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The role of constitutively active signal transducer and activator of transcription 3 in ovarian tumorigenesis and prognosis
Version of Record online: 24 OCT 2006
Copyright © 2006 American Cancer Society
Volume 107, Issue 11, pages 2730–2740, 1 December 2006
How to Cite
Rosen, D. G., Mercado-Uribe, I., Yang, G., Bast, R. C., Amin, H. M., Lai, R. and Liu, J. (2006), The role of constitutively active signal transducer and activator of transcription 3 in ovarian tumorigenesis and prognosis. Cancer, 107: 2730–2740. doi: 10.1002/cncr.22293
- Issue online: 17 NOV 2006
- Version of Record online: 24 OCT 2006
- Manuscript Accepted: 5 SEP 2006
- Manuscript Revised: 29 AUG 2006
- Manuscript Received: 12 MAY 2006
- American Cancer Society Research Scholar. Grant Number: RSG-04-028-1-CCE
- signal transducer and activator of transcription 3;
- tissue microarray;
- prognostic marker;
- ovarian cancer
Signal transducer and activator of transcription 3 (Stat3), which is a latent transcription factor that participates in the transcriptional activation of apoptosis and cell cycle progression, has been implicated as an oncogene in several neoplastic diseases. However, the specific role of Stat3 in ovarian carcinogenesis remains poorly understood. The objectives of the current study were to examine the effect of Stat3 activation on the phenotypic transformation of an immortalized, nontumorigenic ovarian epithelial cell line and to evaluate the expression of tyrosine-activated Stat3 (pStat3) in tissue microarrays from 303 ovarian carcinomas to determine its prognostic relevance and to correlate its expression with several upstream oncogenes of Stat3 and with the oncogenes involved in apoptosis and proliferation.
Overexpression of pStat3 was weakly tumorigenic and produced measurable tumors in mice in 1 of 3 clones. Using tissue microarrays from a large group of patients with primary ovarian carcinoma, the expression of pStat3 was correlated with the expression of growth factor receptors (HER-2/neu and epidermal growth factor receptor [EGFR]), interleukin 6, and the proliferation and apoptosis markers Ki-67, Bcl-2, and Bcl-xL and with clinicopathologic variables and patient survival.
High pStat3 expression in the tumor tissue microarray was associated with high levels of HER-2/neu, EGFR, and Ki-67. No correlation was observed between overall pStat3 levels and any other clinicopathologic variables tested. High nuclear expression of pStat3 (>10% of positive-stained cells) was linked with poor overall survival.
The activation and translocation of pStat3 to the nucleus are frequent events in ovarian carcinoma that are associated with a poor prognosis. Further studies are needed to elucidate the mechanism of activation of Stat3, its effects on downstream targets, and its role in the neoplastic transformation of epithelial ovarian cells. Cancer 2006. © 2006 American Cancer Society.
Ovarian cancer is the most lethal of the gynecologic malignancies, and the epithelial variants are the most common form of the disease. Approximately 66% of patients with ovarian cancer are diagnosed only at advanced disease stages, contributing to the poor overall survival associated with this disease. The introduction of cisplatin-based treatments in the early 1990s has led to improvements in survival; however, long-term survival is achieved in only 15% of patients who have late-stage disease, and the remaining patients experience persistent or recurrent disease.1
Major advances in understanding the molecular basis of various forms of cancer in recent years hold promise for the development of novel anticancer therapies. Interest has focused recently on signal transducer and activator of transcription 3 (Stat3), a latent transcription factor that has been shown to act as an oncogene in several neoplastic diseases.2 Stat3 normally resides in the cytoplasm and can be activated through phosphorylation by a wide range of cytokines, hormones, and growth factors, all of which utilize Stat3 signaling to control a remarkable variety of biologic responses, including cell development, differentiation, proliferation, motility, and survival.3, 4 Tyrosine-activated Stat3 (pStat3) dimerizes and translocates to the nucleus, where its occupation of specific DNA-binding sites results in the increased transcription of several molecules that are involved directly in cell survival and proliferation.5, 6
It has been demonstrated that Stat3 is activated constitutively in many hematologic and solid tumors.2 Increasing evidence indicates that tumor cells express constitutively activated Stat proteins, particularly Stat3, independent of dysregulation of upstream molecules, disabled inhibitory mechanisms, or identifiable ligand stimulation.7 Stat3 overexpression also may promote cell proliferation and transformation to a tumor phenotype. For example, the introduction of Stat3-C, a molecule engineered to promote dimerization, resulted in accumulation in the nucleus, transformed cells in culture, and formed tumors in nude mice.5
Although Stat3 has been implicated in the initiation and progression of cancer in humans, the regulation and clinical significance of Stat3 protein signaling in human ovarian carcinoma largely remains unknown. The objectives of this study were to examine the effect of Stat3 activation on the phenotypic transformation of an immortalized, nontumorigenic ovarian epithelial cell line and to evaluate the expression of pStat3 in tissue microarrays of 303 ovarian carcinomas to determine its prognostic relevance and correlate its expression with several oncogenes upstream of Stat3 and with the oncogenes involved in apoptosis and proliferation.
