mice have also
suggested a role for STAT5A as a survival factor during mammary gland
development and tumorigenesis (50)
.
v-Src is capable of stimulating quiescent cells out of G0 and progression through the G1 phase of the cell cycle (38) . Our results demonstrate that STAT5B significantly enhances this stimulation, which is detectable as early as 24 h after serum withdrawal. After 5 days of serum starvation, the percentage of cells in S + G2 in v-src-transformed cells decreased by 3-fold, whereas it did not change significantly in cells expressing STAT5B and v-src.
The distinction between oncogenesis and tumor progression is critical when one is determining whether a gene is important in controlling steps associated with malignancy or is simply involved in tumor formation (39 , 51) . One of incontrovertible hallmarks of malignancy is the ability to invade through the basement membrane and/or to metastasize. Motility is a required step in the acquisition and maintenance of tumor cell invasion and metastasis. Accordingly, approximately a 3-fold increase in cell motility was observed as a consequence of the expression of STAT5B in v-Src-transformed cells.
In some cases, STAT5 activation is both necessary and sufficient for disease pathology. For example, activation by Tel/JAK2 of STAT5 target genes can significantly affect cell proliferation. However, in other situations (Bcr/Abl or v-Abl), STAT5 may not play a critical role, although its activation is a hallmark of these leukemias (52) . In this situation, STAT5 may potentiate the malignant phenotype but is not required for transformation. In agreement with these in vivo observations, the present experiments also provide evidence that STAT5B can serve to accelerate and enhance tumor formation but is not necessarily required for the initiation of transformation. STAT5B has the ability to potentiate tumor progression in certain cell types and may therefore be required for malignant progression. In addition, these studies suggested that participation of STAT5B in these processes is due to increased cell cycle progression. Recent in vivo studies have indicated that T cells from STAT5 knockout mice display impaired proliferation and failed to undergo cell cycle progression or to express some of the genes controlling cell cycle progression (52 , 53) .
Based on previous observations (33) and the present results, we hypothesize that inappropriate regulation of STAT5 may play an important role in the etiologies of certain cancers. If this occurs by aberrant regulation of JAK kinase activity, both STAT5A and STAT5B may be involved in signal transduction and possibly in potentiation of cell transformation. On the other hand, if the cancer arises due to the inappropriate activation of Src/Abl kinases, STAT5B may be primarily involved in potentiating transformation. STAT5B may not be required for the initiation of transformation but instead may help facilitate malignant progression.
Materials and Methods
Plasmids.
STAT5B, STAT5B, and STAT5A were cloned into the bicistronic vector
pEFIRES-N (34)
. v-Src cDNA was kindly provided by Drs.
Hiromitsu Hanafusa and D. Besser (The Rockefeller University, New York,
NY) and cloned into pEFIRES-P vector (34)
. Bicistronic
vectors were kindly provided by Dr. Steven Hobbs (Cancer Research
Campaign Center for Cancer Therapeutics, Institute of Cancer
Research, London, United Kingdom).
Tissue Culture.
NIH-3T3 cells were grown in DMEM (JRH Biosciences) supplemented with
10% BCS. LipofectAMINE Plus reagent (Life Technologies, Inc.) was used
for all transfections. Transformants were selected with 2 ng/ml
puromycin (Sigma) and 800 ng/ml G418 sulfate (Geneticin; Life
Technologies, Inc.).
Focus Formation Assay.
Stably cotransfected NIH-3T3 cells (105
cells/35-mm-diameter dish) were plated and seeded for 40 h
and for 3 days in medium supplied with puromycin and G418 (see above).
Cells were photographed using phase-contrast microscopy.
Soft Agar Colony Formation Assay.
The soft agar assay was performed mainly as described previously
(37)
. Stably cotransfected NIH-3T3 cells
(104 cells) were plated into 2 ml of soft agar
(BiTek; Difco) containing DMEM, 10% BCS, 2 µg/ml puromycin, and 800
µg/ml G418 on top of 0.6% agar containing DMEM, 10% BCS, 2 µg/ml
puromycin, and 800 µg/ml G418. These assays were carried out in
parallel with three replicates. Colonies were counted and photographed
after 11 days of incubation.
Cell Proliferation Assay.
Cell proliferation assays of stably transfected NIH-3T3 cells
were performed using the calorimetric MTS assay CellTiter 96 AQ
(Promega, Madison, WI). Cells were plated in 98-well plates (2 x
103 cells/well). Cells were analyzed after
12 h of growth in complete media and 36 h in serum-free media
according to the manufacturer’s protocol.
Cell Cycle Analysis.
Cell cycle distribution experiments were performed with an EPICS XL
flow cytometer (Beckman Coulter). A total of 3–5 x
105 cells were pelleted by centrifugation and
resuspended in 0.5 ml of PBS. During vortexing, 5 ml of ice-cold
ethanol were added. The cell suspension was incubated overnight at
4°C. The fixed cells were pelleted by centrifugation and resuspended
in 0.75 ml of PBS. RNase A (Boehringer Mannheim) was added to a final
concentration of 0.1 mg/ml, and cells were incubated at 37°C
for 30 min. After the addition of propidium iodide (Boehringer
Mannheim) to a final concentration of 0.05 mg/ml, the DNA content of
cells was analyzed by flow cytometry (fluorescence-activated cell
sorting). The distribution of cells in different phases of the cell
cycle was quantitated with the computer program for this flow cytometer
(System II version 3.0) and is shown in a bar diagram (see Fig. 5
) presenting the percentage of cells in S +
G2 phases. The experiment was repeated twice,
with each run performed in duplicate.
Cell Migration Assay.
Cell migration was determined using a modified Boyden chamber
containing a polycarbonate membrane (6.5-mm diameter; 10-um pores;
Transwell; Costar, Cambridge, MA; Ref. 54
). The membrane
was coated with gelatin. Cell suspensions (5 x
105 cells/500 µl DMEM) were added to the top
compartment. The lower compartment was filled with DMEM containing 10%
BCS. The chamber was then incubated at 37°C in a 5%
CO2 atmosphere. After 12 h, the number of
cells that had migrated to the lower compartment was counted under a
phase-contrast microscope after trypsinization. The experiment was
performed twice, and each run was performed in triplicate.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be
addressed, at Department of Molecular and Cellular Biology, Baylor
College of Medicine, Houston, TX 77030-3498. E-mail: jrosen{at}bcm.tmc.edu 
2 The abbreviations used are: STAT, signal
transducers and activators of transcription; JAK, Janus-activated
kinase; BCS, bovine calf serum.
Received for publication 7/19/00. Revision received 11/ 3/00. Accepted for publication 11/ 6/00.
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