MATERIALS AND METHODS
Cell Lines and Stable Transfectants that Express Constitutively Active Stat3
T29 cells, a human ovarian epithelial cell line that has been immortalized with the simian virus 40 (SV40) large-T antigen and with human telomerase reverse transcriptase,8, 9 were cultured at 37°C under 5% carbon dioxide in normal ovarian surface epithelial cell medium (a 1:1 mixture of MCDB105 and M199 media; Sigma Aldrich, St. Louis, MO). All media were supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 2 mM of L-glutamine (Life Technologies, Inc., Gaithersburg, MD). To establish Stat3-C stable transfectants, we transfected T29 cells either with RcCMV-Stat3-C-neo5 or with pBabe-enhanced green fluorescent protein (EGFP)-neo as a negative control and selected stable transfectants that expressed Stat3-C or EGFP by treatment with neomycin (50 μg/mL) at 37°C for 10 days. Clones that contained either the control EGFP or the Stat3 construct were selected individually by using glass cloning cylinders (Sigma Aldrich). Cells that were grown in medium without neomycin were used as controls in various analyses.
Western Blot Analysis
Cells were washed twice with phosphate-buffered saline (PBS) and homogenized in lysis buffer (150 of mM NaCl, 50 mM of N-2-hydroxyethyl piperazine-N′-2-ethane sulfonate [pH 7.2], 1 mM of ethylenediamine tetraacetic acid, 1 mM of ethylene glycol bis-2-aminoethyl ether-N,N′,N′′,N′-tetraacetic acid; 1 mM of dithiothreitol; 0.1% Tween-20; 0.1 mM of phenyl methyl sulfonyl fluoride; 2.5 μg/mL of leupeptin; and 0.1 mM of sodium orthovanadate [Sigma Aldrich]) at 4°C for 30 minutes. Lysates were then spun at × 10,000g for 10 minutes at 4°C. The resulting supernatant fluid was transferred to a clean tube, and the total protein concentration was determined using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The samples were then boiled in polyacrylamide gel sample buffer that contained sodium dodecyl sulfate (SDS) for 5 minutes. Equal amounts (40 μg) of total protein were separated on 7.5% SDS-polyacrylamide electrophoresis gels and transferred to Hybond-C nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ) by electroblotting. The membranes were blocked in 5% milk overnight and were incubated sequentially with the primary antibodies anti-Stat3 (SC-8019; 1:200 dilution) or anti-pStat3 (SC-7993-R; 1:500 dilution; both from Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Sigma; 1:40,000 dilution), followed by secondary antibody (NA 934V; Amersham Biosciences), and visualized with an electrochemiluminescence detection system (Amersham Biosciences). The signal intensity was quantified by image analysis (Alpha Innotech Corp., San Leandro, CA) and was normalized to that of β-actin to obtain arbitrary units of relative intensity.
Tumor Growth in Nude Mice
To study the tumor growth in vivo, 2 × 106 T29 cells that had been infected with the RcCMV-Stat3-C-neo construct or the pBabe-EGFP-neo construct were harvested by trypsinization, washed twice with 1 × PBS, and resuspended in 0.15 mL PBS. The suspensions were then injected subcutaneously and intraperitoneally into BALB/c athymic nude mice aged 4 to 6 weeks (Jackson Laboratory, Bar Harbor, ME). The control cells (T29-EGFP) were injected into separate mice. The mice were kept in pathogen-free environments and were checked every 3 days for 6 months. The date when macroscopically visible tumor first appeared and the size of the tumor were recorded. The mice were killed either when tumors reached 1.5 cm in greatest dimension or after 6 months. All animal experiments were approved by the Institutional Animal Care and Use Committee of The University of Texas M. D. Anderson Cancer Center.
Samples from 322 women with primary epithelial ovarian cancer who had undergone initial surgery at The University of Texas M. D. Anderson Cancer Center between 1990 and 2003 for whom specimens and medical records were available were included. Follow-up information was updated through June 2005 by reviewing medical records. Demographic and survival data were entered into a comprehensive data base that was created with Microsoft Access (version 97; Microsoft Corporation, Redmond, WA). Histopathologic diagnosis was based on World Health Organization criteria, tumor grade was based on Gynecologic Oncology Group criteria, and disease stage was assigned according to the International Federation of Gynecology and Obstetrics system.10–14 Serous carcinomas were graded by using a 2-tier (low-grade/high-grade) system according to criteria recently proposed by Malpica et al.15 For statistical analysis, Grade 2 and Grade 3 endometrioid carcinomas were grouped as “high grade”, and Grade 1 endometrioid carcinomas were considered “low grade.” Disease-specific survival rates were calculated as the percentage of patients who survived with disease for defined periods and are reported as the time since diagnosis or treatment; only deaths from ovarian cancer were counted. The extent of cytoreduction was considered optimal if residual disease after surgery was smaller than 1 cm or suboptimal if residual disease was larger than 1 cm.16, 17 The use of tissue blocks and chart review was approved by the Institutional Review Board of The University of Texas M. D. Anderson Cancer Center.
Construction of the Tissue Microarrays
Tissue blocks were stored under ambient conditions at approximately 24°C. Hematoxylin and eosin-stained sections were reviewed by a pathologist to select representative areas of tumor from which to acquire cores for microarray analysis. Tissue microarray blocks were constructed by taking core needle biopsy samples from morphologically representative areas of paraffin-embedded tumor tissues and assembling them on a recipient paraffin block. This was performed with a precision instrument (Beecher Instruments, Silver Spring, MD) that uses 2 separate core needles for punching the donor and recipient blocks and a micrometer-precise coordinate system for assembling tissue samples on a block. For each patient, 2 replicate, 1-mm core-diameter samples were collected, and each was placed on a separate recipient block. The final tissue microarray consisted of 4 blocks; the first pair (blocks 1a and 1b) contained duplicates of 158 cores and the second pair (blocks 2a and 2b) contained duplicates of 164 cores. All samples were spaced 0.5 mm apart. Five-micrometer sections were obtained from the microarray and stained with hematoxylin and eosin to confirm the presence of tumor and to assess the tumor histology. Tumor samples were arranged randomly on the blocks.
Sample tracking was based on coordinate positions for each tissue core in the tissue microarray block; the cores were transferred onto tissue microarray slides for staining. This sample tracking system was linked to a Microsoft Access database that contained demographic, clinicopathologic, and survival data on the patients who provided the samples, thereby allowing rapid links between histologic data and clinical features. The array was read according to the given tissue microarray map; each core was scored individually, and the results were presented as the means of the 2 replicate core samples. Patients for whom no tumor was found or no cores were available were excluded from the final data analysis.
The tissue microarray and xenograft tumor slides were subjected to immunohistochemical staining as follows. After initial deparaffinization, endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. Deparaffinized sections were microwaved in 10 mM citrate buffer (pH 6.0) to unmask the epitopes. The slides were then incubated with the following primary antibodies: HER-2/neu (A0485; 1:250 dilution [DakoCytomation, Carpenteria, CA]), epidermal growth factor receptor (EGFR) (H11; 1:50 dilution [DakoCytomation]), interleukin 6 (IL-6) (M19; 1:50 dilution [Santa Cruz Biotechnology]), Bcl-2 (100/D5; 1:50 dilution [Biocare Medical, Concord, CA]), Bcl-xL (YTH-2H12; 1:50 dilution [Biocare Medical]), Ki-67 (DVB-2; 1:100 dilution [Biocare Medical]), Stat3 (SC-8019; 1:50 dilution [Santa Cruz Biotechnology]), and pStat3 (SC-7993-R; 1:200 dilution [Santa Cruz Biotechnology]). The xenograft tumor slides also were stained for vimentin (V9; 1:100 dilution [Biocare Medical]), pancytokeratin (LU-5; 1:100 dilution [Biocare Medical]), SV40 large-T antigen (Pab101; 1:200 dilution [Santa Cruz Biotechnology]), and p53 (D07; 1:1000 dilution [Santa Cruz Biotechnology]) followed by biotin-labeled secondary antibody for 20 minutes and, finally, a 1:40 solution of streptavidin:peroxidase for 20 minutes. Tissues were then stained for 5 minutes with 0.05% 3′,3-diaminobenzidine tetrahydrochloride, which had been prepared fresh in 0.05 M Tris buffer (pH 7.6) containing 0.024% hydrogen peroxide; the tissues were then counterstained with hematoxylin, dehydrated, and mounted. All dilutions of antibody, biotin-labeled secondary antibody and streptavidin:peroxidase were made in PBS (pH 7.4) that contained 1% bovine serum albumin. Negative controls were made by replacing the primary antibody with PBS. All controls produced satisfactory results.
For the xenograft tumor samples, a mouse-on-mouse biotinylation kit (MMBK-G; Biocare Medical) was used for the antibodies raised in mice. Briefly, primary antibodies against EGFR, Stat3, pStat3, vimentin, pancytokeratin, and p53 were mixed with the biotinylation reagent followed by the mopping solution for 30 minutes each. After the complexes formed, the primary biotinylated antibody was incubated for 2 hours at room temperature, after which the samples were incubated with streptavidin:peroxidase solution (Biocare Medical) and developed with diaminobenzidine.
The immunoreactivity of EGFR and HER-2/neu was scored on a scale from 0 to 3 + as follows: no staining or membrane staining of <10% of tumor cells was scored as negative (0); weak, noticeable, membranous staining in >10% of tumor cells was scored as 1 +; weak-to-moderate, complete membrane staining in >10% of tumor cells was scored as 2 +; and strong, complete membrane staining in >10% of tumor cells was scored as 3 +. Nuclear expression of the proliferation marker Ki-67 was determined by counting the total number of cells that exhibited nuclear staining, and the Ki-67 labeling index was defined as the percentage of nuclear area that was stained for Ki-67. An index value >15% was considered high, and an index value ≤15% was considered low.18 Immunostaining for IL-6, Bcl-2, and Bcl-xL was performed semiquantitatively on a scale from 0 to 3 in which negative expression (the total absence of staining) was scored as 0, weak staining was scored as 1, moderate staining was scored as 2, and strong staining was scored as 3. Cytoplasmic and nuclear staining for the phosphorylated form of Stat3 (pStat3) were scored separately. Negative staining was defined as the absence of cytoplasmic stain and staining of <10% of the nuclei. Cytoplasmic staining was scored on a 3-point scale based on intensity: negative (no stain; 0), weakly positive (1 +), and strongly positive (2 +). When staining was present but was not present in the cytoplasm, the sample was scored as “nuclear staining only.” Nuclear staining was judged positive if >10% of nuclei in the sample demonstrated staining for the factor of interest and negative if <10% of nuclei demonstrated staining. Staining for pStat3 also was assessed in terms of overall intensity, regardless of the subcellular location of the marker, using the same scale from 0 to 3 described above for assessing IL-6, Bcl-2, and Bcl-xL.
For statistical purposes, negative and weak staining was grouped as “low expression,” and moderate and strong staining was grouped as “high expression.” In all specimens, 10 high-power fields were examined. Normal ovarian epithelial cells were used as a comparison for the intensity and pattern of staining. In all immunostaining results, the means of the findings from the 2 replicate core samples from each tumor specimen were considered. Quantification of cells was performed on the whole core tissue at ×40 magnification, and only tumor epithelial cells were considered. Counting criteria and software settings were identical for all slides. Quantification was performed by pathologists who were blinded to the clinicopathologic information.
Differences in proportions were evaluated using the chi-square test of independence and the Spearman rank-order correlation, as appropriate. Disease-specific survival rates and progression-free survival were calculated using the method of Kaplan and Meier and were compared using log-rank tests. Statistica for Windows (version 6.0; Statsoft Inc., Tulsa, OK) was used for the statistical analyses. Results were considered statistically significant at the P < .05 level.
Constitutive Activation of Stat3 Is Weakly Tumorigenic
To study the role of Stat3 in the transformation of ovarian surface epithelial cells, we used the well-characterized human ovarian immortalized (but nontumorigenic) T29 cell line, as described previously.19 After those cells were transfected with constitutively active Stat3-C vector, 3 viable clones were identified (T29-Stat3-C1, T29-Stat3-C2, and T29-Stat3-C3), and basal levels of Stat3 and pStat3 were assessed by Western blot analysis. Two of the 3 clones (T29-Stat3-C1 and T29-Stat3-C3) expressed considerably more pStat3 than control cells (Karpas 299 and untransfected T29 cells) (Fig. 1A). T29-Stat3-C1 cells also demonstrated lower levels of Stat3 than T29-Stat3-C3 cells (Fig. 1A). We examined the growth rate and cell migration assay on the 3 individual clones and observed no statistically significant difference among the clones in in vitro assays (data not shown).
Only 1 of the 3 transfected clones (T29-Stat3-C1) produced measurable tumors after injection into nude mice, and those tumors did not appear consistently: Only 1 of 4 subcutaneous injections led to tumor formation (Fig. 1C), and only 1 of 3 intraperitoneal injections led to tumor formation (Fig. 1D). Neither the T29 control cells nor the T29-Stat-3C2 or T29-Stat3-C3 clones led to measurable subcutaneous or intraperitoneal tumors at 6 months after injection (Fig. 1B).
Morphologically, the tumors resembled poorly differentiated or undifferentiated carcinoma of the ovary. Hematoxylin and eosin-stained sections of the subcutaneous tumor demonstrated solid neoplastic growth with poor cellular differentiation (Fig. 2A). The intraperitoneal tumor demonstrated the typical histologic growth patterns of ovarian carcinoma. Papillary projections formed around a fibrous core lined by epithelial cells growing into the peritoneal cavity (Fig. 2B). The epithelial tumor cells demonstrated nuclear atypia, increased mitotic activity (>25 mitoses per 10 high-power fields), and stratification.
To verify that the T29-Stat3-C1 cells forming xenograft tumors were derived from the subcutaneously or intraperitoneally injected cells, we stained for pancytokeratin, vimentin, SV40, and p53. Expression of all 4 of these markers (Fig. 2C-F) indicated that the tumor cells indeed were derived from the injected cells. Next, we assessed the expression of HER-2/neu, EGFR, and IL-6, all of which are upstream activators of Stat3. The staining was negative for HER-2/neu and EGFR and weakly positive for IL-6 (Fig. 2G-I). It is noteworthy that staining for 2 common antiapoptosis markers demonstrated no expression of Bcl-2 but demonstrated strong staining for Bcl-xL (Fig. 2J, K). Finally, staining for Stat3 and pStat3 demonstrated high expression of both (Fig. 2L, M).
Expression of Growth Factor Receptors Correlates With pStat3 Expression in Human Ovarian Tumor
Next, we used tissue microarrays to analyze the expression of pStat3 in a large cohort of patients with ovarian cancer. The distribution of staining intensity for the different growth factors and cytokine receptors is shown in Table 1. Because pStat3 staining demonstrated wide variation in both intensity and subcellular location (nuclear, cytoplasmic, or a combination of both) in the ovarian tumor tissue microarray (data not shown), we elected to analyze each subcellular expression pattern separately. Using this method, we found that increased levels of Her2/neu and EGFR were correlated with higher overall levels of pStat3 regardless of the subcellular location of the pStat3 (Table 2). No correlation was observed between pStat3 expression and IL-6 expression (Table 2).
|Score||No. of patients (%)|
|Marker||No. of patients||pStat3 expression|
|Any expression||Nuclear or cytoplasmic expression||Nuclear expression only|
|Spearman ρ||P||Spearman ρ||P||Spearman ρ||P|
Activation of pStat3 Correlates With Proliferation but Not With Apoptosis
We next assessed pStat3 expression compared with expression of the proliferation marker Ki-67 and the antiapoptosis markers Bcl-2 and Bcl-xL in our human ovarian carcinoma tissue microarrays. High expression of overall pStat3 was found to be associated with a high Ki-67 proliferation index (P<.0001) but not with Bcl-2 levels (P = .32) or Bcl-xL levels (P = .61) (Table 2).
pStat3 Expression and Patient and Tumor Characteristics
Of 322 patients who were identified, 303 patients (94%) could be scored for pStat3 staining; the remaining patients either were lost during the sectioning procedure or did not meet the criteria for inclusion. The mean age of the 303 evaluable patients was 58.2 years (range, 20–86 years), the mean follow-up interval was 64 months (range, 1–120 months), and the overall 5-year survival rate was 38%. The results from pStat3 staining sorted according to clinicopathologic characteristics are presented in Table 3. The pStat3 staining patterns were heterogeneous and varied considerably among tumors; a fraction of tumors had only nuclear expression, others had only cytoplasmic expression, and some had both nuclear and cytoplasmic staining (Fig. 3). In 107 patients (35%), no cytoplasmic expression was observed, but nuclear expression varied from 0% to 100% of nuclei. Forty-six tumors (15%) demonstrated weak to strong cytoplasmic expression of pStat3 and no nuclear expression. Overall, pStat3 protein was expressed in 261 tumors (86%) regardless of subcellular location. In general, constitutive pStat3 expression was found to be associated with poorly differentiated (75%), clear cell (73%), and serous carcinoma (63%) histotype (P = .01) but not with any of the other clinicopathologic variables tested (Table 3).
|Characteristic||Overall expression (N = 303)||Subcellular expression (N = 303)||Nuclear expression Only (N = 303)|
|Clear cell carcinoma||4||11||2||1||5||7||3||12|
|Poorly differentiated carcinoma||2||6||1||1||3||3||2||6|
|Transitional cell carcinoma||0||2 1||1||0||2|
|Response to primary therapy|
pStat3 Expression and Survival
Overall survival was compared according to the subcellular localization of pStat3 (Fig. 4A) and according to nuclear expression regardless of the cytoplasmic localization of the marker (Fig. 4B). Results indicated that women who had tumors with high nuclear expression of pStat3 (with >10% of nuclei stained), regardless of cytoplasmic expression level, had poorer survival rates and shorter survival compared with women who had tumors with low nuclear pStat3 expression (<10% of nuclei stained; P = .03).
In the current study, we have demonstrated that the expression of phosphorylated Stat3 is increased in primary human ovarian carcinoma and that its nuclear localization predicts a poor prognosis. We also have demonstrated that increased levels of pStat3 are correlated with increased expression of HER-2/neu, EGFR, and proliferation but not with apoptosis markers. In addition, we demonstrated that, in our tumor xenograft model, the introduction of Stat3 was weakly tumorigenic.
Stat3 has been implicated in the pathogenesis of different human malignancies. Several studies have shown that the constitutive activation of Stat3 can induce neoplastic transformation of normal cell lines and is associated strongly with tumor development and progression in various solid tumors and hematologic neoplasms.2, 20, 21 Likewise, our results indicate that the introduction of Stat3 into the immortalized (but nontumorigenic) T29 ovarian cell line has oncogenic potential, albeit weak. Even though it is believed generally that aberrant Stat3 is important for the initiation, maintenance, and progression of different malignancies,2, 20, 21 to our knowledge the mechanisms by which these take place are poorly understood. Unlike other molecules involved in oncogenesis, no genetic mutations or amplifications have been identified for Stat3, suggesting that persistent Stat3 activity is caused mostly by the dysregulation of upstream molecules, such as receptors with intrinsic tyrosine kinase activity (e.g., EGFR or HER-2/neu), cytokine receptors by endogenous or exogenous IL-6, or nonreceptor tyrosine kinases (e.g., Src or Abl).3, 4 Moreover, the regulation and functions of Stat proteins are highly dependent on the type of cell, the activating stimulus, and the cellular context, especially the activity of other signaling pathways and transcription factors that interact with the Stat proteins.3 Consequently, depending on the cellular context, Stat3 may mediate conflicting responses in terms of cell proliferation, differentiation, or apoptosis. This concept is supported by our current finding that only 1 of 3 Stat3-expressing clones led to tumor development in mice and that this effect was not universal; the relatively weak oncogenic potential of Stat3 suggests that it may require additional genetic events gained in vitro or in vivo to transform human ovarian surface epithelial cells. For example, the concurrent coexpression of dominant-negative Stat3 and the oncoprotein Ras does not arrest Ras-induced transformation,22 suggesting that Stat3 signaling is only 1 of several pathways required for cell transformation induced by this oncogenic tyrosine kinase. In addition, Stat3 demonstrated a histotype-specific pattern of expression. High levels of expression were observed most commonly in those histotypes with more aggressive biologic behavior (undifferentiated, clear cell, and serous carcinomas) than in those histotypes with less aggressive behavior (mucinous and endometrioid carcinomas). The nature of this finding is unclear and should be a matter of further investigation.
Stat proteins can be activated by several different cytokines and growth factors, and 1 of the most common is IL-6. Numerous cells, including those from the ovary, respond to stimulation by IL-6, the secretion of which can be induced by many different factors, including IL-1β, tumor necrosis factor-α, prostaglandin E2, vascular endothelial factor, and growth factor receptors.23 It has been demonstrated that the dysregulation or excessive production of IL-6 in the setting of chronic inflammation is strongly correlated with many types of cancer24; however, whether this also is true in ovarian cancer is controversial. Results from a recent study by Syed et al.25 suggested that IL-6 secretion increases during malignant progression of ovarian epithelial cells. Conversely, results from a study by Ziltener et al.26 indicated that IL-6 expression was reduced in cultures of immortalized and malignant ovarian cells compared with normal ovarian surface epithelial cells. In the current study, approximately 37% of microarray samples demonstrated moderate to high expression of IL-6, and the remaining samples demonstrated negative to low expression. IL-6 expression levels also were not found to be correlated with the expression or subcellular location of pStat3. These results bring into question the possibility that the frequency of IL-6 expression may not be correlated with its biologic activity and also support the finding that IL-6 is involved in other signaling pathways, independent of Stat3, in ovarian carcinoma.27
Other Stat3 upstream molecules with intrinsic tyrosine kinase activity, such as growth factor receptors, can activate Stat3. It has been proposed that the basis of the constitutive activation of Stat3 in cancer cells is because of aberrant EGFR signaling.28 In agreement with this possibility, we observed a significant correlation between high overall levels of pStat3 expression and the overexpression of EGFR and HER-2/neu in human ovarian carcinoma samples. However, relatively few ovarian cancers (<30%) express HER-2/neu29 or EGFR.30, 31 Given the observations that cancer cells can constitutively express Stat3 in the absence of stimulation by any known ligand5 and that expression of Stat3 is higher in ovarian carcinoma than in normal ovarian tissue,4, 32 we speculate that Stat3 may be activated by HER-2/neu or EGFR in ovarian cancer, at least in some patients. Alternatively, we observed previously that the constitutive activation of Stat3 in ovarian cancer may be caused, at least in part, by the elevation of Src and focal adhesion kinase levels.4 Nevertheless, to our knowledge, the correlation between these molecules and Stat3 has yet to be determined. Therefore, in the great majority of patients, other yet to be determined Stat3 activators or dysregulated Stat3-inhibitory mechanisms most likely are responsible for the elevation or persistence of Stat3 activity.
Elevated levels of pStat3 can lead to altered cell function. Depending on the cellular context, activated Stat3 can regulate cell proliferation, apoptosis, or differentiation. The current results indicate that increased Stat3 expression, both in xenograft tumors and in human ovarian carcinoma tissue microarrays, correlated directly with increased proliferation but not with the apoptosis markers.
Finally, the results of our survival analysis indicate that the nuclear expression of Stat3 is associated with poor survival, an observation that constitutes further, indirect evidence that constitutively activated Stat3 may be related to disease progression in ovarian carcinoma. We conclude that the activation of Stat3 and its translocation to the nucleus are frequent events in ovarian carcinoma that are associated with a poor prognosis. Further studies will be needed to elucidate the mechanism of activation of Stat3, its effects on downstream targets, and its role in the neoplastic transformation of epithelial ovarian cells.